The Casali Lab's Research

The adaptive immune system is a sophisticated system in the targeting and destruction of harmful, disease-causing agents such as bacteria, viruses, parasites and any other microorganisms collectively called pathogens. The adaptive immune system can be separated into two parts: the cellular response and the humoral response. The cellular response involves the actions of cells such as helper and killer T lymphocytes, which are responsible for the proliferation of the immune signal and destruction of infected cells in the body, respectively. The humoral immune response, on the other hand, involves B lymphocytes and their ability to produce and secrete immunoglobulins (Igs), or antibodies, to recognize and detect antigenic agents. Overall, the adaptive immune response is crucial to the development of a long lasting immunological memory towards previously encountered pathogens to prevent future infections.

Antibodies consist of a heavy (H) chain and a light (L) chain and are capable of recognizing an unlimited number of foreign particles through their variable binding domains. B lymphocytes are able to create clones capable of generating unique antibodies from gene recombination of the variable (V), diversity (D), and joining (J), or V(D)J segments, as mediated by recombination activating gene-1 (RAG-1) and RAG-2. During the humoral adaptive immune response, Ig genes undergo further genetic recombination processes, namely somatic hypermuation (SHM) and class switch DNA recombination (CSR), to generate antibodies with a higher affinity to pathogens and new effector functions, respectively (Figure 1).

Figure 1. SHM and CSR in the mouse IgH locus (chromosome 12). Each CH gene comprises 4 or 5 exons (CH1, hinge, CH2, CH3, CH4) and contains a switch (S) region, an evolutionary conserved sequence (ECS) and an intervening (I) region 5’ of the CH1 exon, as depicted by three vertical bars. SHM inserts mismatches (depicted as “x”) in VHDJH region DNA. CSR entails generation of DSBs, S-S region recombination and looping out of the intervening DNA. Depicted is CSR from Cμ to Cγ2a, which leads to generation of IgG2a, one major Ig isotype of pathogenic autoantibodies in lupus patients. SHM and CSR are RAG-independent and AID-dependent and, as we argue, consist of two sequential stages: generation of DNA lesions, as initiated by AID, and lesion repair, as effected by DNA abasic site bypass and BER, and MMR pathways.

SHM emerged before CSR in phylogeny, first appearing in sharks. It introduces mainly point-mutations, with rare deletions or insertions into rearranged V(D)J region DNA, at a rate of 10-3 change per base per cell generation, which is a million-fold higher than the rate of spontaneous mutations in the genome at large. SHM is limited to the Ig locus and few other genes, as abnormal and widespread mutations in the genome would be detrimental to cell homeostasis and would favor emergence of neoplasia and autoimmunity. Point-mutations inserted in the V(D)J region DNA are subjected to selection pressure by the antigen driving the immune response, be it an exogenous antigen on microbial pathogens or a self-antigen, such as DNA in lupus patients. SHM is initiated by V(D)J-C transcription of the target DNA and eventually inserts mismatches in both the top and bottom (transcribed) DNA strands, while preferentially targeting the RGYW/WRCY mutational hotspot, mainly in its RGYW (AGCT and AGCA) iterations.

CSR replaces the constant μ (Cμ) region of the IgH chain with a downstream Cγ, Cα, Cε region, thereby changing expressed antibodies from IgM to other Ig isotypes, i.e., IgGs, IgA and IgE, with new biological effector functions. While isotype-switched antibodies effectively eradicate pathogens, they can also mediate tissue injury or autoimmunity. CSR entails the generation of double-strand DNA breaks (DSBs) in switch (S) regions located 5’ of each constant (CH) gene and a subsequent recombination event ligating the free ends of the upstream and downstream S regions.

Figure 2. Illustration of germinal centers (GCs) in the mouse spleen. Spleen sections of C57BL/6 mice immunized with alum-precipitated 4-hydroxy-3-nitrophenyl acetyl coupled to chicken gamma globulin (NP16-CGG) for fourteen days were stained with phycoerythrin (PE)-conjugated antibody for B220, a B cell-specific marker, and fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), a marker for GCs, which formed within B cell follicles. Marginal zone (MZ) B cells, metalophillic macrophages lining between follicles and MZ, as well as the T zone are also shown.

SHM and CSR are highly regulated and unfold mainly in germinal centers (GCs) of secondary lymphoid organs (Figure 2). Antigen-primed B cells are activated in a T cell-dependent fashion to undergo SHM and CSR upon engagement of surface CD40 and CD80/CD86 by CD154 and CD28/CTLA-4 expressed on the surface of activated CD4+ T cells. Exposure to T cell-secreted cytokines, particularly IL-4 or TGF-β, is critical for CSR to IgG1 and IgE or IgA, respectively. Crosslinking of B cell receptor (BCR) is also critical for the induction of SHM. SHM and CSR require the intervention of activation-induced cytidine deaminase (AID), which is involved in early steps in both reactions. We have shown that CD154:CD40-engagement and BCR crosslinking upregulate AID and selected (lesion bypass) translesion synthesis (TLS) DNA polymerases, which are critical mediators of SHM (Figure 3).

Figure 3. BCR crosslinking and T:B cell contact through CD154:CD40 engagement and CD86/CD80:CD28 co-engagement are required for the induction of SHM. CD154:CD40 engagement and BCR crosslinking upregulate the expression of AID (large red circles) and the error-prone TLS pol (large pink ovals) and pol ζ catalytic subunit Rev3 (brown ovals). Pol θ and pol ζ, together with other TLS polymerases (pol η, dark blue ovals; Rev1, light green circles; and, perhaps, pol ι, orange ovals) are recruited into the DNA repair process to replace high fidelity DNA polymerases ε and/or δ, a process called the “polymerase switch” that results in the insertion of mutations. The small inset depicts the mutation frequency in V(D)J and C regions. As depicted in the large inset, somatic mutations (red crosses) are introduced by TLS polymerase(s) during DNA synthesis, while bypassing an abasic site or while copying undamaged DNA in patch DNA re-synthesis of MMR or, perhaps, a mutagenic long-patch BER. Abasic site bypass requires the sequential action of two DNA polymerases: one, such as pol θ, pol η, Rev1 or, perhaps, pol ι, that inserts a nucleotide opposite the damaged template nucleotide (inserter), and the other, such as pol θ or pol η, that extends from the inserted nucleotide (extender). Pol θ is the first DNA polymerase known to bypass abasic sites efficiently by functioning as a mispair inserter and a mispair extender. The ability of pol η in inserting nucleotides opposite to abasic sites is weak alone, but can be greatly enhanced upon recruitment and stimulation by PCNA.

SHM and CSR are highly regulated and unfold mainly in germinal centers (GCs) of secondary lymphoid organs (Figure 2). Antigen-primed B cells are activated in a T cell-dependent fashion to undergo SHM and CSR upon engagement of surface CD40 and CD80/CD86 by CD154 and CD28/CTLA-4 expressed on the surface of activated CD4+ T cells. Exposure to T cell-secreted cytokines, particularly IL-4 or TGF-β, is critical for CSR to IgG1 and IgE or IgA, respectively. Crosslinking of B cell receptor (BCR) is also critical for the induction of SHM. SHM and CSR require the intervention of activation-induced cytidine deaminase (AID), which is involved in early steps in both reactions. We have shown that CD154:CD40-engagement and BCR crosslinking upregulate AID and selected (lesion bypass) translesion synthesis (TLS) DNA polymerases, which are critical mediators of SHM (Figure 3).

Both SHM and CSR would entail two sequential stages: (i) generation of DNA lesions, as initiated by AID and Ung dU glycosylase, and (ii) DNA lesion repair, as dealt with by the cell abasic site bypass and base-exicision repair (BER) (Phase 1b) or mismatch repair (MMR) (Phase 2) machineries, leading to introduction of mutations or DSBs, the obligatory intermediates in CSR, and their resolution (Figure 4). AID initiates SHM and CSR by directly deaminating dC in DNA, thereby yielding dU:dG mispairs. The generation of this and other lesions, such as DSBs, by direct AID-mediated DNA deamination and/or by intervention of yet to be identified endonuclease(s) would constitute the initial step in SHM and CSR. The second step, DNA repair, would be responsible for the insertion of mismatches (mutations) and DNA recombination. In this step, DNA lesions are resolved through a process entailing intervention of "DNA replication/repair factors", including PCNA, replication protein A (RPA), BER proteins that include Ung, MMR proteins such as—as we showed—the MutL homolog Mlh3 and, finally, error-prone TLS DNA polymerases, including pol θ, pol ζ, pol η, pol ι and Rev1. This woukd eventually lead to the assembly of a "mutasome", which would be instrumental in facilitating the “polymerase switch”, by effecting the faulty DNA resynthesis inherent to the lesion repair process. The Casali Lab is attempting to define the role of V and S region DNA sequences in recruiting specific trans-factors in SHM and CSR, and to determine the role of DNA replication/repair factors, such as TLS polymerases and PCNA and PCNA-mediated "polymerase switch" in SHM and CSR.

Figure 4. An integrated model of SHM and assembly of the "mutasome". The process depicted here is based on the assumption that AID deaminates dC in both DNA strands. dU is not germane to DNA and the dU:dG mismatch is "replicated over" or dealt with by the DNA repair machinery. Replicating over dU results in a dC/dT transition mutation (Phase 1a), whereas dU deglycosylation by Ung gives rise to an abasic site. In the presence of PCNA (orange ring), DNA synthesis opposite the abasic site by TLS pol θ, which has both nucleotide inserter and extender activity, or by the nucleotide inserter pol ι, Rev1 or, perhaps, pol δ, followed by the nucleotide extender pol ζ or pol η, yields dC/dT transitions and dC/dA or dC/dG transversions (Phase 1b). Alternatively, the abasic site can be recognized and excised by APE or the Mre11-Rad50 glycosylase to create a DNA nick. This nick can be repaired by DNA pol β (light pink circles) in an error-free fashion (short-patch BER) or repaired in an error-prone fashion by a TLS polymerase through a long-patch BER also involving PCNA and Fen1. dU:dG mispairs can also be recognized by the MMR machinery, resulting in a DNA-gap formation through the intervention of an unidentified endonuclease or MRN and Exo I. Subsequently, TLS pol θ, pol η, Rev1, pol ζ and, perhaps, pol ι, can effect DNA re-synthesis as part of a patch repair, thereby inserting mismatches (Phase 2). In the long-patch BER or MMR, RPA (large brown ovals) and PCNA would recruit other repair proteins to the lesion and co-ordinate their actions in an multi-factor complex (mutasome). MMR proteins are indicated as large green ovals. Mutated nucleotides are shown in red.

References:

  1. Zan, H. and P. Casali. 2008. AID- and Ung-dependent generation of resected double-strand DNA breaks in immunoglobulin class switch DNA recombination: a postcleavage role for AID. Mol. Immunol., in press.
  2. Elkon, K. and P. Casali. 2008. Nature and functions of autoantibodies. Nature Clin. Pract. Rheum., in press.
  3. Al-Qhatani, A., Z. Xu, H. Zan, C. Walsh and P. Casali. 2008. A role for Drak2 in the germinal center reaction and the antibody response. Autoimmunity 41:341-352.
  4. Atassi, M.Z. and P. Casali. 2008. Molecular mechanisms of autoimmunity. Autoimmunity 41:122-132.
  5. Xu, Z., E.J. Pone, A. Al-Qahtani, S.-R. Park, H. Zan and P. Casali. 2007. Regulation of aicda expression and AID activity: Relevance to somatic hypermutation and class switch DNA recombination. Crit. Rev. Immunol. 27:367-397.
  6. Xu, Z., S. Pal, H. Zan and P. Casali. 2007. DNA replication to aid somatic hypermutation. Adv. Exp. Med. Biol. 596:111-128.
  7. Duquerroy, S., E.A. Stura, S. Bressanelli, S.M. Fabiane, M.C. Vaney, D. Beale, M. Hamon, P. Casali, F.A. Rey, B.J. Sutton and M.J. Taussig. 2007. Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation. J. Mol. Biol. 368:1321-1331.
  8. Casali, P., S.Pal, Z. Xu and H. Zan. 2006. DNA repair in antibody somatic hypermutation. Trends Immunol. 27: 313-321.
  9. Wu, X., C.Y. Tsai, M.Patam, H. Zan, S.M. Lipkin and P. Casali. 2006. A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation. J. Immunol. 176:5426-5437.
  10. Xu, Z., E. J. Pone, S.-R. Park, H. Zan, and P. Casali. 2007. Regulation of aicda expression and AID activity. Crit. Rev. Immunol. 27: 367-397.
  11. Xu, Z., H. Zan, Z. Pal and P. Casali. 2007. DNA replication to aid somatic hypermutation. Adv. Exp. Med. Biol. 596: 111-128.
  12. Casali, P., Z. Pal, Z. Xu and H. Zan. 2006. DNA repair in antibody somatic hypermutation. Trends Immunol. 27:313-321.
  13. Wu, X., C.Y. Tsai, M. Patam, H. Zan, S.M. Lipkin and P. Casali. 2006. A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation. sJ. Immunol. 176: 5426-5437.
  14. Komori, A., Z. Xu, X. Wu, H. Zan and P. Casali. 2006. Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEm enhancer and 3'Ea enhancers in human lymphoblastoid B cells. Mol. Immunol. 43: 1817-1826.
  15. Zan, H., N. Shima, Z. Xu, A.J. Evinger, III, Y. Zhong, J.C. Schimenti and P. Casali. 2005. The translesion DNA polymerase θ plays a dominant role in immunoglobulin gene somatic hypermutation. EMBO J. 24:3757-3769.
  16. Xu, Z., Z. Fulop, Y. Zhong, A.J. Evinger, III, H. Zan, and P. Casali. 2005. DNA lesions and repair in class switch DNA recombination and somatic hypermutation. Ann. N.Y. Acad. Sci. 1050: 146-162.
  17. Casali, P. and H. Zan. 2004. Class switching and myc translocation: how does DNA break? Nat. Immunol. 5: 1101-1103.
  18. Kim, E., C.R. Edmonston, X. Wu, A. Schaffer and P. Casali. 2004. The HoxC4 homeodomain protein mediates activation of the immunoglobulin heavy chain 3' hs1,2 enhancer in human B cells: relevance to class switch DNA recombination. J. Biol. Chem. 279: 42258-42269.
  19. Liu, S., A. Cerutti, P. Casali* and M.K. Crow*. 2004. Ongoing immunoglobulin class switch DNA recombi-nation in lupus B cells: analysis of switch regulatory regions. Autoimmunity 37: 431-443. [*equal contrib.]
  20. Wu, X., H. Zan, A. Komori, J. Feng, E.C. Kim and P. Casali. 2003. Immunoglobulin somatic hypermutation: double-strand DNA breaks, AID and error-prone DNA repair. J. Clin. Immunol. 23: 235-246.
  21. Zan, H., X. Wu, A. Komori, W.K. Holloman and P. Casali. 2003. AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/Rad51 in somatic hypermutation. Immunity 18: 727-738.
  22. Schaffer, A, E. Kim, H. Zan, L. Testoni, S. Salamon, A. Cerutti and P. Casali. 2003. Selective inhibition of class switching to IgG and IgE by recruitment of the HoxC4 and Oct-1 homeodomain proteins and Ku70/Ku86 to newly identified and conserved ATTT cis-elements. J. Biol. Chem. 278: 23141-23150.
  23. Diaz, M. and P. Casali. 2002. Somatic hypermutation. Curr. Opin. Immunol. 14: 235–240.
  24. Zan, H., A. Komori, Z. Li, A. Cerutti, M. Flajinik, M. Diaz and P. Casali. 2001. The translesion DNA polymerase z plays a major role in Ig and bcl-6 somatic hypermutation. Immunity 14: 643-653.
  25. Zan, H., Z. Li, K. Yamaji, P. Dramitinos, A. Cerutti and P. Casali. 2000. BCR engagement and T cell contact induce bcl-6 hypermutation in human B cells: identity with Ig hypermutation. J. Immunol. 165: 830-839.
  26. Zan, H., A. Cerutti, P. Dramitinos, A. Schaffer, Z. Li and P. Casali. 1999. Induction of Ig somatic hypermutation and class switch DNA recombination in a human monoclonal IgM+ IgD+ cell line in vitro: definition of the requirements and the modalities of hypermutation. J. Immunol. 162: 3437-3447.
  27. Cerutti, A., H. Zan, A. Schaffer, L. Bergsagel, N. Harindranath, E.E. Max and P. Casali. 1998. CD40L and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center-like phenotype differentiation in a human monoclonal IgM+ IgD+ B cell line. J. Immunol. 160: 2145-2157.
  28. Cerutti, A., A. Schaffer, S. Shah, H. Zan, H.-C. Liou, R.G. Goodwin and P. Casali. 1998. CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non antigen-selected human B cells. Immunity 9: 239-246.
  29. Zan, H., A. Cerutti, A. Schaffer, P. Dramitinos and P. Casali. 1998. CD40 engagement triggers class switch DNA recombination to IgA1 and IgA2 in human B cells through induction of endogenous TGF-β. Evidence for TGF-β but not IL-10-dependent direct Sμ→Sα and sequential Sμ→Sγ, Sγ→Sα DNA recombination. J. Immunol. 161: 5217-5225.