Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Type I conventional dendritic cells (cDC1s) are an essential Ag-presenting population required for generating adaptive immunity against intracellular pathogens and tumors. While the transcriptional control of cDC1 development is well understood, the mechanisms by which extracellular stimuli regulate cDC1 function remain unclear. We previously demonstrated that the cytokine-responsive transcriptional regulator STAT3 inhibits polyinosinic:polycytidylic acid [poly(I:C)]-induced cDC1 maturation and cDC1-mediated antitumor immunity in murine breast cancer, indicating an intrinsic, suppressive role for STAT3 in cDC1s. To probe transcriptional mechanisms regulating cDC1 function, we generated novel RNA sequencing datasets representing poly(I:C)-, IL-10–, and STAT3-mediated gene expression responses in murine cDC1s. Bioinformatics analyses indicated that poly(I:C) stimulates multiple inflammatory pathways independent of STAT3, while IL-10–activated STAT3 uniquely inhibits the poly(I:C)-induced type I IFN (IFN-I) transcriptional response. We validated this mechanism using purified cDC1s deficient for STAT3 or IFN signaling. Our data reveal IL-10–activated STAT3 suppresses production of IFN-β and IFN-γ, accrual of tyrosine phosphorylated STAT1, and IFN-stimulated gene expression in cDC1s after poly(I:C) exposure. Moreover, we found that maturation of cDC1s in response to poly(I:C) is dependent on the IFN-I receptor, but not the type II IFN receptor, or IFN-λ. Taken together, we elucidate an essential role for STAT3 in restraining autocrine IFN-I signaling in cDC1s elicited by poly(I:C) stimulation, and we provide novel RNA sequencing datasets that will aid in further delineating inflammatory and anti-inflammatory mechanisms in cDC1s.

STAT3 is pivotal for governing myeloid responses to inflammatory stimuli to prevent hyperinflammation in vivo, yet whether STAT3 mediates pathogen control and clearance by myeloid cells remains unclear. In this study, we identified significant enrichment of IFN-stimulated transcriptional pathways in Stat3-deficient bone marrow–derived macrophages (BMDMs) at steady state. This was accompanied by activation of autocrine type I IFN (IFN-I) and aberrant autocrine IL-6 signaling associated with increased STAT1 activity. Despite exaggerated baseline IFN-STAT1 signaling, Stat3-deficient BMDMs were significantly impaired in their ability to induce expression of key pathogen defense genes and specific immune mediators upon LPS stimulation. STAT3 was also required in Citrobacter rodentium–infected BMDMs for expression of pathogen defense genes and effective bacterial killing. Moreover, bone marrow chimeric mice with 20% Stat3-deficient hematopoietic cells were more susceptible to infection with C. rodentium and showed increased bacterial dissemination and reduced production of specific cytokines. IL-6 blockade subdued the intrinsic IFN response and rescued expression of select pathogen defense genes in Stat3-deficient BMDMs upon LPS stimulation yet was unable to restore bacterial killing activity. Taken together, our results identify novel functions for STAT3 in orchestrating an optimal microbial defense response and suggest this is regulated by discrete mechanisms including modulation of autocrine IL-6 signaling in macrophages.

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3 -deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3 -sufficient (CreER) controls. Single-cell RNA sequencing of Lin ⁻ ckit ⁺ Sca1 ⁺ BM cells revealed altered transcriptional responses in Stat3 -deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3 -deficient Lin ⁻ ckit ⁺ Sca1 ⁺ cells accumulated γH2AX over time. Following secondary BM transplantation, Stat3 -deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs. Key Points: STAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditions STAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells

Type 1 conventional dendritic cells (cDC1s) are important for generating and sustaining antitumor immunity. Accordingly, the abundance of cDC1s in human tumors correlates with improved outcomes in cancer. Capitalizing on this role, we previously demonstrated that vaccination with murine cDC1s, generated in culture from bone marrow cells (termed here “in vitro–derived cDC1s”), elicits durable tumor control in multiple preclinical models; however, the immunological mechanisms underlying the efficacy of cDC1 vaccination remain unclear. Here, we examined whether in vitro–derived cDC1s resemble tumor-infiltrating DC populations and whether MHC-I and MHC-II antigen presentation contribute to cDC1-mediated tumor control following vaccination in melanoma. As expected, MHC-I or MHC-II deficiency had minimal impact on the transcriptional state of cDC1s in homeostasis or following stimulation with the adjuvant poly dI:dC. Moreover, in vitro–derived cDC1s cultured under steady-state conditions closely resembled tumor-infiltrating cDC1s, whereas their poly dI:dC–stimulated counterparts resembled CCR7+ tumor-infiltrating DC populations, also referred to as mregDCs or LAMP3+ DCs. Our data further show that both MHC-I and MHC-II contribute to tumor control upon cDC1 vaccination and that coexpression of MHC-I and MHC-II on the same cDC1 is necessary for a robust vaccine response. We also identified an important function for host cDC1s in supporting the efficacy of vaccination with in vitro–derived cDC1s, as judged by impaired tumor control in Irf8 + 32−/− mice, which lack endogenous cDC1s. Overall, these results indicate that effective antitumor responses depend on MHC-I and MHC-II antigen presentation by vaccine-delivered cDC1s, with additional contributions from host cDC1s.