Upon activation, naive T cells give rise to a heterogeneous cell population of effector and memory T cells that mediate antigen clearance and provide long-lived protection from rechallenge. Many of the transcriptional regulators that direct the differentiation of naive T cells to acquire the range of phenotypic and functional characteristics of effector and memory T cells have been described. However, the active programs that maintain the specific subsets of memory T cells are less clear. Here, we discuss recent studies that suggest effector and memory CD8⁺ T cells exist in cellular 'states' with inherent plasticity. Further, we consider the newly identified role of active transcriptional and epigenetic programming in maintaining the identity of the distinct subsets within the memory population.

T cells are essential mediators of immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive to effector T cells and the subsequent differentiation and persistence of memory T cell populations in response to infection is a highly regulated process. E protein transcription factors and their inhibitors, Id proteins, are important regulators of both CD4⁺ and CD8⁺ T cell responses; however, their regulation at the protein level has not been explored. Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas. In this study, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in murine T cells. We show that Usp1 was upregulated in T cells following activation in vitro or following infection in vivo, and the extent of Usp1 expression correlated with the degree of T cell expansion. Usp1 directly interacted with Id2 and Id3 following T cell activation. However, Usp1 deficiency did not impact Id protein abundance in effector T cells or alter effector T cell expansion or differentiation following a primary infection. Usp1 deficiency resulted in a gradual loss of memory CD8⁺ T cells over time and reduced Id2 protein levels and proliferation of effector CD8⁺ T cell following reinfection. Together, these results identify Usp1 as a player in modulating recall responses at the protein level and highlight differences in regulation of T cell responses between primary and subsequent infection encounters. Finally, our observations reveal differential regulation of Id2/3 proteins between immune versus nonimmune cell types.

CD8⁺ T cells responding to infection differentiate into a heterogeneous population composed of progeny that are short-lived and participate in the immediate, acute response and those that provide long-lasting host protection. Although it is appreciated that distinct functional and phenotypic CD8⁺ T cell subsets persist, it is unclear whether there is plasticity among subsets and what mechanisms maintain subset-specific differences. Here, we show that continued Id2 regulation of E-protein activity is required to maintain the KLRG1^hi CD8⁺ T cell population after lymphocytic choriomeningitis virus infection. Induced deletion of Id2 phenotypically and transcriptionally transformed the KLRG1^hi "terminal" effector/effector-memory CD8⁺ T cell population into a KLRG1^lo memory-like population, promoting a gene-expression program that resembled that of central memory T cells. Our results question the idea that KLRG1^hi CD8⁺ T cells are necessarily terminally programmed and suggest that sustained regulation is required to maintain distinct CD8⁺ T cell states.

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.

Upon infection with an intracellular pathogen, cytotoxic CD8⁺ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated that circulating memory cells can be further divided into effector memory (Tem) and central memory (Tcm) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was terminally fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally differentiated effector memory CD8 T cell population (terminal-Tem) was conflated within the conventional Tem population, prompting redefinition of the classical characteristics of Tem cells. Murine terminal-Tem were transcriptionally, functionally, and developmentally unique compared to Tem cells. Through mass cytometry and single-cell RNA sequencing (RNA-seq) analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-Tem population of CD8 T cells that was transcriptionally distinct from Tem and Tcm Key findings from this study show that parsing of terminal-Tem from conventionally defined Tem challenge the reported characteristics of Tem biology, including enhanced presence in lymphoid tissues, robust IL-2 production, and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-Tem and clarification of Tem biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

Tissue-resident memory T cells (T_RM cells) provide protective immunity, but the contributions of specific tissue environments to T_RM cell differentiation and homeostasis are not well understood. In the present study, the diversity of gene expression and genome accessibility by mouse CD8⁺ T_RM cells from distinct organs that responded to viral infection revealed both shared and tissue-specific transcriptional and epigenetic signatures. T_RM cells in the intestine and salivary glands expressed transforming growth factor (TGF)-β-induced genes and were maintained by ongoing TGF-β signaling, whereas those in the fat, kidney and liver were not. Constructing transcriptional-regulatory networks identified the transcriptional repressor Hic1 as a critical regulator of T_RM cell differentiation in the small intestine and showed that Hic1 overexpression enhanced T_RM cell differentiation and protection from infection. Provision of a framework for understanding how CD8⁺ T_RM cells adapt to distinct tissue environments, and identification of tissue-specific transcriptional regulators mediating these adaptations, inform strategies to boost protective memory responses at sites most vulnerable to infection.

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 Blimp1^hiId3^lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1^loId3^hi 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.

ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in epithelial-mesenchymal transition-dependent tumor metastasis. Although the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is up-regulated by activated T cells, specifically in the KLRG1(hi) effector CD8(+) T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8(+) T cells after primary and secondary infection with a severe impairment in the generation of the KLRG1(hi) effector memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress Il7r and Il2 in CD8(+) T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box sites in the Zeb2 gene and that T-bet and ZEB2 regulate similar gene expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Collectively, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8(+) T cells.

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8⁺ tissue-resident memory T cells (T_RM), the developmental origins and transcriptional regulation of CD4⁺ T_RM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4⁺ T_RM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating T_H1 and the progressive acquisition of a mature T_RM program. Single-cell RNA sequencing identified heterogeneity among established CD4⁺ T_RM, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. T_H1-associated Blimp1 and Id2 and T_FH-associated Bcl6 were required for early T_RM formation and development of a mature T_RM population in the SI. These results demonstrate a developmental relationship between T_H1 effector cells and the establishment of early T_RM, as well as highlighted differences in CD4⁺ versus CD8⁺ T_RM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4⁺ T_RM in response to viral infection.