Abstract LB079: Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4
Authors: Saha Akata , Shavkunov Alexander S. , Singh Shailbala , Salmon Avery J. , Keshari Sunita , Miao Qi , Edelson Brian T. , Yee Cassian , Chen Ken , Gubin Matthew 
Published: April 2024

Abstract
T cell-dependent anti-tumor activity unleashed by immunotherapies such as immune checkpoint therapy (ICT) can generate durable clinical responses. However, not all patients benefit, and epigenetic and transcriptional features required by T cells for effective ICT remain to be fully established. We previously documented that the transcription factor Bhlhe40 is upregulated by ICT in tumor specific CD4 and CD8 T cells in preclinical tumor models. We validated the relevance of this by ablating Bhlhe40 in both CD4 and CD8-T cells which rendered αPD-1 or αCTLA-4 ICT ineffective in tumor models normally responsive to ICT. Our findings extended beyond preclinical models as we recently found that human tumor-specific T cells recognizing mutant neoantigens display upregulation of Bhlhe40 and CRISPR-mediated Bhlhe40 deletion in human CD8 T cells recognizing the MART-1 tumor-antigen abrogates IFNG expression. Since we previously used CD4-Cre Bhlhe40f/f (B40ΔT) mice where Bhlhe40 was deleted in both CD4 and CD8 T cells, we could not definitively delineate how Bhlhe40 was regulating CD4 versus CD8 T cells under ICT. We thus used a model where Bhlhe40 is deleted specifically in CD8 T cells (B40ΔCD8). We found that like B40ΔT mice, B40ΔCD8 mice bearing the 1956 sarcoma were insensitive to αPD-1 ICT. While B40ΔT mice were also insensitive to αCTLA-4 ICT, interestingly, B40ΔCD8 mice displayed nearly normal tumor rejection upon αCTLA-4 ICT. Thus, Bhlhe40 is required in CD8 T cells for αPD-1 response, whereas is dispensable in CD8 T cells for αCTLA-4 efficacy. Single-cell protein and transcriptomic analysis of intratumoral immune-cells from ICT-treated control B40f/f, B40ΔCD8 and B40ΔT mice revealed overlapping and distinct changes to CD4 and CD8 T cells depending on which cells had Bhlhe40 deleted and which treatment was employed. With αPD-1 or αCTLA-4, defects in expression of IFN-γ, glycolytic enzyme transcripts and cytotoxic molecules, along with increased IL-10 production in CD4 and CD8 T cells from B40ΔT mice and in B40−/− CD8 T cells in B40ΔCD8 mice was observed. Whereas αCTLA-4-treated B40ΔT mice failed to reject tumors, addition of αIL-10R led to modest tumor growth inhibition, while with αPD-1 treatment, IL-10R blockade had little effect on tumor growth. We also noted that in control B40f/f mice, αPD-1 predominately altered CD8 T cells, whereas αCTLA-4 modified CD8 and CD4 T cells, including induction of an ICOS+ IFNγ+ CD4 T cell population expressing Bhlhe40. This population was largely preserved in B40ΔCD8 mice undergoing αCTLA-4 ICT. Analysis of intratumoral macrophages revealed profound defects in ICT-induced macrophage remodeling from CX3CR1+CD206+ to iNOS+ macrophages in B40ΔT mice lacking Bhlhe40 in CD4 and CD8 T cells. Although αCTLA-4-treated B40ΔCD8 mice reject their tumors, we observed a modestly higher frequency of CX3CR1+CD206+ macrophages and lower frequency of iNOS+ macrophages as compared to αCTLA-4-treated B40f/f mice. The highest percentage of CX3CR1+CD206+ macrophages and the fewest iNOS+ macrophages was observed in αPD-1-treated B40ΔCD8 mice that completely fail to reject tumors. These results reveal distinct requirements of Bhlhe40 in CD4 and CD8 T cells that depend on which ICT is used and further highlight non-redundant mechanisms of effective αPD-1 and αCTLA-4
Citation Format: Akata Saha, Alexander S. Shavkunov, Shailbala Singh, Avery J. Salmon, Sunita Keshari, Qi Miao, Brian T. Edelson, Cassian Yee, Ken Chen, Matthew Gubin. Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB079.