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

Cell Surface Free Thiols Increase On Activated CD8⁺ T Cells Prior To Division.

<p>Splenocytes were isolated from naïve mice and CD8⁺ T cells were purified and labeled with CFSE. Cells were then stimulated with either 10μg/mL isotype or anti-CD3 and anti-CD28. At the indicated timepoint, cells were harvested and incubated with maleimide-Alexa Fluor 647. Proliferation (A) was assessed by loss of CFSE fluorescence after activation. (B) The fold increase in cell surface free thiols relative to those found on isotype stimulated cells was determined and the average and standard deviation are plotted. Six mice were analyzed in two independent experiments. *, significant difference, P<u><</u>0.05.</p

Cell Surface Free Thiols Are Elevated On Antigen-Specific Cells Undergoing Recent TCR Stimulation.

<p>Naïve mice were infected with either LCMV-Armstrong (Acute) or LCMV-Clone 13 (Chronic) and harvested 8 days postinfection. To determine if cell surface free thiol levels correlated with expression of granzyme B (A), splenocytes were stained with anti-CD8α, D^bGP33-41, isotype control or anti-Granzyme B and maleimide-Alexa Fluor 488. The histogram is gated on CD8⁺D^bGP33-41⁺ T cells and the shaded area indicates isotype staining. For each population the Granzyme B expression was divided into negative (neg), intermediate (int) , and high (high) levels of staining. (B) The cell surface free thiol levels compared to those on CD8⁺CD44^loGranzyme B^- T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. To determine whether cell surface free thiols were elevated on antigen specific CD8⁺ T cells that were actively proliferating (C) splenocytes were stained with anti-CD8α, D^bGP33-41, isotype control or anti-Ki-67, and maleimide-Alexa Fluor 488 . Dot plots are gated on total CD8⁺ T cells and the values in each corner indicate the percent of cells that are in that quadrant. (D) The cell surface free thiol levels compared to those on CD8⁺CD44^loKi-67^- T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. (E) To determine recent TCR stimulation splenocytes were stained with anti-CD8α, anti-CD69, anti-PD-1, D^bGP33-41 and maleimide-Pacific Blue. The dot plots are gated on either CD8⁺CD44^lo (naïve) or CD8⁺D^bGP33-41⁺ (infected) T cells. The value in each corner indicates the percent of cells in that quadrant. (F) The cell surface free thiol levels compared to those on CD8⁺PD-1^-CD69^- T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. Six mice were analyzed in two independent experiments. *, significant difference between acute and chronic infection, P<u><</u>0.05.</p

Cell Surface Free Thiols Are Higher On Effector Compared To Memory CD8⁺T Cells During Acute Infection.

<p>(A) Mice were infected with LCMV-Armstrong and on day 8 postinfection splenocytes were harvested and stained with anti-CD8α, anti-CD44, the indicated MHC Class I tetramer and maleimide-Alexa Fluor 488. The maleimide-Alexa Fluor 488 levels of the gated populations are plotted in the histogram format with the shaded area indicating the staining of CD8⁺CD44^lo T cells from naïve mice. (B) Mice were examined at multiple times following LCMV- Armstrong (1°) infection or Clone 13 rechallenge (2°) and the fold increase in cell surface free thiols relative to naïve phenotype cells from uninfected mice was determined and the average and standard deviation are plotted. Six mice were analyzed in two independent experiments. *, significant difference, P<u><</u>0.05.</p

Cell Surface Free Thiols Remain Elevated On Antigen-Specific CD8⁺ T Cells For Extended Periods During Chronic Viral Infection.

<p>(A) Mice were infected with LCMV-Clone 13 and on day 8 postinfection, splenocytes were harvested and stained with anti-CD8α, anti-CD44, the indicated MHC Class I tetramer and maleimide-Alexa 488. The maleimide-Alexa Fluor 488 level of the gated populations is plotted in the histogram format with the shaded area indicating the staining of CD8⁺CD44^lo T cells from naïve mice. (B) Mice were examined at multiple times following LCMV-Clone 13 infection and the fold increase in cell surface free thiols relative to naïve phenotype cells from uninfected mice was determined and the average and standard deviation are plotted. Six mice were analyzed in two independent experiments. *, significant difference, P<u><</u>0.05.</p

Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation.

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins