Background

We previously documented that the transcription factor Bhlhe40 is upregulated by immune checkpoint therapy (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. However, the mechanisms underlying how Bhlhe40 regulates CD8 T cell function in the context of different ICTs is yet to be fully delineated.

Methods

We used CD4-Cre Bhlhe40^f/f (B40^DCD4/8) conditional knockout (KO) mice where Bhlhe40 was deleted in both CD4 and CD8 T cells, as well as a model where Bhlhe40 is deleted specifically in CD8 T cells [E8I CD8 Cre Bhlhe40^f/f (B40^DCD8)] and compared αPD-1/αCTLA-4 ICT-induced anti-tumor responses.

Results

We found that like B40^DCD4/8 mice (Bhlhe40 KO in CD4 and CD8 T cells), tumors in B40^DCD8 mice (Bhlhe40 KO in CD8 T cells only) were insensitive to αPD-1 ICT. Interestingly, while B40^DCD4/8 mice were also insensitive to αCTLA-4, B40^DCD8 mice displayed nearly normal tumor rejection upon αCTLA-4. Importantly, both CD4 and CD8 T cells are required for αPD-1- or αCTLA-4-mediated tumor rejection in the models we assessed. Intratumoral CD8 T cells from either αPD-1 or αCTLA-4-treated B40^DCD8 mice displayed defects in expression of IFN-g and glycolytic enzyme transcripts, with the latter being particularly reduced in αPD-1-treated B40^DCD8 mice. As compared to control mice, αPD-1 or αCTLA-4 failed to induce expansion of activated effector Cxcr1⁺CD8 T cells in B40^DCD8mice. αPD-1 notably expanded a dysfunctional CD39⁺CD8 T cell population in B40^DCD8 mice and this expansion was not observed in αPD-1-treated control mice with nor αCTLA-4-treated control or B40^DCD8 mice. Profound defects in ICT-induced macrophage remodeling from CX3CR1⁺CD206⁺ macrophages to iNOS⁺ macrophages was observed in B40^DCD4/8 mice lacking Bhlhe40 in CD4 and CD8 T cells. Although αCTLA-4-treated B40^DCD8 mice reject their tumors, we nevertheless observed a modestly higher frequency of CX3CR1⁺CD206⁺ macrophages and lower frequency of iNOS⁺ macrophages as compared to αCTLA-4-treated B40^f/f mice. The highest percentage of CX3CR1⁺CD206⁺ macrophages and the fewest iNOS⁺ macrophages was observed in αPD-1-treated B40^DCD8 mice that completely fail to reject tumors

Conclusions

Bhlhe40 is required in CD8 T cells for αPD-1 response, but dispensable in CD8 T cells for αCTLA-4 efficacy. 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.

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