Solid tumors are infiltrated by effector T cells (Teff) with the potential to control or reject them, as well as by regulatory T cells (Treg) that restrict the function of Teff and thereby promote tumor growth.1 The anti-tumor activity of Teff can be therapeutically unleashed and is now being exploited for the treatment of some forms of human cancer. However, weak tumor-associated inflammatory responses and the immune-suppressive function of Treg remain major hurdles to broader effectiveness of tumor immunotherapy.2 Here we show that upon disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, the majority of tumor-infiltrating Treg produce IFN-γ, followed by stunted tumor growth. Remarkably, genetic deletion of both or even just one allele of Carma1 in only a fraction of Treg, which avoided systemic autoimmunity, was sufficient to produce this anti-tumor effect, showing that not mere loss of suppressive function, but gain of effector activity by Treg initiates tumor control. Treg-production of IFN-γ was accompanied by macrophage activation and up-regulation of MHC-I on tumor cells. However, tumor cells also up-regulated expression of PD-L1, indicating activation of adaptive immune resistance.3 Consequently, PD-1 blockade concomitant with CARMA1-deletion caused rejection of tumors that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFN-γ-secretion in the preferentially self-reactive Treg pool does not cause systemic autoimmunity but is sufficient to prime the tumor environment for successful immune checkpoint therapy.

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Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (Trm) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 Trm and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK Trm-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 Trm homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.