Jan D. Haas

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24low CD44high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ γδ T cells produced IL‐17A, while NK1.1+ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1+ or CCR6+ γδ T cells, respectively. Importantly, we show that CCR6+ γδ T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1+ γδ T cells.

CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24low CD44high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ γδ T cells produced IL‐17A, while NK1.1+ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1+ or CCR6+ γδ T cells, respectively. Importantly, we show that CCR6+ γδ T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1+ γδ T cells.

High TCR diversity ensures optimal function andhomeostasis of Foxp3+ regulatory Tcells

Dominant tolerance to self‐antigen requires the presence of sufficient numbers of CD4+Foxp3+ Treg cells with matching antigen specificity. However, the size and role of TCR repertoire diversity for antigen‐specific immuno‐regulation through Treg cells is not clear. Here, we developed and applied a novel high‐throughput (HT) TCR sequencing approach to analyze the TCR repertoire of Treg cells and revealed the importance of high diversity for Treg‐cell homeostasis and function. We found that highly polyclonal Treg cells from WT mice vigorously expanded after adoptive transfer into non‐lymphopenic TCR‐transgenic recipients with low Treg‐cell diversity. In that system, we identified specific Treg‐cell TCR preferences in distinct anatomic locations such as the mesenteric LN indicating that Treg cells continuously compete for MHC class‐II‐presented self‐, food‐, or flora‐antigen. Functionally, we showed that high TCR diversity was required for optimal suppressive function of Treg cells in experimental acute graft versus host disease (GvHD). In conclusion, we suggest that efficient immuno‐regulation by Treg cells requires high TCR diversity. Thereby, continuous competition of peripheral Treg cells for limited self‐antigen shapes an organ‐optimized, yet highly diverse, local TCR repertoire.

Intra- and Intercompartmental Movement of γδ T Cells: Intestinal Intraepithelial and Peripheral γδ T Cells Represent Exclusive Nonoverlapping Populations with Distinct Migration Characteristics

Unlike the ∼1% of γδ TCR-positive T cells being regularly present in blood and secondary lymphoid organs (peripheral γδ T cells), ∼50–60% of small intestinal intraepithelial lymphocytes (iIELs) in the mouse express the γδ TCR (γδ iIELs). In this study, we investigated the overlap and exchange of γδ iIELs and γδ T cells found in peripheral secondary lymphoid organs. Using two-photon laser-scanning microscopy, we found γδ T cells within peripheral lymph nodes to be highly motile, whereas γδ iIELs were characterized by a locally confined scanning behavior. Our results implied a strict separation of peripheral γδ T cells and γδ iIELs. Nevertheless, γδ iIELs could be efficiently regenerated from bone marrow-derived precursors in irradiated or T cell-deficient adult mice. However, outside the intestinal epithelium, survival of γδ iIELs was very poor. In CCR9-deficient mice, homing of γδ iIELs was impaired, but did not lead to an accumulation of γδ iIEL-like cells in the periphery. Conversely, in situations in which specific γδ iIEL niches were empty, adoptive transfer of isolated γδ iIELs led to a sustained engraftment of transferred γδ iIELs in the intestinal epithelium for at least 100 d. Furthermore, we demonstrated by heterotopic intestinal transplantation experiments that an exchange of γδ iIELs only rarely happens in the steady state of adult mice. We therefore conclude that peripheral versus intestinal intraepithelial γδ T cells are exclusive, nonoverlapping populations that virtually do not exchange with each other.

Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

Differentiation of T helper 17 cells (Th17) is a multistep process that involves the cytokines IL-6, TGF-β, and IL-23 as well as IL-1β, IL-21, and TNF-α. Thereby, robust induction of the capacity to produce IL-17 involves epigenetic modifications of the syntenic Il17a/f locus. Using inbred mouse strains, we identified co-regulation of gene transcription at the Il17a/f locus with the nearby microRNAs miR-133b and miR-206 that are clustered approximately 45 kb upstream of Il17a/f. Expression of these microRNAs was specific for Th17 as compared to other CD4+ T cell subsets and this was equally valid for in vitro polarized and ex vivo derived cells. From all factors analyzed, IL-23 was the most important cytokine for the in vitro induction of miR-133b and miR-206 in naive CD4+ T cells of wild type mice. However, analysis of IL-23R deficient mice revealed that IL-23R signaling was not essential for the induction of miR-133b and miR-206. Importantly, we found a similar co-regulation in CCR6+ and other γδ T cell subsets that are predisposed to production of IL-17. Taken together, we discovered a novel feature of T cell differentiation towards an IL-17-producing phenotype that is shared between αβ and γδ T cells. Notably, the specific co-regulation of miR-133b and miR-206 with the Il17a/f locus also extended to human Th17 cells. This qualifies expression of miR-133b and miR-206 in T cells as novel biomarkers for Th17-type immune reactions.

Induction of BALT in the absence of IL-17.

1 and Il17a–/–Il17f –/– mice in follicle formation, as described above. In summary, our data show that BALT can be efficiently induced in the complete absence of IL-17A and its homolog IL-17F. Therefore, we believe that the general conclusion reached by Rangel-Moreno et al. that iBALT formation depends on IL-17 (ref. 1) is inappropriate. The function of IL-17 in the induction of BALT needs clarification for understanding of the role of this cytokine in the biology of ectopic lymphoid structures.

CCR7-mediated migration in the thymus controls γδ T-cell development

αβ T‐cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C‐C chemokine receptor (CCR)7‐deficient (Ccr7−/−) and CCR9‐deficient mice (Ccr9−/−) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T‐cell development. γδ T‐cell frequencies and numbers were decreased in CCR7‐deficient and increased in CCR9‐deficient mice. Transfer of CCR7‐ or CCR9‐deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell‐intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T‐cell output in CCR7‐deficient mice. In vitro, CCR7‐deficient precursors showed normal γδ T‐cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T‐cell localization within thymic medulla or cortex, respectively. However, γδ T‐cell motility was unaltered in CCR7‐ or CCR9‐deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T‐cell development.

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial γδ T cells is functional in vivo.

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK‐receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T‐cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR‐crosslinking. In contrast to systemic γδ T cells, CD8αα+ γδ iIEL showed high basal calcium levels and were refractory to TCR‐dependent calcium‐flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN‐γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα+ γδ iIEL. This suggests that the γδ TCR of CD8αα+ γδ iIEL is constantly being triggered and therefore functional in vivo.

CCR6 and NK1.1 distinguish between IL-17A and IFN-γ-producing γδ effector T cells: Innate immunity

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24low CD44high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ γδ T cells produced IL‐17A, while NK1.1+ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1+ or CCR6+ γδ T cells, respectively. Importantly, we show that CCR6+ γδ T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1+ γδ T cells.