Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.     

Vdelta1+ T cells are crucial for repertoire formation of gammadelta T cells in the lung

The pulmonary resident T lymphocytes (RPLs) expressing a nearly invariant T cell receptor γδ heterodimer (γδTCR) migrate from fetal thymus to the lung epithlium, followed by RPL subsets expressing diverse sets of γδTCRs after birth. However, it remains unclear whether the fetal type Vγ6/Vδ1⁺ RPLs are essential for γδ T cell repertoire formation in the lung epithelium. In this study, we found a marked decrease in the number of γδRPLs at 4 weeks of age in Vδ1^−/− mice and they predominantly expressed Vγ6 and Vδ4 genes. The skewed diversity towards the Vδ4-(Dδ1)-Dδ2-Jδ2 junctional region was observed only in γδ RPLs from 4-week-old Vδ1^−/− mice, compared with those from 8-week-old Vδ1^−/− mice and the both ages of wild-type mice. These results suggest that the invariant Vδ1⁺ T cells are crucial not only for optimal γδ T cell expansion but also for affecting the migration or microenvironment for other γδ T cells in the lung epithelium.     

Microbial isoprenoid biosynthesis and human gammadelta T cell activation

Human Vgamma9/Vdelta2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non-peptide antigens has been enigmatic until recently. The break-through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) (HMB-PP), turned out to be by far the most potent Vgamma9/Vdelta2 T cell activator known, with an EC(50) of 0.1 nM.     

Infiltration of canonical Vgamma4/Vdelta1 gammadelta T cells in an adriamycin-induced progressive renal failure model

We have previously reported an infiltration of renal interstitial gammadelta T cells in Adriamycin-induced progressive glomerulosclerosis in the rat kidney. The TCR repertoire and sequences used by these gammadelta T cells have now been studied. Two injections of Adriamycin 14 days apart caused segmental glomerulosclerosis, massive interstitial infiltration of mononuclear cells, and end-stage renal failure. Flow cytometry of lymphocyte subpopulations with Abs to CD3, the gammadelta TCR, and the alphabeta TCR showed that gammadelta T cells as a proportion of CD3(+) cells were increased in Adriamycin-treated kidneys (8.5 +/- 5.4%), but not in lymph nodes (1.3 +/- 0.4%). A semiquantitative score of glomerular damage (r = 0.65; p < 0.01) and creatinine (r = 0.62; p < 0.01) correlated significantly with the presence of gammadelta T cells. TCR Vgamma repertoire analysis by RT-PCR and Southern blotting showed that Vgamma2 was the dominant subfamily in lymph nodes, whereas Vgamma4 became the predominant subfamily in advanced stages of the rat Adriamycin-treated kidney. Sequencing of the Vgamma4-Jgamma junctional region showed an invariant sequence. The amino acid sequence of the junctional region of the Vgamma4 TCR was the same as the reported mouse canonical Vgamma4 TCR sequence. Analysis of the kidney Vdelta repertoire showed dominant expression of Vdelta1, and sequencing again revealed the selective expression of a canonical Vdelta1 gene. Semiquantitative RT-PCR for cytokine gene expression showed that gammadelta T cells from the kidneys expressed TGF-beta, but not IL-4, IL-10, or IFN-gamma. These results suggest that the predominant gammadelta T cells in the Adriamycin kidney use an invariant Vgamma4/Vdelta1 receptor.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.     

Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that gammadelta T cell population was the major source of IL-17. Mice depleted of gammadelta T cells by mAb treatment or mice genetically lacking Vdelta1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vdelta1(+) gammadelta T cells as the source of IL-17. It was further revealed that gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although gammadelta T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of gammadelta T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.     

Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

Induction of T cell activation by monoclonal anti-Thy-1 antibodies

We have analyzed the requirements for T cell activation by monoclonal anti-Thy-1 antibodies (MAb). A large panel of unselected anti-Thy-1 MAb was capable of inducing a strong proliferative response in resting peripheral T cells and a rise in cytoplasmic free calcium ([Ca2+]i) in both peripheral T cells and a T cell hybridoma. Both of these responses required the interaction of a MAb bound to Thy-1 with a second layer of anti-Ig antibody. Induction of T cell proliferation also required an additional signal, which could be provided by PMA. T cell activation in this system was specific for the Thy-1 molecule, independent of the epitope on Thy-1 recognized by a given MAb, with the anti-Ig reagent was also independent of the type of anti-Ig used, as both polyvalent rabbit anti-rat Ig sera and a mouse MAb to rat Ig functioned as effective cross-linkers. All signals provided by the interaction of anti-Thy-1 MAb with anti-Ig preparations could also be reproduced by the simultaneous binding of two MAb recognizing independent epitopes on Thy-1. Although the physiological role of Thy-1 remains unknown, the model system described here should prove to be very useful in further analysis of the steps involved in the polyclonal activation of murine T cells.     

Regulation of T cell-dependent autoantibody production by a gammadelta T cell line derived from lupus-prone mice

Lupus-prone (MRLxC57BL/6) F(1) mice lacking gammadelta T cells show more severe lupus than their T cell-intact counterparts, suggesting that gammadelta T cells down-modulate murine lupus. To determine the mechanisms for this effect, we assessed the capacity of gammadelta T cell lines derived from spleens of alphabeta T cell-deficient MRL/Mp-Fas(lpr) (MRL/Fas(lpr)) mice to down-regulate anti-dsDNA production generated by CD4(+)alphabeta T helper cell lines and activated B cells from wild-type MRL/Fas(lpr) mice. One line, GD12 (gd TCR(+), CD4(-)CD8(-)), had the capacity to reduce anti-dsDNA production in a contact-dependent manner. GD12 also killed activated MRL/Fas(lpr) (H-2(k)) B cells, with less cytolysis of resting B cells than that generated by in comparison to cytokine-matched gammadelta T cell lines. In addition, GD12 also killed activated B cells derived from C57BL/6-Fas(lpr) (H-2(b)) or beta(2)-microglobulin (beta(2) M)-deficient MRL/Fas(lpr) mice, suggesting cytolysis was neither MHC- nor CD1-restricted. Killing by GD12 was inhibited by anti-TNFalpha and anti-TNF-R1, and partially blocked by anti-gd TCR Fab fragments, but not by anti-FasL, anti-TNF-R2 (p75) or concanamycin A. IL-10 produced by GD12 also partially inhibited alphabeta Th1-dependent but not alphabeta Th2-dependent autoantibody production. These findings prove that we have identtified a gammadelta T cell line that suppresses autoantibody synthesis by alphabeta T-B cell collaboration in vitro.     

Contact-dependent T cell activation and T cell stopping require talin1

T cell-APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell-APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell-APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.     

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.     

Vdelta1+ gammadelta T cells producing CC chemokines may bridge a gap between neutrophils and macrophages in innate immunity during Escherichia coli infection in mice

An influx of neutrophils followed a short time later by an influx of macrophages to the infected site plays a key role in innate immunity against Escherichia coli infection. We found in this study that Vdelta1-/- mice exhibited impaired accumulation of peritoneal macrophages but not neutrophils and delayed bacterial clearance after i.p. inoculation with E. coli. Peritoneal gammadelta T cells from E. coli-infected wild-type mice produced CCL3/MIP-1alpha and CCL5/RANTES in response to gammadelta TCR triggering in vitro, whereas such production was not evident in gammadelta T cells from E. coli-infected Vdelta1-/- mice. Neutralization of CCL3/MIP-1alpha by a specific mAb in vivo significantly inhibited the accumulation of macrophages in the peritoneal cavity after E. coli infection, resulting in exacerbated bacterial growth in the peritoneal cavity. These results suggest that Vdelta1+ gammadelta T cells bridge a gap between neutrophils and macrophages in innate immunity during E. coli infection mediated by production of CC chemokines, enhancing macrophage trafficking to the site of infection.     

Immunoregulation in the tissues by gammadelta T cells

For a T-cell subset to be classified as immunoregulatory, it might reasonably be predicted that in its absence, animals would experience pathological immune dysregulation. Moreover, reconstitution of the subset should restore normal immune regulation. So far, these criteria have been satisfied by only a few of the candidate regulatory T-cell subsets, but among them is the intraepithelial gammadelta T-cell receptor (TCR)+ subset of mouse skin. In this article, we look at immunoregulatory gammadelta T cells, and the growing evidence for tissue-associated immunoregulation mediated by both gammadelta T cells and alphabeta T cells.     

Liver autoimmunity triggered by microbial activation of natural killer T cells

Humans with primary biliary cirrhosis (PBC), a disease characterized by the destruction of small bile ducts, exhibit signature autoantibodies against mitochondrial Pyruvate Dehydrogenase Complex E2 (PDC-E2) that crossreact onto the homologous enzyme of Novosphingobium aromaticivorans, an ubiquitous alphaproteobacterium. Here, we show that infection of mice with N. aromaticivorans induced signature antibodies against microbial PDC-E2 and its mitochondrial counterpart but also triggered chronic T cell-mediated autoimmunity against small bile ducts. Disease induction required NKT cells, which specifically respond to N. aromaticivorans cell wall alpha-glycuronosylceramides presented by CD1d molecules. Combined with the natural liver tropism of NKT cells, the accumulation of N. aromaticivorans in the liver likely explains the liver specificity of destructive responses. Once established, liver disease could be adoptively transferred by T cells independently of NKT cells and microbes, illustrating the importance of early microbial activation of NKT cells in the initiation of autonomous, organ-specific autoimmunity.     

T cell Ig and mucin domain-1-mediated T cell activation requires recruitment and activation of phosphoinositide 3-kinase

Ligation of the transmembrane protein T cell Ig and mucin domain (Tim)-1 can costimulate T cell activation. Agonistic Abs to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or costimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in a lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI3K. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation.     

Non-T cell activation linker promotes mast cell survival by dampening the recruitment of SHIP1 by linker for activation of T cells

The linker for activation of T cells (LAT) and the non-T cell activation linker (NTAL) are two transmembrane adapters which organize IgE receptor (FcepsilonRI) signaling complexes in mast cells. LAT positively regulates, whereas NTAL negatively regulates mast cell activation. We previously found that the four distal tyrosines of LAT can generate negative signals. We show here that two of these tyrosines provide two binding sites for SHIP1, that LAT recruits SHIP1 in vivo, and that SHIP1 recruitment is enhanced in NTAL-deficient cells. We show that NTAL negatively regulates mast cell activation by decreasing the recruitment, by LAT, of molecules involved in FcepsilonRI-dependent positive signaling. We show that NTAL also decreases the recruitment of SHIP1 by LAT, leading to an increased phosphorylation of the antiapoptotic molecule Akt, and positively regulates mast cell survival. We finally show that the positive effect of NTAL on Akt phosphorylation and mast cell survival requires LAT. Our data thus document the mechanisms by which LAT and NTAL can generate both positive and negative signals which differentially regulate mast cell activation and survival. They also provide molecular bases for the recruitment of SHIP1 in FcepsilonRI signaling complexes. SHIP1 is a major negative regulator of mast cell activation and, hence, of allergic reactions.