Analysis of the Function of DNA Polymerase Kappa and M17 in the Immune System

In T cell-dependent immune responses, activated B cells undergo a phase of rapid expansion and form distinct histological structures, the germinal centers (GC). GCs are the sites of secondary antibody diversification. Somatic hypermutation (SHM) introduces mutations into the rearranged V genes, whereas class switch recombination (CSR) alters the IgH constant region to modulate effector function. The current model of SHM postulates cytidine deamination by AID, followed by error-prone repair that involves short-patch DNA synthesis by error-prone DNA polymerases. The Pol k (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase k. The mouse Pol k gene is expressed in most tissues of the body including B cells. The ability of Pol k to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a candidate for the introduction of nucleotide exchanges during SHM. Here, I show that Polk-deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4-hydroxy-3-nitrophenyl) acetyl-chicken globulin (NPGC). Polk-deficient mice mutate their Ig genes normally. Pol k-/- Pol l-/- Pol i-/- mice also show no defects in SHM, indicating that these error-prone DNA polymerases do not substitute for each other's function during SHM. However, Polk-deficient embryonic fibroblasts are sensitive to cell death following exposure to ultraviolet radiation, suggesting a role for Polk in translesion DNA synthesis. The human gene HGAL serves as marker in the prognosis of patients with GC-derived diffuse large B cell lymphomas (DLBCL). The mouse gene M17 is the homologue of HGAL. M17 is predominantly expressed in the GCs, indicating a role in GC function. In the present study, I analyzed M17-/- mice to investigate the role of M17 in the GC reaction. M17-/- mice form normal GCs, undergo efficient CSR and SHM and mount a T cell-dependent immune response. Thus, M17 is dispensable for the GC reaction and the current data support a rather indirect role for HGAL as a prognostic marker in the biology of DLBCL

MyD88 signalling in colonic mononuclear phagocytes drives colitis in IL-10-deficient mice

Commensal bacterial sensing by Toll-like receptors is critical for maintaining intestinal homeostasis, but can lead to colitis in the absence of interleukin-10. Although Toll-like receptors are expressed in multiple cell types in the colon, the cell type(s) responsible for the development of colitis are currently unknown. Here we generated mice that are selectively deficient in MyD88 in various cellular compartments in an interleukin-10[superscript −/−] setting. Although epithelial expression of MyD88 was dispensable, MyD88 expression in the mononuclear phagocyte compartment was required for colitis development. Specifically, phenotypically distinct populations of colonic mononuclear phagocytes expressed high levels of interleukin-1β, interleukin-23 and interleukin-6, and promoted T-helper 17 responses in the absence of interleukin-10. Thus, gut bacterial sensing through MyD88 in mononuclear phagocytes drives inflammatory bowel disease when unopposed by interleukin-10.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (NIH grant DK071754)National Institutes of Health (U.S.) (NIH grant AI046688)National Institutes of Health (U.S.) (NIH grant AI055502)National Institutes of Health (U.S.) (NIH grant RO1OD011141)National Institutes of Health (U.S.) (Training grant)National Cancer Institute (U.S.) (Irvington Fellowship

A20-Deficient Mast Cells Exacerbate Inflammatory Responses In Vivo

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-kappa B negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/Fc epsilon RI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function

Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining

Signaling through the Adaptor Molecule MyD88 in CD4+ T Cells Is Required to Overcome Suppression by Regulatory T Cells

Innate immune recognition controls adaptive immune responses through multiple mechanisms. The MyD88 signaling adaptor operates in many cell types downstream of Toll-like receptors (TLRs) and interleukin-1 (IL-1) receptor family members. Cell-type-specific functions of MyD88 signaling remain poorly characterized. Here, we have shown that the T cell-specific ablation of MyD88 in mice impairs not only T helper 17 (Th17) cell responses, but also Th1 cell responses. MyD88 relayed signals of TLR-induced IL-1, which became dispensable for Th1 cell responses in the absence of T regulatory (Treg) cells. Treg cell-specific ablation of MyD88 had no effect, suggesting that IL-1 acts on naive CD4(+) T cells instead of Treg cells themselves. Together, these findings demonstrate that IL-1 renders naive CD4(+) T cells refractory to Treg cell-mediated suppression in order to allow their differentiation into Th1 cells. In addition, IL-1 was also important for the generation of functional CD4(+) memory T cells

M17, a gene specific for germinal center (GC) B cells and a prognostic marker for GC B-cell lymphomas, is dispensable for the GC reaction in mice

In T-cell–dependent antibody responses, antigen-specific B cells undergo a phase of secondary antibody diversification in germinal centers (GCs). Somatic hypermutation (SHM) introduces mutations into the rearranged immunoglobulin (Ig) variable (V) region genes, and class-switch recombination (CSR) alters the Ig heavy (H) chain constant region. Aberrant SHM or CSR is thought to contribute to the development of GC-derived B-cell malignancies. Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of such GC-derived tumors. Based on their gene expression profile, DLBCLs can be divided into activated B-cell–like and GC-like subgroups. The human gene HGAL is predominantly expressed in GCs. It is also part of the gene expression signature of GC-like DLBCL, and its high expression in DLBCL has been associated with a better clinical prognosis. We have generated mice deficient of the HGAL homologue M17 in order to investigate its functional significance. The mutant animals form normal GCs, undergo efficient CSR and SHM, and mount T-cell–dependent antibody responses similar to wild-type controls. Thus, M17 is dispensable for the GC reaction, and its potential function in the pathogenesis of DLBCL remains elusive