THE SIGNIFICANCE OF HEAVY CHAIN CDR3 DIVERSITY IN THE ANTIBODY RESPONSE TO POLYSACCHARIDES TAMER I. MAHMOUD

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1 THE SIGNIFICANCE OF HEAVY CHAIN CDR3 DIVERSITY IN THE ANTIBODY RESPONSE TO POLYSACCHARIDES by TAMER I. MAHMOUD JOHN F. KEARNEY, COMMITTEE CHAIR PETER BURROWS DAVID D. CHAPLIN CHRISTOPHER KLUG HARRY W. SCHROEDER, JR. A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2009

2 THE SIGNIFICANCE OF HEAVY CHAIN CDR3 DIVERSITY IN THE ANTIBODY RESPONSE TO POLYSACCHARIDES TAMER I. MAHMOUD MICRBIOLOGY ABSTRACT An understanding of the molecular mechanisms involved in the generation of protective antibody responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design. The heavy chain third complementarity-determining region (HCDR3) of an antibody molecule is at the center of its antigen-binding site and plays a determinative role in antigen recognition. The goal of this dissertation was to investigate the role of HCDR3 diversity and composition on the antibody response to the polysaccharide α 1 3 Dextran (DEX). In the first study, we investigated the role of TdT, a DNA polymerase that plays a major role in generating diversity of lymphocyte antigen receptors during V(D)J recombination. We show that the DEX-specific antibody response is lower and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT-/- mice when compared to wild type BALB/c (WT) mice. Complementation of TdT expression in TdT-/- mice by early forced expression of TdT restored WT expression of J558 Id+ and also abrogated the development of the minor M104E Id+ clones. These data suggest that TdT is essential for the generation of the higher affinity DEX-responsive J558 clone. In the second study, we investigated the DEX-specific antibody response of genetargeted mice limited to use one D H gene segment in different reading frames (Dlimited mice). We show that the DEX-specific antibody response is comparable in D- ii

3 limited mice to that of WT mice. D-limited mice forced to use D H reading frame 2 ( D-DµFS) showed higher antibody responses to DEX compared to other D-limited mice and higher levels of J558 Id compared to WT mice. D-DµFS DEX-specific plasma cells contained greater numbers of V(D)J rearrangements that encoded for expression of J558 Id compared to other D-limited mice. D-limited mice had a higher frequency of D H -less HCDR3 regions compared to WT. These data suggest that constraints on HCDR3 diversity by limiting D H usage do not preclude the generation of a proper anti-dex response and that altered D H reading frame usage could enhance the generation of the anti-dex clone that is predominant in the WT. iii

4 DEDICATION To my parents, Drs. Ibrahim Mahmoud and Suheir Ali, my wife Enas, my twin children Yusuf and Noor and to my best friend, Mohamed Farid, may he rest in peace. iv

5 ACKNOWLEDGEMENTS None of this work would have been possible without the incredible support I received from many individuals. I thank John for taking a student with no significant experience, like myself, as part of his research group. John makes it hard for anyone to blame him if the work does not go so well; he is always available and provides each and everyone of us with privileges I believe many students and postdocs in Ivy League schools do not have. I hope that I have met his expectations as a student and I look forward to a lasting relationship with him and his research group. I would like to thank past and present members of the Luckie lab: Jeremy Foote, whom I had the privilege of sharing lab space with, has contributed significantly to my work through his discussions, suggestions and insightful comments. We have also had very thought-provoking discussions about life, politics, sociology and even theology. Our camaraderie has been instrumental in the battle against frequent research frustrations. I wish him the very best in his endeavor to become a DVM. I am very grateful to Nic Kin for the time he took to teach me some basic techniques such as injecting and bleeding mice. He spent a significant amount of time teaching me how to write a manuscript and I thank him for that. I am also indebted to Marilyn Wright and Jeffrey Sides for their assistance with animal husbandry; To-Ha Thai for her hands-on training during my rotation in the lab; Thiago Carvalho for his suggestions and critical comments; Brian v

6 Dizon for injecting mice before I started injecting by myself and the rest of the Luckie lab members for their discussions at our weekly lab meetings. I also thank the labs of Drs. Harry Schroeder and David Briles for their limitless support. Dr. Schroeder has been like a second advisor to me and I have consulted with him very often in my work. He has shared data and reagents, no strings attached. We have also collaborated on the work I report in this dissertation on the DEX-specific antibody response elicited by D-limited mice. Members of his lab have also been generous with me and I am obliged to Ivaylo Ivanov, Bob Schelonka and Yingxin Zhuang. Drs. David Briles and Glenn Ulett have adopted me for about a month in their lab as I was trying to establish a urinary tract infection model. Although our work together did not culminate into publishable data, I have learned a lot and I thank them for their support. Last but not least I would like to thank Dr. Rizk Elgalley who has had a significant impact on my life experience here. I thank him for hosting us in his home every Sunday afternoon where we learned so much from his vast knowledge and experience. vi

7 TABLE OF CONTENTS Page ABSTRACT...ii DEDICATION...iv ACKNOWLEDGEMENTS...v LIST OF TABLES...viii LIST OF FIGURES...viii INTRODUCTION...1 Beginnings of Vaccinology...1 Antibodies...2 Antibody structure and function...3 The antibody variable Ig domain...3 Generation of HCDR3 diversity by TdT and its physiological significance...6 Other factors that contribute to HCDR3 diversity...10 Role of D H gene segments in generating HCDR3 diversity...14 Scope and objective of dissertation...16 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IS REQUIRED FOR AN OPTIMAL RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN...20 THE ANTIBODY RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN IS ENHANCED IN D-LIMITED MICE FORCED TO USE D H READING FRAME CONCLUSIONS AND FUTURE PERSPECTIVES...80 GENERAL LIST OF REFERENCES...86 APPENDIX: IACUC APPROVAL...99 vii

8 LIST OF TABLES Table Page INTRODUCTION 1 Vκ light chain CDR3 (LCDR3) junctions are comparable between TdTL transgenic mice and controls...13 LIST OF FIGURES Figure Page INTRODUCTION 1 VH 7183 HCDR3 is comparable between TdTL transgenic mice TdT-/- controls...12 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IS REQUIRED FOR AN OPTIMAL RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN 1 Ontogeny of DEX-responding B cell clones TdT-/- mice elicit a lower antibody response to DEX and fail to generate the J558 Id+ clone TdTS x TdT-/- restore J558 Id+ antibody levels to DEX Adult WT and TdT-/- pre-immune mice have similar numbers of DEX-binding B cells WT mice response to DEX shows higher J558 Id expression and greater diversity of anti-dex antibodies TdTS expression correlates with J558 expression when J558 Id starts to emerge...37 viii

9 LIST OF FIGURES (Continued) Figure Page THE ANTIBODY RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN IS ENHANCED IN D-LIMITED MICE FORCED TO USE D H READING FRAME 2 1 ΔD-DµFS generate a higher J558 Id+ anti-dex antibody response Adult D-limited and WT pre-immune mice have similar numbers of DEX-binding B cells in the spleen but not the peritoneal cavity The anti-dex antibody repertoire of D-limited mice, except ΔD-DiD, is comparable to that of the WT HCDR3 regions of DEX-specific plasma cells from D-limited have similar hydrophobicities to those of the WT ΔD-DµFS generate more nucleotide sequences encoding for J558 Id compared to other D-limited mice A significant percentage of DEX-specific HCDR3 regions from D-limited mice have D H -less V(D)J joins...68 ix

10 1 INTRODUCTION Beginnings of vaccinology The specific antitoxins which represent the active principle of blood serum therapy have only been found in the blood of immunized animals. (Behring, 1894) The attempts to prevent infectious diseases by means of boosting the host s immune system predate the science of vaccinology and Immunology. Smallpox was the first disease people tried to prevent by purposely inoculating themselves with other types of live infections. Smallpox inoculation was started in China or India before 200 BC. However it was not until 1796 when British physician Edward Jenner tested the possibility of using the cowpox vaccine as an immunization for smallpox in humans for the first time did the science of vaccination emerge (1). Louis Pasteur furthered the concept through his pioneering work in microbiology by establishing the germ theory of disease and by generating live attenuated or weakened virus to make the first vaccine to rabies (2, 3). Emil Von Behring, Shibasaburo Kitasato and Paul Ehrlich, at the Institute for Infectious Diseases, headed by Robert Koch, developed the beginning of the theory of humoral immunity or passive vaccination in their search for successful serum therapies to Diphtheria and Tetanus toxins (2, 3). The concept of receptor-ligand binding was introduced and defined immunologic specificity as we know it today. The component of serum that mediates protection was not exactly known.

11 2 Behring believed that the antitoxin effect was mediated by the protein fraction of serum. He could show that ammonium sulphate precipitated a fraction of serum that retained the anti-toxin effect (now known as gammaglobulin). Paul Ehrlich proposed that preformed side chains on the surface of cells interacted with the toxins and that immunity arose because of an over production of these side chains. We know now that antibodies, also known as immunoglobulins, are the active components of gammaglobulin that mediated protection in these early pioneering studies (4). Antibodies Protection conferred by most successful vaccines in use today is predominantly antibody based (5). Among the earliest studies that demonstrated the capability of antibodies to protect against viral disease was the study that showed protection from measles upon administration of concentrated human immunoglobulin derived from normal adults (6). Patients with immunoglobulin deficiencies lack immunity and are routinely given purified immunoglobulin preparations, pooled from the blood of random donors, to protect them from infection. Maternal transfer of antibody to the fetus has been shown to give protection against Herpes simplex virus (7). Together with several demonstrations of protective immunity conferred by passive administration of vaccineinduced antibodies against bacterial toxins, encapsulated bacteria and viruses (8-16), the significance of antibody-mediated protection against infection is indisputable.

12 3 Antibody structure and function Antibodies are glycoproteins consisting of two identical heavy chain-light chain heterodimers that are linked by disulfide bonds. Each heavy chain is made up of one N- terminal variable (V) immunoglobulin (Ig) domain and three or four constant (C) C- terminal Ig domains, whereas the light chain is made up of one V and one C domain. The V domains of the heavy and light chain constitute the antigen-binding site of the antibody. The heavy chain C (C H ) domains of the antibody are responsible for the antibody s effector functions, which vary according to the antibody isotype (17, 18). Antibodies exert their effector function through a variety of mechanisms. First, V domains recognize a certain determinant or epitope on the surface of the invading pathogen. Then, C H domains can promote the phagocytosis of the pathogen by binding to Fc receptors expressed by professional phagocytes such as macrophages and neutrophils. Alternatively, C H domains can activate the complement system, whose components can either promote phagocytosis or induce cell membrane lysis by activation of the membrane attack complex (17, 18). The antibody variable Ig domain The discovery of multiple myeloma, a cancer of antibody-producing plasma cells, allowed for the purification and isolation of large quantities of clonal isolated Ig light chains, called Bence-Jones myeloma proteins, from the urine of patients. This finding permitted the sequencing of individual myelomas and hence the rational analysis of antibody structure and function (19). Aligning the sequences obtained from several

13 4 individuals with the disease allowed for the detection of highly variable V domains and extremely conserved C domains. Further examination revealed that there were three regions within the heavy and light chain V domain that were more variable than others and were termed hypervariable regions. Intervals between hypervariable regions where the sequences were relatively stable were called framework regions. Studies that examined the three-dimensional structure of the antigen-binding site could show the hypervariable regions binding to antigen and were therefore called complementaritydetermining regions (CDRs) (20-23). Therefore, it is generally accepted that CDRs of heavy and light chains encode for the receptor function and monovalent specificity of the antibody molecule. The mechanism of generation of diversity in the V domain of the antibody was a puzzle for many years. If a separate gene encoded for each antibody V domain, then these incredibly diverse molecules would be encoded by a large segment of the genome. This led Dreyer and Bennett to propose the two genes, one polypeptide postulate (24). This hypothesis was experimentally confirmed by Tonegawa (25-27) and others (28, 29) whose seminal work was among the first to show that somatic rearrangement of Variable (V), Diversity (D) and Joining (J) gene segments generated antibody diversity in a process called V(D)J recombination. V(D)J recombination is initiated in the Ig locus by the lymphoid-specific RAG-1 and RAG-2 proteins (30, 31) which introduce DNA double strand breaks at specific sites called recombination signal sequences (RSSs) that flank rearranging gene segments. RSSs consist of conserved nonamer and heptamer sequences separated by a spacer of either 12 or 23 nucleotides (32, 33). V(D)J recombination is only efficient between RSSs

14 5 with 12 and 23 nucleotide-long spacers. The RAG proteins bind to an RSS and introduce a single strand nick to generate a 3 hydroxyl group. The RAG-RSS complex synapses with another RSS and uses the 3 hydroxyl group as a nucleophile to attack the opposite strand in a trans-esterification reaction to generate a DNA double strand break. The result of these reactions are two hairpin gene coding ends and two blunt 5 phosphorylated signal ends, which are associated with the RAG proteins in a post-cleavage complex. The intervening DNA between the coding gene hairpins, which contains the 5 phosphorylated RSSs, is joined, with the RSSs head to head, together to form a signal joint (32, 33). The gene coding hairpins are resolved by an endonuclease, called Artemis, whose enzymatic activity is dependent on phosphorylation by DNA-dependent Protein Kinase (DNA-PK) (34). The gene coding ends now have 3 overhangs, which undergo extensive nucleotide modifications (see below) before they are ligated together using the non-homologous end joining (NHEJ) DNA repair proteins (32, 33). Recombination begins at the heavy chain locus in developing pro-b cells of mice with D H J H rearrangements resulting from the joining of one of 13 D H gene segments to one of 4 J H gene segments. This is followed by V H D H J H joining, where one of 110 functional V H gene segments is recombined (35-37). Therefore, diversity from heavy chain recombination alone generates 3.4 x 10 4 possible germline combinations. Heavy chain framework regions 1 through 3, CDR1 and CDR2 are encoded entirely by the V H gene segment, whereas the J H segment encodes framework region 4. CDR3 contains the entire D H gene segment, portions of the 3 V H and 5 J H regions as well as from nucleotide modifications that take place during V(D)J joining (17). After the successful rearrangement of the heavy chain, recombination takes place at the κ or λ light chain

15 6 locus where V L J L joining takes place. The κ locus includes 4 Jκ, 73 Vκ functional gene segments in addition to 1 Cκ (38-41). The λ locus includes 3 Vλ, 3 Jλ and 2 or 3 Cλ gene segments (42, 43). Any heavy chain can combine with any one light chain thus increasing the potential germline combinations to 1 x 10 7 (17). These potential combinations are based on minimal calculations and are not a reflection of the actual estimate of different B cell receptors that can be generated. Diversity afforded by recombination of germline segments is enhanced several fold by junctional diversity, the non-germline modifications that occur at the heavy and light chain CDR3. Junctional diversity, which is most significant in heavy chain CDR3 (HCDR3) regions, can result from imprecise V(D)J joining, exonucleolytic loss, palindromic addition as well as non-templated random nucleotide addition mediated by the DNA Polymerase Terminal deoxynucleotidyl Transferase (TdT) (26, 28). This seemingly limitless diversity at the HCDR3 is reflected in its length, which can range from 5-20 amino acids in mouse bone marrow B cells (44). In contrast, the length of the light chain CDR3 seems to be under tight control, limiting the potential for light chain junctional diversity (43, 45). Together with its position at the center of the antigenbinding site (46), HCDR3 represents the greatest focus for the primary somatic diversification of the antibody repertoire. Generation of HCDR3 diversity by TdT and its physiological significance One of the most significant factors in generating HCDR3 diversity is the nontemplated nucleotide addition (N-addition) mediated by TdT (47, 48). TdT is a nuclear enzyme expressed in developing B cells and T cells. TdT transcripts are also detected in

16 7 myeloid progenitor cells in the mouse bone marrow (T.H Thai and John F. Kearney, unpublished observations). TdT expression is absent in fetal and neonatal hematopoietic progenitors in mice. In humans, however, TdT activity is detected in fetal life albeit at much lower levels than in the adult (49). In developing B cells, its activity is limited to the junctions of rearranging heavy chain V(D)J joins (50, 51). Adult TdT-deficient (TdT-/-) mice show many features of the fetal and neonatal antigen receptor repertoires. Homology-directed recombination and canonical V(D)J joins are enhanced in adult TdT-/- to the levels seen in fetal Ig and T cell receptor (TCR) repertoires (50). Homology-mediated recombination products contain 1 6 nucleotides that could have been encoded by either of the two joined gene segments. This phenomenon leads to the increased likelihood that certain CDR3 regions are repeatedly generated leading to an overall significant decrease in diversity. The catalytic activity of TdT was shown to significantly decrease the frequency of homology-mediated joining in a recombination assay using episomal substrates (52). Using the human Ig heavy chain minilocus transgenic mice, TdT has been shown to influence V H, D H and J H gene segment usage (53). Adult TdT-/- minilocus transgenic mice showed V H gene usage similar to that of fetal TdT-sufficient transgenic mice. The authors proposed a mechanism that is independent of increased propensity of homology-mediated joining, where N-addition influences the ligation efficiency mediated by NHEJ repair factors in the post-cleavage complex (53). The activity of TdT has been shown to be a major contributor to TCR diversity. Sequence analysis of the TCR α/β repertoire in TdT-/- mice showed a 90% decrease in diversity (54). Despite this dramatic decrease in diversity, studies have suggested that

17 8 TdT is not required in generating efficient T-cell and antibody responses (48, 55). In addition, the public α/β TCR repertoire, which is the repertoire observed in response to immunodominant and subdominant peptides common to all mice or humans of the same MHC haplotype, is conserved in TdT-/- mice (56). However, there is mounting evidence that the advantage offered by the diversifying activity of TdT is subtle and is better measured at the level of clonal populations. Heterosubtypic immunity to Influenza A virus infection, which affords cross-protection against a different virus subtype, has been shown to be impaired in TdT-/- mice (57). This observation may be explained in part by the finding that TdT is required for the generation of private influenza virus-specific CD8+ repertoires that contribute to enhance diversity in TCRs across species as a whole (58). Yewdell et al have also shown that TdT-/- mice mount a total CD8+ response to influenza and vaccinia viruses that is lower in magnitude and breadth and that responses to immunodominant viral determinants are reshuffled (59). From these studies, it is clear that TdT plays a significant role in the expression of a fully immuno-competent B and T cell repertoire. The significant role for TdT in B cell repertoire diversity has been demonstrated using TdT transgenic mice in addition to TdT-/- mice. However, some of these studies show, interestingly, an advantage for the host that is afforded by the fetal N-less repertoire. Ann Feeney has shown a high prevalence of the T15 anti-phosphorylcholine (PC) junctional sequence in neonatal pre-b cells (60). Normal mice challenged with Streptococcus pneumoniae produce high levels of the protective T15 clone (61, 62). In addition, adult TdT-/- mice generate a more robust T15 antibody response than normal animals, suggesting that T15 dominance increases in the absence of TdT (63, 64). Further

18 9 evidence to support this hypothesis was shown by the finding that transfer of adult bone marrow cells to irradiated hosts could not rescue the T15 anti-pc response (65). In this study, the transfer of peritoneal cavity B cells or CD5+ IgM+ B cells restored the T15 anti-pc response. TdT transgenic mice, in which TdT exerted its diversifying activity in fetal and neonatal repertoires, were not capable of establishing the dominant T15 anti-pc antibody and could not generate a protective antibody response to Streptococcus pneumoniae (66). However, there are clearly other clones that require TdT for their generation, such as the M603 anti-pc antibody (64). In this study it was shown that TdT is responsible for the Asn/Asp substitution at the V-D junction of the T15 Id to generate the M603 Id+ clones. In summary, the presence or absence of TdT clearly plays a significant role in the generation of fine antibody specificities that confer an immunological advantage to the host. Recent studies have addressed the question of whether HCDR3 diversity plays a role in the selection of B cells into different compartments in the adult mouse spleen. The marginal zone B cell HCDR3 length was shown to be shorter than the follicular and transitional counterparts possibly due to lower N-addition (67). These findings were further confirmed by bone marrow chimera experiments that showed preferential selection of cells derived from TdT-/- bone marrow cells to the marginal zone B cell compartment (68). These studies confirm that the B cell receptor, and possibly HCDR3 diversity, plays a determinative role in deciding the fate and anatomical location of mature B cells (69, 70). The high frequency of natural autoantibodies in neonatal mice (71) prompted many researchers to investigate the connection between HCDR3 composition and

19 10 autoimmunity. HCDR3 was shown to be essential in distinguishing polyreactive and monoreactive combining sites in natural and antigen-induced antibodies in mice (72). Site-directed mutagenesis studies of the HCDR3 of an IgM polyreactive antibody, showed that one amino acid substitution within the HCDR3 could significantly alter the specificity of this autoantibody (73). Similar studies on polyreactive and monoreactive IgM rheumatoid factors and anti-insulin IgG confirmed the significant role of HCDR3 in binding specifically to self-antigen (74, 75). In light of these studies, Thierry Martin and his colleagues examined the role of TdT and N-addition in the affinity and polyreactive nature of anti-dna antibodies and rheumatoid factors. By stimulating TdT-/- and littermate controls with LPS, they found that TdT is not critical for the generation of these autoreactive B cells. However, the frequency of these cells was reduced in TdT-/- mice due to a lower incidence of polyreactivity (76). The notion that TdT deficiency may protect against autoimmunity was more evident when non-obese diabetic mice (77), (NZB x NZW) F1 mice (78) and MRL-Fas lpr mice (77, 79, 80) were crossed to TdT-/- mice. There was a decreased incidence of insulitis in the first model and autoimmune nephritis in the latter two models. Other factors that contribute to HCDR3 diversity Gain of palindromic nucleotides (P-addition) at the V(D)J coding ends results when the coding end hairpin is resolved by Artemis at a position other than its tip. Resolution of hairpin ends has been shown to occur within several nucleotides from the tip (81, 82), preferentially 3 from it (34). Opening distal from the hairpin tip results in nucleotide overhangs, which if incorporated into the V(D)J join generate these non-

20 11 germline P-additions further contributing to CDR3 diversity (83). The immense heterogeneity in HCDR3 lengths implies that variable trimming of the V(D)J coding ends takes place before ligation, however the enzyme(s) responsible for such activity has not been unequivocally characterized (84). Biochemical primer modification assays and recombination assays have shown that a splice variant of TdT (TdTL) in mice and humans exhibits 3 5 exonuclease activity (85, 86). Mice transgenic for both TdT splice variants were shown to exhibit less N-additions and more homology-mediated joining, compared to TdTS transgenic mice alone, upon examination of DFL16.1-J H 1 gene rearrangements from fetal livers. These data suggested that the exonuclease activity of TdTL regulates the activity of TdTS (the shorter splice variant that mediates N-addition) (63). The exonuclease function attributed to TdTL has been challenged by in vitro biochemical and functional assays (52, 87). To test for an exonuclease activity for TdTL in vivo we examined the expressed repertoire in two transgenic mouse lines that overexpress TdTL in B cells (63). We crossed the two transgenic lines to TdT-/- mice, so they only express TdTL in their B cells. We then amplified V H 7183 V(D)J rearrangements from Hardy fraction B (pro-b cells) cells from the bone marrow of these mice and compared them to TdT-/- mice and wild type BALB/c mice and examined their junctional diversity. V(D)J junctions from TdTL transgenic mice were found to be similar to TdT-/- controls and we could not find any evidence for enhanced exonuclease activity in these junctions (Figure 1). The results were similar upon examination of V H J558 V(D)J rearrangements. Since TdTL, but not TdTS, was shown to be expressed in pre-b cells (85), a developmental stage where B cells rearrange their light chains, we amplified Vκ rearrangements from TdTL transgenic mice.

21 12 Examination of Vκ light chain junctional sequences showed no differences compared to those amplified from TdT-/- controls (Table 1). Taken together, the function of TdTL as an exonuclease warrants further investigation and may be more evident upon examination of an antigen specific repertoire. Figure 1: V H 7183 HCDR3 is comparable between TdTL transgenic mice and TdT-/- controls. Hardy fraction B bone marrow cells were FACS sorted from wild type BALB/c (WT), TdT-/-, and 2 lines of TdTL transgenic mice crossed to TdT-/- (TdTL1 Lines H and I). RNA was extracted and VH 7183 V(D)J Cu transcripts were amplified as described elsewhere (44). Deconstruction of the components contributing to HCDR3 length in sequences containing identifiable D H gene segments is shown. The potential contribution of the germ-line sequence of the V H gene segment, P junctions, N region addition, the D H gene segment, and the J H gene segment to HCDR3 length is illustrated. The length of DFL16.2 is identical to that of DSP family members. Bone marrow Hardy fraction B is compared. Mice from which the B cell subset was sorted are identified in the left column, followed by the number of sequences analyzed in the flanking column to the right. All components are shown to scale.

22 13 Artemis, upon phosphorylation by DNA-PK, is a likely candidate for nucleotide nibbling at the 5 and 3 V(D)J coding ends by means of its endonucleolytic activity (34). The involvement of other factors that contribute to nucleotide loss at V(D)J junctions, such as Exo1 and TREX1, has not been ruled out (84, 88). A role for PolX polymerases, other than TdT, in modulating heavy and light chain CDR3 lengths has been demonstrated recently. Mice deficient for Polµ were shown to have shorter light chain CDR3 lengths and impaired transition from the pre-b to the B- cell stage which leads to a modest B cell deficiency (89). The size of the heavy chain CDR3 was not affected in these mice. In contrast, mice that were deficient for Polλ exhibited shorter HCDR3 regions and the light chain junctions were not affected (90). The mechanism by which these two DNA polymerases exert their role in V(D)J recombination is not known. It has been proposed that they could protect V(D)J coding ends from an exonuclease, or fill in DNA gaps at junctions with minimal homology or both (84). Table 1 Vκ light chain CDR3 (LCDR3) junctions are comparable between TdTL transgenic mice and controls LCDR3 length V L length N 5 J P-addition J L length TdTL x TdT-/ TdT-/ Deconstruction of the components contributing to Vκ LCDR3 length in sequences amplified from whole spleens of TdTL transgenic mice crossed to TdT-/- mice (TdTL x TdT-/-) and TdT-/- mice as described by Ramsden et al (91). The potential contribution of the germ-line sequence of the V L gene segment, P addition, N region addition and the J L gene segment to LCDR3 length is illustrated. n= 75 and 47 sequences for TdTL x TdT-/- and TdT-/- respectively.

23 14 Role of D H gene segments in generating HCDR3 diversity Another major contributor to HCDR3 composition and diversity is the D H gene segment. The D H gene segment can generate six possible reading frames (RFs), three by inversion and three by deletion recombination reactions (92). One of these RFs, RF1 by deletion is the preferred product of V(D)J recombination in mice (35, 93, 94). Several mechanisms have been proposed to explain this preference: 1) there is a preference for D H gene segments to rearrange by deletion, therefore decreasing the chances of generating the three RFs by inversion (i-rfs). This preference could be accounted for by sequence homology between the 5' terminus of the J H and the 3' terminus of the D H that promotes rearrangement into RF1, or that following surface expression of IgM, antigen receptor-based selection favors B cells expressing RF1-encoded HCDR3 regions; 2) RF3 by deletion tends to encode termination codons in the germline sequence therefore precluding its use; 3) in RF2 by deletion, there is an ATG translation start site upstream of the D H that encodes for a truncated protein, called Dµ protein (92). Dµ protein has been shown to trigger allelic exclusion, therefore precluding subsequent V DJ rearrangement and selecting against the usage of this RF (95). The allelic exclusion mechanism seems to depend on the expression of the pre-b cell receptor, because mice that lack the surrogate light chain component, λ5, express RF2 (96). This enhanced usage of RF1 affects the composition of the HCDR3 such that the amino acids glycine and tyrosine tend to be more preferentially used (92). This consequently generates HCDR3 regions that have an average hydrophobicity, as calculated by the Kyte-Doolilttle hydropathy index (97), that ranges from neutral to hydrophilic. These constraints on the composition of the HCDR3 regions are established

24 15 early in the mouse bone marrow during adult B cell development (44) as well as the fetal liver (92). Usage of RF2 and RF3 by deletion tends to encode hydrophobic amino acids in the HCDR3 regions. RF1 by inversion tend to encode highly charged amino acids, such as arginine, and have been associated with HCDR3 regions from autoantibodies that bind to DNA (98-100) contributing to autoimmune diseases. RF2 by inversion encodes hydrophobic amino acids and RF3 by inversion is neutral but, like its deletional counterpart, is embedded with translation termination stop codons. Similar conclusions were made about D H RF1 usage upon examination of human fetal liver sequences isolated from the second trimester. Despite the absence of an upstream ATG translation start site in RF2 in humans, the frequency of RF2 usage that encode for hydrophobic amino acids is similar to that in the mouse. Taken together, we can conclude that the maintenance of a slightly hydrophilic nature is an essential property of the developing HCDR3 repertoire (92). Using gene-targeted mice, Schroeder and colleagues have examined the role of D H gene segments on the mature HCDR3 repertoire and on B cell biology. They firstly reported that mice limited to use of one D H gene segment, the DFL16.1 gene in RF1, had normal numbers of mature B cells, with HCDR3 properties comparable to wild type mice, and were able to mount robust humoral immune responses to T-dependent and T- independent antigens (101). Therefore, limiting HCDR3 diversity by limiting D H gene segment usage was not detrimental to the host immune response. To test whether the genetic make-up or antigen receptor mediated selection was responsible for the pattern of D H RF usage seen in the developing repertoire, they generated the following: 1) mice limited to the use of single inverted D H gene segment, therefore encoding for highly

25 16 charged HCDR3 regions (termed D-DiD) and 2) mice limited to the use of a single frameshifted DFL16.1 gene segment to force RF2 usage, therefore encoding for hydrophobic HCDR3 regions (termed D-DµFS). D-DiD mice showed an HCDR3 repertoire that deviated from normal littermate control. There was an increased usage of highly charged amino acids and a decreased use of neutral amino acids. This altered repertoire led to a decrease in the numbers of mature B cells, with the exception of the marginal zone B cells which showed an increase in numbers, and compromised total and antigen-specific antibody levels (102). The HCDR3 repertoire in D-DµFS mice deviated from controls as well, where hydrophobic amino acids were used in the majority of the mature B cells. Total B cell numbers were reduced and antibody production to various T-indpendent and T-dependent antigens were variably impaired (103). These studies show that the forces of antigen receptor mediated selection could not return the HCDR3 repertoire to that normally found in wild type mice and underscore the significance of the sequence of the D H in regulating the composition of HCDR3. Scope and objective of dissertation Despite the availability of highly effective vaccines against pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis, about 1 million young children die from pneumonia and meningococcal diseases every year (104, 105). Targeting polysaccharide capsules associated with these pathogens, by inducing polysaccharide-specific antibodies through neonatal immunization, is a major factor in protection from disease ( ). An understanding of the molecular

26 17 mechanisms involved in the generation of the antigen-binding sites of protective polysaccharide-specific antibodies will provide insights on which antibodies protect and why. Polysaccharides from these life-threatening microorganisms are virulence determinants that are composed of repeating saccharide units. Mice respond to polysaccharides in a T-cell independent manner and the antibody response is characterized by the rapid production of IgM and IgG3 isotypes (109). The response is typically oligoclonal and is orders of magnitude of lower affinity in comparison to T-celldependent antibody responses ( ). Polysaccharides induce poor antibody responses in human and mouse neonates ( ), and are generally poor inducers of memory, although long-lasting antibody responses to polysaccharides have been recently demonstrated ((116); Foote J. and Kearney J.F., manuscript submitted). Although conjugating polysaccharides with carrier proteins has improved the immunogenicity and seroconversion in susceptible individuals and infants, there are numerous concerns about the persistence of the protective antibody levels and hence long-term protection from disease. Studies have shown that induction of potent initial B cell responses after vaccine administration in infants correlates with better persistence of protective antibodies (104). While several mechanisms have been put forward and tested to account for the immaturity of the responses to vaccines (and hence potential persistence of protection) (117, 118), few studies have examined the role of the HCDR3 repertoire of B cells as a possible mechanism. Antibodies to some of the well-studied polysaccharide antigens have several common attributes: HCDR3 is implicated in binding to antigen (119, 120); its length is

27 18 strictly maintained (121, 122) and almost invariably characterized by the presence of hydrophilic tyrosine residues ( ). Although Davis et al. (126) have shown that HCDR3 diversity is sufficient for specific antibody responses to a variety of antigens, they could not extend this observation to two bacterial polysaccharide antigens they tested. Work by Casadevall and colleagues (127) show that in addition to HCDR3 regions, some amino acids in the HCDR1 and HCDR2 may be required for the fine specificity to the polysaccharide GXM. These studies highlight the significant role of HCDR3 region diversity to generate polysaccharide-specific antibodies. In this work, our objective was to ask the following questions: 1) what are the effects of limiting HCDR3 diversity on the antibody response to polysaccharides? 2) what are the effects of enforcing the usage of altered genetically-altered HCDR3 repertoires on this antibody response? To answer these questions, we examined the molecular characteristics of the antibody response to α 1 3 Dextran (DEX). DEX is a branched polymer of α 1 3 glucose epitopes expressed by a variety of organisms such as Enterobacter cloacae (128), Histoplasma capsulatum yeast cell wall (129) and Aspergillus fumigatus (Kearney J.F. and Dizon B.L., unpublished observations). The antibody response of adult BALB/c mice to DEX is oligoclonal and consists entirely of antibodies bearing the λ light chain (130). The majority of anti-dex antibodies express idiotypic determinants cross-reactive with the BALB/c myeloma proteins J558 and M104E (131). Amino acid sequence analysis of DEX binding hybridoma proteins showed many sequence similarities, with diversity existing in the putative D region of the HCDR3. This region contributes to the individual idiotype identity expressed by individual B cell clones (122).

28 19 To test for the effect of limiting HCDR3 diversity on the antibody response to DEX we examined the DEX-specific response elicited by TdT-\- and by mice limited to the use of a single D H gene segment, when immunized with DEX-expressing Enterobacter cloacae. We also investigated the effect of altering the normal HCDR3 composition to highly charged or hydrophobic amino acid content on the DEX-specific response. Our results show that TdT is required for an optimal response to DEX and for normal levels of J558-expressing anti-dex antibodies. We also show that limiting HCDR3 to a single D H gene segment, regardless of its amino acid content, does not significantly impair the antibody response to DEX. Limiting D H usage to RF2-encoded amino acids enhanced the levels of antibodies that express the J558 idiotype. Taken together, our work shows the impact of limiting and altering HCDR3 diversity on the antibody response to DEX. HCDR3 diversity fine-tunes the antibody response to DEX and allows for the generation of clones that are of higher affinity to antigen, those that express J558 idiotype. Extending these findings to other polysaccharide-specific responses will further develop our understanding of the molecular mechanisms associated with the generation of high affinity polysaccharidespecific antibody clones.

29 20 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IS REQUIRED FOR AN OPTIMAL RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN 1 TAMER I. MAHMOUD AND JOHN F. KEARNEY 2 In preparation for The Journal of Immunology Format adapted for dissertation

30 21 ABSTRACT An understanding of antibody responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of antigens. In this study, we have investigated the role of the lymphoid specific enzyme TdT in generating B cell clones responsive to α 1 3 Dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte antigen receptors during V(D)J recombination. In this study we show that the DEX-specific antibody response is lower and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT-/- mice when compared to wild type BALB/c (WT) mice. Nucleotide sequencing of heavy chain CDR3s of DEX-specific plasma cells, sorted post-immunization, showed that TdT-/- mice generate a lower frequency of the predominant adult molecularlydetermined clone J558. Complementation of TdT expression in TdT-/- mice by early forced expression of the short splice variant of TdT restored WT levels of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 days after birth in IgM negative B cell precursors in the liver and spleen of WT and TdT transgenic mice but not in TdT- /- mice. These data suggest that TdT is essential for the generation of the higher affinity DEX-responsive J558 clone.

31 22 INTRODUCTION Polysaccharides serve as important virulence factors for many pathogenic microorganisms (1). The induction of polysaccharide-specific antibodies is a major factor in successful vaccination against pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis (2-4). An understanding of the molecular mechanisms involved in the generation of the antigen-binding sites of protective polysaccharide-specific antibodies will provide clues to the nature of these antibodies and the means by which they afford protection. Many antibody responses to polysaccharides in mice are T-cell independent and characterized by the rapid production of IgM and IgG3 (5), oligoclonality and low affinity (6-8). Polysaccharides are generally poor inducers of memory, although features of memory antibody responses to polysaccharides have been recently demonstrated ((9); Foote J. and J.F. Kearney, manuscript submitted). Polysaccharides induce poor antibody responses in neonatal humans and mice (10-13) and several mechanisms have been proposed to account for this relative unresponsiveness compared to adults (reviewed in (14, 15)). One possible mechanism is that the neonate, in contrast to the adult, does not contain B cells with the appropriate polysaccharide-reactive immunoglobulin (Ig) receptors (8, 13). The neonatal B cell repertoire differs significantly from that of the adult with respect to Ig V H, V L, D H and J H gene usage (16-19). In addition, in-frame rearrangements predominate, as a result of enhanced homology-mediated recombination, leading to increased representation of certain CDR3 sequences (20-22). One notable difference between neonatal versus adult Ig repertoire is that heavy chain CDR3 lengths are shorter in the neonate due to the lack

32 23 of or lower Terminal deoxynucleotidyl Transferase (TdT) activity in mice (23, 24) and humans (25) respectively. TdT is a lymphoid-specific DNA polymerase that plays a major role in the generation of B and T cell antigen receptor diversity (26-28). TdT is conserved among vertebrate species (29, 30) and of the TdT alternative splice variants, the short form of TdT (TdTS) has been shown to exert its diversifying activity by adding non-templated nucleotides (N-addition) at the V(D)J junctions of rearranging B and T cell receptors (27, 28, 31-33). The presence or absence of TdT functional activity has been shown to play a significant role in mouse antibody responses to T-independent antigens. The germlineencoded T15 antibody specific for phosphoryl choline (PC), expressed on the surface of Streptococcus pneumoniae, is generated early in life in the absence of TdT and protects against infection with this pathogen. Forced expression of TdT during this period leads to the loss of the canonical T15 antibody in adulthood and hence loss of protection (34). In contrast, the activity of TdT is required for the generation of the M603 idiotype+ (Id+) B cell clone, responsive to PC expressed on Proteus morganii (35). Both of these studies provide examples of the significant role that TdT plays in modulating the B cell repertoire. In this study we investigated the role of TdT during the generation of B cell clones involved in the antibody response to the Enterobacter cloacae-associated polysaccharide α 1 3 Dextran (DEX) (36). DEX is a branched polymer containing α 1 3 glucose epitopes which are also expressed in glucans associated with a variety of organisms such as, Histoplasma capsulatum yeast cell wall (37) and Aspergillus

33 24 fumigatus (Dizon B.L. and J.F. Kearney, unpublished observations). The antibody response of adult BALB/c mice to DEX is oligoclonal and consists almost entirely of antibodies bearing the λ light chain (38). The majority of anti-dex antibodies possess idiotypic determinants cross-reactive with the BALB/c plasmacytoma proteins J558 and M104E (39). Amino acid sequence analysis of DEX binding hybridoma proteins showed V H region homology, with most diversity existing in the putative D H region of the heavy chain CDR3, the structure of which depends significantly on the activity of TdT during Ig gene recombination. This region contributes to the individual idiotype identity expressed by individual B cell clones (40). We show by examining the anti-dex antibody response of TdT-deficient and TdT transgenic mice that TdTS is required to give rise to the dominant anti-dex J558 idiotypic determinant and the generation of an optimal anti- DEX antibody response in adult BALB/c mice. MATERIALS AND METHODS Mice: BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). TdT-/- were originally obtained from Dr. Diane Mathis (27) and crossed for 10 generations or more to the BALB/c background. TdTS transgenic mice were developed in our laboratory (34) and were crossed to TdT-/- mice in this study (TdTS x TdT-/-). All mice were bred and housed within the specific pathogen-free facility at The University of Alabama at Birmingham and used at 8 to 12 weeks of age according to protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

34 25 Immunization and serum analysis: Mice were immunized i.v. with 10 8 DEXexpressing heat-killed Enterobacter cloacae (strain MK7). ELISA was used to quantify serum antibodies by methods previously described (36). Plates were coated with either DEX at 2 µg /ml (Dr. Slodki, USDA, Peoria, IL) or the anti-j558 idiotype antibody EB3-7 at 1 µg /ml (41). Bound serum immunoglobulins were developed with alkalinephosphatase conjugated anti-igm for DEX-coated plates or anti-λ for EB3-7 coated plates (Southern Biotechnology Associates, Birmingham, AL). Flow Cytometry and Cell Sorting: Single cell suspensions of spleen cells or peritoneal cavity cells were treated with Ab 93 (42) to block Fc receptors then stained with CD19 PE, DEX antigen labeled with Alexa 488 and anti- λ JC5-1 for staining DEXbinding B cells. The dump channel included anti-cd3, anti-cd4, anti-cd8, anti- CD11c and anti-gr-1 biotin labeled antibodies, which were then developed with SA- PerCP. DEX+ λ+ plasma cells (B220 +/- Syndecan-1+) were sorted 7 days postimmunization with Enterobacter cloacae. All samples were analyzed using a FACSCalibur flow cytometer or FACSAria cell sorter (BD Biosciences, San Jose, CA). The data were analyzed using FLOWJO software (Tree Star, Inc.). All antibodies were purchased from BD Biosciences except for Ab 93 and JC5-1 that were developed in our laboratory. Polymerase Chain Reaction: V(D)J rearrangements were amplified by PCR from genomic DNA extracted from sorted plasma cells using primers specific for gene segments J558.3 V H 5 -AGCTGCAACAATCTGGACCT-3 and J H 1 5 -

35 26 CCCCAGACATCGAAGTACCA-3. PCR conditions were 95 for 3 min then 95 for 1 min, 58 for 1 min and 72 for 1 min for 35 cycles then 72 for 7 min. RT-PCR for detection of TdTS and Actin was done as described (32). Cloning and Sequencing: PCR products were subcloned into TOPO-TA vector and transformed into TOP10 competent bacteria (Invitrogen, Carlsbad, CA). Templiphi Amplification Kits were used to isolate plasmid DNA from colonies (Amersham Biosciences, Piscataway, NJ). Plasmid DNA was sequenced at the Center for AIDS Research sequencing facility at the University of Alabama at Birmingham. Sequence analysis: Any nucleotides that could not have been derived from a coding sequence or P addition were considered as N nucleotides. To assign D H gene segments utilized by HCDR3 at least five nucleotides had to match germline D H gene sequence. In the case of D H -J H homology joining, where nucleotides could be assigned to either D H or J H, nucleotides were assigned to D H except when P-addition to J H gene was identified, in which case the nucleotides were assigned to J H. HCDR3 was identified as the region between the 3' V H encoded conserved cysteine (TGT) at Kabat position 92 (IMGT 104) and the 5' J H -encoded conserved tryptophan (TGG) at Kabat position 103 (IMGT 118). Duplicate sequences (identical V H, D H, and J H segments as well as junctional sequences) derived from individual PCR reactions were only counted once. Statistics - Data comparing three or more groups were analyzed by a one-way ANOVA test for data with normal distribution and Kruskal-Wallis test for data that did

36 27 not distribute normally. Statistical significance was determined by a p value of <0.05. Data comparing two groups were analyzed by a two-tailed unpaired t test and statistical significance was determined by a p value of <0.05. RESULTS Ontogeny of B cell precursors responding to DEX In previous ontogenetic studies, we showed that DEX-responsive B cells were first detected in BALB/c mice between 5-11 days of age and gradually increased to adult frequencies, which peaked at 30 days of age. By using idiotypic markers, it was determined that the M104E Id+ clone was predominant during the emergence of the anti- DEX repertoire but was eventually largely replaced by the J558 Id+ clone which normally dominates the adult anti-dex repertoire (43) (Figures 1A, B). The J558 and M104E B cell clones were originally defined by the amino acid sequences from corresponding plasmacytomas and have identical heavy and light chain amino acid sequences, except for two amino acids constituting the heavy chain CDR3 region (Figure 1C) (44, 45). Nucleotide sequence alignment of the two clones show that the difference lies in four nucleotides where TACGAC (encoding amino acids YD) in the M104E heavy chain CDR3 is substituted by CGCTAC or AGGTAC (encoding amino acid RY) in the J558 heavy chain CDR3 (46). The heavy chain CDR3 of M104E is germline and is encoded by the D H gene segment DSP2.2, whereas that of the J558 could be encoded by DSP2.11 (AGGTAC) or by an unassignable DH segment (CGCTAC). In summary, the presence of clones with the ability to respond to DEX as well as the pattern of idiotype expression on anti-dex antibodies is age-dependent with the major responding clones

37 28 Figure 1: Ontogeny of DEX-responding B cell clones. Figure adapted from data described in ref. (43). (A) Percent of DEX-responsive spleen precursors in BALB/c mice from donors of the indicated ages was determined using the splenic focus assays. (B) The idiotype expression profile in each assay was determined by ELISA using anti-id antibodies to detect J558 Id, M104E and IdX. IdX, the cross-reactive idiotype, is expressed by most DEX responding precursors. (C) Amino acid sequence alignment of the heavy chain of the two dominant anti-dex clones M104E and J558 shows that the two clones are identical except for the HCDR3 region. The two asparagines at positions 54, 55 define thee cross-reactive idiotype, IdX.

38 29 differing only in their heavy chain CDR3 regions. The antibody response to DEX is lower in TdT-/- mice due to loss of J558 Id expressing clones that can be restored by transgenic TdTS expression Since the amino acid content of the heavy chain CDR3 is the major contributor to differences between J558 Id and M104E Id, we examined the antibody response to DEX in wild type (WT) versus TdT-/- BALB/c mice. TdT-/- mice showed a marked reduction in the total IgM anti-dex response compared to WT mice, at day 7 post-immunization (Figure 2A) and J558 Id+ antibody was barely detectable (Figure 2B). To determine whether J558 Id+ antibodies could be restored by the expression of TdTS, we next immunized transgenic TdTS x TdT-/- mice. Although the total DEXspecific antibody response elicited in TdTS x TdT-/- mice was lower than in WT mice (Figure 3A), J558 Id+ anti-dex antibody levels were restored to that of the WT (Figure 3B). We suspect that the lower anti-dex antibody level is partly caused by N-additions in the λ light chains that occurs in these TdTS transgenic mice, which may contribute to the partial loss of the expression of J558 Id, as shown previously in other studies with the 81X Id and the T15 Id (34, 47). In addition, early forced expression of TdTS may have prevented the generation of the M104E Id, which is expressed by almost 25% of anti- DEX antibodies elicited by the adult (Figure 1B). Taken together, these results suggest that the activity of TdTS is involved in the formation of the heavy chain CDR3 expressed by J558 Id+ anti-dex antibodies.

39 30 Figure 2: TdT-/- mice elicit a lower antibody response to DEX and fail to generate the J558 Id+ clone. Adult WT and TdT-/- mice were immunized i.v. with DEX-expressing Enterobacter cloacae and serum was collected at the indicated time points. ELISA was used to determine (A) DEX-specific IgM antibodies and (B) J558-specific antibodies (detected using EB3-7 anti-j558 Id).

40 31 Figure 3: TdTS x TdT-/- restore J558 Id+ antibody levels to DEX. Adult WT, TdT-/- and TdTS x TdT-/- mice were immunized i.v. with DEX-expressing Enterobacter cloacae and serum collected day 7 post-immunization. ELISA was used to determine (A) DEX-specific IgM antibodies and (B) J558-specific antibodies. *** p < , * p < 0.05 by One Way ANOVA post-hoc test, ns; not significant by Kruskal Wallis post-hoc analysis.

41 32 TdT-/- and WT mice have similar numbers of DEX-specific B cells We next asked whether the frequency of DEX-specific B cells in adult mice is dependent on the expression of TdTS. If this were true then we would expect TdT-/- animals to have a lower number of DEX-specific B cells than WT mice. Flow cytometric analysis showed that, within the limits of detection of this assay, the frequency of λ light chain expressing B cells that bind to DEX was similar in the spleens and peritoneal cavity cells of TdT-/- compared to WT mice (Figures 4A, B). As a negative control we showed that B cells from the C57BL/6 strain, which is a DEX non-responder strain, did not stain with DEX and anti-λ light chain antibody (Figure 4A). These results show that the lack of TdTS does not affect the frequency of DEX-binding B cells in the spleen and peritoneal cavity. TdT-/- mice express a neonatal-like anti-dex repertoire To compare idiotype expression at the molecular level in DEX-responding B cells of WT, TdT-/- and TdTS x TdT-/- mice, we amplified and sequenced heavy chain V(D)J rearrangements from genomic DNA of DEX+ λ+ plasma cells sorted from the spleens of Enterobacter cloacae-immunized animals. As expected, WT mice expressed mostly the J558 V(D)J rearrangement, containing the amino acids arginine and tyrosine in HCDR3, and in agreement with serum antibody idiotype expression TdT-/- animals expressed a much lower proportion of this V(D)J rearrangement in DEX+ λ+ plasma cells (Figure 5A). The frequency of J558-expressing DEX+ λ+ plasma cells was completely restored in TdTS x TdT-/- animals. The frequency of M104E CDR3 sequences detected in TdT-/- DEX+ λ+ plasma cells was similar to that of the WT, but barely detectable in TdTS x

42 33 TdT-/- DEX+ λ+ plasma cells (Figure 5A). WT sequences showed a greater number of non-j558 non-m104e, but J558.3-J H 1 expressing, DEX-responsive rearrangements than sequences from TdT-/- mice. The frequency of non-j558, non-m104e sequences in TdTS x TdT-/- is also reduced, which could explain the lower total DEX antibody response of these animals. These data show that the altered DEX-responsive repertoire in TdT-/- mice, as shown by the decreased expression of J558 and decreased frequencies of non- J558, non-m104e sequences, is an important factor in the overall decreased antibody response to DEX. Although TdTS x TdT-/- DEX+ λ+ plasma cells showed a restoration of the WT frequency of the dominant J558 sequence, examination of the deduced amino acid sequences of the V(D)J rearrangements amplified from DEX+ λ+ plasma cells shows that diversity at the heavy chain CDR3 region was lower than the WT (Figure 5B). Comparison of nucleotide sequences from WT and TdTS x TdT-/- heavy chain CDR3 regions showed that although some of the WT sequences require one or two N-additions for their formation, over-expression of TdTS in TdTS x TdT-/- mice led to the abolition of these sequences and the emergence of other sequences with longer N-additions. As expected, the diversity in TdT-/- sequences was also lower compared to WT sequences with the M104E sequence the most frequently used.

43 34 Figure 4: Adult WT and TdT-/- pre-immune mice have similar numbers of DEX-binding B cells. (A) A representative FACS density plot showing the percentage of CD19+ B cells that are DEX+ JC5-1+ (λ+) in adult WT BALB/c and TdT-/- mice. C57BL/6 mice were used as negative control. (B) A graphical representation of the frequency of DEX+ λ+ B cells in the spleens and peritoneal cavity cells (PEC) of WT and TdT-/- mice.

44 35 Figure 5: WT mice response to DEX shows higher J558 Id expression and greater diversity of anti-dex antibodies. DEX+ λ+ plasma cells were sorted 7 days post-immunization with Enterobacter cloacae and V(D)J rearrangements were amplified from genomic DNA from WT, TdT-/- and TdTS x TdT-/- mice. (A) Frequency of DEX+ λ+ plasma cells utilizing J558, M104E or other sequences. ND; not detected. The frequency of plasma cells expressing J558 or M104E was back calculated from the frequency of total DEX+ λ+ plasma cells (as determined by FACS analysis, 7 days post-immunization) and the percentage of J558+ or M104E+ sequences obtained out of the total V(D)J sequences amplified from bulk DEXspecific plasma cells. (B) Representative nucleotide and deduced amino acid sequences of heavy chain CDR3 regions amplified from WT, TdT-/- and TdTS x TdT-/-. N- nucleotides are underlined. Data is representative of four independent FACS sorts for WT, five for TdT-/- and three for TdTS x TdT-/-.

45 36 J558 HCDR3 expression correlates with TdTS expression early in ontogeny of DEXspecific repertoire To further confirm the requirement for TdTS for the generation of the J558 sequence, we examined the expression of TdTS in WT BALB/c day 7 neonates at the time when DEX-responsive B cells are first beginning to appear. TdTS mrna message levels were detected in B220+ IgM- B cell progenitors in the liver, spleen and bone marrow (Figure 6A). We then amplified antibody heavy chain V(D)J rearrangements that utilize VH J558.3 and J H 1, the gene segments used in both M104E, J558 and other reported DEX+ sequences, from these B cell progenitor populations. With the exception of the bone marrow compartment, the expression of the J558 sequence correlated with the expression of TdTS as shown in Figure 6B. DEX-responsive V(D)J rearrangements were barely detected in TdT-/- neonates, however they were readily detectable in neonates from TdTS x TdT-/- mice with J558 being the dominant sequence amplified. No other DEX-responsive B cell associated molecular clones were detected at this time point. These data show that the expression of TdTS coincides with the expression of the J558 V(D)J rearrangement when DEX-responding clones start to emerge.

46 37 Figure 6: TdTS expression correlates with J558 HCDR3 expression when J558 Id+ B cells starts to emerge. B220+ IgM- B cell progenitors were sorted from the spleens, liver and bone marrow of WT, TdT-/- and TdTS x TdT-/- mice at 7 days of age. (A) RT-PCR showing the expression of TdTS and Actin in the liver, spleen and bone marrow. (B) Percent of J558 sequence expression from J558.3-J H 1 V(D)J rearrangements amplified from sorted neonatal B cell progenitors in the liver and spleen. The figure is representative of two independent FACS sorts (one sort for TdTS x TdT-/-) from an entire litter of 7-day-old pups. ND: not detected. N= 17 for WT liver, 11 for WT spleen, 25 for TdT-/- liver, 18 for TdT-/- spleen, 9 for TdTS x TdT-/- liver and 18 for TdTS x TdT-/- spleen. The J558 sequence was not detected in bone marrow B cell progenitors at this time point.

47 38 DISCUSSION Immune protection against encapsulated bacteria, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilis influenzae is provided by antibodies against the bacterial capsular polysaccharides. Elucidation of factors required for the generation of these protective antibodies is of utmost importance in understanding mechanisms by which a competent immune system combats disease. We investigated the molecular mechanisms involved in generating an oligoclonal response to a typical type-2 T-cell independent antigen (DEX) and demonstrate that TdTS is important for the optimal antibody responses to DEX. We show that TdTS is required to generate a diverse DEX-responsive repertoire including optimal numbers of the J558 DEX-responsive clone involved in the adult response to DEX. Earlier studies suggested that TdT is not required in generating efficient polyclonal T-cell and antibody responses to selected complex protein antigens (28, 48). However, there is mounting evidence that the advantage offered by the diversifying activity of TdT is subtle and is better measured at the level of clonal populations. Heterosubtypic immunity to Influenza A virus infection, which affords protection against a virus subtype differing from that used for immunization, is impaired in TdT-/- mice (49). This observation may be explained in part by the finding that TdT is required for the generation of private influenza virus-specific CD8+ repertoires that contribute to enhance diversity in TCRs across species as a whole (50). Yewdell et al have also shown that TdT-/- mice mount a total CD8+ response to influenza and vaccinia viruses that is lower in magnitude and breadth compared to WT and that responses to immunodominant viral determinants are reshuffled (51). From these studies, as well as our findings, it is clear

48 39 that TdT plays a significant role in the expression of a fully immuno-competent B and T cell repertoire in a given host population. Alternatively it has been shown that the absence of TdT expression in neonatal life and the window of limited diversity that ensues is required to generate certain antibody clones with protective, germline-encoded specificity. This was clearly demonstrated in TdTS transgenic mice, where forced TdT expression exerted its diversifying activity in fetal and neonatal repertoires, with the result that the mice were incapable of establishing the dominant T15 Id+ anti-pc antibody that is protective against Streptococcus pneumoniae (34). TdT-/- mice have also been shown to generate a more robust T15 antibody response than WT animals (35, 52). However, there are clearly other clones that require the activity of TdT for their generation, such as the M603 anti- PC antibody (35). In this study it was shown that TdT is responsible for the Asn/Asp substitution at the V-D junction of the T15 Id to generate the M603 Id+ clones. In our present study, we reveal another example of the requirement for TdT to generate the J558 Id instead of the germline M104E Id, both of which are responsive to the polysaccharide DEX. The finding that TdT is required for the expression of the J558 Id may partially explain the observation in our earlier study that: 1) the frequency of anti-dex spleen precursors, as detected by the splenic focus assays, gradually increases to adult levels subsequent to the onset of TdTS expression; 2) The idiotype pattern of the DEXresponding precursors gradually changes from a high M104E/J558 ratio to a much lower one. The difference between the two idiotypes lies in the heavy chain CDR3 region, where TdT exerts its diversifying activities. Our data confirm that the M104E sequence is

49 40 germline as evidenced by its absence in TdTS x TdT-/- mice, where TdT is expressed constitutively in fetal and neonatal B cell precursors. The J558 Id+ heavy chain CDR3 could be encoded by DSP2.11 (AGGTAC) or by the nucleotides (CGCTAC) which cannot be assigned to a known D H segment. We recover predominantly J558 Id+ sequences that express the latter CDR3 from our DEX+ λ+ plasma cells after immunization with Enterobacter cloacae. This CDR3 region is GC rich, which is suggestive that TdT is involved in its formation. However the generation of the J558 Id CDR3 is not absolutely dependent on TdT since we recover some sequences from TdT-/- mice that express this idiotype. There may be other ways in which the J558 Id is generated independent of TdT activity: 1) there may be no nucleotide loss from J H 1 (J H 1 gene segment starts with the nucleotides CTAC) thus making the G nucleotide a product of palindromic addition and the remaining nucleotides remnants of a D H -segment that suffered extensive nucleotide loss; 2) the CDR3 of J558 Id+ clones could be generated by the activity of other DNA polymerases, such as DNA Polymerase λ (Pol λ) (53), that has been shown to be implicated in modifying gene segment ends during heavy chain V(D)J rearrangement. Examination of antibody responses by Pol λ-/- x TdT-/- would test this possibility. However, the two genes are on the same chromosome and it would be very difficult to obtain double knockout animals. The alternative would be to test Pol λ-/- neonates before TdTS is expressed, however B cells responsive to DEX in BALB/c mice do not emerge until 5 days of age, at which time point TdT is already beginning to be expressed (23, 24). Regardless of the mechanism by which the J558 sequence is generated in the absence of TdTS, it is not as efficient in generating this clone as in WT mice.

50 41 The possibility exists that the absence of TdT alters the repertoire of other B cells and T cells that would have otherwise contributed to the selection of the J558 clone. There is evidence supporting a role for B cell idiotypic interactions early in life that modulate the development of B cell clones responsive to DEX and to other T- independent antigens (54, 55). Neonatal injection of the anti-pc antibodies, such as the T15 antibody that is presumably more abundant in TdT-/- mice, leads to a reduction in J558 idiotype expression when these mice were immunized with DEX as adults (56). Therefore, the mechanism by which J558 Id expression requires TdTS may be indirect involving the selection of J558-expressing clones as a result of B cell interactions with other B cells or other immune cells. However, the most likely explanation of our findings is that the J558 clone, along with HDEX9 and HDEX37 (all of which use RY amino acids at their CDR3 regions) show the highest affinity to DEX (57, 58). HDEX9 and HDEX37 sequences were not detected in our PCR assays because they use different V H and J H segments respectively. The difference between J558 and the germline M104E Ka to DEX is almost 100-fold (1.4 x 10 5 versus 1.3 x 10 3 ). This illustrates the importance of TdT in the generation of clones of higher affinity and which are likely involved in the higher antibody response in WT versus TdT-/- mice. The higher affinity of the J558 clone may also be responsible for its selective expansion in the splenic marginal zone and the peritoneal B1b cell compartment by environmental or self antigens (J. Foote and J.F. Kearney, manuscript submitted). The diversity of the anti-dex antibody response contributed by TdT is also evident in the increased number of non-j558, non-m104e sequences recovered from WT versus TdT-/- animals. The activity of TdT promotes a more diverse usage of D H

51 42 segments and less homology-mediated joining that is characteristic of fetal and neonatal sequences. Overexpression of TdTS in TdTS x TdT-/- mice did not restore this element of antibody diversity. However, TdTS x TdT-/- mice are capable of harboring N- additions in their light chains, which in turn might compromise the ability of clones to bind to DEX, thus decreasing the frequency of overall DEX binding plasma cells that were sorted. Therefore, sequences recovered from these plasma cells might not show the same level of diversity as those from WT mice. An examination of the nucleotide sequences of V(D)J rearrangements amplified post-immunization from DEX-specific plasma cells of these mice reveals that it is also possible that supra-physiological levels of TdTS activity might obliterate the generation of some of the DEX-specific sequences normally recovered from WT animals. Therefore, normal physiological levels of TdTS may be required for a diverse anti-dex antibody repertoire. Examination of neonatal B cell progenitors in the liver and spleen revealed a correlation between TdTS and J558 expression. This correlation did not hold true for neonatal bone marrow progenitors, perhaps because the bone marrow has not been colonized with precursors capable of generating J558 at that point. Our assay did not detect other non-j558 DEX+ sequences in all neonatal B cell progenitors examined, perhaps because the ontogenetic development of these clones is different and they may have been generated at an earlier or later time point than 7 days after birth when the samples were taken. However, J558 contributes to about 70% of the adult response and detection of other DEX-responsive non-j558 sequences may require a very large sample size.

52 43 This report sheds light on the role of TdT on the host antibody response to a polysaccharide antigen. Antibodies vary in their protective abilities and our findings highlight another factor that may be considered to explain the molecular mechanism of these differences. A number of DNA polymerases have been shown to play a role in modulating heavy and light chain junctional diversity (53, 59). It would be of interest to examine the role of these factors in modulating the antibody response to polysaccharides. These findings have further implications for the observed differences in the ability of neonatal versus adult B cell repertoires to respond to polysaccharide antigens. The results lend support to the hypothesis that in some instances the poor neonatal response to polysaccharides may be due to the lack of B cell clones expressing the appropriate B cell receptor. ACKNOWLEDGEMENTS The authors are very grateful to Jeremy Foote (University of Alabama at Birmingham) for very helpful discussions and Dr. Nicholas Kin (University of Alabama at Birmingham) for critically reading the manuscript. FOOTNOTES 1 This work was supported by research funds from the National Institutes of Health (NIH) Grant AI T.I.M. is a recipient of the grant T32 AI from the National Institute of Allergy and Infectious Diseases, NIH.

53 44 2 Address correspondence and reprint requests to Dr. John F. Kearney 410 Shelby Biomedical Research Building 1825 University Blvd Birmingham, AL Office: (205) Fax: (205) address: jfk@uab.edu REFERENCES 1. Comstock, L. E., and D. L. Kasper Bacterial glycans: key mediators of diverse host immune responses. Cell 126: Mazmanian, S. K., and D. L. Kasper The love-hate relationship between bacterial polysaccharides and the host immune system. Nat Rev Immunol 6: Lesinski, G. B., and M. A. Westerink Vaccines against polysaccharide antigens. Curr Drug Targets Infect Disord 1: Weintraub, A Immunology of bacterial polysaccharide antigens. Carbohydr Res 338: Vos, Q., A. Lees, Z. Q. Wu, C. M. Snapper, and J. J. Mond B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms. Immunol Rev 176: Insel, R. A., E. E. Adderson, and W. L. Carroll The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide. Int Rev Immunol 9: Zhou, J., K. R. Lottenbach, S. J. Barenkamp, A. H. Lucas, and D. C. Reason Recurrent variable region gene usage and somatic mutation in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F. Infect Immun 70:

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57 Stohrer, R., and J. Kearney Ontogeny of B cell precursors responding to alpha 1- greater than 3 dextran in BALB/c mice. J Immunol 133: Schilling, J., D. Hansburg, J. M. Davie, and L. Hood Analysis of the diversity of murine antibodies to dextran B1355: N-terminal amino acid sequences of heavy chains from serum antibody. J Immunol 123: Schilling, J., B. Clevinger, J. M. Davie, and L. Hood Amino acid sequence of homogeneous antibodies to dextran and DNA rearrangements in heavy chain V-region gene segments. Nature 283: Clemens, A., A. Rademaekers, C. Specht, and E. Kolsch The J558 VH CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response. Int Immunol 10: Martin, F., and J. F. Kearney Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 12: Gilfillan, S., M. Bachmann, S. Trembleau, L. Adorini, U. Kalinke, R. Zinkernagel, C. Benoist, and D. Mathis Efficient immune responses in mice lacking N-region diversity. Eur J Immunol 25: Nguyen, H. H., M. Zemlin, Ivanov, II, J. Andrasi, C. Zemlin, H. L. Vu, R. Schelonka, H. W. Schroeder, Jr., and J. Mestecky Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire. J Virol 81: Kedzierska, K., P. G. Thomas, V. Venturi, M. P. Davenport, P. C. Doherty, S. J. Turner, and N. L. La Gruta Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses. J Immunol 181: Haeryfar, S. M., H. D. Hickman, K. R. Irvine, D. C. Tscharke, J. R. Bennink, and J. W. Yewdell Terminal deoxynucleotidyl transferase establishes and broadens antiviral CD8+ T cell immunodominance hierarchies. J Immunol 181: Benedict, C. L., S. Gilfillan, and J. F. Kearney The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform. J Exp Med 193: Bertocci, B., A. De Smet, J. C. Weill, and C. A. Reynaud Nonoverlapping functions of DNA polymerases mu, lambda, and terminal

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59 50 THE ANTIBODY RESPONSE TO THE POLYSACCHARIDE α 1 3 DEXTRAN IS ENHANCED IN D-LIMITED MICE FORCED TO USE D H READING FRAME 2 1 TAMER I. MAHMOUD, HARRY W. SCHROEDER, JR. AND JOHN F. KEARNEY In preparation for The Journal of Immunology Format adapted for Dissertation

60 51 ABSTRACT An understanding of the molecular mechanisms to generate antibody responses to polysaccharides associated with pathogenic microorganisms has implications for characterizing the nature of antigen-antibody interactions that lead to protection from many life-threatening diseases. In this study, we investigated the antibody response of gene-targeted mice limited to the usage of one D H gene segment (D-limited mice) to α 1 3 Dextran (DEX). We show that with the exception of D-DiD mice, the DEXspecific antibody response is fairly comparable in D-limited mice to that of wild type BALB/c (WT) mice. D-limited mice forced to use D H reading frame 2 ( D-DµFS) showed a higher antibody response to DEX compared to other D-limited mice and higher levels of the dominant DEX-specific J558 idiotype (Id) compared to WT mice. Nucleotide sequencing of HCDR3s of DEX-specific plasma cells post-immunization showed that D-DµFS and D-DFL mice generated about 30% of the same clones generated by WT, including the predominant adult molecularly-determined clone J558. The average HCDR3 hydrophobicity was very similar among all mouse groups. D-limited mice had a higher frequency of D H -less HCDR3 regions compared to WT. D-DµFS and WT DEX-specific plasma cell sequences showed a greater number of V(D)J rearrangements that encoded for expression of J558 Id in comparison to other D-limited mice. These data suggest that constraints on HCDR3 diversity by limiting D H usage do not preclude the generation of a proper anti-dex response and that altered D H reading frame usage could enhance the generation of the anti-dex clone that is predominant in the WT.

61 52 INTRODUCTION Despite the availability of highly effective vaccines against pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis, about 1 million young children die from pneumonia and meningococcal diseases every year (1, 2). Targeting polysaccharide capsules associated with these pathogens, by inducing polysaccharide-specific antibodies by early neonatal immunization, is a major factor in protection from disease (3-5). An understanding of the molecular mechanisms involved in the generation of the antigen-binding sites of protective polysaccharidespecific antibodies will provide insights on the nature of the antigen-antibody interactions that lead to such protection. Mice respond to many polysaccharides in a T-cell independent manner and the antibody response is characterized by the rapid production of IgM and IgG3 isotypes (6). The response is often oligoclonal and the antibody produced is lower in affinity by several orders of magnitude in comparison to T-cell-dependent antibody responses (7-9). Polysaccharides induce poor antibody responses in human and mouse neonates (10-12), and are generally poor inducers of memory, although evidence of memory antibody responses to polysaccharides have been recently demonstrated ((13); Foote J. and Kearney J.F., manuscript submitted). The antigen-binding site of an antibody is generated by the combination of three hypervariable complementarity determining regions (CDR) from the heavy chain and three CDRs from the light chain (14). The third CDR of the heavy chain (HCDR3) has been shown to be sufficient for the generation of most antibody specificities to proteins, but surprisingly not to carbohydrates (15). HCDR3 is created by the rearrangement of

62 53 Variable (V H ), Diversity (D H ), and Joining (J H ) segments and by the addition of random N nucleotides, P nucleotides as well as exonucleolytic loss during the process of somatic recombination (16). Being at the core of the antigen-binding site, HCDR3 plays a decisive role in the recognition and binding of the antigen to antibody (15, 17). In jawed vertebrates most HCDR3 regions use only one of six available D H reading frames, three by deletion and three by inversion. Reading frame (RF) 1 by deletion, the preferred reading frame, tends to encode neutral amino acids such as tyrosine and glycine, whereas the other five RFs tend to be enriched for either hydrophobic or charged amino acids. Anti-dsDNA and anti-nucleosome HCDR3 loops tend to contain positively charged amino acids, especially arginine and/or lysine (18-21) in human lupus patients and in lupus-prone mouse models. In contrast, HCDR3 regions of the most effective neutralizing antibodies against HIV have a propensity to contain hydrophobic amino acids such as those encoded by RF2 (22-24). We have studied the effect of D H reading frame usage on the antibody response to α 1 3 Dextran (DEX) in mice limited to the usage of one D H gene segment (referred to as D-limited mice in this manuscript), that respectively either encode neutral amino acids (ΔD-DFL), charged amino acids (ΔD-DiD) or hydrophobic amino acids (ΔD-DµFS). DEX is a branched polymer of α 1 3 glucose sugar moieties expressed by a variety of organisms such as Enterobacter cloacae (25), Histoplasma capsulatum yeast cell wall (26) and Aspergillus fumigatus (Dizon B.L. and J.F. Kearney, unpublished observations). The antibody response of adult normal BALB/c mice to DEX is oligoclonal and consists entirely of antibodies bearing the λ light chain (27). The majority of anti-dex antibodies express idiotypic determinants cross-reactive with the BALB/c myeloma proteins J558

63 54 and M104E (28). Amino acid sequence analysis of DEX binding antibodies showed many sequence similarities, with diversity existing in the putative D region of the HCDR3 regions. Examination of the anti-dex antibody response of D-limited mice show that, with the exception of ΔD-DiD mice, such a restriction in HCDR3 diversity does not significantly impair the anti-dex response. We also show that mice forced to use RF2 (ΔD-DµFS), elicit a superior J558 Id expressing anti-dex response, in comparison to other D-limited and WT mice, by expanding a minor population (about 20% of recirculating mature B cells) containing neutral and hydrophilic amino acid residues. These responding B cells tend to possess several different V(D)J rearrangements that express the J558 Id. Taken together, despite genetic constraints to encode HCDR3 regions of different hydrophobicities, the antibody response to DEX maintains the presence of hydrophilic residues in its heavy chain HCDR3 regions. MATERIALS AND METHODS Mice: ΔD-DFL16.1, ΔD-iD and Δ DµFS were generated as described on the BALB/c background (29-31). Wild type BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were bred and housed within the specific pathogen-free facility at The University of Alabama at Birmingham and used at 8 to 12 weeks of age according to protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

64 55 Immunization and serum analysis: Mice were immunized i.v. with 10 8 DEXexpressing heat-killed Enterobacter cloacae (strain MK7). ELISA was used to quantify serum antibodies by methods previously described (25). Plates were coated with either DEX at 2 µg /ml (Dr. Slodki, USDA, Peoria, IL) or the anti-j558 idiotype EB3-7 at 1 µg /ml (32). Bound serum immunoglobulins were developed with alkaline-phosphatase conjugated anti-igm for DEX-coated plates or anti-λ for EB3-7 coated plates (Southern Biotechnology Associates, Birmingham, AL). Flow Cytometry and Cell Sorting: Single cell suspensions of spleen cells or peritoneal cells were treated with Ab 93 (33) to block Fc receptors and then stained with CD19 PE, DEX antigen labeled with Alexa 488 and Alexa 647 labeled anti- λ JC5-1 for staining DEX-binding B cells. The dump channel included anti-cd3, anti-cd4, anti- CD8, anti-cd11c and anti-gr-1 biotin labeled antibodies, which were then developed with SA-PerCP. DEX+ λ+ plasma cells (B220 +/- Syndecan-1+) were sorted from mice 7 days post-immunization with DEX-expressing Enterobacter cloacae. All samples were analyzed using a FACSCalibur flow cytometer or FACSAria cell sorter (BD Biosciences, San Jose, CA). The data were analyzed using FLOWJO software (Tree Star, Inc.). All antibodies were purchased from BD Biosciences except for Ab 93 and JC5-1 that were developed in our laboratory. Polymerase Chain Reaction: V(D)J rearrangements were amplified by PCR from genomic DNA extracted from sorted plasma cells using primers specific for gene segments J558.3 VH 5 -AGCTGCAACAATCTGGACCT-3 and JH1 5 -

65 56 CCCCAGACATCGAAGTACCA-3. PCR conditions were 95 for 3 min then 95 for 1 min, 58 for 1 min and 72 for 1 min for 35 cycles then 72 for 7 min. Cloning and Sequencing: PCR products were cloned into TOPO-TA vector and transformed into TOP10 competent bacteria (Invitrogen, Carlsbad, CA). Templiphi Amplification Kits were used to isolate plasmid DNA from colonies (Amersham Biosciences, Piscataway, NJ). Plasmid DNA was sequenced at the Center for AIDS Research sequencing facility at the University of Alabama at Birmingham. Sequence analysis: Any nucleotides that could not have been derived from a coding sequence or P addition were considered as N nucleotides. To assign D H gene segments utilized by HCDR3 at least five nucleotides had to match germline D H gene sequence. In the case of D H -J H homology joining, where nucleotides could be assigned to either D H or J H, nucleotides were assigned to D H except when P-addition to J H gene was identified, in which case the nucleotides were assigned to J H. HCDR3 was identified as the region between the 3' V H encoded conserved cysteine (TGT) at Kabat position 92 (IMGT 104) and the 5' J H -encoded conserved tryptophan (TGG) at Kabat position 103 (IMGT 118). Duplicate sequences (identical V H, D H, and J H segments as well as junctional sequences) derived from individual PCR reactions were only counted once. Statistics - Data comparing three or more groups were analyzed by a one-way ANOVA test, followed by Tukey s post test, for data with normal distribution and Kruskal-Wallis test, followed by Dunn s post test, for data that did not distribute

66 57 normally. Data comparing two groups were analyzed by a two-tailed unpaired t test. To determine statistical significance for data comparing proportions, we calculated the 95% confidence interval of the mean for each group. Statistical significance was determined by a p value of <0.05. RESULTS ΔD-DµFS mice elicit a superior J558 antibody response to DEX To test the role of limiting D H usage and hence HCDR3 diversity in the antibody response to DEX, we immunized adult D-limited mice with DEX-expressing Enterobacter cloacae. Seven days after immunization ΔD-DµFS mice show a higher DEX-specific antibody response compared to ΔD-DiD and ΔD-DFL mice (Figure 1A). We next determined whether the anti-dex antibodies from D-limited mice express the J558 idiotype that normally predominates in the adult WT BALB/c response. Anti-DEX antibodies from ΔD-DµFS show the highest level of J558 idiotype expression compared to ΔD-DiD, ΔD-DFL and wild type (WT) BALB/c mice (Figure 1B). ΔD-DiD and ΔD- DFL mice showed DEX-specific and J558-specifc antibody responses comparable to WT mice. These results show that; 1) limiting HCDR3 diversity by limiting D H segments availability does not impair the antibody response to DEX upon bacterial challenge and that 2) restricting the repertoire to use a single D H gene segment forced to use RF2, which generates predominantly hydrophobic amino acids, rather than precluding the generation of J558, whose HCDR3 is hydrophilic, enhances its levels.

67 58 Figure 1: D-DµFS generate a higher J558 Id+ anti-dex antibody response. Adult WT, D-DµFS, D-DiD and D-DFL mice were immunized i.v. with DEXexpressing Enterobacter cloacae and serum collected seven days post-immunization. ELISA was used to determine (A) DEX-specific IgM antibodies and (B) J558-specific antibodies. * p < 0.05

68 59 Similar numbers of DEX-binding B cells detected in WT and D-limited mice in the spleen but not the peritoneal cavity We next asked whether the enhanced anti-dex response shown by ΔD-DµFS mice is a result of the increased frequency of DEX-specific B cells in pre-immune adult mice. Flow cytometric analysis showed that, within the limits of detection of this assay, the frequency of λ+ light chain B cells binding to DEX was similar in the spleens of all mouse groups (Figure 2B). However, the frequency of these DEX-specific B cells was higher in peritoneal cavity cells of ΔD-DµFS and ΔD-DFL mice compared to WT mice (Figure 2B). As a negative control we showed that B cells from the C57BL/6 strain, which is a DEX non-responder strain, did not stain with DEX and anti-λ light chain antibody (Figure 2B). These results suggest that the increased frequency of DEX-binding B cells in the peritoneal cavities of ΔD-DµFS mice does not account for their capacity to generate a superior J558 antibody response to DEX compared to all mouse groups, since ΔD-DFL control mice have an increased frequency of DEX-binding B cells in peritoneal cavity cells compared to WT, as well. D-limited mice, except ΔD-DiD, express an anti-dex repertoire comparable to that of the wildtype The majority of mature recirculating B cells in ΔD-DµFS utilize D H RF2 in their HCDR3 regions encoding for hydrophobic amino acids, except for about 20% of these B cells that utilize D H RF1 (31). However, several reports show that antibodies to polysaccharides, such as polysaccharides associated with the capsules of Cryptococcus

69 60 Figure 2: Adult D-limited and WT pre-immune mice have similar numbers of DEXbinding B cells in the spleen but not the peritoneal cavity. (A) Representative FACS density plot showing the percentage of CD19+ B cells that are DEX+ JC5-1+ (λ+) in spleen cells of adult WT BALB/c and D-limited mice. C57BL/6 mice were used as negative control. (B) A graphical representation of the absolute cell numbers of DEX+ λ+ B cells in the spleen (left panel) and peritoneal cavity (right panel) of WT BALB/c, and D-limited mice. * p < 0.05

70 61 neoformans and Neisseria meningitidis, have HCDR3 that are tyrosine rich and hydrophilic in nature (34, 41). To determine the amino acid composition of HCDR3 regions in the responding anti-dex repertoire, we amplified and sequenced heavy chain V(D)J rearrangements from genomic DNA of DEX+ λ+ plasma cells sorted from the spleens of Enterobacter cloacae-immunized D-limited and BALB/c WT mice. Examination of deduced amino acid sequences showed that HCDR3 regions amplified from DEX-specific plasma cells use mostly hydrophilic amino acids in all mouse groups (Figure 3). The average Kyte-Doolittle hydropathy index, which we used to determine the average HCDR3 region hydrophobicity, was comparable in all mouse groups (Figure 4A). Distribution of average HCDR3 hydrophobicities in the DEX-specific sequences shows that there is a slight shift to the neutral side in sequences amplified from ΔD-DµFS and ΔD-DFL DEX-specific plasma cells compared to WT sequences (Figure 4B). HCDR3 sequences from ΔD-DiD DEX-specific plasma cells show a distribution of hydrophobicity on both extremes, with some sequences that are hydrophilic and others that are hydrophobic and almost none that are neutral. These results show and confirm that HCDR3 regions of DEX-specific antibodies are hydrophilic in nature and that they enrich in the minority B cell population that utilize D H RF1 in ΔD-DµFS mice. With the exception of sequences from ΔD-DiD plasma cells, HCDR3 amino acid sequences from D-limited mice match many of those amplified from the WT. For example, ΔD-DµFS sequences as well as ΔD-DFL sequences each had 3 unique HCDR3 sequences that matched those of the WT, while ΔD-DiD HCDR3 sequences had only one. Some sequences were unique to each mouse group though; for example sequences unique to ΔD-DµFS plasma cells contained KY, GS, GG and AH amino acids in their

71 62 Figure 3: The anti-dex antibody repertoire of D-limited mice, except D-DiD, is comparable to that of the WT. DEX+ λ+ plasma cells were sorted 7 days post-immunization with Enterobacter cloacae and V(D)J rearrangements were amplified from genomic DNA from D-limited and WT mice. Deduced unique amino acid sequences of heavy chain CDR3 regions amplified from WT, D-DµFS, D-DiD and D-DFL are shown in comparison to the prototypical predominant J558 heavy chain CDR3. n is number of unique V(D)J sequences.

72 63

73 64 Figure 4: HCDR3 regions of DEX-specific plasma cells from D-limited mice have similar hydrophobicities to those of the WT. (A) Average hydrophobicity index of HCDR3 regions amplified from DEX-specific plasma cells post-immunization with Enterobacter cloacae are shown for each of the D- limited mice and WT BALB/c. (B) Distribution of average HCDR3 hydrophobicities from all D-limited mice and WT BALB/c. Average hydrophobicity index was calculated using the normalized Kyte-Doolittle hydrophobicity scale (57). This scale ranges from to +1.7, however all of our sequences were in the range from -1.0 to Unique HCDR3 regions are shown.

74 65

75 66 putative D H regions. Sequences from ΔD-DiD plasma cells suggest that the response is more oligoclonal and less diverse at the HCDR3 regions as we were able to amplify a fewer number of unique HCDR3 sequences from their plasma cells (Figure 3). However, they were unique in their ability to generate four independent sequences that encode for RN, RH, RI and VN in their putative D H region. Sequences unique to ΔD-DFL plasma cells had GD, YS and SH in their D H regions. Taken together, these results show that all D-limited mice, apart from ΔD-DiD mice, are capable of generating a heterogeneous anti-dex repertoire that is rather comparable to that of the WT at the genetic level. Since J558 idiotype expression was highest in ΔD-DµFS mice, as determined using the anti-j558 idiotype EB3-7, after immunization with Enterobacter cloacae, we also compared J558 idiotype-determining HCDR3s in these DEX-specific sequences. All D-limited mice were capable of generating the J558 sequence (RY in putative D H region) (Figure 3), which predominates in the adult WT BALB/c response, confirming our ELISA data (Figure 1B). However, other HCDR3 sequences that weakly express the J558 idiotype were also recovered from HCDR3 regions of all groups such as those that express NY (HDEX 2), SY (HDEX 25), SH (HDEX 6), AH, GN (HDEX 13) in their putative D H region as determined by reactivity with the anti-j558 idiotype EB3-7 ((36) and Stohrer and Kearney, unpublished observations). ΔD-DµFS and WT mice DEXspecific HCDR3 sequences showed the highest expression of J558 Id expression in comparison to ΔD-DiD and ΔD-DFL mice (Figure 5). These data suggest that ΔD-DµFS mice are more capable of generating the J558 Id than other D-limited mice.

76 67 Figure 5: D-DµFS generate more nucleotide sequences encoding for J558 Id compared to other D-limited mice. Percent of total V(D)J nucleotide sequences from D-limited and WT mice that encode for J558 Id. Expression of the J558 Id was determined based on reactivity with the anti-j558 idiotype EB3-7. * p < 0.05

77 68 Nucleotide sequence analysis could not identify D H segments in a large proportion of HCDR3 regions amplified from all D-limited mice ( 40-80%) in contrast to those in WT ( 20%) (Figure 6). D H RF1 was predominantly used in WT HCDR3 regions ( 80% versus 20-60% in D-limited mice) as expected (Figure 6). These observations suggest that extensive nucleotide deletion and addition at the V(D)J joins have occurred in DEX-specific B cells in D-limited mice. Figure 6: A significant percentage of DEX-specific HCDR3 regions from D-limited mice have D H -less V(D)J joins. HCDR3 sequences amplified from DEX-specific plasma cells from each of the D-limited mice and WT were examined for D H reading frame preference. Percent of total unique HCDR3 sequences utilizing each D H reading frame is shown. Sequences where D H gene segment could not be assigned to a germline D H gene segment (see Material and Methods) are denoted as No D H.

78 69 DISCUSSION Antibodies to a number of polysaccharide antigens have several common attributes: HCDR3 is implicated in binding to antigen (37, 38); its length is strictly maintained (39, 40) and almost invariably characterized by the presence of hydrophilic tyrosine residues (34, 35, 41). Although Davis et al. (15) have shown that HCDR3 diversity is sufficient for specific antibody responses to a variety of antigens, they could not extend this observation to two bacterial polysaccharide antigens they tested. They interpreted the latter finding that the single V H gene used by their mouse model could not accommodate the polysaccharide-specific response, suggesting that antibody responses to the polysaccharides they tested depended on the V H region. Work by Casadevall and colleagues (42) show that in addition to HCDR3 regions, some amino acids in the HCDR1 and HCDR2 may be required for the fine specificity to the polysaccharide GXM. These studies highlight the significant role of V H and HCDR3 region diversity to generate polysaccharide-specific antibodies. We restricted HCDR3 diversity in this study by using gene-targeted mice that are limited to use one D H gene segment and investigated the effect of this limited D H diversity on the antibody response to DEX. We have shown that limited HCDR3 diversity in TdT-/- mice leads to a lower anti-dex response and limited expression of the J558 idiotype (T.I. Mahmoud and J.F. Kearney, manuscript in preparation). We show here that forcing the use of a D H segment that encodes for hydrophilic (ΔD-DFL), charged (ΔD-DiD) or hydrophobic (ΔD-DµFS) amino acids does not impair the DEXspecific antibody response and that ΔD-DµFS mice are capable of generating higher levels of J558 idiotype expressing antibodies. This study shows that despite genetic

79 70 constraints on D H usage, the diversity and magnitude of the antibody response was not greatly affected. Compared to the other D-limited mice ΔD-DµFS mice show the most optimal response to DEX. DEX-specific B cells were found to be higher in number in peritoneal cavity exudates in ΔD-DµFS, as well as in D-DFL controls, compared to WT. This suggests that the increase in pre-immune DEX-specific B cells could not explain this enhanced responsiveness. Holmberg et al. have shown that B-1b cells frequently express D H genes in RF2, the reading frame used by the majority of ΔD-DµFS B cells suggesting that DEX-specific B cells utilizing this reading frame could enrich there (43). However, Schelonka et al. show that the total B1b population numbers in ΔD-DµFS mice do not differ much from littermate controls (44). The increased number of pre-immune DEXbinding B cells in two of our D-limited mice suggests that the DEX-specific repertoire in these mice could be self-reactive and therefore occupy a niche in the peritoneal cavity to prevent any inadvertent autoimmune reaction. If this were true, then we can speculate that there are increased numbers of DEX-binding B cells in the spleen of these mice, compared to the WT, that are of the marginal zone B cell phenotype. On the other hand, these D-limited mice could have an altered mucosal Ig repertoire allowing for increased numbers of DEX-expressing commensal bacteria, which in this case would select for increased numbers of DEX-specific B cells in the peritoneal cavity. A fecal count of DEX-expressing bacteria in ΔD-DµFS and D-DiD compared to control mice would test this hypothesis. At the molecular level, D-limited mice were more or less capable of recapitulating the anti-dex antibody response elicited by WT mice to Enterobacter cloacae. However,

80 71 a large proportion of the DEX-specific HCDR3 sequences amplified from these immunized mice contained no identifiable D H gene segments. It seems that extensive nucleotide modifications, such as nucleotide deletions or additions, were very common in HCDR3 regions of DEX-specific plasma cells isolated from D-limited mice. These junctional alterations seemed to be required to diverge the repertoire away from its genetically enforced D H gene segment in order to respond properly to DEX and generate a more or less HCDR3 region of hydrophilic nature. It is of interest to investigate the DEX response elicited by D-limited mice crossed to TdT-/- mice, due to extensive role of TdT in generating junctional diversity. Other mechanisms of generating D H -less HCDR3 regions could also be playing a role. These mechanisms are thoroughly discussed in a recent report by Rohatgi et al (45). Briefly, these D H -less HCDR3 regions can be generated by 1) Recombination Signal Sequence replacement, hence permitting direct V H to J H joining (46) 2) violation of the 12/23 bp rule as demonstrated in the D H gene deficient mouse (47). Taken together, several different mechanisms are in play to ensure that the heavy chain antibody repertoire conforms to certain architecture despite genetic manipulations. ΔD-DµFS mice were capable of generating higher levels of J558-expressing antibodies compared to the other mouse groups. The anti-dex clone J558 is of the highest affinity among all anti-dex antibodies studied (48) and is the predominant clone in the adult BALB/c response. Limited expression of this clone, as is evident in TdT-/- mice (T.I. Mahmoud and J.F. Kearney, manuscript in preparation), is among the factors that lead to a diminished antibody response to DEX. Our sequence analysis of HCDR3 regions amplified from DEX-specific plasma cells 7 days after immunization suggest that

81 72 the ΔD-DµFS mouse is more capable of generating nucleotide sequences encoding for the antibodies that express the J558 idiotype compared to other D-limited mice. This possibly provides an explanation for the capability of ΔD-DµFS mice to generate elevated serum levels of the J558 idiotype post-immunization. It is possible that the D H sequence of ΔD-DµFS mice is a better substrate for exonucleases and for TdT-mediated N-nucleotide addition, thus increasing the chances of generating J558 idiotype by alternative nucleotide arrangements. A TdT splice variant, TdTL, has been described as an exonuclease trimming 3 coding ends during V(D)J recombination (49, 50) The exonuclease activity attributed to this splice variant has been challenged by several reports based on in vitro assays (51-53). It may be that this putative exonuclease activity for TdTL is only apparent in the context of a specific immune response where constraints on HCDR3 lengths is a recurrent feature, such as the case with the antibody response to many polysaccharides. Past studies have shown that D-limited mice elicit a lower antibody response to purified DEX (29-31). In this study, we analyzed the DEX-response elicited by the gramnegative commensal Enterobacter cloacae and it is very likely that pathogen associated molecular patterns, such as LPS, engaged Toll-like receptors and other receptors involved in the innate arm of the immune response, possibly boosting the antibody response. This report shows that mechanisms that control the nature of the antibody response to DEX are operating to ensure that antibodies generated are of certain characteristics, regardless of the available HCDR3 that could be generated as dictated by the genetic make-up. These restrictive mechanisms are not very well understood and have received little attention in immunological research (42). Possible mechanisms include

82 73 strict structural constraints for the antigen antibody interaction such that only some V regions can be selected, idiotypic regulation and deletion of self reactive clones resulting in the emergence of a dominant clone (54-56). Better understanding of these mechanisms has very significant implications in the struggle to make vaccines that are effective in protection for the lifetime of the individual. ACKNOWLEDGEMENTS The authors are very grateful to Jeremy Foote (University of Alabama at Birmingham) for very helpful discussions and Yingxin Zhuang (University of Alabama at Birmingham) for animal husbandry. FOOTNOTES 1 This work was supported by research funds from the National Institutes of Health (NIH) Grant AI T.I.M. is a recipient of the grant T32 AI from the National Institute of Allergy and Infectious Diseases, NIH. 2 Address correspondence and reprint requests to Dr. John F. Kearney 410 Shelby Biomedical Research Building 1825 University Blvd Birmingham, AL Office: (205) Fax: (205) address: jfk@uab.edu

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89 80 CONCLUSION AND FUTURE PERSPECTIVES The focus of the work presented here was to investigate the role of limiting HCDR3 diversity in the antibody response to polysaccharides. We established in the first paper a significant role for TdT in generating the DEX-specific high affinity clone J558. TdT is also important for the generation of a set of diverse DEX-specific clones that contribute to an enhanced overall antibody response to DEX. Our findings also provide some molecular details for the late ontogenetic development of DEX-specific precursors. Fetal and neonatal repertoires lack the expression and activity of TdT (94, 132). Therefore, our findings add another factor to consider when accounting for the immaturity of the neonatal response to T-independent and T-dependent antigens. This is significant, because this age group is most susceptible to a number of life-threatening microorganisms using polysaccharides as virulence determinants and it is therefore important to induce a protective immune response upon vaccination. Several reasons have been put forward to explain the relatively feeble neonatal response to antigenic challenge (reviewed in (118)): 1) maternal antibodies circulating in the neonate which preclude the infant s immune system from sampling these antigens (133); 2) mouse and human B cells express lower levels of activation makers such as CD80, CD86, CD40 (134); 3) both human and mouse B cells express lower levels of TACI, the ligand for APRIL which is a significant survival factor for plasma cells, the B cell subset that produces antibodies (134, 135); 4) the mouse neonatal bone marrow compartment has a

90 81 limited capacity to support the establishment of long-lived plasma cells (136, 137); 5) impaired complement-mediated reactions in humans and mice (138, 139); 6) deficient organization of the human splenic marginal zone (140); 7) a delay in germinal center induction leading to impaired antibody switching to the IgG isotype as a result of the immature state of the follicular dendritic cell network (141, 142). Our work shows that selection of B cell clones by intrinsic or extrinsic factors also play a role in the immaturity of the response. The idiotype expression profile of anti-dex antibodies elicited upon immunization with a T-dependent form of DEX was very similar to that of T-independent DEX (143). Therefore our findings for the requirement of TdT for an optimal anti-dex response can likely be further extended to T-dependent antigens. It is expected that the presence of high affinity responsive B cell clones, would generate a more robust initial response upon vaccination. This robust antibody and memory B cell response upon primary vaccination has been correlated with longer persistence of levels of protective antibodies and the intensity of secondary booster responses (141). Persistence of primary vaccine-induced antibody is a major concern among vaccinologists and has prompted calls for adopting multiple booster doses to ensure persistence of protective antibodies for the first decade of life. Protecting this susceptible age group leads to better overall defense against pathogens through herd immunity. The other alternative is to have a better understanding of the mechanisms that lead to the induction of the best neonatal response possible and attempt to enhance it. The implications of TdT deficiency go far beyond the absence of its activity in neonatal mice. Adult mice that lack TdT-/- have been shown to lack heterosubtypic immunity to influenza viruses (57). Examination of the CD8+ T cell repertoire has shown

91 82 that TdT-/- mice, despite preserving their public TCR repertoires (56), have diminished responding private TCR repertoires (58). Private TCR repertoires confer an immunological advantage to the population as a whole and its loss risks the demise of the population in the case that the public repertoires are incapable of handling a new antigenic threat. I predict that genome-wide association studies with large cohorts will one day uncover a population with mutations in their TdT genes and demonstrate that this mutation is genetically linked to susceptibility to certain infections. Studies that address the function of TdTL, a splicing variant of TdT, are relevant in this context. TdTL follows the same expression pattern as TdT and therefore its activity is absent from neonatal and fetal repertoires. Studies to date that have looked at the activity of TdTL using biochemical assays (52, 85-87) or by examining the effects of overexpressing TdTL on the global repertoire ((63), T.I. Mahmoud and J.F. Kearney, unpublished observations) have failed to definitively demonstrate its function. Mice transgenic for both TdT splice variants show that TdTL may regulate the activity of TdT. Although, examination of neonatal sequences of mice transgenic for TdTS and therefore do not express TdTL show a normal frequency and size of N-additions at V(D)J junctions, mice transgenic for both TdT splice variants were shown to exhibit less N- additions and more homology-mediated joining, compared to TdTS transgenic mice alone, upon examination of DFL16.1-J H 1 gene rearrangements from fetal livers (63). We have preliminary evidence that the activity of TdTL can be demonstrated more clearly in the context of a specific antibody response. We show that mice transgenic for both TdT splice variants generate DEX-specific antibody levels comparable to wild type BALB/c but fail to express the J558 idiotype. Therefore the activity of TdTL could very subtle in

92 83 that it fine-tunes the nature of the clonal response to antigen in a manner that must be of an advantage to the host; although that advantage remains to be demonstrated. In our second paper we demonstrate that limiting the contribution of D H gene segments to HCDR3 diversity does not significantly impair the antibody response to DEX. We also show that regardless of the hydropathic nature of the starting HCDR3 repertoire, the DEX-specific clones maintain their hydrophilic nature as well as the expression of idiotypes that correlate with high affinity to antigen. The expression of the J558 idiotype was shown to be increased in ΔD-DµFS mice compared to other D-limited mice. Although the antibody response to T-dependent and T-independent antigens elicited by D-limited mice has been studied, our paper examines the response at the molecular level. Essentially, we demonstrate that in the context of a bacterial antigenic challenge, a minor population whose HCDR3 composition is hydrophilic expands to dominate the response to DEX. These data suggest that mechanisms involved in diversification of the HCDR3 repertoire ensure that a certain proportion of mature recirculating B cells have wild type-like HCDR3 properties. It will be of great interest to study those mechanisms, especially in antibody responses to polysaccharides, which often show remarkably oligoclonal responses with restricted heterogeneity. Few studies have addressed the mechanisms of that lead to the expression of a certain set of antibody genes that predominate in a response. This restriction has considerable implications to host defense because homogeneity in the response can limit the biological potential of antibody responses and render the host vulnerable to loss of the relevant B cell clones. It is very likely that selection based on the antigen receptor plays a major role in this

93 84 process. To test this hypothesis, it is possible to examine the DEX-specific repertoire of D-limited mice crossed to Bcl-xL transgenic mice. Bcl-xL is an anti-apoptotic protein and Bcl-xL transgenic mice show an increased frequency of aberrant V(D)J rearrangements as well as an increase in self-reactive B cells that have escaped negative selection (144, 145). Alternatively, an examination of D J rearrangements, which are not subject to antigen-receptor mediated selection, in DEX-specific B cells isolated from D-limited mice can address this question. However, we find that most of the sequences amplified from DEX-responding plasma cells in these mice had no identifiable D H gene segments therefore precluding the amplification of these rearrangements. The high frequency of DEX-responding plasma cells with D H -less joins suggests that extensive nucleotide modifications have occurred to generate these sequences. These could be N-additions or nucleotide deletions to V(D)J gene coding ends. This prompts an investigation into the role for TdT, Polλ and Polµ in generating D H -less joins in DEXresponding B cells of D-limited mice. Mice that are deficient for each of these Pol X polymerases are available and we suspect that crossing D-limited mice to TdT-/- mice will significantly reduce the anti-dex antibody response. The antibody response of Polλ and Polµ-deficient mice to DEX is worth investigating as well, given the significant role contribution of the HCDR3 region to the nature of the DEX-response. Finally, I propose to further investigate the role of HCDR3 diversity in the antibody response to polysaccharides and other T-cell independent antigens derived from microorganisms that pose a major health threat. Group A carbohydrate (GAC) is associated with Group A Streptocooci, and is actively studied in our research group. Examination of the primary V(D)J heavy chain sequence of the HGAC39 antibody clone

94 85 that binds to GAC shows an HCDR3 regions with extensive N-addition at the V DJ join. The D J in the HGAC39 clone shows homology-mediated joining suggesting that this rearrangement may have been formed just prior to the expression of TdT and therefore maybe generated early in life. HGAC39 uses DST4 D H gene segment in RF1, as expected. Another antibody clone of interest to our group, SMB19, which recognizes the sialyllacto-n-tetraose polysaccharide associated with Group B Streptocooci, shows N- addition at both V DJ and D J junctions suggesting that the clone is of mature onset. SMB19 uses DSP2.2 D H gene segment in RF3, which shows extensive 5 nucleotide deletions that nibbled away the termination codon that would have been encoded by this reading frame. The HCDR3 loop of this clone interestingly encodes two arginines, two leucines and one proline amino acid. I expect that investigating the role of limiting HCDR3 diversity, using TdT-/- and D-limited mice, on the antibody response to these two polysaccharides will hold promising results that will further substantiate the findings we report in this work.

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105 Haas, K. M., J. C. Poe, D. A. Steeber, and T. F. Tedder B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 23: Landers, C. D., R. L. Chelvarajan, and S. Bondada The role of B cells and accessory cells in the neonatal response to TI-2 antigens. Immunol Res 31: Siegrist, C. A., and R. Aspinall B-cell responses to vaccination at the extremes of age. Nat Rev Immunol 9: Casadevall, A., and M. D. Scharff The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies. J Exp Med 174: Kimura, H., R. Cook, K. Meek, M. Umeda, E. Ball, J. D. Capra, and D. M. Marcus Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine. J Immunol 140: Rudikoff, S Antibodies to beta(1,6)-d-galactan: proteins, idiotypes and genes. Immunol Rev 105: Schilling, J., B. Clevinger, J. M. Davie, and L. Hood Amino acid sequence of homogeneous antibodies to dextran and DNA rearrangements in heavy chain V-region gene segments. Nature 283: Otteson, E. W., W. H. Welch, and T. R. Kozel Protein-polysaccharide interactions. A monoclonal antibody specific for the capsular polysaccharide of Cryptococcus neoformans. J Biol Chem 269: Evans, S. V., D. R. Rose, R. To, N. M. Young, and D. R. Bundle Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idiotope Fab. J Mol Biol 241: Hutchins, W. A., A. R. Adkins, T. Kieber-Emmons, and M. A. Westerink Molecular characterization of a monoclonal antibody produced in response to a group C meningococcal polysaccharide peptide mimic. Mol Immunol 33: Xu, J. L., and M. M. Davis Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities. Immunity 13: Nakouzi, A., and A. Casadevall The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan. Mol Immunol 40:

106 Kearney, J. F., M. T. McCarthy, R. Stohrer, W. H. Benjamin, Jr., and D. E. Briles Induction of germ-line anti-alpha 1-3 dextran antibody responses in mice by members of the Enterobacteriaceae family. J Immunol 135: Rappleye, C. A., L. G. Eissenberg, and W. E. Goldman Histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the betaglucan receptor. Proc Natl Acad Sci U S A 104: Blomberg, B., W. R. Geckeler, and M. Weigert Genetics of the antibody response to dextran in mice. Science 177: Hansburg, D., R. M. Perlmutter, D. E. Briles, and J. M. Davie Analysis of the diversity of murine antibodies to dextran B1355. III. Idiotypic and spectrotypic correlations. Eur J Immunol 8: Carlsson, L., and D. Holmberg Genetic basis of the neonatal antibody repertoire: germline V-gene expression and limited N-region diversity. Int Immunol 2: Siegrist, C. A Mechanisms by which maternal antibodies influence infant vaccine responses: review of hypotheses and definition of main determinants. Vaccine 21: Kaur, K., S. Chowdhury, N. S. Greenspan, and J. R. Schreiber Decreased expression of tumor necrosis factor family receptors involved in humoral immune responses in preterm neonates. Blood 110: Kanswal, S., N. Katsenelson, A. Selvapandiyan, R. J. Bram, and M. Akkoyunlu Deficient TACI expression on B lymphocytes of newborn mice leads to defective Ig secretion in response to BAFF or APRIL. J Immunol 181: Pihlgren, M., M. Friedli, C. Tougne, A. F. Rochat, P. H. Lambert, and C. A. Siegrist Reduced ability of neonatal and early-life bone marrow stromal cells to support plasmablast survival. J Immunol 176: Pihlgren, M., N. Schallert, C. Tougne, P. Bozzotti, J. Kovarik, A. Fulurija, M. Kosco-Vilbois, P. H. Lambert, and C. A. Siegrist Delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life. Eur J Immunol 31: Johnston, R. B., Jr., K. M. Altenburger, A. W. Atkinson, Jr., and R. H. Curry Complement in the newborn infant. Pediatrics 64: Pihlgren, M., A. Fulurija, M. B. Villiers, C. Tougne, P. H. Lambert, C. L. Villiers, and C. A. Siegrist Influence of complement C3 amount on IgG responses

107 98 in early life: immunization with C3b-conjugated antigen increases murine neonatal antibody responses. Vaccine 23: Timens, W., A. Boes, T. Rozeboom-Uiterwijk, and S. Poppema Immaturity of the human splenic marginal zone in infancy. Possible contribution to the deficient infant immune response. J Immunol 143: Blanchard Rohner, G., M. D. Snape, D. F. Kelly, T. John, A. Morant, L. M. Yu, A. Borkowski, F. Ceddia, R. Borrow, C. A. Siegrist, and A. J. Pollard The magnitude of the antibody and memory B cell responses during priming with a protein-polysaccharide conjugate vaccine in human infants is associated with the persistence of antibody and the intensity of booster response. J Immunol 180: Pihlgren, M., C. Tougne, P. Bozzotti, A. Fulurija, M. A. Duchosal, P. H. Lambert, and C. A. Siegrist Unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to T-dependent antigens. J Immunol 170: Kearney, J. F., B. A. Pollok, and R. Stohrer Analysis of idiotypic heterogeneity in the anti-alpha 1-3 dextran and anti-phosphorylcholine responses using monoclonal anti-idiotype antibodies. Ann N Y Acad Sci 418: Fang, W., D. L. Mueller, C. A. Pennell, J. J. Rivard, Y. S. Li, R. R. Hardy, M. S. Schlissel, and T. W. Behrens Frequent aberrant immunoglobulin gene rearrangements in pro-b cells revealed by a bcl-xl transgene. Immunity 4: Fang, W., B. C. Weintraub, B. Dunlap, P. Garside, K. A. Pape, M. K. Jenkins, C. C. Goodnow, D. L. Mueller, and T. W. Behrens Self-reactive B lymphocytes overexpressing Bcl-xL escape negative selection and are tolerized by clonal anergy and receptor editing. Immunity 9:35-45.

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