Pancreatic ductal adenocarcinoma (PDAC) is the deadliest major cancer and has a profoundly immunosuppressive tumor microenvironment (TME). Previous studies have shown that inhibition of the E1 enzyme, which catalyzes the small ubiquitin-like modifiers (SUMO), with the small molecule TAK-981, can reprogram the TME to enhance immune activation and suppress tumor growth. We found that the CD-155/TIGIT pathway, a key regulator of immune evasion in PDAC, is influenced by SUMOylation. We hypothesized that the combination of SUMO E1 and TIGIT inhibition would synergistically induce anti-tumor immune effects. We used a clinically relevant orthotopic mouse model that consistently develops liver metastases to study this combination therapy alone and in the perioperative setting with surgical resection. The combination of SUMO E1 and TIGIT inhibition significantly prolonged survival. Complete responders exhibited protective immunity and enhanced T cell reactivity to model-specific alloantigens. Complementary immune analyses of resected tumors demonstrated that combination therapy more significantly reduces the abundance of regulatory FOXP3+CD4+ T cells than each monotherapy alone. The findings suggest that SUMO E1 inhibition enhances antibody-mediated elimination of Tregs through innate immune cells, potentially by activation of type I interferon responses. Our results highlight a mechanism to enhance the efficacy of anti-TIGIT therapy. Brief Summary SUMOylation is a post-translational modification process critical for cancer. Inhibition of SUMOylation can improve the sensitivity of pancreatic cancer to immune checkpoint inhibition.

Commensal microbes induce cytokine-producing effector tissue-resident CD4+ T cells, but the function of these T cells in mucosal homeostasis is not well understood. Here, we report that commensal-specific intestinal Th17 cells possess an anti-inflammatory phenotype marked by expression of interleukin (IL)-10 and co-inhibitory receptors. The anti-inflammatory phenotype of gut-resident commensal-specific Th17 cells was driven by the transcription factor c-MAF. IL-10-producing commensal-specific Th17 cells were heterogeneous and derived from a TCF1+ gut-resident progenitor Th17 cell population. Th17 cells acquired IL-10 expression and anti-inflammatory phenotype in the small-intestinal lamina propria. IL-10 production by CD4+ T cells and IL-10 signaling in intestinal macrophages drove IL-10 expression by commensal-specific Th17 cells. Intestinal commensal-specific Th17 cells possessed immunoregulatory functions and curbed effector T cell activity in vitro and in vivo in an IL-10-dependent and c-MAF-dependent manner. Our results suggest that tissue-resident commensal-specific Th17 cells perform regulatory functions in mucosal homeostasis.
Copyright © 2023 Elsevier Inc. All rights reserved.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.
Copyright © 2023 by The American Association of Immunologists, Inc.

Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models.
Copyright © 2023 Moi, Zeng, Minnie, Bhatt, Wood, Sester, Mazzieri and Dolcetti.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.
© 2022. The Author(s).