Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient Tcrd -/- mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd -/- mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.

Publisher Correction: Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

In the version of this article initially published, a source of funding (Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.)) was not included in the Acknowledgments section. The correct statement is as follows: "Supported by Deutsche Forschungsgemeinschaft, (SFB900/B8 to C.K. and I.P.; and PR727/4-1 to I.P.), Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.) and the German Federal Ministry of Education and Research (01EO1302 to C.S.-F., C.K. and I.P.)." The error has been corrected in the HTML and PDF versions of the article.

Eomes expression reports the progressive differentiation of IFN-γ-producing Th1-like γδ T cells.

The transcription factor Eomesodermin (Eomes) plays a crucial role in regulating cytotoxic function, development, and survival of immune cells. γδ T cells can express Eomes, but its contribution to their differentiation is unknown. Using Eomes-IRES-GFP mice, we show that Eomes+ γδ T cells are unequally distributed among organs, with the highest proportion in spleen. While the majority of Eomes+ γδ T cells expressed Vγ1+ and Vγ4+ TCRs, Eomes was absent in Vγ5+ , Vγ6+ , and Vγ7+ subsets. Moreover, Eomes was co-expressed in γδ T cells with Th1 lineage-related factors such as CD27, T-bet, and Ly6C, but not with Th17 lineage-related genes. Eomes+ and Eomes- γδ T-cell populations showed distinct gene expression profiles, with an increase of cytotoxic-related genes in Eomes+ γδ T cells. Furthermore, Eomes could be induced in peripheral γδ T cells by IL-12 and IL-4, and Eomes+ γδ T cells presented a higher proliferation rate and IFN-γ production when stimulated in vitro with IL-12 and IL-18. However, γδ T cells with very high Eomes levels displayed an exhausted phenotype with high levels of PD-1, and were less capable of IFN-γ production. Together, this study highlights Eomes as a marker for the differentiation of Th1-like effector γδ T cells.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

Disruption of de novo fatty acid synthesis via acetyl-CoA carboxylase 1 inhibition prevents acute graft-versus-host disease.

Upon antigen-specific or allogeneic activation, T cells sharply increase their metabolic activity to cope with augmented needs for proliferation and effector functions. Therefore, enzymes involved in energy metabolism constitute attractive targets to modulate the activity of pathogenic effector T cells in the setting of graft-versus-host-disease (GVHD). Here, we show that T cells deficient for acetyl-CoA carboxylase 1 (TACC1) are dramatically less pathogenic than wild-type (WT) T cells in a lethal C57BL/6 into BALB/c model of acute GVHD and permitted sustained survival of recipient mice. In line with this clinical observation, higher frequencies of GVHD-suppressing Foxp3(+) regulatory T (Treg) cells were detected in the colon of TACC T-cell recipients. In vitro, T-cell stimulation with allogeneic DCs induced higher proportions of Treg cells but also led to diminished proliferation of TACC1 T cells compared to WT T cells. Furthermore, TACC1 T cells activated by allogeneic DCs showed impaired glycolysis and lipid synthesis. Thus, targeting de novo fatty acid synthesis via acetyl-CoA carboxylase inhibition may be a promising new strategy to prevent GVHD.

Interleukin-23-Dependent γ/δ T Cells Produce Interleukin-17 and Accumulate in the Enthesis, Aortic Valve, and Ciliary Body in Mice.

OBJECTIVE: The spondyloarthritides (SpA) are a group of rheumatic diseases characterized by ossification and inflammation of entheseal tissue, the region where tendon attaches to bone. Interleukin-23 (IL-23) is involved in the pathogenesis of SpA by acting on IL-23 receptor (IL-23R) expressed on enthesis-resident lymphocytes. Upon IL-23 binding, CD3+CD4-CD8- tissue-resident lymphocytes secrete IL-17A and IL-22, leading to inflammation, bone loss, and ossification. Knowledge about enthesis-resident lymphocytes remains fragmentary, and the contribution of entheseal γ/δ T cells in particular is not clear. This study was undertaken to investigate the presence of γ/δ T cells in the enthesis. METHODS: We used 2-photon microscopy and flow cytometry to analyze entheseal lymphocytes from C57BL/6, Tcrd-H2BeGFP, Rorc-GFP, and IL-23R-eGFP mice. To analyze entheseal γ/δ T cells in IL-23-induced inflammation, Tcrd-H2BeGFP mice were crossed with mice of the susceptible B10.RIII background. Hydrodynamic injection of IL-23 minicircle DNA was performed for overexpression of IL-23 and induction of inflammation. Light-sheet fluorescence microscopy was used to visualize arthritic inflammation. RESULTS: Activated Vγ6+CD27- γ/δ T cells were abundant in uninflamed entheseal tissue and constituted the large majority of retinoic acid receptor-related orphan nuclear receptor γt (RORγt)+IL-23R+ enthesis-resident lymphocytes. Fetal thymus-dependent γ/δ T cells were the main source of IL-17A at the enthesis. Under inflammatory conditions, γ/δ T cells increased in number at the Achilles tendon enthesis, aortic root, and adjacent to the ciliary body. CONCLUSION: Entheseal γ/δ T cells are derived from fetal thymus and are maintained as self-renewing tissue-resident cells. As main IL-17A producers within tissues exposed to mechanical stress including enthesis, γ/δ T cells are key players in the pathogenesis of IL-23-induced local inflammation.

MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development.

Thymic development of αß T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1-/-mice, which show impaired agonistic T cell selection of invariant αß NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αß NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.

A clonotypic Vγ4Jγ1/Vδ5Dδ2Jδ1 innate γδ T-cell population restricted to the CCR6⁺CD27⁻ subset.

Here we investigate the TCR repertoire of mouse Vγ4(+) γδ T cells in correlation with their developmental origin and homeostasis. By deep sequencing we identify a high frequency of straight Vδ5Dδ2Jδ1 germline rearrangements without P- and N-nucleotides within the otherwise highly diverse Trd repertoire of Vγ4(+) cells. This sequence is infrequent in CCR6(-)CD27(+) cells, but abundant among CCR6(+)CD27(-) γδ T cells. Using an inducible Rag1 knock-in mouse model, we show that γδ T cells generated in the adult thymus rarely contain this germline-rearranged Vδ5Dδ2Jδ1 sequence, confirming its fetal origin. Single-cell analysis and deep sequencing of the Trg locus reveal a dominant CDR3 junctional motif that completes the TCR repertoire of invariant Vγ4(+)Vδ5(+) cells. In conclusion, this study identifies an innate subset of fetal thymus-derived γδ T cells with an invariant Vγ4(+)Vδ5(+) TCR that is restricted to the CCR6(+)CD27(-) subset of γδ T cells.

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

αß 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.

Differential postselection proliferation dynamics of αß T cells, Foxp3+ regulatory T cells, and invariant NKT cells monitored by genetic pulse labeling.

The thymus generates two divergent types of lymphocytes, innate and adaptive T cells. Innate T cells such as invariant NKT cells provide immediate immune defense, whereas adaptive T cells require a phase of expansion and functional differentiation outside the thymus. Naive adaptive T lymphocytes should not proliferate much after positive selection in the thymus to ensure a highly diverse TCR repertoire. In contrast, oligoclonal innate lymphocyte populations are efficiently expanded through intrathymic proliferation. For CD4(+)Foxp3(+) regulatory T cells (Tregs), which are thought to be generated by agonist recognition, it is not clear whether they proliferate upon thymic selection. In this study, we investigated thymic and peripheral T cell proliferation by genetic pulse labeling. To this end, we used a mouse model in which all developing αß thymocytes were marked by expression of a histone 2B-enhanced GFP (H2BeGFP) fusion-protein located within the Tcrd locus (TcrdH2BeGFP). This reporter gene was excised during TCR α-chain VJ-recombination, and the retained H2BeGFP signal was thus diluted upon cell proliferation. We found that innate T cells such as CD1d-restricted invariant NKT cells all underwent a phase of intense intrathymic proliferation, whereas adaptive CD4(+) and CD8(+) single-positive thymocytes including thymic Tregs cycled, on average, only once after final selection. After thymic exit, retention or loss of very stable H2BeGFP signal indicated the proliferative history of peripheral αß T cells. There, peripheral Tregs showed lower levels of H2BeGFP compared with CD4(+)Foxp3(-) T cells. This further supports the hypothesis that the Treg repertoire is shaped by self-Ag recognition in the steady-state.

Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave.

γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

CCR7 essentially contributes to the homing of plasmacytoid dendritic cells to lymph nodes under steady-state as well as inflammatory conditions.

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7(-/-) mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.