Rika Ouchida

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM–antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.

DNA polymerases η and θ function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase η (POLH) exhibited an ∼80% reduction of the mutations at A/T, whereas absence of polymerase θ (POLQ) resulted in ∼20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq–/–Polh–/– mice, however, did not show a further decrease of A/T mutations as compared with Polh–/– mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene Hypermutation

REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. To explore the role of its catalytic activity in Ig gene hypermutation in mammalian cells, we have generated mice expressing a catalytically inactive REV1 (REV1AA). REV1AA mice developed normally and were fertile on a pure C57BL/6 genetic background. B and T cell development and maturation were not affected, and REV1AA B cells underwent normal activation and class switch recombination. Analysis of Ig gene hypermutation in REV1AA mice revealed a great decrease of C to G and G to C transversions, consistent with the disruption of its deoxycytidyl transferase activity. Intriguingly, REV1AA mice also exhibited a significant reduction of C to T and G to A transitions. Moreover, each type of nucleotide substitutions at A:T base pairs was uniformly reduced in REV1AA mice, a phenotype similar to that observed in mice haploinsufficient for Polh. These results reveal an unexpected role for REV1 in the generation of C:G transitions and A:T mutations and suggest that REV1 is involved in multiple mutagenic pathways through functional interaction with other polymerases during the hypermutation process.

A Role for Lysosomal-Associated Protein Transmembrane 5 in the Negative Regulation of Surface B Cell Receptor Levels and B Cell Activation

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.

Role of DNA polymerase θ in tolerance of endogenous and exogenous DNA damage in mouse B cells

DNA polymerase θ (Polθ) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Polθ in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Polθ polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, γ‐irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Polθ is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Polθ in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross‐links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.

Deficiency of the oxidative damage-specific DNA glycosylase NEIL1 leads to reduced germinal center B cell expansion

Mammalian cells possess multiple DNA glycosylases, including OGG1, NTH1, NEIL1, NEIL2 and NEIL3, for the repair of oxidative DNA damage. Among these, NEIL1 and NEIL2 are able to excise oxidized bases on single stranded or bubble-structured DNA and has been implicated in repair of oxidative damage associated with DNA replication or transcription. We found that Neil1 was highly constitutively expressed in the germinal center (GC) B cells, a rapidly dividing cell population that is undergoing immunoglobulin (Ig) gene hypermutation and isotype switching. While Neil1(-/-) mice exhibited normal B and T cell development and maturation, these mice contained a significantly lower frequency of GC B cells than did WT mice after immunization with a T-dependent antigen. Consistent with the reduced expansion of GC B cells, Neil1(-/-) mice had a decreased frequency of Ig gene hypermutation and produced less antibody against a T-dependent antigen during both primary and secondary immune responses. These results suggest that repair of endogenous oxidative DNA damage by NEIL1 is important for the rapid expansion of GC B cells and efficient induction of humoral immune responses.

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4+ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.

LAPTM5 Protein Is a Positive Regulator of Proinflammatory Signaling Pathways in Macrophages

Background: The late endosomal/lysosomal transmembrane protein LAPTM5 is expressed in hematopoietic cells. Results: LAPTM5 facilitates activation of NF-κB and MAPK signaling and proinflammatory cytokine release mediated by cytokine and pattern recognition receptors. Conclusion: LAPTM5 is a positive regulator of proinflammatory responses by macrophages. Significance: The late endosomal/lysosomal system plays a role in the regulation of inflammatory responses by macrophages. LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane. Here we investigated the function of LAPTM5 in macrophages. We demonstrate that expression of LAPTM5 is required for the secretion of proinflammatory cytokines in response to Toll-like receptor ligands. We also show that RAW264.7 cells knocked down for LAPTM5 or macrophages from LAPTM5−/− mice exhibit reduced activation of NF-κB and MAPK signaling pathways mediated by the TNF receptor, as well as multiple pattern recognition receptors in various cellular compartments. TNF stimulation of LAPTM5-deficient macrophages leads to reduced ubiquitination of RIP1 (receptor-interacting protein 1), suggesting a role for LAPTM5 at the receptor-proximate level. Interestingly, we find that macrophages from LAPTM5−/− mice display up-regulated levels of A20, a ubiquitin-editing enzyme responsible for deubiquitination of RIP1 and subsequent termination of NF-κB activation. Our studies thus indicate that, in contrast to its negative role in T and B cell activation, LAPTM5 acts as a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. They also highlight a role for the endosomal/lysosomal system in regulating signaling via cytokine and pattern recognition receptors.

DNA polymerase η is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation

DNA polymerase η (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh+/− mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh+/+ and Polh−/− mice. These observations suggest that Polh is haplo‐insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh+/− mice as compared with WT mice. Polh−/− mice produced decreased serum titers of high‐affinity antibodies against a T‐dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high‐affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh−/− mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.