The impact of KLF2-KLF3 regulated cellular localization on exhausted T cell differentiation [scMultiome-seq]

During chronic viral infection, CD8 T cells lose effector function and acquire an exhausted phenotype, a process accompanied by changes in migratory behavior and tissue residency. Exhausted CD8 T cells differentiate from stem-like progenitor (Tpro) cells into either circulating, cytotoxic effector-like (Teff) cells or tissue-resident terminally exhausted (Texh) cells. However, whether these migratory changes passively reflect cell state or actively influence exhaustion trajectories remains unclear. Here, we identify a transcriptional circuit involving Krüppel-like factor 2 (KLF2) and its homolog KLF3 that links migratory program to CD8 T cell differentiation during exhaustion. Using multi-module approaches, we show that KLF2 primarily promotes the expression and chromatin accessibility of migratory genes in Tpro and Teff subsets, while KLF3 limits these programs by repressing Klf2 transcription and competing for shared genomic binding sites. Enhancing circulation through enforced S1PR1/S1PR5 expression or CD69 deletion biases differentiation toward effector-like fates and away from terminal exhaustion, suggesting that cellular localization can influence, not just reflect, CD8 T cell fate decisions. We further describe an integrated feed-forward and feedback loop in which KLF2 induces KLF3, which in turn constrains KLF2 transcription and binding to shared genomic loci. This reciprocal regulatory circuit modulates the balance between migration and tissue residency, thereby tuning the differentiation of CD8 T cells in chronic infection. These findings provide mechanistic insight into how transcriptional regulation of migratory behavior contributes to cell fate decisions during T cell exhaustion.