Background CD8⁺ T cells are a critical component of the immune response to intracellular infections and malignancies. Recently, tissue-resident CD8⁺ memory cells (Trm) have been shown to provide a first line of defense against reinfection at barrier tissues such as the intestine. However, the transcriptional networks regulating tissue-adaptation processes are incompletely understood. Here, we sought to define the transcriptional orchestration of small intestinal Trm cells.

Methods To identify gene-expression and genome-accessibility changes that arise in Trm cells in response to acute LCMV infection, we used scRNA-seq and ATAC-seq of cells from small intestine (SI), kidney, liver, salivary glands, as well as the spleen and blood. To assess the importance of the transcription factor Hic1 for the differentiation of intestinal Trm cells we used short-hairpin RNAs, inducible genetic deletion mouse models, pharmacological inhibition and Cut&Run sequencing.

Results RNA sequencing of tissue Trm reveals shared and tissue-specific gene-expression programs, along with intra-tissue heterogeneity of Trm populations. Using transcriptional-regulatory networks, we identify the transcription factor Hic1 as a regulator for intestinal Trm differentiation. We observe that knockdown of Hic1 hinders Trm formation in the SI epithelium in response to both LCMV Armstrong and Listeria monocytogenes infection. In contrast, overexpression of Hic1 promotes Trm formation in the SI upon infection and provides protection upon reinfection. Consistent with Hic1 being a transcriptional repressor, we observe that Hic1 binding sites at genes downregulated by SI Trm lose accessibility in SI Trm. However, Hic1 enhances P2rx7 expression, an eATP receptor previously shown to be important for memory T cell differentiation and Trm homeostasis, by directly binding to its promoter, suggesting that Hic1 acts as both an activator and a repressor during SI Trm formation. Surprisingly, deletion of Hic1 after establishment of intestinal Trm cells does not reduce Trm cell numbers. In line with that, retinoic acid (RA) induces expression of Hic1 and pharmacological blockade of the RA receptor alpha reduces Trm formation, but does not impair Trm maintenance. Further, we find that SI Trm are genetically imprinted to reenter the intestine upon secondary infection, whereas this tissue-specific bias was not observed for liver and kidney Trm cells.

Conclusions In summary, our work highlights the broad transcriptional adaptations of Trm to a range of tissue environments and highlights the importance of Hic1 as a regulator for SI Trm cell differentiation, which may be used as a framework for identifying targets that influence tissue-specific Trm populations in therapeutic contexts.