Interleukin-17 is disease promoting in early stages and protective in late stages of experimental periodontitis.

Periodontitis is one of the most common infectious diseases in humans. It is characterized by a chronic inflammation of the tooth-supporting tissue that results in bone loss. However, the role and source of the pro-inflammatory cytokine interleukin-17 (IL-17) and of the cells producing it locally in the gingiva is still controversial. Th17 αß T cells, CD4+ exFoxP3+ αß T cells, or IL-17-producing γδ T cells (γδ17 cells) seem to be decisive cellular players in periodontal inflammation. To address these issues in an experimental model for periodontitis, we employed genetic mouse models deficient for either γδ T cells or IL-17 cytokines and assessed the bone loss during experimental periodontal inflammation by stereomicroscopic, histological, and µCT-analysis. Furthermore, we performed flow-cytometric analyses and qPCR-analyses of the gingival tissue. We found no γδ T cell- or IL-17-dependent change in bone loss after four weeks of periodontitis. Apart from that, our data are complementary with earlier studies, which suggested IL-17-dependent aggravation of bone loss in early periodontitis, but a rather bone-protective role for IL-17 in late stages of experimental periodontitis with respect to the osteoclastogenicity defined by the RANKL/OPG ratio.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer's Patches of Mice.

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer's patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

Maturation of the neonatal oral mucosa involves unique epithelium-microbiota interactions.

Postnatal host-microbiota interplay governs mucosal homeostasis and is considered to have life-long health consequences. The intestine monolayer epithelium is critically involved in such early-life processes; nevertheless, the role of the oral multilayer epithelium remains ill defined. We demonstrate that unlike the intestine, the neonate oral cavity is immensely colonized by the microbiota that decline to adult levels during weaning. Neutrophils are present in the oral epithelium prenatally, and exposure to the microbiota postnatally further recruits them to the preamble neonatal epithelium by γδT17 cells. These neutrophils virtually disappear during weaning as the epithelium seals. The neonate and adult epithelium display distinct turnover kinetics and transcriptomic signatures, with neonate epithelium reminiscent of the signature found in germ-free mice. Microbial reduction during weaning is mediated by the upregulation of saliva production and induction of salivary antimicrobial components by the microbiota. Collectively, unique postnatal interactions between the multilayer epithelium and microbiota shape oral homeostasis.

Microbiota-dependent expansion of testicular IL-17-producing Vγ6+ γδ T cells upon puberty promotes local tissue immune surveillance.

γδT cells represent the majority of lymphocytes in several mucosal tissues where they contribute to tissue homoeostasis, microbial defence and wound repair. Here we characterise a population of interleukin (IL) 17-producing γδ (γδ17) T cells that seed the testis of naive C57BL/6 mice, expand at puberty and persist throughout adulthood. We show that this population is foetal-derived and displays a T-cell receptor (TCR) repertoire highly biased towards Vγ6-containing rearrangements. These γδ17 cells were the major source of IL-17 in the testis, whereas αß T cells mostly provided interferon (IFN)-γ in situ. Importantly, testicular γδ17 cell homoeostasis was strongly dependent on the microbiota and Toll-like receptor (TLR4)/IL-1α/IL-23 signalling. We further found that γδ17 cells contributed to tissue surveillance in a model of experimental orchitis induced by intra-testicular inoculation of Listeria monocytogenes, as Tcrδ-/- and Il17-/- infected mice displayed higher bacterial loads than wild-type (WT) controls and died 3 days after infection. Altogether, this study identified a previously unappreciated foetal-derived γδ17 cell subset that infiltrates the testis at steady state, expands upon puberty and plays a crucial role in local tissue immune surveillance.

Microbial exposure drives polyclonal expansion of innate γδ T cells immediately after birth.

Starting at birth, the immune system of newborns and children encounters and is influenced by environmental challenges. It is still not completely understood how γδ T cells emerge and adapt during early life. Studying the composition of T cell receptors (TCRs) using next-generation sequencing (NGS) in neonates, infants, and children can provide valuable insights into the adaptation of T cell subsets. To investigate how neonatal γδ T cell repertoires are shaped by microbial exposure after birth, we monitored the γ-chain (TRG) and δ-chain (TRD) repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of TRG and TRD sequences that were shared by all children from Europe and Africa. These were primarily public clones, characterized by simple rearrangements of Vγ9 and Vδ2 chains with low junctional diversity and usage of non-TRDJ1 gene segments, reminiscent of early ontogenetic subsets of γδ T cells. Further profiling revealed that these innate, public Vγ9Vδ2+ T cells underwent an immediate TCR-driven polyclonal proliferation within the first 4 wk of life. In contrast, γδ T cells using Vδ1+ and Vδ3+TRD rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1+ and Vδ3+TRD sequences in the majority of African children. In summary, we show how dynamic γδ TCR repertoires develop directly after birth and present important differences among γδ T cell subsets.

TCR repertoire analysis reveals phosphoantigen-induced polyclonal proliferation of Vγ9Vδ2 T cells in neonates and adults.

The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.

Single-Cell Transcriptomics Identifies the Adaptation of Scart1+ Vγ6+ T Cells to Skin Residency as Activated Effector Cells.

IL-17-producing γδ T cells express oligoclonal Vγ4+ and Vγ6+ TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6+ T cells as a highly homogeneous Scart1+ population in contrast to their Scart2+ IL-17-producing Vγ4+ T cell counterparts. Parabiosis demonstrates that Vγ6+ T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1+ Vγ6+ T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1+ Vγ6+ T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.

Styk1 is specifically expressed in NK1.1+ lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.

Innate T cells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αß iNKT, and γδ NKT cell lineages. To investigate the role of Styk1 in the development and function of NK1.1+ innate T-cell subsets, we generated and analyzed a novel Styk1null mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKT cells and in thymic, but not in peripheral invariant αß iNKT cells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKT cells, and thymic αß iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.

Mutual interplay between IL-17-producing γδT cells and microbiota orchestrates oral mucosal homeostasis.

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6+ cells, and to a lesser extent Vγ1+ and Vγ4+ cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6+ cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6+ and Vγ4+ cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.

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.

Human papillomavirus oncoproteins induce a reorganization of epithelial-associated γδ T cells promoting tumor formation.

It has been shown that γδ T cells protect against the formation of squamous cell carcinoma (SCC) in several models. However, the role of γδ T cells in human papillomavirus (HPV)-associated uterine cervical SCC, the third-leading cause of death by cancer in women, is unknown. Here, we investigated the impact of γδ T cells in a transgenic mouse model of carcinogenesis induced by HPV16 oncoproteins. Surprisingly, γδ T cells promoted the development of HPV16 oncoprotein-induced lesions. HPV16 oncoproteins induced a decrease in epidermal Skint1 expression and the associated antitumor Vγ5+ γδ T cells, which were replaced by γδ T-cell subsets (mainly Vγ6+ γδlowCCR2+CCR6-) actively producing IL-17A. Consistent with a proangiogenic role, γδ T cells promoted the formation of blood vessels in the dermis underlying the HPV-induced lesions. In human cervical biopsies, IL-17A+ γδ T cells could only be observed at the cancer stage (SCC), where HPV oncoproteins are highly expressed, supporting the clinical relevance of our observations in mice. Overall, our results suggest that HPV16 oncoproteins induce a reorganization of the local epithelial-associated γδ T-cell subpopulations, thereby promoting angiogenesis and cancer development.