461. Classical Antigen Presenting Cell Activation Correlates with T Cell Immunity and COVID-19 Severity

Background The initial response of immune cells against respiratory viruses often determines the severity and duration of disease. The early trajectory of the immune response during infection with SARS-CoV-2 remains poorly understood. Dysregulation of innate immune factors that facilitate viral clearance and the adaptive response, such as type I interferons, have been implicated in severe COVID-19. However, collection of biological samples during the first seven days post-symptom onset has posed a logistical challenge, limiting our knowledge surrounding the immune responses that drive protection versus immunopathology. Methods From March 2020, Military Health System beneficiaries presenting with a positive SARS-CoV-2 test, a COVID-19 like illness, or a high-risk SARS-CoV-2 exposure at nine military medical treatment facilities across the United States were eligible for enrollment in our longitudinal cohort study, which included collection of respiratory sample, sera, plasma, and peripheral blood mononuclear cells (PBMCs). Twenty-five SARS-CoV-2 infected study participants provided samples with in the first seven days of symptom onset, fifteen of whom were hospitalized with COVID-19. We employed multiparameter spectral flow cytometry to comprehensively analyze the early trajectory of the innate and adaptive immune responses. Results We discovered that early activation of critical antigen presenting cell subsets was impaired upon comparing inpatients with outpatients, correlating with decreased antigen-experienced T cell responses. Specifically, we noted reduced expression of key costimulatory molecules, CD80 and CD86, on conventional dendritic cells that are required for viral antigen-specific T cell priming. Reduction in CD38, a marker of activation was also observed on inpatient dendritic cell subsets. Conclusion Reduced antigen presenting cell activation and expression of ligands that facilitate T cell engagement may impede the efficient clearance of SARS-CoV-2, coinciding with more severe disease in our cohort. Further analysis of the functional activation of early innate immune responses triggered by SARS-CoV-2 may unveil new immune biomarkers and therapeutic targets to predict and prevent severe disease associated with inadequate T cell immunity. Disclosures Simon Pollett, MBBS, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work))

T cell infiltration on local CpG-B delivery in early-stage melanoma is predominantly related to CLEC9A+CD141+ cDC1 and CD14+ antigen-presenting cell recruitment

BackgroundWe previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8+ T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration.MethodsRe-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles.ResultsSignificant increases in CD83+, CD14+, CD68+, and CD123+ APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123+BDCA2+ plasmacytoid dendritic cells (DCs) and BDCA3/CD141+CLEC9A+ type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14+ APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14+ monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration.ConclusionThe CpG-B-induced concerted recruitment of cDC1 and CD14+ APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites.

S-Layer From Lactobacillus brevis Modulates Antigen-Presenting Cell Functions via the Mincle-Syk-Card9 Axis

C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle−/−, CARD9−/− or conditional Syk−/− mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly decreased. Importantly, this was accompanied by an altered CD4+ T cell priming capacity of Mincle−/− BMDCs resulting in an increased CD4+ T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions.