Extrafollicular plasmablast responses (EFRs) are considered to generate antibodies of low affinity that offer little protection from infections. Paradoxically, high avidity antigen-B cell receptor engagement is thought to be the main driver of B cell differentiation, whether in EFRs or the slower-developing germinal centers (GCs). This study demonstrates that influenza infection rapidly induced EFRs generating protective antibodies in a B cell intrinsic and extrinsic Toll-like receptor (TLR)-dependent manner. B cell-intrinsic TLR signals supported antigen-stimulated B cell survival, clonal expansion, and the differentiation of B cells via induction of IRF4, the master regulator of B cell differentiation, through activation of NF-kB c-Rel. Provision of sustained TLR4 stimulation after immunization altered the fate of virus-specific B cells towards EFRs instead of GCs, accelerating rapid antibody production and improving their protective capacity over antigen/alum administration alone. Thus, inflammatory signals act as B cell fate-determinants for the rapid generation of protective, antiviral EF responses.

Tick-borne infection with Borrelia burgdorferi (Bb) causes Lyme disease in humans and persistent infection of mice, its natural reservoir host. IgG responses critically control Bb tissue burden but do not clear the infection. We previously showed induction of germinal centers (GC) in mice after Bb infection followed by their rapid collapse within 30 days, despite ongoing infection. This was consistent with the lack of long-lived and high-affinity antibody responses in these mice. Our recent experiments showed that serum IgG from Bb-infected mice bound more strongly to the inhibitory FcγRIIb on B cells, as well as recombinant FcγRIIb than those from uninfected mice, as assessed by flow cytometry and ELISA, respectively. The data prompted us to investigate whether the interaction between Bb infection-induced IgG and FcγRIIb results in the suppression of GC. Indeed, Bb-infected mice lacking FcγRIIb signaling (FcγRIIb−/−) maintained GC B cells and CD4 TFH for at least 90 days after infection, and GC B cells showed reduced apoptosis, as assessed by staining for Annexin V. Furthermore, transfer of serum from Bb-infected wild type but not AID−/−sIgM−/− mice lacking IgG, IgM and from non-infected wild type mice, into 14-day Bb-infected wild type mice resulted in reduced GC at day 21. However, despite GC maintenance and the resulting increases in Bb-specific, T cell-dependent IgG responses, enhanced antibody affinity maturation was not observed and Bb tissue burdens of FcγRIIb−/− and control mice were comparable. The data show the importance for FcγRIIb in the regulation of GC during Bb infection, while additional mechanisms currently under investigation seem to underlie their functional deficits, enabling the establishment of Bb persistence.

Regulatory B cells (Bregs) are critical for maintaining immune tolerance, and alterations in Breg numbers has detrimental effects in inflammatory diseases, infection, and cancer. Bregs are found among most B cell subsets and act primarily by producing IL-10. Despite their importance, the signals that control Breg differentiation and maintenance are not well defined. Here, we demonstrate that mice incapable of secreting IgM (sIgM−/−) have drastically increased IL-10+ Bregs in lymphoid organs compared to wildtype (WT) mice. These IL-10+ Bregs are polyclonal and increased among all major B cell subsets. While newborn WT mice (<24 hr of age) harbor detectable serum IgM, IL-10+ B cell expansion occurs early after birth in both WT and sIgM−/− mice. However, IL-10+ Bregs contract only in WT but not in sIgM−/− mice, which is in parallel to rising levels of serum IgM in WT mice. SIgM−/− B cells that develop in the presence of circulating IgM express IL-10 similar to that of WT B cells within the same mouse indicating that the expansions in IL-10+ B cell populations are not due to B cell-intrinsic effects based on the inability to secrete IgM, but rather the presence of sIgM. Mice that lack B cell-expressed high-affinity IgM receptor (FcμR) mirror the IL-10 phenotype in their B-1 and marginal zone B cells, but not follicular B cells, suggesting that secreted IgM can act as a negative regulator of IL-10 expression in B cell subsets via binding to surface-expressed FcμR, and potentially other IgM binding receptors. Our data indicate that sIgM controls IL-10 competence in B cells via IgM binding receptors thereby revealing potential novel avenues for altering IL-10+ Breg induction in disease settings. Funding: T32AI134646, R01AR067751, R01AI127389, R01AI148652. Supported by grants from NIH (T32AI134646, R01AR067751, R01AI127389, R01AI148652)

Appropriate polarization of effector CD4 T cells is essential for eliminating invading pathogens. Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, evades immune clearance in infected mice and humans, establishing bacterial persistence. Here we aimed to explore the functionality of CD4 T cell as a possible target of Borrelia-induced immune evasion. In response to Bb infection, CD4 T cell polarization is considered to be Th1 dominant. However, when we stimulated CD4 T cells from Bb infected mice ex vivo with Bb surface Ag Arthritis Related Protein (Arp), we were unable to detect IFNγ, prompting us to re-examine the polarization state of lymph node and blood effector CD4 T cells throughout Bb infection by quantitative RT-PCR and flow cytometry. In contrast to prevailing views, we found only modest numbers of Th1-polarized activated CD4 T cells, as measured by expression of Tbet (Tbx21), and little to no induction of Ifng, including in Bb-specific CD4 T cells, identified by a novel I-Ab tetramer to Arp. This could not be explained by enhanced polarization to Th2 or Th17, as GATA-3 and RORγt expression were also largely absent. Nor could it be explained by polarization at a T cell effector site, the skin, as skin-resident T cells also showed no polarization even 4 months after infection. Furthermore, Th1 polarization was not necessary for controlling bacterial dissemination, as Bb infection of Tbet−/− mice showed significant reductions in Borrelia numbers in the tibiotarsal joints, which correlated with enhanced Th17 polarization as measured by RORγt expression. Together, our data demonstrate a failure of CD4 T cell polarization after Bb infection, indicating effector CD4 T cell polarization as a novel target of Bb immune evasion.

FcμR is present on the cell surface of B cells as well as other antigen presenting cells where they bind the secreted form of IgM (sIgM). Our previous studies have shown that follicular B cells internalize sIgM continuously in vivo and that lack of sIgM or the FcμR on B cells results in diminished IgG responses following influenza virus infection. We aim to determine the mechanisms underlying the need for sIgM in the development of IgG responses. For this we generated HELB-FcmR−/− and HELB-FcmR+/+ mice. In vitro stimulation of purified B cells from these mice with HEL-OVA conjugate in the presence of OVA-specific OTII CD4 T cells showed an antigen-dose-dependent stimulation of both types of B cells. However, FcmR−/− B cells proliferated less at intermediate and low doses of antigen compared to controls, while they proliferated similar as wild type B cells following high-dose antigen stimulation. Intravenous injection of CD45.1-expressing C57BL/6 mice with B cells from either FcmR-deficient or -sufficient CD45.2+ HELB mice together with sheep red blood cells conjugated to HEL showed no significant differences in HEL-specific serum IgM or IgG, or frequencies of HEL-specific B cells between mice receiving either type of B cell, as assessed by ELISA and FACS, respectively. Ongoing studies are aimed at determining whether antigen-dose affects the need for FcmR expression in vivo. For that we have created mixed bone marrow (BM) irradiation chimeras with 2% BM from CD45.2 HELB or HELB-FcmR−/− mice, and 98% CD45.1 wild type BM. To-date our studies suggest that FcmR-IgM interaction enhances B cell proliferation when antigen is limiting. Future studies are aimed at testing this conclusion and explore how sIgM-FcmR interaction enhanced B cell responses.