31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

γδ T Cells Acquire Effector Fates in the Thymus and Differentiate into Cytokine-Producing Effectors in a Listeria Model of Infection Independently of CD28 Costimulation

<div><p>Both antigen recognition and CD28 costimulation are required for the activation of naïve αβ T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. Notably, this two-signal paradigm holds true for all αβ T cell subsets, regardless of whether they acquire their effector function in the periphery or the thymus. Because of contradictory results, however, it remains unresolved as to whether CD28 costimulation is necessary for γδ T cell activation and differentiation. Given that γδ T cells have been recently shown to acquire their effector fates in the thymus, it is conceivable that the contradictory results may be explained, in part, by a differential requirement for CD28 costimulation in the development or differentiation of each γδ T cell effector subset. To test this, we examined the role of CD28 in γδ T cell effector fate determination and function. We report that, although IFNγ-producing γδ T (γδ-IFNγ) cells express higher levels of CD28 than IL-17-producing γδ T (γδ-17) cells, CD28-deficiency had no effect on the thymic development of either subset. Also, following Listeria infection, we found that the expansion and differentiation of γδ-17 and γδ-IFNγ effectors were comparable between CD28^+/+ and CD28^−/− mice. To understand why CD28 costimulation is dispensable for γδ T cell activation and differentiation, we assessed glucose uptake and utilization by γδ T cells, as CD28 costimulation is known to promote glycolysis in αβ T cells. Importantly, we found that γδ T cells express higher surface levels of glucose transporters than αβ T cells and, when activated, exhibit effector functions over a broader range of glucose concentrations than activated αβ T cells. Together, these data not only demonstrate an enhanced glucose metabolism in γδ T cells but also provide an explanation for why γδ T cells are less dependent on CD28 costimulation than αβ T cells.</p></div

Comparison of CD28 expression levels on γδ T cell subsets in the thymus and periphery.

<p>Analysis of CD28 expression on various gated subsets in the thymus (<b>A</b>) and pLNs (<b>B</b>) of CD28^+/+ (i.e., IL-23R^gfp/+) mice. Black histograms show representative staining of CD28 on total, IL-23R⁺ (GFP⁺) and CD27⁺ DN TCRγδ⁺ subsets as well as on CD4⁺ TCRαβ⁺ subsets. Staining of thymocytes (<b>A</b>) and pLN cells (<b>B</b>) from CD28^−/− mice are shown as negative controls (shaded histograms). Numbers in the plots represent the mean fluorescent intensity (MFI) of CD28 expression. Data are representative of six mice in three independent experiments.</p