Ellen A. Robey

Regulatory T cells (Tregs) are a suppressive subset of CD4 T cells that maintain immune homeostasis. Most Tregs develop in the thymus but how thymic selection impacts peripheral Treg fate remains unknown. Here we show that the strength of TCR signal experienced during positive selection in the thymic cortex impacts how CD4 SP thymocytes respond to Treg inducing signals in the medulla. Thymocytes that experienced weak signals during positive selection (identified as CD5 ^LO CD4 SP) show a greater dependence on TGFβ than IL-2 for Treg induction and produce Foxp3+ cells that lack constitutive expression of the classic Treg marker CD25. Moreover, CD4 clones that give rise to CD25- thymic Tregs also produce CD25- peripheral Tregs. These data indicate that positive selection provides critical context for how CD4 T cells respond to TCR agonist and cytokine signals, leading to an alternative lineage of Tregs that lacks constitutive CD25 expression.

Modulation of T cell receptor (TCR) sensitivity during positive selection is critical to avoid negative selection and direct thymocytes into their appropriate lineage. Thymocytes just prior to positive selection (preselection) are highly responsive to low affinity self-ligands and are also actively rearranging their TCRα locus as they await a positive selection signal. Preselection double-positive thymocytes were thought to be relatively homogeneous, and TCR modulation during this stage had not been previously described. Here, we provide evidence for progressive gene expression changes within the preselection double-positive thymocyte population that correlates with a gradual reduction in TCR responsiveness and reduced upregulation of TCR target genes associated with the CD4 fate. We relate these observations to the link between positive selection and T cell lineage commitment.

MHC‐E‐restricted CD8 T cells are emerging as an attractive therapeutic mechanism due to their strong protective capacity and ability to respond to cells with defects in antigen processing; however, their thymic development remains poorly understood. Here, we explore the MHC ligand requirement for thymic development of T cells reactive to a self‐peptide (FL9) presented by mouse MHC‐E (Qa1 ^b ) under conditions of deficiency in the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP), called QFL T cells. We show that, while QFL T cells can develop in the absence of the restricting Qa1 ^b molecule, their development was abrogated in the combined absence of classical MHC Ia and Qa1 ^b . Interestingly, QFL thymocytes did not recognize classical MHC Ia molecules through their TCR but instead used non‐cognate CD8‐MHC Ia interactions to boost responses to MHC Ib ligands. Furthermore, we also identify an alternative ligand for the QFL TCR, the MHC Ib molecule H2‐T11. Our data provide evidence that both cognate and non‐cognate MHC interactions contribute to the development of a Qa1 ^b ‐specific T cell population.

Modulation of TCR sensitivity during positive selection is critical to avoid negative selection and direct thymocytes into their appropriate lineage. Thymocytes just prior to positive selection (preselection) are highly responsive to low affinity self-ligands and are also actively rearranging their TCRα locus as they await a positive selection signal. Preselection DP thymocytes were thought to be relatively homogeneous and TCR modulation during this stage had not been previously described. Here we provide evidence for progressive gene expression changes within the preselection DP thymocyte population that correlates with a gradual loss of TCR responsiveness and a defect in upregulating TCR target genes associated with the CD4 fate. We relate these observations to the link between positive selection and T cell lineage commitment.

Glycans regulate cellular function, yet how aging affects the glycocalyx remains unclear. Here, we investigate changes in immune cell glycocalyx with age and find that α2,6-linked sialic acid, a glycan epitope associated with inhibitory signaling, is down-regulated in T cells from old animals. This reduction is tightly correlated with age-associated accumulation of effector T cells, which have little to no α2,6-linked sialic acid. To understand how α2,6-linked sialic acid affects T cell physiology, we generated a mouse model with T cell–specific deletion of sialyltransferase gene St6gal1 . The lack of α2,6-linked sialic acid leads to reduced responsiveness in naïve T cells, leading to impaired T cell responses against Listeria monocytogenes infection and tumor growth. PD-1 pathway blockade partially restores St6gal1 -deficient T cells’ ability to control tumor growth. These findings suggest that α2,6-linked sialic acid is critical for maintaining long-term T cell responsiveness, and its loss may contribute to decreased T cell function with age.

Cell surface glycans, termed the glycocalyx, are essential regulators of cellular signaling and thus cellular development and functions, but how aging impacts the glycocalyx remains poorly understood. Here, using immune cells as a model system for studying the relationship between aging and glycocalyx remodeling, we show that α2,6-linked sialic acid – a terminal glycan epitope typically associated with inhibitory signaling – becomes downregulated in T cells from older animals. This downregulation is tightly correlated with age-associated accumulation of effector T cells, which are decorated with little to no α2,6-linked sialic acids. T cell aging renders older individuals more vulnerable to infections and cancers. To understand the role of α2,6-linked sialic acids in T cell physiology, we generated a mouse model with T cell-specific deletion of the sialyltransferase gene St6gal1 . The chronic depletion of α2,6-linked sialic acids led to naïve T (T _N ) cells expansion in the periphery and premature T cell exhaustion. As a result, these mice were less able to control acute Listeria infection and chronic tumor growth. Blockade of the PD-1 pathway can partially restore the ability of St6gal1 -deficient T cells to control tumor growth. Together, these data suggest that α2,6-linked sialic acids are critical for maintaining long-term T cell responsiveness, and the loss of α2,6-linked sialic acids may directly contribute to age-related T cell exhaustion.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ⁺ and CD8αα⁺TCRαβ⁺ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

During thymic development, thymocytes adjust their TCR response based on the strength of their reactivity to self-peptide MHC complexes. This tuning process allows thymocytes with a range of self-reactivities to survive positive selection and contribute to a diverse T cell pool. In this review, we will discuss recent advances in our understanding of how thymocytes tune their responsiveness during positive selection, and we present a “sequential selection” model to explain how MHC specificity influences lineage choice. We also discuss recent evidence for cell type diversity in the medulla and discuss how this heterogeneity may contribute to medullary niches for negative selection and regulatory T cell development.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8⁺ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1^b-restricted CD8⁺ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1^b-dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigens, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1^b-restricted IEL landscape.