Marka Crittenden

Transforming growth factor beta (TGFβ) is an immunosuppressive cytokine that is overexpressed in tumor microenvironments. We have shown that CD8+ T cells with genetic ablation of the TGFβ type I receptor, Alk5 (CD8ΔALK5), were more sensitive to αCD3 stimulation resulting in enhanced proliferation and cytokine production. Based on these data, we hypothesized that TGFβ impaired T-cell receptor (TCR) signaling. We tested in vitro cytotoxicity of wild-type (WT) and CD8ΔALK5 OT-I T cells against murine oral carcinoma models transduced with ovalbumin altered peptide ligands (APLs) of differing affinities and found that loss of TGFβ renders CD8+ T cells more cytotoxic, but with diminishing effect at lower TCR agonism. TGFβ limits proximal TCR signaling intensity and duration, mediated by an interaction between the TGFβ type II receptor, PTPN22, and Zap70 that requires the Alk5 receptor. Downstream TCR signal integration is impaired by TGFβ following high and moderate, but not low TCR agonism. In vitro and in vivo models of chronic antigen stimulation demonstrate that TGFβ promotes both stem-like differentiation and terminal exhaustion, with loss of the more cytotoxic transitory exhausted population. Tumors of mixed APL clonality were implanted into Rag−/− animals followed by adoptive cell transfer of WT or CD8ΔALK5 OT-I T cells and monitored for clonal outgrowth. CD8ΔALK5 OT-I T cells were better able to control tumor clones with moderate TCR agonism compared to WT OT-I T cells. Targeting TGFβ signaling is one approach to enhance TCR signaling following strong or moderate agonism, alter differentiation toward more cytotoxic transitory exhaustion, and reduce terminal exhaustion, to improve antitumor immunity.

Radiation therapy (RT) activates multiple immunologic effects in the tumor microenvironment (TME), with diverse dose-response relationships observed. We hypothesized that, in contrast with homogeneous RT, a heterogeneous RT dose would simultaneously optimize activation of multiple immunogenic effects in a single TME, resulting in a more effective antitumor immune response. Using high-dose-rate brachytherapy, we treated mice bearing syngeneic tumors with a single fraction of heterogeneous RT at a dose ranging from 2 to 30 gray. When combined with dual immune checkpoint inhibition in murine models, heterogeneous RT generated more potent antitumor responses in distant, nonirradiated tumors compared with any homogeneous dose. The antitumor effect after heterogeneous RT required CD4 and CD8 T cells and low-dose RT to a portion of the tumor. At the 3-day post-RT time point, dose heterogeneity imprinted the targeted TME with spatial differences in immune-related gene expression, antigen presentation, and susceptibility of tumor cells to immune-mediated destruction. At a later 10-day post-RT time point, high-, moderate-, or low-RT-dose regions demonstrated distinct infiltrating immune cell populations. This was associated with an increase in the expression of effector-associated cytokines in circulating CD8 T cells. Consistent with enhanced adaptive immune priming, heterogeneous RT promoted clonal expansion of effector CD8 T cells. These findings illuminate the breadth of dose-dependent effects of RT on the TME and the capacity of heterogeneous RT to promote antitumor immunity when combined with immune checkpoint inhibitors.

SM16 efficacy and radiosensitivity of CT26 and Panc02.

Quantified effect of tumor size and epithelial-to-mesenchymal transition.

Multiple preclinical studies have shown improved outcomes when radiation therapy is combined with immune modulating antibodies. However, to date, many of these promising results have failed to translate to successful clinical studies. This led us to explore additional checkpoint and co-stimulatory pathways that may be regulated by radiation therapy. Here, we demonstrate that radiation increases the expression of inducible T cell co-stimulator (ICOS) on both CD4 and CD8 T cells in the blood following treatment. Moreover, when we combined a novel ICOS agonist antibody with radiation we observed durable cures across multiple tumor models and mouse strains. Depletion studies revealed that CD8 T cells were ultimately required for treatment efficacy, but CD4 T cells and NK cells also partially contributed to tumor control. Phenotypic analysis showed that the combination therapy diminished the increased infiltration of regulatory T cells into the tumor that typically occurs following radiation alone. Finally, we demonstrate in a poorly immunogenic pancreatic tumor model which is resistant to combined radiation and anti-PD1 checkpoint blockade that the addition of this novel ICOS agonist antibody to the treatment regimen results in tumor control. These findings identify ICOS as part of a T cell pathway that is modulated by radiation and targeting this pathway with a novel ICOS antibody results in durable tumor control in preclinical models.