STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment.

The degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Enhancement of tumor infiltrating lymphocyte (TIL) is a critical element of efficacious therapy and one that may be achieved via administration of agents that promote tumor vascular normalization (VN) and/or induce the development of tertiary lymphoid structures (TLS) within the tumor microenvironment (TME). Low-dose stimulator of interferon genes (STING) agonist ADU S-100 (5 µg/mouse) was delivered intratumorally to established subcutaneous B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation. Treated and control tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via quantitative PCR (qPCR), with corollary immune cell composition changes in isolated tissues determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 µg/mL ADU S-100 or CD11c+ DCs isolated from tumor digests and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For T cell repertoireβ-CDR3 analyses, T cell CDR3 was sequenced from gDNA isolated from splenocytes and enzymatically digested tumors. We report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of antiangiogenic factors including Tnfsf15 (Vegi) and Cxcl10, and TLS-inducing factors including Ccl19, Ccl21, Lta, Ltb and Light. Therapeutic responses resulting from intratumoral STING activation were characterized by improved VN, enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neogenesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), interleukin (IL)-36, inflammatory chemokines and type I interferons in vitro and in vivo. TLS formation in ADU S-100-treated mice was associated with the development of a highly oligoclonal TIL repertoire enriched in expanded T cell clonotypes unique to the TME and not detected in the periphery. Our data support the premise that i.t. delivery of low-dose STING agonist promotes VN and a proinflammatory TME supportive of TLS formation, enrichment in the TIL repertoire and tumor growth control.

Chelvanambi M ，Fecek RJ ，Taylor JL ，Storkus WJ ... -  《Journal for ImmunoTherapy of Cancer》

STING Agonist Combined to a Protein-Based Cancer Vaccine Potentiates Peripheral and Intra-Tumoral T Cell Immunity.

Combining different immunotherapy approaches is currently building the future of immunotherapy, with the view to maximize anti-tumoral efficacy for larger patient population. The KISIMA™ platform allows the development of protein-based cancer vaccines able to induce tumor-specific T cell response resulting in anti-tumoral efficacy in various mouse models. Intra-tumoral administration of stimulator of interferon gene agonists (STINGa) was shown to induce a potent inflammatory response leading to the development of tumor-specific immunity. Here, we explored the efficacy and mechanisms of action of subcutaneous STINGa treatment combined with therapeutic vaccination in various mouse tumor models. This combinatory treatment highly enhanced frequency and effector function of both peripheral and intra-tumoral antigen-specific CD8 T cells, promoting potent IFNγ and TNFα production along with increased cytotoxicity. Moreover, combination therapy favorably modulated the tumor microenvironment by dampening immune-suppressive cells and increasing CD4 T cell infiltration together with their polarization toward Th1 phenotype. Combination with STINGa treatment improved the effect of therapeutic vaccination, resulting in a prolonged control and slower growth of B16-OVA and TC-1 tumors. Altogether, the results presented here highlight the potential of combining STINGa with a therapeutic protein vaccine for cancer treatment.

Rossi M ，Carboni S ，Di Berardino-Besson W ，Riva E ，Santiago-Raber ML ，Belnoue E ，Derouazi M ... -  《Frontiers in Immunology》

STINGing the Tumor Microenvironment to Promote Therapeutic Tertiary Lymphoid Structure Development.

Tertiary lymphoid structures (TLS), also known as ectopic lymphoid structures (ELS) or tertiary lymphoid organs (TLO), represent a unique subset of lymphoid tissues noted for their architectural similarity to lymph nodes, but which conditionally form in peripheral tissues in a milieu of sustained inflammation. TLS serve as regional sites for induction and expansion of the host B and T cell repertoires via an operational paradigm involving mature dendritic cells (DC) and specialized endothelial cells (i.e. high endothelial venules; HEV) in a process directed by TLS-associated cytokines and chemokines. Recent clinical correlations have been reported for the presence of TLS within tumor biopsies with overall patient survival and responsiveness to interventional immunotherapy. Hence, therapeutic strategies to conditionally reinforce TLS formation within the tumor microenvironment (TME) via the targeting of DC, vascular endothelial cells (VEC) and local cytokine/chemokine profiles are actively being developed and tested in translational tumor models and early phase clinical trials. In this regard, a subset of agents that promote tumor vascular normalization (VN) have been observed to coordinately support the development of a pro-inflammatory TME, maturation of DC and VEC, local production of TLS-inducing cytokines and chemokines, and therapeutic TLS formation. This mini-review will focus on STING agonists, which were originally developed as anti-angiogenic agents, but which have recently been shown to be effective in promoting VN and TLS formation within the therapeutic TME. Future application of these drugs in combination immunotherapy approaches for greater therapeutic efficacy is further discussed.

Filderman JN ，Appleman M ，Chelvanambi M ，Taylor JL ，Storkus WJ ... -  《Frontiers in Immunology》

In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models.

Dendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT. Mouse iPSCs were differentiated to iPSC-DCs on OP9 stromal cells expressing the notch ligand delta-like 1 in the presence of granulocyte macrophage colony-stimulating factor. Phenotype and the capacities of iPSC-DCs to traffic tumor-draining lymph nodes (TdLNs) and prime antigen-specific T cells were evaluated by flow cytometry and imaging flow cytometry. Antitumor efficacy of intratumoral injection of iPSC-DCs and RT was tested in syngeneic orthotopic mouse tumor models resistant to anti-PD-1 ligand 1 (PD-L1) therapy. Mouse iPSC-DCs phenotypically resembled conventional type 2 DCs, and had a capacity to promote activation, proliferation and effector differentiation of antigen-specific CD8+ T cells in the presence of the cognate antigen in vitro. Combination of in situ administration of iPSC-DCs and RT facilitated the priming of tumor-specific CD8+ T cells, and synergistically delayed the growth of not only the treated tumor but also the distant non-irradiated tumors. Mechanistically, RT enhanced trafficking of intratumorally injected iPSC-DCs to the TdLN, upregulated CD40 expression, and increased the frequency of DC/CD8+ T cell aggregates. Phenotypic analysis of tumor-infiltrating CD8+ T cells and myeloid cells revealed an increase of stem-like Slamf6+ TIM3- CD8+ T cells and PD-L1 expression in tumor-associated macrophages and DCs. Consequently, combined therapy rendered poorly immunogenic tumors responsive to anti-PD-L1 therapy along with the development of tumor-specific immunological memory. Our findings illustrate the translational potential of iPSC-DCs, and identify the therapeutic efficacy of a combinatorial platform to engage them for overcoming resistance to anti-PD-L1 therapy in poorly immunogenic tumors.

Oba T ，Makino K ，Kajihara R ，Yokoi T ，Araki R ，Abe M ，Minderman H ，Chang AE ，Odunsi K ，Ito F ... -  《Journal for ImmunoTherapy of Cancer》

Modulation of lactate-lysosome axis in dendritic cells by clotrimazole potentiates antitumor immunity.

Dendritic cells (DCs) play a critical role in antitumor immunity, but the therapeutic efficacy of DC-mediated cancer vaccine remains low, partly due to unsustainable DC function in tumor antigen presentation. Thus, identifying drugs that could enhance DC-based antitumor immunity and uncovering the underlying mechanism may provide new therapeutic options for cancer immunotherapy. In vitro antigen presentation assay was used for DC-modulating drug screening. The function of DC and T cells was measured by flow cytometry, ELISA, or qPCR. B16, MC38, CT26 tumor models and C57BL/6, Balb/c, nude, and Batf3-/- mice were used to analyze the in vivo therapy efficacy and impact on tumor immune microenvironment by clotrimazole treatment. By screening a group of small molecule inhibitors and the US Food and Drug Administration (FDA)-approved drugs, we identified that clotrimazole, an antifungal drug, could promote DC-mediated antigen presentation and enhance T cell response. Mechanistically, clotrimazole acted on hexokinase 2 to regulate lactate metabolic production and enhanced the lysosome pathway and Chop expression in DCs subsequently induced DC maturation and T cell activation. Importantly, in vivo clotrimazole administration induced intratumor immune infiltration and inhibited tumor growth depending on both DCs and CD8+ T cells and potentiated the antitumor efficacy of anti-PD1 antibody. Our findings showed that clotrimazole could trigger DC activation via the lactate-lysosome axis to promote antigen cross-presentation and could be used as a potential combination therapy approach to improving the therapeutic efficacy of anti-PD1 immunotherapy.

Wang Z ，Xu F ，Hu J ，Zhang H ，Cui L ，Lu W ，He W ，Wang X ，Li M ，Zhang H ，Xiong W ，Xie C ，Liu Y ，Zhou P ，Liu J ，Huang P ，Qin XF ，Xia X ... -  《Journal for ImmunoTherapy of Cancer》