Bhlhe40 Coordinates T Cell Programs with Distinct CD4 and CD8 T Cell Requirements for Anti-PD-1 Versus Anti-CTLA-4

The transcriptional programs that enable CD4 and CD8 T cells to mediate effective anti-tumor immunity remain incompletely defined. Here, we identify distinct, therapy-specific roles for the transcriptional regulator Bhlhe40 in CD4 and CD8 T cells, revealing divergent requirements during anti-PD-1 versus anti-CTLA-4 immune checkpoint therapy (ICT). Using conditional knockout mice, we show that anti-PD-1 efficacy depends on CD8 T cell-intrinsic Bhlhe40 and may also require its expression in CD4 T cells, whereas anti-CTLA-4 relies primarily on CD4 T cell-intrinsic Bhlhe40 and remains effective without Bhlhe40 in CD8 T cells. Loss of Bhlhe40 skews CD8 T cells toward TCF-1-expressing naive and progenitor exhausted-like states, particularly in the absence of ICT. Bhlhe40 sustains CD8 effector and exhausted phenotypes, promotes IFN-{gamma} production, and supports glycolytic and mitochondrial programs, with Bhlhe40 deficiency leading to impaired glycolysis under either anti-PD-1 or anti-CTLA-4 ICT, and reduced mitochondrial function primarily during anti-PD-1. In addition, CD8 T cell-intrinsic Bhlhe40 is required for full ICT-induced remodeling of the tumor myeloid compartment from CX3CR1 macrophages to iNOS macrophages. In contrast, anti-CTLA-4 can drive tumor rejection and partial macrophage remodeling in the absence of CD8 T cell-intrinsic Bhlhe40, provided CD4 T cell function is intact. Analysis of human cancer datasets revealed that BHLHE40 is enriched in tumor-reactive and activated/exhausted CD8 T cells, where its expression is inversely correlated with TCF7 (TCF-1) and positively associated with TOX and IFNG. Moreover, subsets of CD8 T cells expressed higher levels of BHLHE40 in basal and squamous cell carcinoma responders compared with non-responders. Together, these findings establish Bhlhe40 not only as a transcriptional coordinator of T cell effector programs and metabolic fitness, but also as a therapy-specific, subset-dependent determinant of ICT efficacy, providing a mechanistic basis for the divergent modes of action of anti-PD-1 versus anti-CTLA-4. HIGHLIGHTSBhlhe40 is essential in CD8 T cells for anti-PD-1 ICT, but dispensable for anti-CTLA-4 due to compensatory CD4 T cell function CD8 T cell-intrinsic Bhlhe40 drives effector/exhausted states over TCF-1+ naive and progenitor-exhausted like phenotypes Bhlhe40 sustains T cell metabolic fitness, supporting glycolysis under both therapies and OXPHOS primarily during anti-PD-1 CD8 T cell-intrinsic Bhlhe40 is required for full ICT-driven macrophage remodeling In human cancers, Bhlhe40 is enriched in tumor-reactive CD4 and CD8 T cells, positively linked to TOX/IFNG and inversely to TCF7 (TCF-1)