The novel roles of BCL6 and BATF3 in regulating human CD8⁺ T cell dysfunction

Abstract

Reduced effector function and elevated inhibitory receptor expression are hallmarks of exhausted CD8⁺ T cells, yet the underlying molecular and epigenetic drivers remain incompletely defined. Here, we developed an in vitro repeated stimulation model to recapitulate features of human CD8⁺ T cell dysfunction and delineate transcriptional and epigenetic landscapes. Our analyses revealed that BCL6 and BATF3 are robustly upregulated in dysfunctional CD8⁺ T cells, with ATAC-seq demonstrating enhanced chromatin accessibility at their gene loci. Transcription factor footprinting shows increased BATF3 motif occupancy in chronically stimulated cells and integrative multi-omic analysis combining footprints, open chromatin regions, RNA-seq and ChIP-seq data revealed that putative BATF3 target genes may include master regulators of exhaustion. Moreover, overexpression of BCL6 or BATF3 markedly upregulates TIM-3 expression and suppressed cytokine release, establishing their capacity to induce T cell dysfunction. We further validated these findings ex vivo in antigen-specific CD8⁺ T cells from patients with advanced melanoma, as well as HCV and HIV infections, where cells were enriched for BCL6^high and BATF3^high subsets co-expressing canonical exhaustion markers such as PD-1, TIM-3 and CD39. Notably, Single-cell RNA sequencing of HIV-specific CD8⁺ T cells identified a distinct BCL6^high PD1⁻ progenitor population that gives rise to two distinct subsets via divergent differentiation trajectories: one branch generates effector-like BCL6^high PD1⁺ cells, whereas the other produces BCL6^high PD1⁺ cells that retain an exhaustion gene signature alongside partial memory-like feature. Collectively, these findings identify BCL6 and BATF3 as key mediators of human CD8⁺ T cell dysfunction and illuminate novel transcriptional and epigenetic pathways that may be leveraged for therapeutic intervention in cancer and chronic viral infections.