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Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome. Supported by NIH Intramural Program

Gut microbiota impacts antitumor immunity and recent studies have shown that manipulating microbiota via fecal transplant can rescue immune checkpoint blockade (ICB) response in refractory melanoma patients. However, not all patients benefited from this approach. We hypothesize that tumor-intrinsic factors (e.g. neoantigen load and immune microenvironment) contribute to the microbiota effect on anticancer response. To test this, we performed preclinical microbiota manipulation studies using dietary intervention. Mice fed high-fiber diet (FD) had improved spontaneous tumor control and response to ICB. Mechanistically, FD-induced changes in microbiota composition skewed the tumor innate immune repertoire towards an antitumor profile marked by increased dendritic cell infiltration. Downstream of the innate compartment, FD reduced the proportion of intratumoral Tregs and exhausted CD8+ T cells. We further assessed the effects of FD across 8 tumor models including EL4 lymphoma, MC38 colon carcinoma, and 6 melanomas (M1–M6) that mimic the variety of human melanoma subtypes. Although FD had a beneficial effect overall, not all models responded to the same extent, and were accompanied by different changes in the tumor immune profile. FD delayed tumor initiation for EL4 and M4, reduced growth rate for M3 and M5, both delayed initiation and reduced growth rate for MC38, M2, and M6, but had no significant effects for M1. Additionally, we found that increasing tumor immunogenicity resulted in a synergistic effect with FD. Our data suggests that tumor-intrinsic factors influence the beneficial effect of microbiota on antitumor immunity. Identifying these factors will help refine the use of microbiota-based therapies in the clinic. This research was supported by the Intramural Research Program of the NIH (CCR-NCI). This research was supported by the Intramural Research Program of the NIH (CCR-NCI).

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14–protein conjugate, are both dependent on CD4+ T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14–protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igha, but not Ighb, allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14–protein conjugate, avidity maturation of the 44.1-Id–dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4+ T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show that pre-existing PS-specific Igs, the bacterial surface or particulation, selective recruitment of B cell subsets, or activation and recruitment of Pn protein-specific CD4+ T cells do not account for the failure of Pn to generate PS-specific IgG memory. Rather, the data suggest that the critical factor may be the lack of covalent attachment of PS to protein in intact Pn, highlighting the potential importance of the physicochemical relationship of PS capsule with the underlying bacterial structure for in vivo induction of PS-specific Igs.

During infections with extracellular bacteria, such as Streptococcus pneumoniae (Pn), the immune system likely encounters bacterial components in soluble form, as well as those associated with the intact bacterium. The potential cross-regulatory effects on humoral immunity in response to these two forms of Ag are unknown. We thus investigated the immunologic consequences of coimmunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS) as a model. Coimmunization of mice with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, as well as primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1-μm latex beads in adjuvant. Inhibition was long-lasting and occurred only during the early phase of the immune response, but it was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated Ags are the preferential immunogen for antibacterial Ig responses.

Although inflammatory monocytes (IM) [CD11b+Ly6Chi] play important roles in cell-mediated host protection against intracellular pathogens, their impact on humoral immune responses to extracellular bacteria are unknown. IM, localized largely in the splenic marginal zone of naïve CD11b-Diphtheria toxin receptor (DTR) bone marrow chimeric mice, were selectively depleted following treatment with DT, including no reduction of peritoneal B1a or B1b cells, which express CD11b. Depletion of IM resulted in a marked reduction in the polysaccharide (PS)-specific, T cell-independent IgM and T cell-dependent IgG responses to heat-killed Streptococcus pneumoniae (Pn) with no effect on the Pn protein-specific IgG response, or on the PS- and protein-specific IgG responses to a pneumococcal conjugate vaccine. IM acted largely within the first 48 h to induce the subsequent PS-specific IgM and IgG response. Adoptive transfer of highly purified IM from WT mice into DT-treated CD11b-DTR mice restored the PS-specific Ig response to Pn. IM were phenotypically and functionally distinct from circulating CD11b+CD11clowLy6G-/C- cells (i.e. “blood DC”) previously described to play a role in Ig responses to Pn. These data are the first to establish a critical role for IM in the induction of an Ig response to an intact extracellular bacterium.