The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γ δ T cell population is due to cellular selection

Thy-1^dull γ δ T cells are an unusual subset of mature TCRγ δ T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vγ1 gene together with that of a member of the Vδ6 subfamily (the Vδ6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRγ δ repertoire, we have cloned all Vδ6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1^dull and Thy-1^bright γ δ thymocyte populations. Furthermore, we have also cloned non-functional Vδ6DδJδ1 rearrangements present in the Thy-1^dull γ δ T cell population and compared their Vδ6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRδ repertoire expressed by the Thy-1^dull γ δ thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRγ δ repertoire analysis of Thy-1^dull γ δ T cells in β2 -microglobulin (β₂m)-deficient mice indicated that these putative ligands are not β₂m-dependent major histocompatibility complex class I or class I-like molecules.     

Role of natural killer cells and TCRγ δ T cells in acute autoimmune encephalomyelitis

To elucidate the role of NK cells and TCRγ δ ⁺ T cells in acute experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats, the distribution, number and function of these cells were studied using several methods. Immunohistochemical and flow cytometric analysis revealed that a certain number of NK cells (17 % of the total inflammatory cells) infiltrated the central nervous system (CNS) at the peak stage of EAE and were mainly located in the perivascular region. On the other hand, virtually no TCRγ δ ⁺ T cells were found in the CNS. NK-T (NKR-P1⁺ TCRα β ⁺ ) cells were few and did not increase in number in the CNS and lymphoid organs. In the cytotoxic assay using YAC-1 cells, effector cells isolated from the spleen of rats at the peak of EAE showed essentially the same cytotoxicity as those isolated from normal controls although the total number of NK cells decreased to one fifth of that of normal rats. Furthermore, in vivo administration of anti-NK cell (3.2.3 and anti-asialo GM1), but not of anti-TCRγ δ (V65), antibodies exacerbated the clinical features of EAE and induced fatal EAE in some rats. These findings suggest that NK cells play a suppressive role in acute EAE whereas TCRγ δ ⁺ T cells are not involved in the development of or recovery from the disease.     

The intestinal expansion of TCRγδ+ and disappearance of IL4+ T cells suggest their involvement in the evolution from potential to overt celiac disease

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt‐CD), while others have a morphologically normal mucosa, despite the presence of CD‐specific autoantibodies (potential‐CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt‐ or potential‐CD and in healthy controls. Density of TCRγδ⁺ T cells was found markedly enhanced in intestinal mucosa of children with overt‐CD compared to potential‐CD or controls. By contrast, very few IL4⁺ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4⁺ T cells were classical CD4⁺ T‐helper cells (CD161^?), producing or not IFN‐γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ⁺ T cells, and concomitant disappearance of IL4⁺ cells. These findings suggest that immunomodulatory mechanisms are active in potential‐CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4⁺ and TCRγδ⁺ T cells as biomarkers of the different CD forms.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

Vγ9Vδ2 TCR‐activation by phosphorylated antigens requires butyrophilin 3 A1 (BTN3A1) and additional genes on human chromosome 6

Pyrophosphorylated metabolites of isoprenoid‐biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen‐presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg‐presentation by BTN3A1‐transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T‐cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg‐mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg‐mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC‐localized genes controlling peptide‐antigen presentation.     

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady‐state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ⁺ and CD8⁺ T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild‐type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ⁺ T‐cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.     

Neutrophils suppress γδ T‐cell function

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN‐γ, and the proliferation of γδ T cells induced by (E)‐1‐hydroxy‐2‐methylbut‐2‐enyl 4‐diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil‐derived ROS. We also show that the ROS‐generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi‐directional cross‐talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.     

Interleukin‐18 activates Vγ9Vδ2+ T cells from HIV‐positive individuals: recovering the response to phosphoantigen

The study aimed to identify an immunoregulatory factor that restores the phosphoantigen response of Vγ9Vδ2⁺ T cells from HIV‐positive individuals on antiretroviral therapy. It was designed to characterize the effects of interleukin‐18 (IL‐18) on proliferation and effector function in Vγ9Vδ2 T cells from HIV‐negative individuals and test whether exogenous IL‐18 reconstitutes the Vγ9Vδ2 T‐cell response to phosphoantigen from HIV‐positive donors. Vγ9Vδ2 T cells from HIV‐negative individuals responded strongly to phosphoantigen or aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMC), whereas cells with similar T‐cell receptor profiles from HIV‐positive individuals only responded to aminobisphosphonate. Interleukin‐18 was higher after aminobisphosphonate stimulation due to activation of the inflammasome pathway. Both IL‐18 and IL‐18 receptor levels were measured and the activity of exogenous IL‐18 on HIV‐negative and HIV‐positive PBMC was evaluated in terms of Vγ9Vδ2 T‐cell proliferation, memory subsets, cytokine expression and CD107a expression. Interleukin‐18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV‐negative controls. When Vγ9Vδ2 T cells from HIV‐positive individuals were stimulated with isopentenyl pyrophosphate in the presence of IL‐18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D and CD107a (markers of cytotoxic effector phenotype). Interleukin‐18 stimulation specifically expanded the Vγ9‐JγP⁺ subset of Vγ9Vδ2 T cells, as was expected for normal responses to phosphoantigen. Interleukin‐18 is a potent stimulator of Vγ9Vδ2 T‐cell proliferation and effector function. Therapies directed at reconstituting Vγ9Vδ2 T‐cell activity in HIV‐positive individuals should include stimulators of IL‐18 or direct cytokine supplementation.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

Liver distribution of γδ‐T‐cells in patients with chronic hepatitis of different etiology

The γδ T cells represent a minor unique T‐cell subpopulation long been considered as innate‐like immune cells. They are found in increased numbers in tissues from various inflammatory conditions. Their role in chronic hepatitis, however, is still discussed controversially. Fresh frozen tissues from 50 patients (18 cases hepatitis B infection, 25 hepatitis C, three cases with co‐infection of hepatitis B and C and four patients with autoimmune hepatitis) were investigated. Immunohistochemistry with primary antibodies detecting αβ and γδ TCR was used to evaluate their incidence and distribution in the different histological structures of the liver. The inflammatory infiltrate in all cases of chronic hepatitis was dominated by αβ T cells and was mainly localized in the portal tracts with formation of an interface hepatitis (95.3%αβ T cells; 4.7%γδ T cells). There were neither significant differences between inflammatory infiltrate nor the amount or percentage of γδ T cells between hepatitis B, C or autoimmune hepatitis. No accumulation of γδ T cells could be observed in cases of chronic hepatitis of different etiologies. The immune‐mediated phenomena in chronic hepatitis are dominated by αβ T cells. Thus, the adapted immune system is responsible for the inflammatory processes in chronic hepatitis.     

Promoting angiogenesis within the tumor microenvironment: The secret life of murine lymphoid IL‐17‐producing γδ T cells

After two decades in the shadow of their αβ counterparts, γδ T cells have recently gathered significant attention following the discovery that they produce IL‐17 in various mouse models of infection and autoimmune disease. In contrast, the secretion of large amounts of IFN‐γ by γδ T cells has long been known, and has been tightly linked to their anti‐tumor function. In this issue of the European Journal of Immunology, a study unexpectedly reports that the lymphoid γδ T cells that infiltrate tumor foci induced in the mouse skin produce very little IFN‐γ, but abundant IL‐17. In fact, these γδ T cells are the major source of IL‐17 within the tumor microenvironment, where they appear to promote angiogenesis, and thus tumor growth. This Commentary discusses the relevance of these interesting findings in the context of the currently paradoxical pro‐ versus anti‐tumor roles of IL‐17 in cancer immunology.     

Dermal IL‐17‐producing γδ T cells establish long‐lived memory in the skin

Conventional αβ T cells have the ability to form a long‐lasting resident memory T‐cell (T_RM) population in nonlymphoid tissues after encountering foreign antigen. Conversely, the concept of ‘innate memory’, where the ability of nonadaptive branches of the immune system to deliver a rapid, strengthened immune response upon reinfection or rechallenge, is just emerging. Using the αβ T‐cell‐independent Aldara psoriasis mouse model in combination with genetic fate‐mapping and reporter systems, we identified a subset of γδ T cells in mice that is capable of establishing a long‐lived memory population in the skin. IL‐17A/F‐producing Vγ4⁺Vδ4⁺ T cells populate and persist in the dermis for long periods of time after initial stimulation with Aldara. Experienced Vγ4⁺Vδ4⁺ cells show enhanced effector functions and mediate an exacerbated secondary inflammatory response. In addition to identifying a unique feature of γδ T cells during inflammation, our results have direct relevance to the human disease as this quasi‐innate memory provides a mechanistic insight into relapses and chronification of psoriasis.     

γδ T-Cell Subset Distribution in Human Semen From Fertile Donors

PROBLEM : γδ T-cell subset distribution has not been fully investigated in normal human semen. METHODS : We therefore carried out experiments by using a direct immunofluorescence staining technique followed by two-color cytofluorimetric analysis on mononuclear cell (MC) suspensions from ejaculates of ten healthy, fertile volunteers. Autologous peripheral blood MC were simultaneously analyzed and the results used for statistical comparison. RESULTS : The proportion of normal human semen lymphocytes bearing the γδ T-cell receptor for antigen was greatly increased compared with autologous circulating counterparts. Interestingly, the rise was mainly due to an overexpansion of cells expressing Vδl gene-encoded determinants on their surface. This contrasts with the normal blood picture, where most γδ T cells express Vδ2 conformational epitopes. CONCLUSIONS : The numerical and phenotypical differences in semen γδ T lymphocytes provide further evidence of a defined migrating lymphocyte subset balance in anatomically and physiologically distinct areas of the body. Their functional role, in terms of both helper and suppressor-cytotoxic activities in the nonsterile proximal portions of the male genital tract, now needs to be explored in detail.     

Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti‐tumour necrosis factor‐α therapy

Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn's disease (CD) who received anti‐tumour necrosis factor (TNF)‐α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti‐TNF‐α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty‐five men and 10 women who were treated with anti‐TNF‐α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR‐γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti‐TNF‐α‐treated patients than naive patients, suggesting that anti‐TNF‐α therapy may promote the clonal selection of γδ T cells in psoriasis patients.