Loss of B1 and marginal zone B cells during ovarian cancer.

Recent advances in immunotherapy have not addressed the challenge presented by ovarian cancer. Although the peritoneum is an "accessible" locus for this disease there has been limited characterization of the immunobiology therein. We investigated the ID8-C57BL/6J ovarian cancer model and found marked depletion of B1 cells from the ascites of the peritoneal cavity. There was also selective loss of the B1 and marginal zone B cell subsets from the spleen. Immunity to antigens that activate these subsets validated their loss rather than relocation. A marked influx of myeloid-derived suppressor cells correlated with B cell subset depletion. These observations are discussed in the context of the housekeeping burden placed on innate B cells during ovarian cancer and to foster consideration of B cell biology in therapeutic strategies to address this challenge.

Monoclonal antibody-mediated neutralization of Clostridioides difficile toxin does not diminish induction of the protective innate immune response to infection.

Clostridioides difficile infection causes pathology that ranges in severity from diarrhea to pseudomembranous colitis. Toxin A and Toxin B are the two primary virulence factors secreted by C. difficile that drive disease severity. The toxins damage intestinal epithelial cells leading to a loss of barrier integrity and induction of a proinflammatory host response. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection. However, the impact of toxin neutralization on the induction and quality of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAbs therapy. At day 2 post-infection, C. difficile-infected, mAbs-treated mice had significantly less disease compared to isotype-treated mice despite remaining colonized with C. difficile. C. difficile-infected mAbs-treated mice still exhibited marked neutrophil infiltration and induction of a subset of proinflammatory cytokines within the intestinal lamina propria following infection that is comparable to isotype-treated mice. Furthermore, both mAbs and isotype-treated mice had an increase in IL-22-producing ILCs in the intestine following infection. MAbs-treated mice exhibited increased infiltration of eosinophils in the intestinal lamina propria, which has been previously reported to promote a protective host response following C. difficile infection. These findings show that activation of host protective mechanisms remain intact in the context of monoclonal antibody-mediated toxin neutralization.

Loss of Interleukin-10 (IL-10) Signaling Promotes IL-22-Dependent Host Defenses against Acute Clostridioides difficile Infection.

Infection with the bacterial pathogen Clostridioides difficile causes severe damage to the intestinal epithelium that elicits a robust inflammatory response. Markers of intestinal inflammation accurately predict clinical disease, however, the extent to which host-derived proinflammatory mediators drive pathogenesis versus promote host protective mechanisms remains elusive. In this report, we employed Il10-/- mice as a model of spontaneous colitis to examine the impact of constitutive intestinal immune activation, independent of infection, on C. difficile disease pathogenesis. Upon C. difficile challenge, Il10-/- mice exhibited significantly decreased morbidity and mortality compared to littermate Il10 heterozygote (Il10HET) control mice, despite a comparable C. difficile burden, innate immune response, and microbiota composition following infection. Similarly, antibody-mediated blockade of interleukin-10 (IL-10) signaling in wild-type C57BL/6 mice conveyed a survival advantage if initiated 3 weeks prior to infection. In contrast, no advantage was observed if blockade was initiated on the day of infection, suggesting that the constitutive activation of inflammatory defense pathways prior to infection mediated host protection. IL-22, a cytokine critical in mounting a protective response against C. difficile infection, was elevated in the intestine of uninfected, antibiotic-treated Il10-/- mice, and genetic ablation of the IL-22 signaling pathway in Il10-/- mice negated the survival advantage following C. difficile challenge. Collectively, these data demonstrate that constitutive loss of IL-10 signaling, via genetic ablation or antibody blockade, enhances IL-22-dependent host defense mechanisms to limit C. difficile pathogenesis.

IgA deficiency destabilizes homeostasis toward intestinal microbes and increases systemic immune dysregulation.

The ability of most patients with selective immunoglobulin A (IgA) deficiency (SIgAD) to remain apparently healthy has been a persistent clinical conundrum. Compensatory mechanisms, including IgM, have been proposed, yet it remains unclear how secretory IgA and IgM work together in the mucosal system and, on a larger scale, whether the systemic and mucosal anti-commensal responses are redundant or have unique features. To address this gap in knowledge, we developed an integrated host-commensal approach combining microbial flow cytometry and metagenomic sequencing (mFLOW-Seq) to comprehensively define which microbes induce mucosal and systemic antibodies. We coupled this approach with high-dimensional immune profiling to study a cohort of pediatric patients with SIgAD and household control siblings. We found that mucosal and systemic antibody networks cooperate to maintain homeostasis by targeting a common subset of commensal microbes. In IgA-deficiency, we find increased translocation of specific bacterial taxa associated with elevated levels of systemic IgG targeting fecal microbiota. Associated features of immune system dysregulation in IgA-deficient mice and humans included elevated levels of inflammatory cytokines, enhanced follicular CD4 T helper cell frequency and activation, and an altered CD8 T cell activation state. Although SIgAD is clinically defined by the absence of serum IgA, the symptomatology and immune dysregulation were concentrated in the SIgAD participants who were also fecal IgA deficient. These findings reveal that mucosal IgA deficiency leads to aberrant systemic exposures and immune responses to commensal microbes, which increase the likelihood of humoral and cellular immune dysregulation and symptomatic disease in patients with IgA deficiency.

Immunological consequences of microbiome-based therapeutics.

The complex network of microscopic organisms living on and within humans, collectively referred to as the microbiome, produce wide array of biologically active molecules that shape our health. Disruption of the microbiome is associated with susceptibility to a range of diseases such as cancer, diabetes, allergy, obesity, and infection. A new series of next-generation microbiome-based therapies are being developed to treat these diseases by transplanting bacteria or bacterial-derived byproducts into a diseased individual to reset the recipient's microbiome and restore health. Microbiome transplantation therapy is still in its early stages of being a routine treatment option and, with a few notable exceptions, has had limited success in clinical trials. In this review, we highlight the successes and challenges of implementing these therapies to treat disease with a focus on interactions between the immune system and microbiome-based therapeutics. The immune activation status of the microbiome transplant recipient prior to transplantation has an important role in supporting bacterial engraftment. Following engraftment, microbiome transplant derived signals can modulate immune function to ameliorate disease. As novel microbiome-based therapeutics are developed, consideration of how the transplants will interact with the immune system will be a key factor in determining whether the microbiome-based transplant elicits its intended therapeutic effect.

IgD ligation allows peritoneal cavity B cell proliferation.

Atypical cytokine production and immune cell subset ratios, particularly those that include high proportions of macrophages, characterize tumor microenvironments (TMEs). TMEs can be modeled by culturing peritoneal cavity (PerC) cells which have a high macrophage to lymphocyte ratio. With TCR or BCR ligation, PerC lymphocyte proliferation is tempered by macrophages. However, PHA (T cells) and anti-CD40 (B cells) are activators that induce proliferation. Herein, we report that ligating IgD, in contrast to IgM, triggers PerC B cell proliferation. IL-4 addition enhanced the IgD response for BALB/c PerC B cells but suppressed that of C57BL/6 mice. Intriguingly, concurrent ligation of IgD and CD3ε rescued a PerC T cell proliferative response. These results serve to expand the list of targets for promoting cellular and humoral immunity in conditions that model macrophage-rich TMEs.

Host immunity modulates the efficacy of microbiota transplantation for treatment of Clostridioides difficile infection.

Fecal microbiota transplantation (FMT) is a successful therapeutic strategy for treating recurrent Clostridioides difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited and the mechanism of action remains poorly understood. Here, we demonstrate a critical role for the immune system in supporting FMT using a murine C. difficile infection system. Following FMT, Rag1 heterozygote mice resolve C. difficile while littermate Rag1-/- mice fail to clear the infection. Targeted ablation of adaptive immune cell subsets reveal a necessary role for CD4+ Foxp3+ T-regulatory cells, but not B cells or CD8+ T cells, in FMT-mediated resolution of C. difficile infection. FMT non-responsive mice exhibit exacerbated inflammation, impaired engraftment of the FMT bacterial community and failed restoration of commensal bacteria-derived secondary bile acid metabolites in the large intestine. These data demonstrate that the host's inflammatory immune status can limit the efficacy of microbiota-based therapeutics to treat C. difficile infection.

Detection and elimination of a novel non-toxigenic Clostridioides difficile strain from the microbiota of a mouse colony.

Clostridioides difficile is an enteric bacterial pathogen that can a cause nosocomial infection leading to debilitating colitis. The development of a murine model of C. difficile infection has led to fundamental discoveries in disease pathogenesis and the host immune response to infection. Recently, C. difficile endogenously present in the microbiota of mice has been reported and was found to complicate interpretation of mouse studies. Here, we report a novel C. difficile strain, named NTCD-035, isolated from the microbiota of our mouse colony. The presence of NTCD-035 in mice prior to challenge with a highly pathogenic C. difficile strain (VPI10463) led to significantly reduced disease severity. Phylogenetic characterization derived from whole genome sequencing and PCR ribotyping identified the isolate as a novel clade 1, ribotype 035 strain that lacks the pathogenicity locus required to produce toxins. Deficiency in toxin production along with sporulation capacity and secondary bile acid sensitivity was confirmed using in vitro assays. Inoculation of germ-free mice with NTCD-035 did not cause morbidity despite the strain readily colonizing the large intestine. Implementation of a culture-based screening procedure enabled the identification of mice harboring C. difficile in their microbiota, the establishment of a C. difficile-free mouse colony, and a monitoring system to prevent future contamination. Taken together, these data provide a framework for screening mice for endogenously harbored C. difficile and support clinical findings that demonstrate the therapeutic potential of non-toxigenic strains in preventing C. difficile associated disease. Abbreviations: PaLoc - Pathogenicity locus, CFUs - Colony forming units, TcdA - toxin-A, TcdB - toxin-B, CdtA - binary toxin A, CdtB - binary toxin B, CdtR - binary toxin R, NTCD - non-toxigenic C. difficile.

PHA eludes macrophage suppression to activate CD8+ T cells.

Tumors may include a high proportion of immune modulatory cells and molecules that restrain the anti-cancer response. Activation of T cells to eliminate cancer cells within the immune-suppressive tumor microenvironment remains a challenge. We have shown that C57BL/6 J peritoneal cell culture models features of macrophage-dense tumors as TCR ligation fails to activate T cells unless IFNγ is neutralized or iNOS is inhibited. We tested other forms of T cell activation and found phytohemagglutinin (PHA) distinctive in the ability to markedly expand CD8 T cells in this model. IFNγ or iNOS inhibition was not necessary for this response. PHA triggered less IFNγ production and inhibitory PD-L1 expression than TCR ligation. Macrophages and CD44hi T cells bound PHA. Spleen T cell responses to PHA were markedly enhanced by the addition of peritoneal cells revealing that macrophages enhance T cell expansion. That PHA increases CD8 T cell responses within macrophage-dense culture suggests this mitogen might enhance anti-tumor immunity.