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T cell receptor (TCR) Vγ4+expressing γδT cells can be divided into IFN-γ and IL-17-producing effector T cell subsets. A bias towards γδ17 effector fate decisions is observed during early ontogeny. In contrast, the existence of Vγ4+γδ17 cells derived from adult thymus is still controversial. In the present work, we used a mouse model where T cells are exclusive generated within an adult thymus. Additionally, we employed single-cell chromatin state analysis from thymocytes of normal mice. A small, but considerable population of immatureCd24+Gzma+Vγ4 cells was found that exhibit molecular programs of γδ17 cells. These adult thymus-derived immatureCd24a+cMaf+Vγ4 cells secrete small amounts of IL-17A and IL-17F. Interestingly, do not reach the periphery under steady-state conditions. Furthermore,de novogenerated γδ17-like cells from adult thymus lack transcriptional activity of the Scart2 encoding gene, suggesting that Scart2 is a distinct trait of fetal γδT cell precursors. Together, this study provides valuable insights into developmental traits of Vγ4 cells during adulthood and raises the question on signals suppressing the full maturation and/or thymic export of γδ17-like cells within the adult thymus.HighlightsTranscriptional and epigenetic profiling identifies developmental plasticity ofGzma+Cd24a+Vγ4 cells in adult thymus.Thymic c-Maf+and RORγt+Vγ4 T cells can be generated during adulthood, but do not reach the periphery under steady-state conditions.Innate CD44highCD45RBnegγδ17 cells are completely absent upon induction of T cell development during adulthood.Scart2 expression might be a key molecule to track developmental traits of fetal-derived γδ17 cell precursors.

SummaryExcessive shedding of enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. However, the mechanisms underlying this phenomenon remain unclear. Intraepithelial lymphocytes (IEL) expressing the γδ T-cell receptor (TCR) provide surveillance of the intestinal mucosa at steady-state, which is regulated, in part, by CD103. Intravital microscopy of lipopolysaccharide (LPS)-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, as CD103 blockade significantly reduces LPS-induced shedding. Furthermore, we find that granzymes A and B, but not perforin, are required for cell shedding, and that these granzymes are released by γδ IELs both constitutively and following CD103/E-cadherin ligation. These findings indicate that extracellular granzyme facilitates shedding, likely through cleavage of extracellular matrix proteins. Our results uncover a previously unrecognized role for γδ IELs in facilitating pathological cell shedding in a CD103- and granzyme-dependent manner.

Accumulating evidence suggests that the human embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process comprises a dedicated subset of IL-17-producing γδ T (γδT17) cells, like reported in mice. Here we present a novel protocol for high-throughput paired γδ TCR-sequencing, which in combination with single-cell RNA-sequencing revealed a high heterogeneity of effector γδ T cell clusters. While immature γδ T cell clusters displayed mixed and diverse TCR, effector cell types in neonatal and adult blood segregated according to γδTCR usage. In adult samples, mature Vδ1+ T cells segregated into exhausted PD-1hi and active PD-1low clusters. Among Vγ9Vδ2+ T cell subsets, we identified distinct PLZF-positive effector γδ T cell clusters with innate type-1 and type-3 T cell signatures that were already detectable in a public dataset of early embryonic thymus organogenesis. Together, this suggests that functionally distinct waves of human innate effector γδ T cells including CCR6+ γδT17 cells develop in the early fetal thymus and persist into adulthood.

Background: Elucidating the role of T cell responses in COVID-19 is of utmost importance to understand the clearance of SARS-CoV-2 infection. Methods: 90 individuals were enrolled in this study, 30 hospitalized COVID-19 patients and 60 age- and gender-matched healthy controls (HC). Using two comprehensive 11-color flow cytometric panels conforming to Good Laboratory Practice (GLP) and approved for clinical diagnostics, we longitudinally examined cell count differences in lymphocyte populations and T cell activation in COVID-19 patients. Findings: Absolute numbers of lymphocyte subsets were differentially decreased in COVID-19 patients according to clinical severity. In severe disease (SD) patients, all lymphocyte subsets were reduced, whilst in mild disease (MD) NK, NKT and γδ T cells were at the level of HC. Additionally, we provide evidence of T cell activation in MD but not SD, when compared to HC. Interestingly, follow up samples revealed a marked increase in effector T cells and memory subsets in convalescing but not in non-convalescing patients. Interpretation: Our data suggest that activation and expansion of innate and adaptive lymphocytes play a major role in COVID-19. Additionally, recovery is associated with formation of T cell memory as suggested by the missing formation of effector and central memory T cells in SD but not in MD. Our data imply that the presence of SARS-CoV-2 responsive T cells contributes to convalescence in MD. Thus, understanding the T cell-response in the context of clinical severity might serve as foundation to overcome the lack of effective anti-viral immune response in severely affected COVID-19 patients and can offer prognostic value as biomarker for disease outcome and control.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T cell differentiation remains inefficient. We generated mice expressing human interleukin (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T cell differentiationin vivoand to generate a human immune system with a better approximation of human lymphocyte ratios.

MAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary disfunction and by defects in function, activation and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases.