Lecture 3. Used anti B cell marker antibodies to deplete in mice

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1 Lecture 3 V-Gene Rearrangement and Expression Used anti B cell marker antibodies to deplete in mice Rat anti mouse CD19, anti mouse B220, and anti mouse CD22. Mice were then injected with a secondary antibody mouse anti rat antibody 1

2 Thymus T-Cells Lymph nodes Spleen } T+B-cells Bone Marrow HSCs and B-cells B-lymphocytes produce antibodies 2

3 Immunoglobulin (Ig) Molecule Antigen Binding Variable Region { { Light-chain Heavy-chain Constant Region Domain Theoretically, antibodies (Abs) can be produced to just about any foreign substance and are highly specific So we would need millions of Abs to do this Ex. An antibody can distinguish one protein from another by a single amino acid difference 3

4 There were two main hypotheses for the origin of this diversity. 1) Germline theory: -Held that there is a separate gene for each different immunoglobulin chain and that the antibody repertoire is largely inherited. 2) Somatic diversification theory: -Proposed that the observed repertoire is generated from a limited number of inherited variable (V)-region sequences that undergo alteration within B cells during the individual's lifetime. Clonal Selection Hypothesis: An individual cell expresses a specific receptor that recognizes a unique antigen- specificity determined prior to the presence of antigen Binding of antigen to receptor induces proliferation with each daughter cell producing the same antibody specificity (to the original activating antigen) Specific Antigen 4

5 Ig receptors genes did not follow 1-gene/1-protein rule To produce the millions of different antibodies necessary to combat disease, millions of antibody genes must have evolved to encode this information Since one gene encodes one protein (generally), this would mean that cells would need more genes than potentially encoded by genome The answer to this problem resulted in a Nobel Prize 1987 Nobel Prize Susumu Tonegawa Using light-chain mrna as probes was able to demonstrate that the variable region and the constant regions were rearranged in B-cell tumors (plasmacytomas) 5

6 Southern Analysis of Immunoglobulin Gene Alleles Liver B-cell tumor germline alleles rearranged alleles identical alleles rearranged alleles V-(D) (D)-J (Joining) Recombination V V D J J J 23 bp-rss 23 bp-rss bp-rss 12 bp-rss ~23 6 V J Cµ Enh ~~~~~~~~~~~~~~~~~~~~~~~~ heavy-chain VDJCµ mrna 6

7 V and J gene segments contain same recombination elements Joining-element coding region 23 bp CACAGTG 7mer ACAAAAACC 9mer Recombination Signal Sequences (RSS) The 12bp/23bp Spacer Rule Regulates V-D-J joining 12 bp RSS HEPTAMER CACAGTG ONE-TURN 12bp-SPACER NONAMER ACAAAAACC 23 bp RSS CACAGTG TWO-TURNTURN 23bp-SPACER ACAAAAACC 7

8 Specific signals are necessary to ensure V to J and prevent V to V and J to J recombination Signals are highly evolutionarily conserved-from sharks to man Recombination can proceed via deletion or inversion V J Deletion Inversion Deleted Circle Inverted DNA VJ VJ

9 Junctional flexibility Junctional flexibility Experimental evidence for junctional flexibility in immunoglobulin-gene rearrangement RSS= Recombination signal-sequence flanking each germline V, D, and J gene segments. 9

10 Seven means of antibody diversification have been identified in mice and humans: 1. Multiple germ-line gene segments 2. Combinatorial V-(D) (D)-J-joining 3. Junctional flexibility 4. nucleotide addition (at junctions) 5. Somatic hypermutation 6. Combinatorial association of light and heavy chains In humans, potential antibody combining-site diversity over (10 billion) 10

11 Immunoglobulin gene family Primordial Receptor Gene recognized self vs non-self? V D J C MHC light-chain κ heavy-chain λ α β γ δ CD4 CD8 Thy-1 Antibody T-Cell Antigen Receptor T-cell Antigen Receptors Resemble Antibody molecules 11

12 T-cell Antigen Receptor Genes T-cell antigen receptor also undergoes Site-specific rearrangement using the same recombination signals TCR-β genes TCR-α/δ genes TCR δ loci TCR-γ genes 12

13 What factors might be involved in VDJ Recombination? Sequence-Specific DNA Binding Factors? Site-Specific Cleavage Activity? DNA Ligase Activity? Other "House-keeping" Factors (DNA Repair)? What are the Recombinase factors that recognize the Recombination Sequences (RSS)? recombinase Deleted DNA 13

14 The Search for the Recombinase Complex Pre-B cell lines were created by Abelson Murine Leukemia transformation of mouse bone marrow cells in vivo+in vitro In early 1980s, retroviral vectors were developed in the laboratory of David Baltimore to study the mechanism of V-(D)-J recombination These retroviral vectors revealed that V-(D)-J recombination can be reproduced in the Pre-B cell lines, but not other cells These results lead to a frantic search for the V(D)J recombinase Retroviral Recombination Reporter Vectors V LTR gpt J LTR V J genomic DNA LTR gpt LTR Rearrangement by Inversion Confers Drug Resistance gpt = xanthine-guanine phosphoribosyltransferase gene 14

15 Recombinase must be expressed early during B-Lymphocyte differentiation Pre-B B-cell Plasma-B stem cell Variable Region Formation Retroviral vectors were found to rearrange only in pro/pre-b cell lines but not in non-lymphoid or mature B cells Chance Favors a Prepared Mind Chance Favors a Prepared Mind Louis Pasteur 15

16 An improbable experiment leads to an incredible result A student performs a series of flawed experiments that leads to the discovery of the V-(D)-J recombinase The premise of the experiment was that a single recombinase gene was responsible for V-(D)-J joining Reasoned that a recombinase gene could be transferred from lymphocyte DNA to a cell that does not contain this activity such as fibroblasts This experiment should never have worked! Why? Lymphocyte-specific genes should not be expressed in non-lymphoid cells - also unreasonable to believe that one gene product could do everything David Schatz,

17 Retroviral vectors used to detect site-specific specific recombination (inversion) LTR Vκ gpt untranscribed gpt gene Ja Jb LTR LTR gpt inversion VJa Jb probe LTR Inversion allows expression of sense gpt gene Fig. 1 Schatz and Baltimore (1987) Table I. Developmental Stage Specificity of V(D)J Recombination Activity Cell type Growth under Selection Rec. Freq. (recombination) (event/cell div.) Lymphocyte precursor None <1/2x10 6 (Ba/F3 cell line) Pre-B cells (38B9, PD31) YES 1/1x10 3 B-Cell (IgM Positive) None <1/4x10 6 (Wehi 231) Fibroblast None <1/2x10 7 (NIH 3T3) As expected, Pre-B cell lines contain a specific recombinase activity Schatz and Baltimore (1987) 17

18 The Search for the Recombinase Gene Sheared Human DNA Retroviral V-D-J Reporter construct mouse fibroblast (no recombinase activity) select with specific drug only drug resistant clones underwent recombination Isolate Human Gene Responsible for Recombination Activity Genomic DNA + psv2-his (histidinol dehydrogenase) Select for uptake of foreign DNA Select for Histidinol resistance Select for recombination by gpt activation with mycophenolic acid A few gpt r clones were obtained 18

19 Southern analysis of drug resistant fibroblasts contain expected recombination events controls gpt r clones + - TR-1 Vκ gpt Ja Jb unrearranged RSSs gpt VκJa V-Ja Inverted rearranged constructs Fig. 2 Schatz and Baltimore (1988) Rearrangements are consistent with site-specific specific V-J joining TR-1 TRX-1 Vκ GGATCCT* * GGATCCTCC Jκ **GGACGTTCGGTGGAGGCAC **GGACGTTCGGTGGAGGCAC *deleted bp Fig. 3 Schatz and Baltimore (1988) 19

20 Identification of V-(D) (D)-J Recombinase Gene Human DNA 3TGR cells (Fibroblasts w/ LTR Recombination Vector) Stable Transfer of Recombination Activity What gene caused this effect? How do you isolate a human gene within a sea of mouse and human genes? Link oligos to genomic DNA after RE digestion 1 transfectant different sized fragments on 3 side 2 transfectants Fig. 2 Schatz et al.,

21 Original RAG-1 Germline Clone Contained Another Gene RAG-1 RAG-2 Fig.1 Oettinger, et al., 1990 Probe J detected 2.2 kb mrna by Northern in pre-b cells RAG-2 and RAG-1 are sufficient for high level recombination RAG-1 cdna RAG-2 cdna Fibroblasts High level recombination 21

22 Table I RAG-1 and RAG-2 Act Synergistically Cell Oligo+ line DNA Amp r Cam r Amp r R 3TGR 0 184, TGR RAG-1 60, TGR RAG-2 50, TGR RAG-1 + RAG-2 70, NIH 3T3 RAG-1 + RAG-2 193,600 2, NIH 3T3 Human RAG , mouse RAG-2 *Total number of independent transfections Efficient Rearrangement only found when both RAG-1 and RAG-2 were cotransfected into fibroblasts Oettinger, et al., 1990 Expression of the RAG-2 is Pro/Pre-Lymphocyte Specific Fibroblast Pro/Pre-B Mature B Pre-T Mature T Fig.4 RAG-1 RAG-2 22

23 Targeted Disruption of RAG-1 and RAG-2 yields conclusive proof that the RAG genes are essential for V-(D) (D)-J recombination Shinkai, Y., et al., (1992). RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J recombination. Cell 68: Mombaerts, P., et al., (1992). RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68: Generation of RAG-1/2 knockouts wt RAG gene Disrupted gene neo Homozygous neo r Embryonic Stem Cells (ES) 23

24 Lymphocyte surface markers Used for isolation and identification (Tyr-PPase) double positive B220+/IgM+ (mature B-cells) FACS Analysis of Lymphocyte Populations B220 Wild-type 55% RAG-2 Knockout 0% Spleen Surface IgM B220 26% 0% Bone marrow Pro/Pre-B Surface IgM 24

25 Wild-type RAG-1 mutant T-cell antigen receptor αβ 0% No functional T-cells CD3 Thymus CD4 No mature T-cells CD8 IL-2R Precursor T-cells Thy-1 RAG -/ - knockout Mutant Mice Normal birth and growth but immunodeficient Have no Mature B/T cells No V-(D)-J recombination Higher levels of precursor-t cells (Thy-1+, IL-2R+) Higher levels of NK+, macrophages, and granulocytes probably filling a void left by loss of lymphocytes 25

26 Important questions: Do the RAGs bind and cut the RSS? Can a recombination system be established or dissected in vitro? Characterization of the Recombination Mechanism in vitro van Gent, D.C. et al.,. (1995). Initiation of V(D)J recombination in a cell-free system. Cell, 81: Martin Gellert s Laboratory at NIH makes initial and consistent discoveries which lead to the establishment of in vitro rec. systems 26

27 Other important findings: RAG mediated cleavage requires intact RSS heptamer and nonamer sequences Cleavage products were identical to those detected in vivoblunt 5 phosphorylated signal-ends and coding-ends contain hairpins (closed covalently) Recombinant Rag-1 and Rag-2 proteins plus house-keeping proteins are sufficient to mediate recombination in vitro RAG Mediated Cleavage nick 32 P -RSS RAG-1 RAG-2 Transesterification reaction hairpin hairpin nick 27

28 Recombinant Rag-1 and Rag-2 were subsequently shown to mediate all the cleavage steps in vitro Rag-1 and Rag-2 forms a large stable cleavage complex that requires an intact heptamer and nonamer These results lead to the following question: Can the in vitro system perform all the steps seen in vivo? Initiation of V(D)J recombination in vitro obeying the 12/23 rule Eastman, Q.E., Leu, T.M, and Schatz, D. G. Nature, 380:85-88, 1996 time of reaction 12bp-RSS 23bp-RSS 12bp-RSS 23bp-RSS For efficient cleavage, needed RAG-1+RAG-2 + Nuclear Extract (additional factors) 28

29 1/12/05 Advanced Immunology Dr. Aguilera Big-Bang Theory of Immunology B/T Lymphocytes 450 x 10 6 years ago RAGs and V-(D)-J recombination appeared early in vertebrate evolution Transposable elements created the first antigen receptor genes RAG Transposon Exon 1 Exon 2 Primitive Receptor Gene 29

30 The RAG genes become part of host s genome lymphocyte-specific promoter defective or absent RSS Furthermore, the RAG genes had to come under the control of lymphocyte-specific promoters Simultaneous Cleavage at both RSSs Mg++ synapsis Housekeeping enzymes ligate and repair breaks 30

31 DNA repair enzymes are also needed Malu el al 2012 RAGs are more complex than anyone thought 31

32 Control of recombination is complicated 32