CD4(+)CD25(+)FOXP3(+) regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the... Show moreCD4(+)CD25(+)FOXP3(+) regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the preferred cell type for adoptive cell therapy (ACT). How human tTreg cells develop is incompletely understood. By combining single-cell transcriptomics and flow cytometry, we in this study delineated three major Treg developmental stages in the human thymus. At the first stage, which we propose to name pre-Treg I, cells still express lineage-inappropriate genes and exhibit signs of TCR signaling, presumably reflecting recognition of self-antigen. The subsequent pre-Treg II stage is marked by the sharp appearance of transcription factor FOXO1 and features induction of KLF2 and CCR7, in apparent preparation for thymic exit. The pre-Treg II stage can further be refined based on the sequential acquisition of surface markers CD31 and GPA33. The expression of CD45RA, finally, completes the phenotype also found on mature recent thymic emigrant Treg cells. Remarkably, the thymus contains a substantial fraction of recirculating mature effector Treg cells, distinguishable by expression of inflammatory chemokine receptors and absence of CCR7. The developmental origin of these cells is unclear and warrants caution when using thymic tissue as a source of stable cells for ACT. We show that cells in the major developmental stages can be distinguished using the surface markers CD1a, CD27, CCR7, and CD39, allowing for their viable isolation. These insights help identify fully mature tTreg cells for ACT and can serve as a basis for further mechanistic studies into tTreg development. Show less
Putten, C. van der; Remmerswaal, E.B.M.; Terpstra, M.L.; Bom, N.D. van der; Kers, J.; Berge, I.J.M. ten; ... ; Aalderen, M.C. van 2021
Background: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However,... Show moreBackground: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. Methods: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). Results: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFN gamma than circulating cells and were frequently polyfunctional. Conclusion: Functional T cells with the characteristic traits of T-RM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6. Show less
Tissue-resident memory CD8(+)T cells (T(RM)cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall... Show moreTissue-resident memory CD8(+)T cells (T(RM)cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall responses is unclear. We developed a unique lineage tracer mouse model exploiting the T-RM-defining transcription factor homolog of Blimp-1 in T cells (Hobit) to fate map the T(RM)progeny in secondary responses. After reinfection, a sizeable fraction of secondary memory T cells in the circulation developed downstream of T(RM)cells. These tissue-experienced ex-T(RM)cells shared phenotypic properties with the effector memory T cell population but were transcriptionally and functionally distinct from other secondary effector memory T cell cells. Adoptive transfer experiments of T(RM)cells corroborated their potential to form circulating effector and memory cells during recall responses. Moreover, specific ablation of primary T(RM)cell populations substantially impaired the secondary T cell response, both locally and systemically. Thus, T(RM)cells retain developmental plasticity and shape both local and systemic T cell responses on reinfection.Van Gisbergen and colleagues show that tissue-resident memory T cells, genetically marked in Hobit reporter mice, can exit tissues upon reinfection and contribute to systemic memory responses. Show less
Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells... Show moreInvariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment. Show less
