Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

Single-cell analysis reveals the origins and intrahepatic development of liver-resident IFN-γ-producing γδ T cells

γδ T cells are heterogeneous lymphocytes located in various tissues. However, a systematic and comprehensive understanding of the origins of γδ T cell heterogeneity and the extrathymic developmental pathway associated with liver γδ T cells remain largely unsolved. In this study, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively catalog the heterogeneity of γδ T cells derived from murine liver and thymus samples. We revealed the developmental trajectory of γδ T cells and found that the liver contains γδ T cell precursors (pre-γδ T cells). The developmental potential of hepatic γδ T precursor cells was confirmed through in vitro coculture experiments and in vivo adoptive transfer experiments. The adoptive transfer of hematopoietic progenitor Lin⁻Sca-1⁺Mac-1⁺ (LSM) cells from fetal or adult liver samples to sublethally irradiated recipients resulted in the differentiation of liver LSM cells into pre-γδ T cells and interferon-gamma⁺ (IFN-γ⁺) but not interleukin-17a⁺ (IL-17a⁺) γδ T cells in the liver. Importantly, thymectomized mouse models showed that IFN-γ-producing γδ T cells could originate from liver LSM cells in a thymus-independent manner. These results suggested that liver hematopoietic progenitor LSM cells were able to differentiate into pre-γδ T cells and functionally mature γδ T cells, which implied that these cells are involved in a distinct developmental pathway independent of thymus-derived γδ T cells.     

The role of 1α,25‐dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL‐10+ CD4+ T cells

1α,25‐Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL‐10‐secreting CD4⁺ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3⁺ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3⁺ and IL‐10⁺ CD4⁺T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL‐10 at more moderate levels, with little coexpression of these molecules. The Foxp3⁺ and IL‐10⁺ T‐cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL‐10. 1α25VitD3 enables the selective expansion of Foxp3⁺ Treg cells over their Foxp3^? T‐cell counterparts. Equally, 1α25VitD3 maintains Foxp3⁺ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4⁺Foxp3⁺ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL‐10⁺ and Foxp3⁺ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.     

Targeting Janus tyrosine kinase 3 (JAK3) with an inhibitor induces secretion of TGF-β by CD4+ T cells

Regulatory T cells (Tregs) are critical for the peripheral maintenance of the autoreactive T cells in autoimmune disorders such as type 1 diabetes (T1D). Pharmacological inhibition of Janus tyrosine kinase 3 (JAK3) has been proposed as a basis for new treatment modalities against autoimmunity and allogeneic responses. Targeting JAK3 with an inhibitor has previously been shown to exhibit protective action against the development of T1D in non-obese diabetic (NOD) mice. As the mechanism of such preventative action has been unknown, we hypothesized that JAK3 inhibition induces generation of Tregs. Here, we show that the JAK3 inhibitor 4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) suppresses proliferation of short-term cultured NOD CD4⁺ T cells through induction of apoptosis, while promoting survival of a particular population of long-term cultured cells. It was found that the surviving cells were not of the CD4⁺CD25⁺FoxP3⁺ phenotype. They secreted decreased amounts of IL-10, IL-4 and interferon (IFN)-γ compared to the cells not exposed to the optimal concentrations of JAK3 inhibitor. However, an elevated transforming growth factor (TGF)-β secretion was detected in their supernatants. In vivo treatment of prediabetic NOD mice with WHI-P131 did not affect the frequency and number of splenic and pancreatic lymph node CD4⁺FoxP3⁺ Tregs, while generating an elevated numbers of CD4⁺FoxP3⁻ TGF-β-secreting T cells. In conclusion, our data suggest an induction of TGF-β-secreting CD4⁺ T cells as the underlying mechanism for antidiabetogenic effects obtained by the treatment with a JAK3 inhibitor. To our knowledge, this is the first report of the JAK3 inhibitor activity in the context of the murine Tregs.     

Role of γδ T lymphocytes in the development of Behçet's disease

Phenotypic and functional properties of γδ T cells, which play an important role in mucocutaneous immunity, were examined to elucidate whether immunological abnormality in Behc¸et's disease may be related to a specific T cell population. We found that CD45RA⁺Vγ9⁺Vδ2⁺γδ T cells, which constitute a minor population of γδ T cells in healthy individuals, were increased in number in Behc¸et's disease irrespective of disease activity. This CD45RA⁺ subset of γδ T cells in the active, but not inactive, phase of this disease expressed IL-2Rβ and HLA-DR, suggesting that they are activated in vivo in active Behc¸et's disease. In addition, the CD45RA⁺γδ T cells produced extreme amounts of tumour necrosis factor and contained perforin granules. These data indicate that a phenotypically distinct subset of γδ T cells, CD45RA⁺CD45RO⁻Vγ9⁺Vδ2⁺, may contribute to immunological abnormalities which may lead to complexity of pathophysiology in Behc¸et's disease.     

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.     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.     

Loss of β‐arrestin 2 exacerbates experimental autoimmune encephalomyelitis with reduced number of Foxp3+ CD4+ regulatory T cells

β-Arrestins are well-known regulators and mediators of G protein-coupled receptor signalling, and accumulating evidence reveals that they are functionally involved in inflammation and autoimmune diseases. Of the two β-arrestins, β-arrestin 1 is documented to play regulatory roles in an animal model of multiple sclerosis (MS), whereas the role of β-arrestin 2 is less clear. Here, we show that β-arrestin 2-deficient mice displayed the exacerbated and sustained symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. At the cellular level, deficiency of β-arrestin 2 led to a decreased number of Foxp3⁺ CD4⁺ regulatory T (Treg) cells in peripheral lymphoid organs of EAE mice. Consistently, our in vitro observations also revealed that loss of β-arrestin 2 impaired the conversion of Foxp3⁻ CD4⁺ T cells into Foxp3⁺ CD4⁺ inducible Treg cells. Taken together, our data suggest that β-arrestin 2 plays a regulatory role in MS, that is opposite to that of β-arrestin 1, in autoimmune diseases such as MS, which is at least partially through regulation of iTreg cell differentiation.     

Expansion of CD56+ NK T and γδ T cells from cord blood of human neonates

A particular T cell population expressing NK cell markers, CD56 and CD57, exists in humans. Many CD56⁺ T and CD57⁺ T cells (i.e. NK T cells) exist in the liver and increase in number in the blood with ageing. They may be a human counterpart of extrathymic T cells, similar to NK1.1⁺ CD3^int cells seen in mice. We investigate here the existence of such NK T cells in human cord blood and the in vitro expansion of these cells by the stimulation of human recombinant IL-2 (rIL-2). There were very small populations (< 1.0%) of CD56⁺ T cells, CD57⁺ T cells, and γδ T cells in cord blood. However, all of these populations increased in number after birth and with ageing. When lymphocytes in cord blood were cultured with rIL-2 (100 U/ml) for 14 days, CD56⁺ T cells expanded up to 25% of T cells. CD57⁺ T cells were never expanded by these in vitro cultures. The expansion of γδ T cells (mainly Vγ9^? non-adult type) also occurred in the in vitro culture. A considerable proportion of CD56⁺ T cells was found to use Vα24 (i.e. equivalent to invariant Vα14 chain used by murine NK T cells) for TCR αβ. These results suggest that neonatal blood contains only a few NK T cells but CD56⁺ NK T cells and γδ T cells are able to expand in vitro.     

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.     

2-Gy whole-body irradiation significantly alters the balance of CD4+CD25? T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice

CD4⁺CD25⁺ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4⁺CD25⁺Foxp3⁺ Treg cells has not been determined. The proportion, phenotypes and function of CD4⁺CD25⁺ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁺Foxp3⁺ Treg cells to CD4⁺ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4⁺CD25⁺ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4⁺CD25⁻ T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4⁺CD25⁺Foxp3⁺ Treg cells than in CD4⁺CD25⁻ T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.     

CD4+ CD25high Foxp3+ regulatory T cells downregulate human Vδ2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

Vδ2⁺ T cells, the major circulating T-cell receptor-γδ-positive (TCR-γδ⁺) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vδ2⁺ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4⁺ T cells. CD4⁺ CD25^high Foxp3⁺ T cells (Tregs) comprise 5–10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-αβ⁺ lymphocytes, Tregs suppress Vδ2⁺ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vδ2⁺ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vδ2⁺ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-γ secretion by Vδ2⁺ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vδ2⁺ T-cell interferon-γ responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vδ2⁺ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vδ2⁺ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vδ2⁺ T-cell functional deficiencies observed in TB.     

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4⁺ and CD8⁺ T cells, especially the CD4⁺CD25⁺Foxp3⁺ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.     

CD4+CD25+ but not CD4+Foxp3+ T cells as a regulatory subset in primary biliary cirrhosis

Increasing evidence indicates a role for regulatory T cells (Tregs) in the immune response and in autoimmune diseases, but the role of Tregs and cytokines in autoimmune hepatic diseases remains largely unclear and controversial, especially in patients with primary biliary cirrhosis (PBC). This study was undertaken to investigate Tregs and different cytokines in the liver and peripheral blood of PBC patients. We found that these patients demonstrated a reduction of CD4⁺CD25⁺ T cells but elevated CD4⁺Foxp3⁺ T cells in peripheral blood mononuclear cells (PBMCs) and CD4⁺ T cells. The percentage of CD4⁺CD25⁺ T cells in PBMCs was negatively correlated with elevated plasma interferon (IFN)-γ levels. A liver-specific analysis showed that the frequency of Foxp3⁺ Tregs, transforming growth factor (TGF)-β1 and IFN-γ were increased in PBC patients. Our findings suggest that an imbalance between CD4⁺CD25⁺ Tregs and cytotoxic cytokines plays a crucial role in the pathogenesis of PBC while the role of Foxp3 needs further investigation.     

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity

CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca²⁺ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca²⁺ influx and membrane potential (V_m)-induced Ca²⁺ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.