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Immune checkpoint molecules play critical roles in regulating the anti-tumor immune response, and tumor cells often exploit these pathways to inhibit and evade the immune system. The B7-family immune checkpoint B7x is widely expressed in a broad variety of cancer types, and is generally associated with advanced disease progression and poorer clinical outcomes, but the underlying mechanisms are unclear. Here, we show that transduction and stable expression of B7x in multiple syngeneic tumor models leads to the expansion of immunosuppressive regulatory T cells (Tregs). Mechanistically, B7x does not cause increased proliferation of Tregs in tumors, but instead promotes the conversion of conventional CD4+ T cells into Tregs. Further, we find that B7x induces global transcriptomic changes in Tregs, driving these cells to adopt an activated and suppressive phenotype. B7x increases the expression of the Treg-specific transcription factor Foxp3 in CD4+ T cells by modulating the Akt/Foxo pathway. B7x-mediated regulation of Tregs reduces the efficacy of anti-CTLA-4 treatment, a therapeutic that partially relies on Treg-depletion. However, combination treatment of anti-B7x and anti-CTLA-4 leads to synergistic therapeutic efficacy and overcomes the B7x-mediated resistance to anti-CTLA-4. Altogether, B7x mediates an immunosuppressive Treg-promoting pathway within tumors and is a promising candidate for combination immunotherapy.

Background: Microsatellite instability (MSI) high colorectal cancers (CRCs) have a deficiency in mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and respond positively to anti-programmed death (PD) therapy. MSI low or microsatellite stable (MSS) CRCs that make up majority of tumors in clinical practice have not seen any benefit with PD inhibition. MSS CRC have higher proportion of KRAS oncogenic mutations as compared to MSI CRC. Reovirus, a naturally occurring oncolytic double-stranded RNA virus, has intrinsic preference for replication in KRAS mutant cells causing apoptosis in CRC. Current study was designed to investigate if reovirus could potentiate a beneficial effect of anti-PD therapy in MSS CRC. Methods: An array of CRC cell lines were screened for sensitivity to reovirus by MTT assay and expression of stem cell markers by RNA-seq and FACS. Based on MSI and KRAS status, four cell lines were explored further. Cells were treated with 5MOI reovirus for 48hr and expression of PD-L1 and PD-L2 with and without the potentiating effect of IFN-γ was assayed using FACS and qPCR. Combinatorial effect of reovirus with anti-PD-1 agent was studied in syngeneic models of BALB/c (CT26; KRASmut, MSS) and C57BL/6 (MC38; KRASwt, MSI) mice. The mice were grouped as control (PBS/IgG2A isotype control), reovirus, anti-mouse PD-1 antibody, and combination. Reovirus was used at a dose of 10 million/100 uL daily and anti PD-1 antibody was given i.p 200 ug/100 uL twice a week. Survival data and tumor volumes were recorded. HCT116 cells were xenografted in nude mice and treated with equivalent dose of reovirus. At the end point IHC was performed on excised paraffin-fixed tumor tissue with CD8 and granzyme antibodies. Results: Administration of reovirus was found to be most effective in cell lines tested. HCT116 (MSI & KRASmut), SW620 (MSS, KRASmut), LIM2405 (MSI, KRASwt) and HT29 (MSS, KRASwt) were chosen based on expression of CD133, CD44 and CD24. HCT116, LIM2405 and SW837 revealed increased and HT29 reduced expression of PD-L1 upon treating with reovirus. Survival data and tumor volume measurements showed better potentiating effect of reovirus on anti-PD-1 therapy in CT26 syngeneic model when compared with MC38. While single-agent therapy did not increase survival, the combination did improve survival with significance vs. control in both BALB/c (median 42 vs. 16 days, p=0.003) and C57BL/6 (median 24 vs. 17 days, p=0.02). The reovirus-treated xenografted tumor tissue showed a higher infiltration of T lymphocytes as confirmed by CD8-positive and intensified granzyme staining. Conclusion: Reovirus as single agent is more potent in KRASmut CRC. Syngeneic mice models proved synergistic anticancer effect of reovirus and anti-PD1 agent combination. Reovirus administration increased PD-L1 expression in CRC cells; possible mechanistic rationale for synergistic efficacy. Citation Format: Titto A. Augustine, Radhashree Maitra, Peter John, Sanjay Goel. Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3917.

Over the last decade, a new understanding of tumor-immune system interplay has been ushered in, lead in large part by the discovery of immune checkpoints mediated through B7-CD28 family interactions. Therapeutic blockade of the PD-L1 immune checkpoint pathway has already shown great success as a cancer immunotherapy for advanced urothelial carcinoma, leading to durable clinical remissions in an otherwise incurable disease. There are newly described members of the B7-CD28 family including B7-H3, B7x, and HHLA2. These ligands are thought to play an essential role in suppressing T-cell response, leading to immune tolerance of tumors. This feature makes them attractive targets for novel immunotherapy treatment paradigms. Here, we review the literature of current strategies and future directions of immune checkpoint blockade therapy for bladder cancer.