M261, Lecture 3  

V(D)J Recombination, Ig Class Switching,  

and Somatic Hypermutation  

April 8, 2005

Clonal Selection

Structure of an Antibody MoleculeAntibody structure as determined by amino acid sequence analyses, X-ray crystallography, and various other biochemical techniques (e.g. protease mapping)

Structure of an Immunoglobulin Domain

1977 Tonegawa Experiment

Genomic Southern Blot (Modern version of Tonegawa experiment):

(1989 Nobel Prize)

V-regions are constructed from gene segments

Organization of Ig and TCR Gene Loci

*Each V segment contains its own promoter

Conserved Nonamer-Heptamer and 12/23 bp Spacer Rule

The same nonamer-spacer-heptamer sequences are found adjacent to all V,D, & J gene segments within the Ig heavy-chain locus, the Ig light chain loci, and all the T-cell receptor loci. The 12/23 bp rule is followed in all these loci as well. Therefore, the same recombinase is almost certainly responsible for recombination of all loci.

Gene Rearrangement Can Occur by Deletion or Inversion of Intervening DNA, Depending on the Orientation of Gene Segments

Recombination Substrates: Coding region-heptamer-spacer-nonamer.

Recombination Products: Heptamers always fused to one another.

 

Strategy• Introduce retroviral/gpt construct into 3T3 fibroblasts.• Cotransfect sheared genomic DNA + pSV2His.• Select cells which carry out rearrangement with mycophenolic acid (gpt resistant). Results

1500 histidinol-resistant clones (Indicates that 1500 cells took up genomic DNA)1 mycophenolic acid-resistant clone (Only one cell carried out a recombination event which allowed gpt gene to be expressed.

Proper rearrangementTransferable (secondary transfectant 1 colony; Tertiary 100 colonies)Stable (i.e. When a second recombination substrate was introduced into this clone, it also recombined.)

Summary of Antigen-Independent and Antigen-Dependent B-cell Development

Ig Isotypes (Classes)

Class Switch Recombination via Looping

IgM and IgD are both produced before switching occurs (by alternative mRNA splicing).

Degree of Variability at Each Position of the Variable Domain within a Mature Antibody Repertoire

• The numerous V segments possess considerable sequence homology with each other.

• The numerous J segments are also homologous with each other.

• The hypervariability at HV1 and HV2 results from a process known as “somatic hypermutation”

HV1, HV2, and to a lesser extent HV3 are generated by somatic hypermutation in peripheral lymphoid organs (e.g. Lymph Nodes)

− During an initial infection, many randomly generated antibodies will bind to the infectious organism with a low affinity, which is barely sufficient for activation of the B-cell and for antibody function.

− The V(D)J regions of the rearranged Ig genes can mutate at a rate of 10-3/nucleotide/cell generation. (The spontaneous mutation rate is 10-9/nucleotide/cell generation.)

− A fraction of the mutated Ig genes will produce Ig proteins with a higher affinity for the antigen, leading to a more effective immune response during a subsequent infection. Affinity Maturation.

Mechanisms of Variable Domain DiversityD

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Mechanisms responsible for combinatorial and junctional diversity very well understood. (1989 – present)

Mechanisms responsible for somatic hypermutation and class switch recombination remain unknown. Discovery of AID in 2000 provides first major advance.

1. Combinatorial Diversity ~107

a. V, D and J gene segmentsb. Heavy chain – Light chain combination

2. Junctional Diversity(HV3/CDR3only) ~104

a. Exonuclease activityb. Nucleotide addition (Terminal transferase – TdT, N nucleotides)c. P nucleotides

3. Somatic Hypermutation (or Gene Conversion) (All 3 HVs/CDRs)

Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing EnzymeMasamichi Muramatsu 1, Kazuo Kinoshita 1, Sidonia Fagarasan 1, Shuichi Yamada 2, Yoichi Shinkai 2, and Tasuku Honjo ‡ 1 1 Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 2 Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-8501, Japan,

Cell, Sept. 1, 2000, vol. 102, 553-563.

Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken -globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

- AID was originally identified using a substractive cloning strategy aimed at the identification of mRNAs expressed in lymphoma cells undergoing class switch recombination, but not in non-recombining cells.- This paper describes the phenotype of AID knockout mice. The phenotype reveals that AID is essential for both class switch recombination and somatic hypermutation of Ig genes.- Although the title states that AID is a potential RNA editing enzyme, it probably acts by modifying DNA.

Figure 3. Absence of Class Switching in AID-Deficient B Cells

(A) Sera from 10-week old AID-/- (open circle) and age-matched AID+/- (closed circle) mice were collected and their Ig levels were determined by ELISA. Ig levels less than 0.01 µg/ml were plotted below the base lines.

(B) AID-/- (open circle) or AID+/- (closed circle) chimeras were immunized with 1 × 108 SRBC on day 0 and 14 (arrows). Anti-SRBC IgM or IgG1 serum levels were measured at day 0, 5, 10, 14, and 21. Arbitrary unit is absorbance at OD405nm. Similar results were obtained from two independent experiments.

(C) Ig levels of culture supernatants of spleen cells from 5-week old littermates (AID+/+, AID+/- , and AID-/-) mice were measured after culture for 7 days in the presence of LPS (closed circle), LPS and IL-4 (open circle), or LPS and TGF (open square). Ig levels less than 0.01 µg/ml were plotted below the base lines.

Ig isotypes other than IgM are absent in immunized and non-immunizedAID-deficient mice, demonstrated a critical role in class switching

Figure 7. Defect of Hypermutation in AID-/- Chimera

(A) Primary and secondary responses against NP in AID-/- chimera. Anti-NP IgM and IgG1 levels were measured 0, 7, 14, and 28 days after AID-/- (open circle) or AID+/- (closed circle) chimeras were immunized with NP-CGG intraperitonealy on day 0 and 21 (arrows). Each circle represents an arbitrary unit (absorbance at OD405 nm) of a single mouse.

(B) Mutation frequencies in the VH186.2 transcripts from MLN of NP-immunized AID-/- and AID+/- chimeras. VH186.2 transcripts of µ and 1 isotypes at day 28 were amplified by RT-PCR and cloned into a plasmid vector before sequencing. Six clones of 1 isotype from AID+/- chimera, and 17 and 16 clones of µ isotype from AID+/- and AID-/- chimeras, respectively, were sequenced. Mutation frequency of clones that have amino acid replacements (closed bar) or silent mutation (open bar) at specific position was plotted along residue number from -11 to 98.

Somatic mutations do not accumulate in rearranged Ig genes during an immune response to a specific antigen (NP)

- Antibodies that recognize the NP antigen are almost always derived from a single V segment in the IgH locus(VH186.1).

Activation-Induced Cytidine Deaminase (AID) Deficiency Causes the Autosomal Recessive Form of the Hyper-IgM Syndrome (HIGM2)

Patrick Revy 1, Taro Muto 2, Yves Levy 3, Frédéric Geissmann 4, Alessandro Plebani 5, Ozden Sanal 6, Nadia Catalan 1, Monique Forveille 1, Rémi Dufourcq-Lagelouse 1, Andrew Gennery 1, Ilhan Tezcan 6, Fugen Ersoy 6, Hulya Kayserili 7, Alberto G. Ugazio 5, Nicole Brousse 4, Masamichi Muramatsu 2, Luigi D. Notarangelo 5, Kazuo Kinoshita 2, Tasuku Honjo 2, Alain Fischer 1,8, and Anne Durandy 9 1

1 Inserm U429, Hôpital Necker-Enfants Malades, Paris, France 2 Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Japan 3 Inserm U474, Hôpital Cochin, Paris, France

The activation-induced cytidine deaminase (AID) gene, specifically expressed in germinal center B cells in mice, is a member of the cytidine deaminase family. We herein report mutations in the human counterpart of AID in patients with the autosomal recessive form of hyper-IgM syndrome (HIGM2). Three major abnormalities characterize AID deficiency: (1) the absence of immunoglobulin class switch recombination, (2) the lack of immunoglobulin somatic hypermutations, and (3) lymph node hyperplasia caused by the presence of giant germinal centers. The phenotype observed in HIGM2 patients (and in AID-/- mice) demonstrates the absolute requirement for AID in several crucial steps of B cell terminal differentiation necessary for efficient antibody responses.

Cell, Sept. 1, 2000, vol. 102, 565-75.

Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas

ALBERTO MARTIN*, PHILIP D. BARDWELL*, CAROLINE J. WOO*, MANXIA FAN*, MARC J. SHULMAN† & MATTHEW D. SCHARFF*

* Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue Chanin 403, Bronx, New York 10461, USA† Immunology Department, Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts1, is required for this process, but its exact role is unknown2. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in GC base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

Nature, Feb. 14, 2002, vol. 415, 802-806.

Figure 2 AID induces SHM in hybridomas. a, Hybridomas N89 (nonsense in leader) and N114 (nonsense in V region) were transfected with empty vector or the hAID construct. The frequency of nonsense revertants, as detected with the ELISA-spot assay, is plotted. Typical ELISA spots for N89 are shown in the inset. The difference in the frequencies of reversion of N114 vector and N114 hAID was statistically significant (P<0.05). b, Northern blots for hAID and GAPDH of N89, N114 and P1-5 transfected hybridoma clones. Clone numbers for N89 and N114 clones correspond to numbers in a.

An AID expression plasmid was stably transfected into two hybridoma lines that are known to contain rearranged endogenous IgH genes with nonsense codons in the sequences encoding theleader or variable domain. An ELISA-spot assay was used to monitor the frequency of reversion to a translatable Ig gene.

- The key to the success of this assay was its tremendous sensitivity.

The AID enzyme induces class switch recombination in fibroblasts

IL-MI OKAZAKI*, KAZUO KINOSHITA*, MASAMICHI MURAMATSU*, KIYOTSUGU YOSHIKAWA*† & TASUKU HONJO*

* Department of Medical Chemistry and Molecular Biology, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan † Radioisotope Research Center, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from C to one of the other CH genes1, 2. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity3. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation4, 5. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

Nature, March 21, 2002, vol. 416, 340-345.

Figure 1 Induction of CSR in an artificial substrate in CH12F3-2 cells by overexpression of AID without stimulation. a, Structure of the CSR substrate SCI(,). pEF-1, elongation factor 1 promoter; pTET, tetracycline-responsive promoter; Neo, neomycin resistance gene with the TK promoter; HyTK, gene for the fusion protein hygromycin phosphotransferase–thymidine kinase, with the CMV promoter; Pre-Tr, pre-switch transcripts; Post-Tr, post-switch transcripts; rectangles, exons; arrows, promoters. EC and TM indicate extracellular and transmembrane domains of CD8, respectively. Open and filled triangles indicate PCR primers used for genomic DNA and transcripts, respectively. Primer sequences are described in the Methods. b, A CH12F3-2 transfectant carrying the tTA and a single and intact copy of the switch substrate SCI(,) was stimulated in the absence or presence of tetracycline for 7 days and analysed for expression of IgA, CD8 and GFP by FACS. Percentages of cells expressing CD8–GFP (top) and IgA (bottom) are indicated. c, CH12F3-2-SCI(,) clones transfected with AID expression vector (AID) or the vector alone (mock) under the control of pTET were cultured for 7 days in the absence or presence of tetracycline and analysed for expression of CD8 and GFP by FACS. d, Percentages of CD8–GFP+ cells in three independent clones of the AID or mock transfectants. e, RT–PCR of pre-switch (Pre-Tr1 and 2), post-switch (Post-Tr) and transgenic (TG) AID transcripts in AID-transfected clones. GAPDH is an internal control. Water, without cDNAs. Stim, stimulation with anti-CD40 monoclonal antibody (HM40-3), IL-4 and TGF.

Switching at an artificial reporter construct in unstimulated cells requires AID and transcription from the pTET promoter

-Switch recombination at the artificial construct is required for expression of a membrane-spanning CD8-GFP fusion protein (EC, extracellular domain, TM, transmembrane domain).

- Panel B shows that switching at the artificial construct works as expected when the cells are stimulated with cytokines to undergo switching (no AID overexpression in this control experiment).

-Panels C and D show that switching can occur in unstimulated cells if AID is overexpressed and if the pTET promoter is induced. The requirement for pTET promoter activity is important because previous studies showed a tight linkage between switchingand transcription through the switch regions.

- Panel E analyzes mRNA transcripts to directly confirm theabove results.

Kiyotsugu Yoshikawa,12 Il-mi Okazaki,1 Tomonori Eto,1 Kazuo Kinoshita,1 Masamichi Muramatsu,1 Hitoshi Nagaoka,1 Tasuku Honjo1*

Activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is indispensable for somatic hypermutation (SHM), class switch recombination, and gene conversion of immunoglobulin genes, which indicates a common molecular mechanism for these phenomena. Here we show that ectopic expression of AID alone can induce hypermutation in an artificial GFP substrate in NIH 3T3 murine fibroblast cells. The frequency of mutations was closely correlated with the level of transcription of the target gene, and the distribution of mutations in NIH 3T3 cells was similar to those of SHM in B lymphocytes. These results indicate that AID is sufficient for the generation of SHM in an actively transcribed gene in fibroblasts, as well as B cells, and that any of the required cofactors must be present in these fibroblasts.

1 Department of Medical Chemistry and Molecular Biology, Graduate School of Medicine; and

2 Radioisotope Research Center, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Science, June 14, 2002, vol. 296, 2033-6.

AID Enzyme-Induced Hypermutation in an Actively Transcribed Gene in Fibroblasts

Fig. 1. Induction of hypermutation in the artificial GFP substrate in NIH 3T3 cells by expression of AID. (A) GFP substrate (pI) under tetracycline-inducible promoter (8). TRE, tetracycline-responsive element; PminCMV, minimal CMV promoter; TATA, TATA box; CMVPF and TetGFPR, primers for PCR amplification and sequencing. (B) Three representative NIH 3T3-pI clones 3, 19, and 20 were infected by retrovirus expressing mock, AIDm-1, or AID in the absence of tetracycline. After 10 days, the cells were analyzed for expression of GFP by fluorescence-activated cell sorting (FACS). Copy numbers of pI in clones 3, 19, and 20 are 4, 1, and 2, respectively, as determined by Southern blot analysis (10). (C to E) GFP+ cells and mutations in GFP (11) were analyzed in NIH 3T3-pI clone 19 cells cultured without tetracycline unless otherwise indicated. (C) Dependency of AID-induced hypermutation on dose of AID. Frequencies of GFP+ cells (line) and mutations in the GFP substrate (bar) on day 7 with different virus units (7) indicated. (D) Frequencies of GFP+ cells (line) and mutations (bar) on day 10 at different concentrations of tetracycline with 500 units of AID-expressing virus. (E) Time-course analysis of hypermutation. Frequencies of GFP+ cells (line) and mutations (bar) at different time points with 500 units of AID-expressing virus.

AID overexpression in fibroblasts induces reversion of a nonsense mutation in a GFP expression construct.

AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification

SVEND K. PETERSEN-MAHRT*, REUBEN S. HARRIS* & MICHAEL S. NEUBERGER

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination. All three processes depend on activation-induced cytidine deaminase (AID)1-7, a B-cell-specific protein that has been proposed (because of sequence homology1) to function by RNA editing. But indications that the three gene diversification processes might be initiated by a common type of DNA lesion8-11, together with the proposal that there is a first phase of hypermutation that targets dC/dG12, suggested to us that AID may function directly at dC/dG pairs. Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner. Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA. We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome—that is, hypermutation phases 1 and 2, gene conversion or switch recombination—dependent on the way in which the initiating dU/dG lesion is resolved.

Nature, July 4, 2002, vol. 418, 99-103

Figure 1 DNA deamination model of immunoglobulin gene diversification. For details, see text. KU70/80 are non-homologous end-joining proteins. AP endonuclease, apyrimidic endonuclease; DNA-PK, DNA-dependent protein kinase; dRPase, deoxyribophosphodiesterase; V, variable.

Hypothesis that AID may be a DNA cytosine deaminase and predictions of this hypothesis

- Deamination of cytosine in DNA creates uracil, which can be repaired by various mechanisms. A simple mechanism is DNA replication or patch repair. If the U strand is used as a template during repair, CG bps will be converted to TA bps because U is recognized as T by DNA polymerases. If the G strand is used as the template, no mutation is introduced. Other repair mechanisms require excision of the U by uracil-DNA glycosylase. After excision, a variety of repair mechanisms can be used.

- In this paper, the authors first show that expression of AID in E. coli can induce mutations. Mutations are highly inefficient, but the authors used an extremely sensitive assay: They monitored the frequency of mutations that arise in the rpoB gene. RpoB mutations confer resistance to rifampicin.

- The authors then sequenced the mutations in the rpoB gene and found a biased toward transitions, as predicted by the cytosine deaminase model.

- When the experiment was repeated in an E. coli strain lacking uracil DNA-glycosylase, the mutation frequency greatly increased, as would be expected if the repair pathways dependent on uracil DNA-glycosylase are eliminated.

Figure 2 Expression of AID in E. coli yields a mutator phenotype that is enhanced by UDG deficiency. a, Frequencies of RifR mutants generated after overnight culture (with (+) or without (-) IPTG) of E. coli KL16 carrying either the AID expression plasmid or the vector control. Each point represents the mutation frequency of an independent overnight culture. The fold enhancement by AID expression is indicated. The slight enhancement of mutation in AID-transformed cells in the absence of IPTG may be due to promoter leakiness. b, Mutation frequency of AID- and vector-transformed, UDG-deficient KL16 ung-1 cells as in a, but note the larger scale on the y axis. c, Representative culture plates. The mutation frequency relative to the vector-transformed wild-type control is indicated. See Table 1 for additional data.

Mutations that induce rifampicin resistance increase when AID is expressedin E. coli, and increase even further in a strain lacking uracil DNA glycosylase.Mutations show a strong bias toward transitions

JAVIER DI NOIA AND MICHAEL S. NEUBERGER

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC dG/dA or dG dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC dT (and dG dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC dU deamination within the immunoglobulin locus itself.

Altering the pathway of immunoglobulin hypermutation by

inhibiting uracil-DNA glycosylase

Nature, Sept. 5, 2002, vol. 419, 43-48.

- The authors provide compelling evidence that inhibition of uracil DNA glycosylase activity in chicken B cellsleads to an increased frequency of transitions during a physiological somatic hypermutation process. These dataprovide strong circumstantial evidence that hypermutation is initiated by cytosine deamination. The next step is todetermine whether, as predicted, AID possesses cytosine deaminase biochemical activity.