Expression o f Human IgG Subclasses*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 17, No. 3 Copyright 1987, Institute for Clinical Science, Inc. Expression o f Human IgG Subclasses* M. H. NAHM, M.D.,t M. G. SCOTT, P h.d., Î and P. G. SHACKELFORD, M.D. fdivision o f Laboratory Medicine, Department o f Pathology, and Division of Infectious Diseases, Department o f Pediatrics, Washington University School o f Medicine, St. Louis, MO ABSTRACT H um an immunoglobulin G (IgG) can be divided into four subclasses th a t are selectively expressed. For instance, carbohydrate antigens p referentially elicit IgG2 antibodies, w hereas protein antigens usually elicit Ig G l and IgG3. Elucidating the biological basis of the selective expression of these IgG subclasses is im portant to our understanding im m unodeficiencies and B lym phocyte developm ent. To investigate clinical im portance of IgG subclass deficiencies, a sensitive and specific assay has been developed for IgG subclasses using particle concentration fluorescence immunoassay. Prelim inary clinical studies have already shown that infection-prone individuals often have selective IgG2 subclass deficiency. N ormal levels of IgG2, however, do not rule out an immunodeficiency in the infection-prone individuals because some individuals have normal levels of IgG subclasses and are poorly responsive to antigens of bacteria. Based on animal studies, two contrasting models of B cell developm ent have been advanced. One model of B cell developm ent proposes a single lineage and proposes that a B cell can successively switch and produce any IgG subclass. The other model proposes m ultiple lineages and proposes that a B cell can express only some IgG subclasses. It has been found by us that anti-pc antibodies are mostly IgG2 with some IgG l, and that the V region of Ig G l anti-pc antibody is different from that of IgG2 antibody. O ur finding, therefore, suggests that B cells producing anti-pc antibodies are progeny of not one ancestral B cell that has successively switched, but two independent ancestral B cells. Cellular studies using polyclonal activators also suggest that regulatory mechanisms for IgG l and IgG3 are differe n t from those of IgG2 and IgG4. Taken together, we favor the m ultilineage m odel b etter than the single lineage m odel of hum an B cell developm ent. * This work was supported by grants from the P ublic H ealth Service (AI-19676, AI-19350, and A I-17962), and from Tobacco Research Council (1902). + Present Address: Mallinckrodt, Inc., 20-3-W, 675 M cdonnell Blvd., St. Louis, MO Introduction Im m unoglobulins (Ig) consist of heavy (H) and light (L) polypeptide chains held to g e th e r by non-covalent forces and /87/ $02.00 Institute for Clinical Science, Inc.

2 184 NAHM, SCOTT, AND SHACKELFORD disulfide bonds.9 The variable region of an immunoglobulin, which contains the antigen binding site, is form ed by the amino term inal amino acids of the heavy and light chains. Tremendous structural diversity of antigen binding sites is achieved by a specialized genetic process for encoding th ese amino term i nal segm ents. T he process involves a presum ably random com bination of m ultiple gene segments; VH, D H, and JH for the heavy chain variable regions, and VL and JL for th e lig h t c h a in v a ria b le regions.29 Since there are hundreds of V genes and many D and J genes, a very large num ber of antigen binding sites can be formed. In contrast, the carboxy term inal segm ents, or constant regions of heavy and light chains, are encoded by only one of nine CH genes for the heavy chain and one of two CL genes for the light chain. Im m unoglobulin m olecules are ca te gorized into various isotypes based on th eir heavy chain constant region. In h um ans, th ese are IgM, IgD, Ig A l, IgA2, Ig E, and four IgG subclasses, IgG l, IgG2, IgG3, and IgG 4.7 In mice, th e IgG subclasses are Ig G l, IgG 2a, IgG2b, and IgG3. The constant region of an Ig is responsible for various biologic p ro p erties of the m olecule, including com plem ent fixation, opsonic properties, placental transfer, and Fc recep to r binding.42'47 In view of the biological differences am ong th e IgG subclasses, it is not surprising that the IgG subclasses are selectively expressed (table I). First, the antibody re sp o n se to d iffe re n t ty p es of antigens seem s to favor certain su b classes. For exam ple, m any carbohyd ra te (C H O ) a n tig en s p re fe re n tia lly elicit IgG responses restricted to IgG3 in mice27,45 and to IgG2 in hum ans.2,31,56 In c o n tra s t, I g G l d o m in a te s th e IgG response to m any p ro tein antigens in both species.38,42'44,45,48 Secondly, acqui- T A B L E I Summary of Selective Expression of IgG Subclasses Mice Humans Dominant IgG subclass to: CHO antigens IgG3 IgG2 Protein antigens IgGl IgGl Ontogeny of: CHO immunocompetence Late Late IgG2 (man) - Late IgG subclass associated immunodeficiencies xid Ataxia- Telangiectasia. IgA-IgG2-IgG4- deficiency. IgG subclass restricted response to mitogens Yes Yes IgG subclass specific lymphokines Yes "> Phenotypic B cell subpopulation antigens Yes j sition of adult levels of IgG2 in hum ans is delayed during ontogeny.24 It is speculated that the delayed m aturation of the ability to produce an im m une response to C H O antigens is linked to this late m aturation of IgG2. Interestingly, the re s p o n s e to C H O a n tig e n s is also d e la y e d d u rin g o n to g en y in m ic e.22 Thirdly, expression of certain IgG subclasses is selectively suppressed in certain im m unodeficiencies. Patients with ataxia-telangiectasia have selective deficiencies of IgA and of the IgG2 and IgG4 subclasses.25 CBA/N m ice have an X- chrom osom e-linked im m unodeficiency that results in selective deficiency of the IgG3 subclass.28,32 Lastly, a subclass specific lym phokine (BSF-1) has b een identified in the m ouse which preferentially facilita te s Ig G l and d e c re a ses IgG 3 expression following m itogen stim ulation.54 These observations, summarized in table I, strongly suggest a m echanism ^) that selectively regulates expression of th e IgG subclasses. To explain sele c tiv e IgG subclass expression, two contrasting models of B cell developm ent have been proposed. O ne m odel proposes th at all B cells

3 EXPRESSION OF HUMAN IGG SUBCLASSES 185 d e v e lo p in a sin g le lin eag e an d can express all IgG subclasses. In this model, selective expression of IgG subclasses is solely the result of exogenous factors, such as T cells, in d ucing successive expression of th e IgG subclasses according to their order in the genome. 12,21,5 A s tric t e x tra p o la tio n of th is m odel to hum an B cells is depicted in figure 1. T he a lte rn a tiv e m odel proposes th at m ultiple B cell lineages exist which are restricted in IgG subclass expression and w hich d e v e lo p at d iffe re n t tim es in ontogeny.1,20 B ecause the two m odels are of fundam ental im portance in understanding B cell developm ent and regulation, they have been intensely studied in experim ental animals. Thus far, neither m odel can fully explain all of the observations. Because of the biological and clinical im p o rta n c e of n o n -ran d o m subclass expression, studying this phenom enon in hum ans was of interest to us. It was felt that hum an studies might provide a fresh perspective on the models which had been proposed on the basis of experim ental animal studies. O ur aims w ere (1) to identify patients w ith specific subclass deficiencies; (2) to characterize the hum an IgG subclass responses to several C H O antigens; and (3) to study th e cellular basis of IgG subclass expression. An essen tial p re -re q u isite to accom plish F ig u r e I. T w o h y p o th e tic a l b u t c o n tr a s tin g m o d e ls o f B c e ll d e v e lo p m e n t.

4 186 NAHM, SCOTT, AND SHACKELFORD these aims was the developm ent of sensitive and specific assays for hum an IgG subclasses. These assays, and the results of our studies of IgG subclass expression are described. D evelopm ent of Sensitive and Specific IgG Subclass Assays The constant regions of hum an IgG subclasses have a very high degree of homology, yet have a large degree of allotypic variation. Consequently, ever since th e serological definition of IgG su b classes,14,51 th e ir study has been h am p ered by difficulties in m easurem ent. In ord er to produce specific antisera to IgG subclasses, a variety of anim als, in c lu d in g m onkeys, has b een im m unized w ith hum an m yelom a prote in s. E x h a u stiv e im m u n o ch e m ic a l m anipulations have b een req u ired to render these antisera specific.37,53 Even when a satisfactory antiserum has been prepared for a particular assay, it often lost its specificity w hen used in a different assay configuration.15 For example, m any of the reagents w ere lim ited for use in the relatively insensitive immunodiffusion assay or in the technically difficult hem agglutination assay. In o rd er to investigate th e cellular basis of isotype restriction, extrem ely sensitive assays w ere req u ired to m easure in vitro IgG subclass production. Thus, sensitive and specific radioim m u noassays (RIA) for the m easurem ent of all four IgG subclasses w ere developed by us.37 O ur initial RIA s used m urine monoclonal antibodies specific for IgG l and IgG 3, and specific, ab so rb ed antisera, raised in monkeys and sheep for IgG2 and IgG4, respectively. T he subclass specific antib o d ies w ere coated onto the wells of 96-well polyvinylchloride m icrotiter plates. To these wells, either samples or myeloma protein standards w ere added and titrated as inhibitors of th e b in d in g of an 125I-lab eled purified m yelom a p rotein of th e appropriate subclass. The concentration of the IgG subclasses was calculated by com paring the inhibition produced by standards and samples. The sensitivity was the m inim um am ount of standard which produced 50 percent inhibition and was generally 50 to 200 ng p er ml. Specificity of th e reag en ts was confirm ed w ith a panel of myeloma proteins. The average in h ib itio n c u rv e s w ith a s e rie s of myeloma proteins from each subclass are shown in figure 2. However, RIA results w ere poorly reproducible from plate to plate, presum ably because of variation in m ic ro tite r plates th a t affected th e efficiency of solid phase antibody coating. Since it had been established by us that th e specificity of some of the subclass-specific reagents was dependent on the assay configuration, it was wished th at the rep ro d u cib ility of our assays would be im proved without altering the configuration. T herefore, a new analytical m ethod called particle concentration fluorescent immunoassay (PCFIA), was u tiliz e d.17,19 In this assay, 0.8 m icron latex particles w ere coated with IgG subclass specific antibodies and placed in specialized m icrotiter plates (figure 3). Samples or standards w ere added to the wells as com petitive inhibitors of the binding of fluorescein labelled myeloma proteins of the appropriate subclass. Following an incubation period, unbound f lu o r e s c e n t m y e lo m a p r o te in was rem oved by washing and filtration. The fluorescence bound to the latex particles was inversely related to the am ount of th at IgG subclass in the sam ple. This assay achieves high sensitivity by concentrating the latex particles to a very small area by filtration, after which the fluorescence is read. Also because specialized p lates w ith m any individual wells are used, a large n u m b er of sam ples can easily be analyzed. W hile the inhibition configuration similar to that u sed in o u r RIA s was generally m ain

5 EXPRESSION OF HUMAN IGG SUBCLASSES 187 CONCENTRATION OF INHIBITOR PROTEIN (ng/ml) F ig u r e 2. Specificity of subclass specific radioimmunoassays. Shown are mean inhibition curves of four IgG l ( ), five IgG2 (O O), three IgG3 (A A), and three IgG4 ( ) proteins which include proteins of each light chain type. Bars denote standard deviation. tained, the PC FIA m ethod is also adaptable to sandwich type double antibody assays. Specificity was confirmed for each of the PCFIA subclass assays with a panel of myeloma proteins.19 Accuracy of the assay system was confirmed in four ways: (1) F o r each subclass, th e curves p ro d u c e d by several d ifferen t m yelom a standards w ere shown to be parallel; (2) The sum of the subclass values was close to total IgG values d eterm in ed by an independent m ethod; (3) The correlation betw een PC FIA and RIA results was excellent; and (4) Using m yelom a standards, correct values w ere obtained for the W H O reference serum. The major advantage of this system over RIA was the increased precision (CV 10 p e r cent). However, an additional advantage was the capability of performing num erous (100) d e te rm in a tio n s in a sh o rt period of tim e (two hours). The sensitivity (0.3 to 3 xg per ml) of these assays was less than that of the RIA s, b ut was sufficient for all purposes, except m easurem ent of secreted IgG subclasses in some in vitro cultures. These assays are now routinely used to study total IgG subclass concentrations in serum and in affinity purified antibody preparations. Subclass-Specific Im m unodeficiency In order to study patients w ith IgG subclass deficiencies, it was first necessary to e s ta b lis h n o rm a l ra n g e s in healthy individuals. Using the subclass specific RIA, IgG subclass concentrations w ere m easured in sera from a large num ber of children and adults and the g e o m etric m eans and norm al ranges

6 188 NAHM, SCOTT, AND SHACKELFORD PCFIA (Particle Concentration Fluorescence Immunoassay) C C ^ plastic particles coated with antibody A labeled analyte (fluorescent) unlabeled analyte F ig u r e 3. Principle of particle concentration fluorescence immunoassay. Solid circles in the right panel represent latex particles that have acquired fluorescent analyte. A.1 and A2 represent excitation and emission wavelengths of the fluorescence. determ ined for various age groups ( ± 2 S.D. about the mean) (table II). These normal ranges were similar to previously published values.24 It was also confirmed by us that, in children, acquisition of adult IgG2 levels is delayed, when compared to the other subclasses.39 Next, IgG subclass concentrations in sera w ere prospectively examined from 29 children of different ages w ith recu r rent bacterial infection.40 Seven of these patients had IgG2 deficiency, defined as a serum concentration which was greater than 3 S.D. below the appropriate agegroup mean. Interestingly, only one of these children also was deficient in IgG l (table III). C linical im provem ent was seen in three of four children with IgG2 deficiency who received replacem ent gam m a globulin therapy. O ther recent rep o rts have em phasized the possible role of IgG 2 deficiency in individuals with recurrent sinopulmonary infections and otitis m e d ia.1052 A lthough th ese studies used different criteria for IgG2 deficiency (greater than 2 S.D. below the mean) and w ere largely based on retrospective analysis, it appears that selective IgG2 deficiency is frequent among children w ith re c u rre n t bacterial infections. These observations suggest that serum concentrations of IgG subclasses should be m easured in individuals with recurrent infections. P re v io u s r e p o r ts o f p r e f e re n tia l expression of IgG 2 in antibody responses

7 E X P R E SSIO N O F H U M A N IG G SUBCLASSES TABLE I I IgG Subclass Concentrations (mg/ml) in Healthy Subjects Age (Years) IGGl (n)* [ bounds'] IGG2 (n) [bounds] IGG3 (n) Abounds! IGG4 (n) Lrangel (7)* 0.77(7) ND ND Cl.5-8.7] [ ] 1 6.0(26) 1.1(26) 0.19(10) 0.030(9) X J [ ] [ ] [ ] 2 5.4(23) 1.3(23) 0.19(10) 0.02(10) [ ] [ ] [ ] [ ] (22) 1.5(22) 0.25(21) 0.11(21) [ ] [ ] [ ] [ ] (26) 1.7(26) 0.32(13) 0.16(12) [ [ ] CO.095-l.ll CO ] >17 6.6(41) 2.6(41) 0.32(19) 0.47(19) [ ] [ ] [ ] [ ] *Values shown are geometric means. The normal bounds for IgGl, IgG2, and IgG3 in brackets were determined by taking the antilog of (mean logarithm ± 2 S.D. of the logarithms). For IgG4, because of extremely wide variation, the normal values in brackets indicate the range. The number in parentheses indicates the number of individuals studied for each group. (Reprinted from Shackelford, P.G., at al., Spectrum of IgG2 deficiency in children with recurrent infections: Prospective study, J. Pediatr. 108: , 1986.) to C H O antigens raised th e possibility that individuals who responded poorly to these antigens may be deficient in IgG2. U m etsu et al52 have shown that the abil- ity to respond to H aem ophilus influenzae type b (Hib), polysaccharide vaccine is im paired in children w ith IgG 2 deficiency. However, patients with W iskott- T A B L E I I I Immunoglobulin Concentrations (mg/ml) in IgG2 Deficient Patients A g e Clinical Patient (Years) F e a t u r e s * IgG IgGl IgG2 XgG3 IgG4 IgA I g M 1 19 P,A $ $ <0.06$ 1.0 (6.6)t (4.0) (0.51) (0.072) (0.010) (0.70) (0.56) ,S $ (4.2) (2.7) (0.49) (0.051) (0.005) (0.14) (0.48) S,P,FTT 4.3$ $ <0.06$ 0.58 (6.3) (3.0) (0.32) (0.058) (0.010) (0.33) (0.48) S,P,FTT $ $ (4.5) (2.4) (0.30) (0.039) (0.005) (0.14) (0.43) Hib $ $ (4.2) (2.7) (0.49) (0.051) (0.005) (0.14) (0.48) ,P,S,FTT 4.1$ 1.5$ 0.23$ (6.3) (3.0) (0.54) (0.058) (0.010) (0.33) 0.48) M $ (4.4) (3.0) (0.54) (0.058) (0.010) (0.22) (0.47) *P = pneumonia; S = sinusitis; 0 = otitis media with drainage; Hib = recurrent invasive Haemophilus disease; M = meningitis; A = chronic polyarthritis; FTT = failure to thrive (<5th percentile). fvalues in parentheses for IgG, IgA, IgM, IgGl, IgG3, and IgG4 are the lower limits of normal as defined in Table 2. Value for IgG2 is 3 SD below the mean for a g e. ^Values below normal range. Patient also has thrombocytopenia. (Reprinted from Shackelford, P.G. et al., Spectrum of IgG2 deficiency in children with recurrent infections: Prospective study, J. Pediatr. 108: , 1986.

8 190 NAHM, SCOTT, AND SHACKELFORD A ldrich syndrom e, an X -chrom osom e lin k ed, in h e rite d d iso rd er associated w ith num erous im m unological abnorm alities, including poor responses to C H O antigens and severe recurrent bacterial infections, have norm al serum levels of IgG2 (figure 4).23 Also, serum IgG2 concentrations w ere normal in 33 ch ild ren w ho d e v elo p ed invasive H. influenzae disease in spite of im m unization with type b polysaccharide vaccine. T hese children had low type b polysaccharide antibody concentrations in conv alescen t serum follow ing d ise a se.13 These studies suggest that deficient anti- C H O -antibody responses may occur in individuals w ith norm al serum concentrations of IgG2. Therefore, evaluation of in fe c tio n -p ro n e in d iv id u a ls sh o u ld include m easurem ent of specific anti- C H O antibodies, as well as serum concentrations of IgG subclasses. Isotype Restriction o f Antibodies As m entioned previously, two models of B cell developm ent have been p ro posed on the basis of animal studies. The single lineage model is supported by the dem onstration that a single B cell can express all isotypes under experim ental conditions.12,49 According to this m odel, Ig G Subclass Levels Among WAS Patients I00r IgG I IgG2 I00r 100 IgG3 IgG4 E io N o> E < ce f o 2o o Ar 10 i 8 JL 10 I / t : F i g u r e 4. IgG 2 su b c l a s s l e v e l s a m o n g p a tie n ts w ith W isk o tt- Aldrich Syndrome ( A). P a t ie n t s w it h c y s t i c fibrosis (X, ) were used as c o n tr o ls w h o h a v e chronic infection without known immunological disease. and represent p a tie n ts y o u n g e r th an seven years old. Normal adult ranges (2SD ) are represented w ith arrows to the right of each panel. O ne patient with WAS has an IgG4 level below the detection lim it (0.01 m g per m l). A dapted from Nahm et al., J. Immunol. 137:3484, } WAS }Cys. Fib

9 EXPRESSION OF HUMAN IGG SUBCLASSES 191 all IgG subclasses can b e expressed successively according to their gene order, w ith T cells facilitating the successive downstream switching. According to this model, C H O antigens induce expression of IgG3 in mice because the response is T cell independent and thus select the constant region gene (C^3) nearest to the Cp, g e n e.50 In hum ans, how ever, the 5' > 3' CH region gene order is C^, C8, G.y3, Cai, G^2, C.y4, C, C0 2*A proximal (5' end) gene cluster contains Cy3, Cyl, and Cal while a distal (3' end) cluster contains the last four genes.7 Thus, in hum ans, the single lineage m odel would p redict that T -independent C H O antigens would preferentially elicit the IgG3 subclass. H ow ever, prior observations, although lim ited, have shown p referen tial expression of IgG 2 by these antigens.31,56 Thus, additional studies were perform ed of th e IgG subclass d istributio n o f a n ti-c H O a n tib o d ie s to te s t m o d e ls o f B c e ll d e v e lo p m e n t in hum ans. H um an antibodies were examined to p h o sp h o c h o lin e (PC), th e im m u n o dom inant antigen in the cell wall CH O of Streptococcus pneumoniae, to group A streptococcal C H O (GAC), and to the polysaccharide capsule of Haemophilus i n flu e n z a e ty p e b (H ib P S ).41 As reported by previous investigators,4,31 it was found by us that IgG2 is th e dom i nant subclass in naturally occurring antib o d ie s to PC (ta b le IV) a n d GAC (unpublished data). These results d em o n s tra te th e lim ita tio n of th e lin e a r m odel of B cell developm ent in hum ans as it cannot readily explain the preferential expression of the distally located C72 g e n e in th e s e a n ti-c H O a n tib o d y responses. However, the restriction of anti-cho antibodies to IgG2 is not absolute. One individual subject produced significant proportions of anti-pc antibodies in the IgG l subclass (46 percent) and another produced significant amounts of anti-pc in IgG2 (44 percent). In addition, when the IgG subclass composition of anti-hib PS antibodies in vaccinees was studied, substantial quantities w ere found of both Ig G l and IgG2 (table IV).41 In children, anti-h ib PS antibodies w ere predom i nantly Ig G l.13 Additional studies have also shown that children produce IgG l antib o d ies to pneum ococcal capsular polysaccharide antigens.10 Thus, a successful m odel of B cell d ev elo p m en t m u st explain th ese exceptions in addition to the apparent IgG 2 restriction noted above with respect to antibodies to PC and GAC. To u n d e rsta n d b e tte r the com plex expression of IgG subclasses, the antigen binding specificity of anti-pc antibodies of IgG l and IgG2 subclasses w ere studied. The single lineage model of B cell developm ent suggests that a particular V T A B L E IV IgG Subclass Distribution of Antibodies to PC and Hib Ps in Sera from Healthy Adults Antibody n Percent IgGl* Percent I g G 2 Percent IgG3 Percent IgG4 Anti-PC, healthy adults Anti-Hib PS, immunized adults ± 10 (1-46)f 83 ± 13 (48-100) 6 ± 10 (1-44) < ± 16 (31-77) 41 ± 16 (23-69) ND ND *To calculate the percentage for anti-pc, total IgG was assumed to be IgGl + IgG2 + IgG3; for anti-hib PS, total I g G was assumed to be IgGl + IgG2. IgG3 and IgG4 anti-hib PS antibodies have been assumed to be negligible. fmean ± S.D. (Range) (Data are adapted from Scott, et al.,1987 and Shackelford, et al., 1987.)

10 192 NAHM, SCOTT, AND SHACK ELFO RD region may associate w ith both Ig G l and IgG 2 a n tib o d ie s. H o w ev er, p re v io u s studies in m ice have shown that different V regions, or bin d in g specificities, are p re fe re n tia lly a sso c ia te d w ith e ith e r Ig G l or IgG 3 an tib o d ies.5,6-30'36'43 O u r studies of anti-pc antibodies in hum ans show ed th a t IgG 2 an ti-p C antib o d ies e x p re sse d V regions w hich b o u n d PC e ith e r w hen it was conjugated to a pro tein carrier or w hen it was as a com po n en t of the C -carbohydrate (C-CHO) of S. pneum oniae (figure 5). In contrast, Ig G l anti-pc antibodies bound PC only w h e n it was c o n ju g ated to a p ro te in. T hus, th e re are differences in the fine specificity of PC -binding V regions asso c iated w ith IgG 2 and those associated w ith I g G l. T his ob serv atio n in d icates that in hum ans, as well as in mice, cer tain V regions preferentially pair w ith certain IgG subclasses and supports the m ultiple B cell lineage model. H ow se le c tiv e pairin g of V an d C H regions may affect protective im m unity was also speculated by us. For example, it was questioned w h eth er IgG2 antibod ies differ from Ig G l antibodies in acti- B Gl W G2 W G2 Gl F i g u r e 5. Agarose isoelectric focusing of anti-pc antibody from a single donor. Gels were overlayed with either 125I-PC-BSA (panel A) or 125I-C-CHO (panel B). Lanes marked W are antibodies enriched by affinity chromatography using PC-conjugated Sepharose 4B. Lanes marked G l and G2 are IgG l and IgG2 fractions obtained by passing the enriched antibody over columns of monoclonal anti-igg l or anti-igg2 conjugated to Sepharose 4B and eluting with 0.1 M glycine-h Cl (ph 2.5). IgG2 anti-pc antibodies bind both PC-BSA and C-CHO. Ig G l anti-pc antibodies bind only PC-BSA.

11 EXPRESSION OF HUMAN IGG SUBCLASSES 193 vating co m plem ent-m ediated b actericidal activity or protective activity. Briles et al3 have shown that m urine anti-pc antibodies w ith th re e different IgG constant regions w ere similar in their capacity to p ro tect m ice from experim ental infection w ith S. pneum oniae. In collaboration with Drs. W inberg and Granoff, it was found by us that the functional capacity of hum an Ig G l and IgG2 anti- H ib PS antibodies was sim ilar as m easured by in vitro com plem ent-m ediated bactericidal assay and in vivo rat protectio n activ ity (figure 6).55 T h u s, differences am ong F c m ed ia te d biological activities cannot be responsible for the p re fe re n tia l e x p ressio n of IgG su b classes. C ellular Events in IgG Subclass E xpression To study the cellular m echanism s of IgG subclass expression in vitro, IgG subclass expression by hum an lym phocytes was exam ined following stim ulation with various m itogens.37 Because m itogens stim ulate large fractions of B cells to p ro life ra te and m a tu re into plasma cells, they can be used to identify a cell subpopulation im portant for the expression of certain IgG subclasses. Studies in m ice have shown that some m itogens preferentially stim ulate a subpopulation of B cells, resulting in secretion of only certain IgG subclasses.20 Although hum ans have a large num ber of F i g u r e 6. P a s s iv e protection o f newborn rats with isolated human IgG l and IgG2 antibodies to the capsule of H. influenzae type b (Hib). The X axis shows the dose of IgG l or IgG 2 anti-capsular antibody injected subcutaneously at tim e 0. Twentyfour hours later, animals were injected intraperitoneally w ith 100 colonyforming units o f Hib. The Y axis shows the percent of rats with bacteremia 24 hours after bacterial challenge. Numbers in parenth esis show bacterim ic rats/rats injected. Adapted from W ein berg et al. J. Immunol. 136:4232, ng of an ti-h ib antibody per rat

12 194 NAHM, SCOTT, AND SHACKELFORD IgG2-bearing B cells among peripheral minal centers are found to contain cells blood lym phocytes,1118 many m itogens bearing Ig G l or IgG3 but not IgG2 or preferentially elicit secretion of hum an IgG 4.16 Taken together, these observa- Ig G l and IgG3 subclasses and are poor tions favor the concept of separate B cell stim ulators of IgG2 and IgG4 (figure 7). lineages that express the IgG l/igg 3 or Thus, it is possible that Ig G l and IgG3 Ig G 2 /Ig G 4 s e ts o f subclasses and subclasses are expressed and regulated respond to different antigens and lymas a s e t w hile IgG 2 and IgG 4 are phokines. However, to delineate clearly included in a different se t. th e cellular m echanism s for the prefer- Several additional observations group ential expression of IgG subclasses, furthe four IgG subclasses similarly into two ther studies are needed. In this regard, sets. T he Ig G l and IgG 3 genes are the study of individuals w ith selective closely linked in one location on chromo- IgG subclass deficiency m ay provide som e 14 w hereas th e IgG 2 and IgG 4 im portant insights, genes are in another, m ore distal locatio n.7 In d iv id u als deficient in IgG 2, Sum m ary also fre q u e n tly have a d eficien cy of IgG Finally, when the histol- S ensitive and specific com p etitiv e ogy of lym ph nodes is exam ined, ger- in h ib itio n assays for h u m an IgG su b classes have b e e n d e v e lo p e d by us. A daptation of PC FIA to IgG subclass. 0...,. analysis has greatly facilitated our stud- Comparison of Stimulation Index 7,,,, i les. ih e s e assays have allow ed us to establish the prevalence of IgG subclass- / specific im m unodeficiency, to exam ine p th e extent of subclass restriction of antibodies to C H O antigens in humans, and to begin to study cellular mechanisms of IgG subclass expression. It is believed that the regulation of IgG subclasses will be complex, involving B cell subpopulations which are relatively, but not lutely, precom m itted to particular IgG subclasses and, possibly, to particular V re g io n s. A lth o u g h th e m ech an ism s I b e h in d th e d e s c rib e d o b se rv a tio n s rem ain to b e elucidated, its elucidation STIM U LA TIO N INDEX (Ig G l) will provide us valuable insights for our F ig u re 7. Comparison of IgG2 and lgg 3 stimu- clinical m anagem ent of the com m on, lation indices with IgG l stimulation index. Stimula- IgG subclass specific im m unodeficiention index is the ratio of amount of Ig detected after mitogen induction to that without mitogen induction. M itogens are denoted by letters P, pokeweed mitogen; H, phytohemagglutinin; S, streptolysin O; R e f e r e n c e s W, wheat germ agglutinin; N, nocardia opaca mitogen; M, salmonella typhimurium proteinaceous cell 1. Arney, E. R., Cooper, M. D., Kearney, J. R., wall mitogen; T, staphylococcus aureus Cowan I; L, Law ton, A. R., and Park house, R. M. E.: LPS; U, Ulex europeus agglutinin I. Plain letters Sequential expression of im m unoflobulin on compare IgG3 with IgGl; Circled letters compare developing mouse B-lymphocytes: A systematic IgG 2 w ith Ig G l. L ines rep resen t the b est fit. survey that suggests a model for the generation A d a p ted from S cott and N ahm, J. Im m u n o l. o f im m u n o g lo b u lin is o ty p e d iv e r sity. J. 133:2454, Immunol. 120:2041, c ie s

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