The Notch signaling pathway is a master regulator of CD8 ⁺ T cell exhaustion and differentiation during chronic infection

During chronic infection, the persistence of antigen and inflammation leads to the differentiation of CD8 ⁺ T cells into an exhausted state characterised by expression of inhibitory receptors (IRs) and the progressive loss of T cell functions. Among the different subsets of exhausted CD8 ⁺ T (Tex) cells, Tex progenitors expressing SLAMF6 and the transcription factor TCF-1 (TCF-1+) give rise to Tex effector-like cells expressing CX3CR1 and Tex terminal cells expressing CD101. PD-1/PD-L1 blockade acts on TCF-1+ Tex progenitor cells and promotes their differentiation into Tex effector-like cells. The molecular events controlling CD8 ⁺ Tex cell differentiation are still poorly defined. As Notch signaling may be sustained during chronic infection by persistent TCR stimulation and inflammation, we tested whether Notch signaling influences CD8 ⁺ T cell exhaustion. Using mice lacking (N1N2 ^Δ/Δ ) or not (N1N2 ^fl/fl ) Notch1/2 expression only in mature CD8 ⁺ T cells, we showed that the absence of Notch signal causes severe CD8+ T cell exhaustion during chronic LCMV infection. N1N2 ^Δ/Δ Tex cells express higher levels of IRs and are less functional when compared to their wild-typee counterpart. In the absence of N1N2 receptors, Tex progenitor and Tex terminal cells accumulate and Tex cells cannot be reinvigorated by PD-1/PD-L1 blockade. We further demonstrated that Notch signaling is essential to promote the differentiation of Tex progenitors into Tex effector-like cells. Moreover, Notch signals, provided by stromal cells expressing the ligands Delta-like 1 and 4, are necessary during all stages of the infection to prevent severe exhaustion. Single-nucleus RNA and ATAC multiome profiling identifies Notch signaling as a critical role on effector transcriptional programming in exhausted CD8 T cells. Loss of Notch signaling impairs transcriptional program associated with migration and perception of CD4 ⁺ T cell help. Together, these alterations drive the differentiation of Tex progenitor cells toward a terminally exhausted Tex fate.