Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Regulation of CD8 T Cell Differentiation by the RNA-Binding Protein DDX5.

The RNA-binding protein DEAD-box protein 5 (DDX5) is a polyfunctional regulator of gene expression, but its role in CD8+ T cell biology has not been extensively investigated. In this study, we demonstrate that deletion of DDX5 in murine CD8+ T cells reduced the differentiation of terminal effector, effector memory T, and terminal effector memory cells while increasing the generation of central memory T cells, whereas forced expression of DDX5 elicited the opposite phenotype. DDX5-deficient CD8+ T cells exhibited increased expression of genes that promote central memory T cell differentiation, including Tcf7 and Eomes. Taken together, these findings reveal a role for DDX5 in regulating the differentiation of effector and memory CD8+ T cell subsets in response to microbial infection.

Systems-level identification of key transcription factors in immune cell specification.

Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions.

The long noncoding RNA Malat1 regulates CD8+ T cell differentiation by mediating epigenetic repression.

During an immune response to microbial infection, CD8+ T cells give rise to short-lived effector cells and memory cells that provide sustained protection. Although the transcriptional programs regulating CD8+ T cell differentiation have been extensively characterized, the role of long noncoding RNAs (lncRNAs) in this process remains poorly understood. Using a functional genetic knockdown screen, we identified the lncRNA Malat1 as a regulator of terminal effector cells and the terminal effector memory (t-TEM) circulating memory subset. Evaluation of chromatin-enriched lncRNAs revealed that Malat1 grouped with trans lncRNAs that exhibit increased RNA interactions at gene promoters and gene bodies. Moreover, we observed that Malat1 was associated with increased H3K27me3 deposition at a number of memory cell-associated genes through a direct interaction with Ezh2, thereby promoting terminal effector and t-TEM cell differentiation. Our findings suggest an important functional role of Malat1 in regulating CD8+ T cell differentiation and broaden the knowledge base of lncRNAs in CD8+ T cell biology.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αß T cell receptors (TCRαß) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

A role for phagocytosis in inducing cell death during thymocyte negative selection.

Autoreactive thymocytes are eliminated during negative selection in the thymus, a process important for establishing self-tolerance. Thymic phagocytes serve to remove dead thymocytes, but whether they play additional roles during negative selection remains unclear. Here, using a murine thymic slice model in which thymocytes undergo negative selection in situ, we demonstrate that phagocytosis promotes negative selection, and provide evidence for the escape of autoreactive CD8 T cells to the periphery when phagocytosis in the thymus is impaired. We also show that negative selection is more efficient when the phagocyte also presents the negative selecting peptide. Our findings support a model for negative selection in which the death process initiated following strong TCR signaling is facilitated by phagocytosis. Thus, the phagocytic capability of cells that present self-peptides is a key determinant of thymocyte fate.