Cytotoxic T-lymphocyte Associated Protein 4 (CTLA-4) is an immune checkpoint molecule highly expressed on regulatory T-cells (Tregs) that can inhibit the activation of effector T-cells. Anti-CTLA-4 therapy can confer long-lasting clinical benefits in cancer patients as a single agent or in combination with other immunotherapy agents. However, patient response rates to anti-CTLA-4 are relatively low, and a high percentage of patients experience severe immune-related adverse events. Clinical use of anti-CTLA-4 has regained interest in recent years; however, the mechanism(s) of anti-CTLA-4 is not well understood. Although activating T-cells is regarded as the primary anti-tumor mechanism of anti-CTLA-4 therapies, mounting evidence in the literature suggests targeting intra-tumoral Tregs as the primary mechanism of action of these agents. Tregs in the tumor microenvironment can suppress the host anti-tumor immune responses through several cell contact-dependent and -independent mechanisms. Anti-CTLA-4 therapy can enhance the priming of T-cells by blockading CD80/86-CTLA-4 interactions or depleting Tregs through antibody-dependent cellular cytotoxicity and phagocytosis. This review will discuss proposed fundamental mechanisms of anti-CTLA-4 therapy, novel uses of anti-CTLA-4 in cancer treatment and approaches to improve the therapeutic efficacy of anti-CTLA-4.

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Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells’ effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using in vitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12– and IL-27–induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27–induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12–mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

Background Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) highly expressed on regulatory T-cells (Tregs) inhibit the activation of pro-inflammatory T-cells responsible for eliminating cancer cells. Anti-CTLA-4 can enhance T-cell activation by increasing CD28 co-stimulatory signaling through CTLA-4 blockade or depletion of Tregs by Fc-dependent effector mechanisms. Strategies to improve its therapeutic efficacy are needed as patient response rates to anti-CTLA-4 are low. Response to anti-CTLA-4 has been positively correlated with tumor mutation burden (TMB). Defects in the DNA mismatch repair (MMR) pathway can increase TMB and the production of neoantigens that promote anti-tumor immune responses.Here we investigate the underlying mechanism(s) to which induced MMR deficiency in an immunologically-cold and low TMB tumor model can enhance the therapeutic effect of anti-CTLA-4. We hypothesize that induced MMR deficiency in tumors enhances anti-CTLA-4-mediated Treg depletion and increases the infiltration and activation of effector T-cells. Methods MMR deficiency was induced in a syngeneic murine neuro-2a neuroblastoma cell line by knocking-out MLH1 expression using CRISPR-Cas9. Wildtype MMR-proficient (pMMR) or induced MMR-deficient (idMMR) neuro-2a cells were inoculated into immunocompetent A/J mice and treated with anti-CTLA-4. Tumors were immunophenotyped by flow cytometry and mixed-lymphocyte reaction assays were used to examine the effects of MMR deficiency and anti-CTLA-4 on T-cell activation and proliferation. Results Induced MMR deficiency in neuroblastoma tumors enhances the anti-tumor immune response induced by anti-CTLA-4. MMR deficiency in neuroblastoma tumors promoted anti-CTLA-4-mediated Treg depletion and increased intratumoral CD3 + T-cells. idMMR neuroblastoma tumors had an increase of ICOS + T-cells compared to pMMR tumors. In addition, ICOS + T-cells were increased further with anti-CTLA-4 treatment. Conclusions Our data show that inducing MMR deficiency in low TMB and immune-cold neuroblastoma tumors can enhance the anti-tumor effect of anti-CTLA-4 by increasing Tcell activation and depletion of Tregs. By understanding the underlying mechanism(s) of anti-CTLA-4 in idMMR tumors, it may justify targeting the MMR pathway to improve the response to immune checkpoint inhibitors in patients with immunologically-cold and/or low TMB tumors that are refractory to immunotherapy. Future studies will assess how inducing MMR deficiency alters the tumor microenvironment to enable anti-CTLA-4-mediated Treg depletion and the significance of ICOS + T-cells in the efficacy of anti-CTLA-4 therapy in this setting.

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