Toll-like receptor 2 deficiency promotes the generation of alloreactive γδT17 cells after cardiac transplantation in mice.

Homograft rejection is the main cause of heart transplantation failure. The role of TLR2, a major member of the toll-like receptor (TLR) family, in transplantation rejection is has yet to be elucidated. In this study, we used a mouse model of acute cardiac transplantation rejection to investigate whether the TLR2 signalling pathway can regulate cardiac transplantation rejection by regulating alloreactive IL-17+γδT (γδT17) cells. We found that the expression of TLR2 on the surface of dendritic cells (DCs) and macrophages increased during acute transplantation rejection. In addition, our investigation revealed that γδT17 cells exert a significant influence on acute cardiac transplantation rejection. TLR2 gene knockout resulted in an increase in alloreactive γδT17 cells in the spleen and heart grafts of recipient mice compared with wild-type recipient mice and an increase in the mRNA expression of IL-17, IL-1ß, CCR6, and CCL20 in the heart grafts. In an in vitro experiment, a mixed lymphocyte reaction was conducted to assess the impact of TLR2 deficiency on the generation of γδT17 cells, which further substantiated a significant increase compared to that in wild-type controls. Furthermore, the mixed lymphocyte reaction showed that TLR2 regulated the production of γδT17 cells by regulating the ability of DCs to secrete IL-1ß. These results suggest that TLR2 signalling is important for regulating the generation of γδT17 cells after cardiac allograft transplantation.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Gut microbial fatty acid isomerization modulates intraepithelial T cells.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4+ intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4+CD8αα+ IELs in gnotobiotic mice. Restoration of CLAs increases CD4+CD8αα+ IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4+CD8αα+ IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4+ T cells that were CD4+CD8αα+.

γδ T cells in autoimmune uveitis pathogenesis: A promising therapeutic target.

Autoimmune uveitis is a non-infectious, inflammatory intraocular disease that affects the uveal and adjacent tissues. It frequently causes varying degrees of visual loss. Evidence for the strong association between activated γδ T cells and the development of autoimmune uveitis is growing. The innate and adaptive immune response are connected in the early phases by the γδ T cells that contain the γ and δ chains. γδ T cells can identify antigens in a manner that is not constrained by the MHC. When activated by various pathways, γδ T cells can not only secrete pro-inflammatory factors early on (such as IL-17), but they can also promote Th17 cells responses, which ultimately exacerbates autoimmune uveitis. Therefore, we review the mechanisms by which γδ T cells affect autoimmune uveitis in different activation and disease states. Moreover, we also prospect for immunotherapies targeting different γδ T cell-related action pathways, providing a reference for exploring new drug for the treatment of autoimmune uveitis.

Single-cell RNA-seq and chromatin accessibility profiling decipher the heterogeneity of mouse γδ T cells.

The distinct characteristics of γδ T cells determine their vital roles in the formation of local immune responses and contribute to tissue homeostasis. However, the heterogeneity of γδ T cells across tissues remains unclear. By combining transcriptional and chromatin analyses with a truly unbiased fashion, we constructed a single-cell transcriptome and chromatin accessibility landscape of mouse γδ T cells in the lymph, spleen, and thymus. We also revealed the heterogeneity of γδ T1 and γδ T17 cells across these tissues and inferred their potential regulatory mechanisms. In the thymus, we reconstructed the developmental trajectory and gained further insights into the signature genes from the mature stage, intermediate stage, and immature stage of γδ T cells on the basis of single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing data. Notably, a novel Gzma+ γδ T cell subset was identified with immature properties and only localized to the thymus. Finally, NR1D1, a circadian transcription factor (TF), was validated as a key and negative regulator of γδ T17 cell differentiation by performing a combined analysis of TF motif enrichment, regulon enrichment, and Nr1d1 knockout mice. In summary, our data represent a comprehensive mapping on the transcriptome and chromatin accessibility dynamics of mouse γδ T cells, providing a valuable resource and reference for future studies on γδ T cells.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

RNA m6A demethylase ALKBH5 regulates the development of γδ T cells.

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

TCR-Vγδ usage distinguishes protumor from antitumor intestinal γδ T cell subsets.

γδ T cells represent a substantial fraction of intestinal lymphocytes at homeostasis, but they also constitute a major lymphocyte population infiltrating colorectal cancers (CRCs); however, their temporal contribution to CRC development or progression remains unclear. Using human CRC samples and murine CRC models, we found that most γδ T cells in premalignant or nontumor colons exhibit cytotoxic markers, whereas tumor-infiltrating γδ T cells express a protumorigenic profile. These contrasting T cell profiles were associated with distinct T cell receptor (TCR)-Vγδ gene usage in both humans and mice. Longitudinal intersectional genetics and antibody-dependent strategies targeting murine γδ T cells enriched in the epithelium at steady state led to heightened tumor development, whereas targeting γδ subsets that accumulate during CRC resulted in reduced tumor growth. Our results uncover temporal pro- and antitumor roles for γδ T cell subsets.

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants.

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein­based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) "unhelped" mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and "helped" CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (ß) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell­targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

Stimulation of a subset of natural killer T cells by CD103+ DC is required for GM-CSF and protection from pneumococcal infection.

Innate-like T cells, including invariant natural killer T cells, mucosal-associated invariant T cells, and γδ T cells, are present in various barrier tissues, including the lung, where they carry out protective responses during infections. Here, we investigate their roles during pulmonary pneumococcal infection. Following infection, innate-like T cells rapidly increase in lung tissue, in part through recruitment, but T cell antigen receptor activation and cytokine production occur mostly in interleukin-17-producing NKT17 and γδ T cells. NKT17 cells are preferentially located within lung tissue prior to infection, as are CD103+ dendritic cells, which are important both for antigen presentation to NKT17 cells and γδ T cell activation. Whereas interleukin-17-producing γδ T cells are numerous, granulocyte-macrophage colony-stimulating factor is exclusive to NKT17 cells and is required for optimal protection. These studies demonstrate how particular cellular interactions and responses of functional subsets of innate-like T cells contribute to protection from pathogenic lung infection.

The Role of γδ T Cells as a Line of Defense in Viral Infections after Allogeneic Stem Cell Transplantation: Opportunities and Challenges.

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αß T cells, we describe that by delaying αß T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

In Acute Dengue Infection, High TIM-3 Expression May Contribute to the Impairment of IFNγ Production by Circulating Vδ2 T Cells.

γδ T cells are innate cells able to quickly eliminate pathogens or infected/tumoral cells by their antiviral and adjuvancy activities. The role of γδ T cells during Dengue Viral Infection (DENV) infection is not fully elucidated. Nevertheless, human primary γδ T cells have been shown to kill in vitro DENV-infected cells, thus highlighting their possible antiviral function. The aim of this work was to characterize the phenotype and function of Vδ2 T cells in DENV patients. Fifteen DENV patients were enrolled for this study and peripheral blood mononuclear cells (PBMC) were used to analyze Vδ2-T-cell frequency, differentiation profile, activation/exhaustion status, and functionality by multiparametric flow cytometry. Our data demonstrated that DENV infection was able to significantly reduce Vδ2-T-cell frequency and to increase their activation (CD38 and HLA-DR) and exhaustion markers (PD-1 and TIM-3). Furthermore, Vδ2 T cells showed a reduced capability to produce IFN-γ after phosphoantigenic stimulation that can be associated to TIM-3 expression. Several studies are needed to depict the possible clinical impact of γδ-T-cell impairment on disease severity and to define the antiviral and immunoregulatory activities of γδ T cells in the first phases of infection.

Role of Palatine Tonsil and Epipharyngeal Lymphoid Tissue in the Development of Glomerular Active Lesions (Glomerular vasculitis) in Immunoglobulin A Nephropathy.

Hematuria is an essential symptom of immunoglobulin A nephropathy (IgAN). Although the etiology of hematuria in IgAN has not been fully elucidated, it is thought that the rupture of the glomerular basement membranes caused by intra-capillary leukocyte influx, so-called glomerular vasculitis, is the pathological condition responsible for severe hematuria. Glomerular vasculitis are active lesions that exist in the glomeruli of acute phase IgAN and it is important because it is suspected to make the transition to segmental glomerular sclerosis (SGS) as a repair scar lesion in the chronic phase, and the progression of SGS would eventually lead to glomerular obsolescence. Worsening of hematuria concomitant with acute pharyngitis is common in patients with IgAN; therefore, elucidating the relationship between the immune system of Waldeyer's ring, including the palatine tonsil and epipharyngeal lymphoid tissue, and the glomerular vasculitis may lead to understanding the nature of IgAN. The epipharynx is an immunologically activated site even under normal conditions, and enhanced activation of innate immunity is likely to occur in response to airborne infection. Hyperactivation of innate immunity via upregulation of Toll-like receptors in the interfollicular area of the palatine tonsil and epipharyngeal lymphoid tissue, followed by enhanced fractalkine/CX3CR1 interactions, appears to play an important role in the development of glomerular vasculitis in IgAN. As latent but significant epipharyngitis is present in most patients with IgAN, it is plausible that acute upper respiratory infection may contribute as a trigger for the innate epipharyngeal immune system, which is already upregulated in a chronically inflamed environment. Given that epipharyngitis and its effects on IgAN are not fully understood, we propose that the so-called "epipharynx-kidney axis" may provide an important focus for future research.

Title IgA Nephropathy and Oral Bacterial Species Related to Dental Caries and Periodontitis.

A relationship between IgA nephropathy (IgAN) and bacterial infection has been suspected. As IgAN is a chronic disease, bacteria that could cause chronic infection in oral areas might be pathogenetic bacteria candidates. Oral bacterial species related to dental caries and periodontitis should be candidates because these bacteria are well known to be pathogenic in chronic dental disease. Recently, several reports have indicated that collagen-binding protein (cnm)-(+) Streptococcs mutans is relate to the incidence of IgAN and the progression of IgAN. Among periodontal bacteria, Treponema denticola, Porphyromonas gingivalis and Campylobacte rectus were found to be related to the incidence of IgAN. These bacteria can cause IgAN-like histological findings in animal models. While the connection between oral bacterial infection, such as infection with S. mutans and periodontal bacteria, and the incidence of IgAN remains unclear, these bacterial infections might cause aberrantly glycosylated IgA1 in nasopharynx-associated lymphoid tissue, which has been reported to cause IgA deposition in mesangial areas in glomeruli, probably through the alteration of microRNAs related to the expression of glycosylation enzymes. The roles of other factors related to the incidence and progression of IgA, such as genes and cigarette smoking, can also be explained from the perspective of the relationship between these factors and oral bacteria. This review summarizes the relationship between IgAN and oral bacteria, such as cnm-(+) S. mutans and periodontal bacteria.

Bcl-2 Is Necessary to Counteract Bim and Promote Survival of TCRαß+CD8αα+ Intraepithelial Lymphocyte Precursors in the Thymus.

The precursors of TCRαß+CD8αα+ intraepithelial lymphocytes (IEL) arise in the thymus through a complex process of agonist selection. We and others have shown that the proapoptotic protein, Bim, is critical to limit the number of thymic IEL precursors (IELp), as loss of Bim at the CD4+CD8+ double-positive stage of development drastically increases IELp. The factors determining this cell death versus survival decision remain largely unknown. In this study, we used CD4CreBcl2f/f mice to define the role of the antiapoptotic protein Bcl-2 and CD4CreBcl2f/fBimf/f mice to determine the role of Bcl-2 in opposing Bim to promote survival of IELp. First, in wild-type mice, we defined distinct subpopulations within PD-1+CD122+ IELp, based on their expression of Runx3 and α4ß7. Coexpression of α4ß7 and Runx3 marked IELp that were most dependent upon Bcl-2 for survival. Importantly, the additional loss of Bim restored Runx3+α4ß7+ IELp, showing that Bcl-2 antagonizes Bim to enable IELp survival. Further, the loss of thymic IELp in CD4CreBcl2f/f mice also led to a dramatic loss of IEL in the gut, and the additional loss of Bim restored gut IEL. The loss of gut IEL was due to both reduced seeding by IELp from the thymus as well as a requirement for Bcl-2 for peripheral IEL survival. Together, these findings highlight subset-specific and temporal roles for Bcl-2 in driving the survival of TCRαß+CD8αα+ IEL and thymic IELp.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

Emerging role of γδ T cells in protozoan infection and their potential clinical application.

γδ T cells are thymus derived heterogeneous and unconventional T- lymphocyte expressing TCR γ (V γ9) and TCRδ (Vδ2) chain and play an important role in connecting innate and adaptive armaments of immune response. These cells can recognize wide ranges of antigens even without involvement of major histocompatibility complex and exert their biological functions by cytotoxicity or activating various types of immune cells. In recent past, γδ T cells have emerged as an important player during protozoa infection and rapidly expand after exposure with them. They have also been widely studied in vaccine induced immune response against many bacterial and protozoan infections with improved clinical outcome. In this review, we will discuss the various roles of γδ T cells in immunity against malaria and leishmaniasis, the two important protozoan diseases causing significant mortality and morbidity throughout the world.

γδ T cells in artiodactyls: Focus on swine.

Vaccination is the most effective medical strategy for disease prevention but there is a need to improve livestock vaccine efficacy. Understanding the structure of the immune system of swine, which are considered a γδ T cell "high" species, and thus, particularly how to engage their γδ T cells for immune responses, may allow for development of vaccine optimization strategies. The propensity of γδ T cells to home to specific tissues, secrete pro-inflammatory and regulatory cytokines, exhibit memory or recall responses and even function as antigen-presenting cells for αß T cells supports the concept that they have enormous potential for priming by next generation vaccine constructs to contribute to protective immunity. γδ T cells exhibit several innate-like antigen recognition properties including the ability to recognize antigen in the absence of presentation via major histocompatibility complex (MHC) molecules enabling γδ T cells to recognize an array of peptides but also non-peptide antigens in a T cell receptor-dependent manner. γδ T cell subpopulations in ruminants and swine can be distinguished based on differential expression of the hybrid co-receptor and pattern recognition receptors (PRR) known as workshop cluster 1 (WC1). Expression of various PRR and other innate-like immune receptors diversifies the antigen recognition potential of γδ T cells. Finally, γδ T cells in livestock are potent producers of critical master regulator cytokines such as interferon (IFN)-γ and interleukin (IL)-17, whose production orchestrates downstream cytokine and chemokine production by other cells, thereby shaping the immune response as a whole. Our knowledge of the biology, receptor expression and response to infectious diseases by swine γδ T cells is reviewed here.

Transferable Immunoglobulin A-Coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. METHODS: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. RESULTS: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. CONCLUSIONS: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384.