Apoptosis-regulated low-avidity cancer-specific CD8(+) T cells can be rescued to eliminate HER2/neu-expressing tumors by costimulatory agonists in tolerized mice

Abstract

A major barrier to vaccines in cancer treatment is their failure to activate and maintain a complete cancer-specific CD8(+) effector T-cell repertoire. Low-avidity T cells are more likely to escape clonal deletion in the thymus when compared with high-avidity T cells, and therefore comprise the major population of effector T cells available for activation in patients with cancer. However, low-avidity T cells fail to traffic into the tumor microenvironment and function in eradicating tumor under optimal vaccination conditions as opposed to high-avidity T cells that escape clonal deletion and function in tumor killing. We used high- and low-avidity T-cell receptor transgenic CD8(+) T cells specific for the immunodominant epitope HER2/neu (RNEU420-429) to identify signaling pathways responsible for the inferior activity of the low-avidity T cells. Adoptive transfer of these cells into tumor-bearing vaccinated mice identified the members of apoptosis pathways that are upregulated in low-avidity T cells. The increased expression of proapoptotic proteins by low-avidity T cells promoted their own cell death and also that of other tumor-specific CD8(+) T cells within their local environment. Importantly, we show that this proapoptotic effect can be overcome by using a strong costimulatory signal that prevents the activation-induced cell death and enables the low-avidity T cells to traffic into the tumor and assist in tumor clearance. These findings identify new therapeutic opportunities for activating the most potent anticancer T-cell responses.

Figure 1. Expression of pro-apoptotic proteins DR5, FasL, and CD24 are increased on low avidity relative to high avidity CD8⁺ T cells

6×10⁶ high or low avidity T cells were adoptively transferred into neu-N mice treated with Cy and vaccine. Three days later, adoptive transfer cells were extracted from the tumor-draining node of the neu-N mice and protein expression was assessed by flow cytometry. These experiments were repeated at least 5 times (n=3–5 mice/group). A. DR5 expression compared between low avidity CD8⁺ T cells and high avidity CD8⁺ T cells. B. FasL expression compared between low avidity CD8⁺ T cells and high avidity CD8⁺ T cells. C. CD24 expression compared between low avidity CD8⁺ T cells and high avidity CD8⁺ T cells.

Figure 2. Expression of DR5, CD24, and FasL is correlated with reduced T cell function and increased apoptosis

High or low avidity CD8⁺ T cells were adoptively transferred into Cy- and vaccine-treated neu-N mice. Cells are taken from the tumor-draining node (TDN) on day 3 for all except for figure 2B where cells were taken on day 5 from the tumor-draining node and tumor. These experiments were repeated at least 3 times (n=3–5 mice/group). A. IFNγ secretion on low avidity T cells without DR5, FasL, and CD24 expression when compared with low avidity T cells expressing these proteins. B. DR5, FasL, and CD24 expression on high avidity T cells from the tumor-draining node compared to tumor-infiltrating lymphocytes. C. Dying low avidity T cells were identified through Annexin V⁺ and 7AAD⁻ staining and levels of DR5, FasL, and CD24 protein expression were compared.

Figure 3. Low avidity T cells are more susceptible to Fas-induced apoptosis than high avidity cells; CD24-crosslinking leads to cell death; and low avidity T cells are able to cause death of high avidity T cells

Low and high avidity T cells extracted from TCR transgenic mice were treated in culture plated at a concentration of 1×10⁵ cells/mL and incubated at 37°C before death was assessed by flow cytometry by staining for Annexin V and 7AAD. These experiments were repeated at least 3 times (n=3–5 mice/group). A. High and low avidity T cells were plated at and treated with 0.1μg/mL Fas-crosslinking antibody and peptide-pulsed T2 cells for three hours before analysis. B. Cells were incubated at 1mg/mL of CD24 crosslinking antibody for 24 hours. C. Low and high avidity T cells were mixed at a ratio of 3:1 and incubated for 24 hours. FasL blocking antibody was added at a concentration of 10μg/mL.

Figure 4. Anti-OX40 agonistic antibody treatment leads to increased function of low avidity T cells and reduced expression of FasL, DR5, and CD24

Cy- and vaccine-treated neu-N mice were adoptively transferred with low avidity T cells and also treated with 300ug of OX40 agonistic antibody or IgG the day of adoptive transfer. OX40 antibody or IgG was also given three days following adoptive transfer for trafficking experiments. These experiments were repeated at least 3 times (n=3–5 mice/group). A. Comparison of IFNγ production between mice treated with anti-OX40 antibody or IgG. B. Comparison of Thy1.2⁺ T cell trafficking between mice treated with anti-OX40 antibody or IgG. C. Comparison of pro-apoptotic protein expression between mice treated with or without anti-OX40 antibody. D. Cell death comparison between mice treated with anti-OX40 antibody or IgG. E. Tumor-free survival comparison between Cy-, vaccine- and/or anti-OX40 antibody-treated mice, with and without low avidity adoptive T cell transfer. P value indicates the difference between the Tumor+Cy+Vacc+OX40 group and the next closest group, Tumor+Cy+Vacc+IgG. F. Comparison of Bcl-2 and survivin expression with and without anti-OX40 antibody on days 5 and 7 for Bcl-2 and days 3 and 5 for survivin. G. Comparison of low avidity T cell activity in mice treated with or without anti-OX40 antibody as compared to high avidity T cell activity.

Figure 5. Low avidity T cell apoptosis is antigen-dependent in the periphery and the most highly activated T cells are undergoing apoptosis; express high levels of CD24, DR5, and FasL; and secrete IFNγ

Three days after adoptive transfer, low avidity T cells were extracted from tumor-bearing, Cy-, and vaccine-treated mice. Cells were CFSE stained by incubating at 37°C with 1.5mM CFSE for 8 minutes. Death was assessed by Annexin V and 7AAD staining. These experiments were repeated at least 3 times (n=3–5 mice/group). A. Comparison of cell death between low avidity T cells from tumor-bearing, Cy-treated, and vaccinated neu-N mice with and without anti-OX40 antibody, and untreated mice. B. Amount of low avidity T cell death over increasing amounts of peptide (20μg = 17.39μM). C. Comparison of low avidity T cell death between early and late divisions. D. Demonstration of DR5, FasL, and CD24 expression in divided cells. E. Demonstration of IFNγ secretion in divided cells.

Figure 6. Enhanced low avidity T cell function observed with anti-OX40 antibody treatment is not CD4⁺ T cell-dependent; costimulation with the agonistic 41BB antibody also increases low avidity T cell function

Tumor-bearing, Cy- and vaccine-treated neu-N mice were given anti-OX40, anti-OX40 and CD4-depleting antibody, or 41BB antibody and cells were extracted for analysis three days after low avidity T-cell adoptive transfer for analysis of cytokine secretion and five days after adoptive transfer for trafficking information. OX40 and 41BB were both given at doses of 300μg on the day of adoptive transfer with OX40 again being given three days after adoptive transfer to mice being analyzed for trafficking. These experiments were repeated at least 3 times (n=3–5 mice/group). A. Comparison of IFNγ secretion in anti-OX40-treated cells with and without CD4 depletion. B–C. Comparison of IFNγ secretion and Thy1.2⁺ T cell trafficking with and without 41BB treatment.