γδ T cells are indispensable for interleukin‐23‐mediated protection against Concanavalin A‐induced hepatitis in hepatitis B virus transgenic mice

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation‐induced hepatitis is unknown. In this study, using HBV transgenic (HBs‐Tg) and HBs‐Tg T‐cell receptor‐δ‐deficient (TCR‐δ^−/−) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon‐γ (IFN‐γ) levels and more extensive necrosis. γδ T‐cell deficiency resulted in elevated IFN‐γ in CD4⁺ T cells but not in natural killer or natural killer T cells. The depletion of CD4⁺ T cells and neutralization of IFN‐γ reduced liver damage in HBs‐Tg and HBs‐Tg‐TCR‐δ^−/− mice to a similar extent. Further investigation revealed that HBs‐Tg mice showed an enhanced interleukin‐17 (IL‐17) signature. The administration of exogenous IL‐23 enhanced IL‐17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs‐Tg mice, but not in HBs‐Tg‐TCR‐δ^−/− mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A‐induced hepatitis by inhibiting IFN‐γ production from CD4⁺ T cells and are indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation‐induced liver damage.     

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.     

Zoledronic acid causes γδ T cells to target monocytes and down‐modulate inflammatory homing

Zoledronic acid (ZA) is a potential immunotherapy for cancer because it can induce potent γδ T‐cell‐mediated anti‐tumour responses. Clinical trials are testing the efficacy of intravenous ZA in cancer patients; however, the effects of systemic ZA on the activation and migration of peripheral γδ T cells remain poorly understood. We found that γδ T cells within ZA‐treated peripheral blood mononuclear cells were degranulating, as shown by up‐regulated expression of CD107a/b. Degranulation was monocyte dependent because CD107a/b expression was markedly reduced in the absence of CD14⁺ cells. Consistent with monocyte‐induced degranulation, we observed γδ T‐cell‐dependent induction of monocyte apoptosis, as shown by phosphatidylserine expression on monocytes and decreased percentages of monocytes in culture. Despite the prevailing paradigm that ZA promotes tumour homing in γδ T cells, we observed down‐modulation of their tumour homing capacity, as shown by decreased expression of the inflammatory chemokine receptors CCR5 and CXCR3, and reduced migration towards the inflammatory chemokine CCL5. Taken together our data suggest that ZA causes γδ T cells to target monocytes and down‐modulate the migratory programme required for inflammatory homing. This study provides novel insight into how γδ T cells interact with monocytes and the possible implications of systemic use of ZA in cancer.     

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.     

Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)‐CD40 signaling by γδ T cells induces protective immunity against the blood‐stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T‐cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1⁺ cells, we saw that Vγ1⁺ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1⁺ γδ T cells preferentially expand and express CD40L, and both Vγ1⁺ and Vγ4⁺ γδ T cells produce IFN‐γ during infection. Although expression of CD40L on Vγ1⁺ γδ T cells is maintained during infection, the IFN‐γ positivity of Vγ1⁺ γδ T cells is reduced in late‐phase infection due to γδ T‐cell dysfunction. In Plasmodium‐infected IFN‐γ signaling‐deficient mice, DC activation is reduced, resulting in the suppression of γδ T‐cell dysfunction and the dampening of γδ T‐cell expansion in the late phase of infection. Our data suggest that Vγ1⁺ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1⁺ γδ T‐cell response is dependent on IFN‐γ‐activated DCs.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Modulation of γδ T‐cell activation by neutrophil elastase

γδ T cells are non‐conventional, innate‐like T cells, characterized by a restricted T‐cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up‐regulation of CD69 expression, and the production of interferon‐γ and tumour necrosis factor‐α induced by anti‐CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase‐1 and neutralization of interleukin‐18 did not affect neutrophil‐mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T‐cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T‐cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T‐cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease‐activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T‐cell activation.     

Differential requirement for CARMA1 in agonist‐selected T‐cell development

Caspase recruitment domain‐containing membrane‐associated guanylate kinase protein‐1 (CARMA1) is a critical component of the NF‐κB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist‐selected T‐cell subsets such as Treg, NKT cells, and CD8‐αα T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1‐deficient mice (CARMA1^?/?) have altered populations of specific subsets of agonist‐selected T cells. Specifically, CARMA1^?/? mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild‐type mice. Interestingly, CD8‐αα T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1^?/? mice, whereas memory‐phenotype CD4⁺ T cells (CD62L^low/CD44^high) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist‐selected T‐cell subsets. Overall, these data reveal an important but differential role for CARMA1‐mediated TCR signaling in T‐cell development.     

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCR^low subset had a higher CD3 to TCR ratio and was enriched in Vδ2⁺ cells, whereas most Vδ1⁺ cells belonged to the γδTCR^high subset. In PB from TB, most γδTCR^high were CD45RA⁺CCR7^‐ and γδTCR^low were CD45RA^+/?CCR7⁺CXCR3⁺. In the pleural space the proportion of CD45RA^‐CCR7⁺CXCR3⁺ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCR^low cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.     

Cytomegalovirus drives Vδ2neg γδ T cell inflation in many healthy virus carriers with increasing age

Cytomegalovirus (CMV) usually causes lifelong asymptomatic infection, but over time can distort immune profiles. Recent reports describe selective expansion of Vδ2^neg γδ T cells in healthy and immunocompromised CMV carriers. Having shown previously that virus‐specific CD8⁺ and CD4⁺ T cell responses are increased significantly in elderly CMV carriers, probably driven by chronic stimulation, we hypothesized that Vδ2^neg γδ T cells may also be expanded with age. Our results show that Vδ2^neg γδ T cells are increased significantly in CMV‐seropositive healthy individuals compared to CMV‐seronegative controls in all age groups. The differences were most significant in older age groups (P < 0·0001). Furthermore, while Vδ2^neg γδ T‐ cells comprise both naive and memory cells in CMV‐seronegative donors, highly differentiated effector memory cells are the dominant phenotype in CMV carriers, with naive cells reduced significantly in numbers in CMV‐seropositive elderly. Although phenotypically resembling conventional CMV‐specific T cells, Vδ2^neg γδ T cells do not correlate with changes in magnitude of CMV‐specific CD4⁺ or CD8⁺ T cell frequencies within those individuals, and do not possess ex‐vivo immediate effector function as shown by CMV‐specific CD4⁺ and CD8⁺ T cells. However, after short‐term culture, Vδ2^neg γδ T cells demonstrate effector T cell functions, suggesting additional requirements for activation. In summary, Vδ2^neg γδ T cells are expanded in many older CMV carriers, demonstrating a further level of lymphocyte subset skewing by CMV in healthy individuals. As others have reported shared reactivity of Vδ2^neg γδ T cells towards tumour cells, the composition of γδ T cell subsets may also have implications for risk of developing cancer in elderly people.     

T helper type 1 polarizing γδ T cells and Scavenger receptors contribute to the pathogenesis of Pemphigus vulgaris

γδ T cells and Scavenger receptors are key parts of the innate immune machinery, playing significant roles in regulating immune homeostasis at the epithelial surface. The roles of these immune components are not yet characterized for the autoimmune skin disorder Pemphigus vulgaris (PV). Phenotyping and frequency of γδ T cells estimated by flow cytometry have shown increased frequency of γδ T cells (6·7% versus 4·4%) producing interferon‐ γ (IFN‐γ; 35·2% versus 26·68%) in the circulation of patients compared with controls. Dual cytokine‐secreting (IFN‐γ and interleukin‐4) γδ T cells indicate the plasticity of these cells. The γδ T cells of patients with PV have shown higher cytotoxic potential and the higher frequency of γδ T cells producing IFN‐γ shows T helper type 1 polarization. The increased expression of Scavenger receptors expression (CD36 and CD163) could be contributing to the elevated inflammatory environment and immune imbalance in this disease. Targeting the inflammatory γδ T cells and Scavenger receptors may pave the way for novel therapeutics.     

A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

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.     

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.     

HIV gag‐specific immune response mediated by double negative (CD3+CD4−CD8−) T cells in HIV‐exposed seronegative individuals

Double negative (DN) T cells are CD3⁺, CD4^?, CD8^? cells with either T‐cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV‐exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV‐serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV‐specific immune responses mediated by DN T‐cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV‐Gag peptides. Thirty‐five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag‐specific DN T‐cell responses were found in 3/13 (23%) HIV‐exposed seronegative individuals (Group A), involving both DN/αβ⁺ and DN/γδ⁺ T‐cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag‐specific responses but were mediated exclusively by DN/γδ⁺ T‐cells, mainly through IFNγ production. DN T‐cells in Group A individuals can display differential HIV‐specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A. J. Med. Virol. 85:200–209, 2013. © 2012 Wiley Periodicals, Inc.     

Changes in thymic export of γδ and αβ T cells during fetal and postnatal development

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5–1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both αβ and γδ T cells, our results demonstrate that the proportion of thymic γδ T cells that are exported per day is much higher than that of thymic αβ T cells. Moreover, the export rate of γδ T cells increased from approximately 1 in every 60 γδ thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5⁺CD4^?CD8^?γδ^? T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both γδ and αβ T cells.