Limited diversity of gamma delta antigen receptor genes of Thy-1+ dendritic epidermal cells

T cells bearing gamma delta antigen receptors constitute minor populations in most peripheral lymphoid tissues but represent the major populations of T cells in certain epithelia, including the epidermis. We show that murine dendritic epidermal cell (dEC) clones express V gamma and V delta gene segments, which are rare in adult T cells but predominate in fetal thymocytes. Analysis of the junctions of the rearranged gamma and delta genes shows a striking homogeneity among the receptors of five dEC clones. Our data support a model in which dECs represent one of perhaps several waves of emigrants from the early fetal thymus, and imply a role for dECs in immune surveillance that is distinct from that of alpha beta- and other gamma delta-bearing T cells.     

T cell receptor gamma delta expression of Thy-1+ dendritic epidermal cells--an update

Thy-1+ dendritic epidermal cells (Thy-1+DEC) are present in the murine epidermis. They are morphologically dendritic and express Thy-1, CD3 and asialoGM1, but not CD4 or CD8. T cell receptor (TCR) of Thy-1+DEC is TCR gamma delta. Allison et al and Tonegawa et al recently found that TCR of Thy-1+DEC is V gamma 5 J gamma C gamma -V delta 1D2J2C delta and has no junctional diversity. This TCR gamma delta of Thy-1+DEC is identical to TCR expressed on the earliest fetal thymocytes. It is distinct from that of other epithelial associated lymphocytes or other thymocytes. The ligand of Thy-1+DEC is not known, although TCR gamma delta of adult type could recognize allogenic major histocompatibility complex(MHC) class I or class II and mycobacterium antigen, especially heat shock protein. The TCR of Thy-1+DEC may not be the homing receptor to epidermis. The further studies are needed to elucidate the ligands or functions of Thy-1+DEC.     

Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice

We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E. coli lipid A.     

Characterization of tumor reactivity of human V gamma 9V delta 2 gamma delta T cells in vitro and in SCID mice in vivo

Human Vgamma9Vdelta2 gammadelta T cells are selectively activated by bacterial phosphoantigens and aminobisphosphonates and exert potent cytotoxicity toward various tumor cells. In this study we have characterized the cytotoxic reactivity of gammadelta T cell lines established from healthy donors by stimulation with aminobisphosphonate alendronate toward melanoma MeWo and pancreatic adenocarcinomas Colo357 and PancTu1 lines in vitro and in vivo upon adoptive transfer into SCID mice. Lysis of all tumor cells was enhanced when gammadelta effector cells were preactivated with phosphoantigens. Recognition of MeWo was TCR dependent, as shown by anti-TCR Ab blockade, whereas only the phosphoantigen-mediated increased, but not the basal, lysis of Colo357 and PancTu1 was inhibited by anti-TCR Ab. Furthermore, lysis of Colo357, but not that of MeWo or PancTu1, was completely inhibited by the pan-caspase inhibitor zVAD, indicating different recognition and effector mechanisms involved in the gammadelta T cell/tumor cell interactions. Upon transfer into SCID mice, alendronate-activated gammadelta T cells given together with IL-2 and alendronate significantly prolonged the survival of SCID mice inoculated with human tumor cells. The best results were thus obtained when gammadelta T cells were repetitively given five times over a period of 30 days. With this protocol, human gammadelta T cells prolonged the mean survival of mice inoculated with MeWo melanoma from 28.5 to 87.3 days (p < 0.0001) and in the case of PancTu1 adenocarcinoma from 23.0 to 48.4 days (p < 0.0001). We conclude that an effective gammadelta T cell-based immunotherapy might require activation of endogenous gammadelta T cells with aminobisphosphonate (or phosphoantigen) and IL-2, followed by adoptive transfer of in vitro expanded gammadelta T cells.     

The interaction of gamma delta T cells with activated macrophages is a property of the V gamma 1 subset

Immunoregulation is an emerging paradigm of gammadelta T cell function. The mechanisms by which gammadelta T cells mediate this function, however, are not clear. Studies have identified a direct role for gammadelta T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages. The aim of this study was to identify macrophage-reactive gammadelta T cells and establish the requirements/outcomes of macrophage-gammadelta T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm). Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta(-/-) mice were incubated with splenocytes from naive and Lm-infected alphabeta/gammadelta T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to gammadelta T cells and the GV5S1 (Vgamma1) subset of gammadelta T cells. Macrophage adherence resulted in a 4- to 10-fold enrichment of Vgamma1(+) T cells. Enrichment of Vgamma1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of gammadelta T cells. Vgamma1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRgammadelta Abs inhibited both Vgamma1 binding and killing activities. These studies establish the identity of macrophage cytotoxic gammadelta T cells, the conditions under which this interaction occurs, and the outcome of this interaction. These findings are concordant with the involvement of Vgamma1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.     

树突状表皮T细胞在胸腺内发育的研究进展

树突状表皮T淋巴细胞(DETC),特异性分布在表皮组织内,在皮肤免疫监视,皮肤伤面愈合中发挥重要作用.小鼠DETC发育仅涉及胚胎14.5 ～ 18 d这一短暂时间窗,而后则不再产生这类细胞.本综述拟从DETC T细胞受体(TCR)基因重排特点及调控机制,DETC在胸腺中的阳性选择及调控机制等方面进行论述,以期对DETC...     

V gamma 4+ gamma delta T cells regulate airway hyperreactivity to methacholine in ovalbumin-sensitized and challenged mice

The Vgamma4(+) pulmonary subset of gammadelta T cells regulates innate airway responsiveness in the absence of alphabeta T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vgamma4(+) cells preferentially increased in number following airway challenge. Depletion of Vgamma4(+) cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vgamma1(+) cells had no effect on AHR, and depletion of all TCR-delta(+) cells was no more effective than depletion of Vgamma4(+) cells alone. Adoptively transferred pulmonary lymphocytes containing Vgamma4(+) cells inhibited AHR, but lost this ability when Vgamma4(+) cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vgamma4(+) cells, although cytokine-producing alphabeta T cells in the lung increased. These findings establish Vgamma4(+) gammadelta T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.     

Phenotypic and functional analysis of gamma delta T cell receptor-positive murine dendritic epidermal clones

Thy-1+ dendritic cells isolated from the epidermis of normal mice (dEC)3 bear the gamma delta TCR associated with the CD3 complex. We have analyzed the effects of antibodies directed against the TCR complex, Ly-6C, and Thy-1, as well as pharmacologic agents which have been shown to activate T cells without engagement of the TCR complex, on levels of intracellular free calcium, activation of protein kinase C, cytolysis, IL-2R expression, and secretion of lymphokines by dEC clones. We have found that the dEC cells express a fully functional TCR complex which can function to transmit signals upon perturbation leading to an increase in IL-2R expression, release of lymphokines, and cytolytic activity. These results indicate that the gamma delta TCR+ dEC are capable of responding to activation signals in the same manner as mature alpha beta TCR+ cells and suggests that they may play a functional role in the skin.     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Extrathymic positive selection of gamma delta T cells. V gamma 4J gamma 1 rearrangements with "GxYS" junctions.

Neither function nor Ag recognition properties of gamma delta T cells are well understood yet. A TCR gamma-chain family, characterized by distinct N region sequences that converge in coding for a "GxYS" VJ junctional sequence, appears late in ontogeny among highly diversified V gamma 4J gamma 1C gamma 1 chains of C57BL/6 and BALB/c mice. The glycine and serine codons are of germline V gamma 4 and J gamma 1 origin, respectively, whereas the N region consists of a variable amino acid residue x, followed by an invariant tyrosine Y. The high expression of V gamma 4 xYJ gamma 1C gamma 1 in the lung of BALB/c mice compared to that of C57BL/6 is apparently due to a novel pattern of strain-dependent positive selection which, unlike for alpha beta TCR, operates extrathymically. This type of selection seems to be determined by strain specific polymorphic ligands encoded outside of the classical H-2 region.     

T cell receptor-gamma allele-specific selection of V gamma 1/V delta 4 cells in the intestinal epithelium

Previous genetic analyses have shown that the relative representation of subsets of gammadelta intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRgamma, TCRdelta, and MHC loci. Here, we have analyzed V-gene use in gammadelta i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F(1) and F(2) progenies with a larger panel of Vgamma- and Vdelta-specific mAbs and have shown that the influence of TCRgamma-linked genes operates at two levels: one influencing the representation of Vgamma1 (or Vgamma7) i-IELs and other acting specifically on the Vgamma1/Vdelta4 i-IEL subset, which represents 3% and 15% of the gammadelta i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vgamma1Jgamma4Cgamma4 chain of B6 origin demonstrated that the TCRgamma-linked genes influencing the representation of the Vgamma1/Vdelta4 i-IEL subset are the structural genes of TCRgamma chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vgamma1/Vdelta4 cells bearing either parental allele in F(1) mice. The representation of Vgamma1/Vdelta4 cells among gammadelta thymocytes is similar in B6 and B10 mice, demonstrating that the Vdelta4 chain can pair well with both alleles of the Vgamma1Jgamma4Cgamma4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vgamma1-Jgamma4 junctional amino acid sequences of B6 Vgamma1/Vdelta4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vgamma1/Vdelta4 cells are the target of this cellular selection event.     

Patterns of chemokine receptor expression on peripheral blood gamma delta T lymphocytes: strong expression of CCR5 is a selective feature of V delta 2/V gamma 9 gamma delta T cells

Gammadelta T lymphocytes play an important role in the immune defense against infection, based on the unique reactivity of human Vdelta2Vgamma9 gammadelta T cells toward bacterial phosphoantigens. Chemokines and their corresponding receptors orchestrate numerous cellular reactions, including leukocyte migration, activation, and degranulation. In this study we investigated the expression of various receptors for inflammatory and homeostatic chemokines on peripheral blood gammadelta T cells and compared their expression patterns with those on alphabeta T cells. Although several of the analyzed receptors (including CCR6, CCR7, CXCR4, and CXCR5) were not differentially expressed on gammadelta vs alphabeta T cells, gammadelta T cells expressed strongly increased levels of the RANTES/macrophage inflammatory protein-1alpha/-1beta receptor CCR5 and also enhanced levels of CCR1-3 and CXCR1-3. CCR5 expression was restricted to Vdelta2 gammadelta T cells, while the minor subset of Vdelta1 gammadelta T cells preferentially expressed CXCR1. Stimulation with heat-killed extracts of Mycobacterium tuberculosis down-modulated cell surface expression of CCR5 on gammadelta T cells in a macrophage-dependent manner, while synthetic phosphoantigen isopentenyl pyrophosphate and CCR5 ligands directly triggered CCR5 down-modulation on gammadelta T cells. The functionality of chemokine receptors CCR5 and CXCR3 on gammadelta T cells was demonstrated by Ca(2+) mobilization and chemotactic response to the respective chemokines. Our results identify high level expression of CCR5 as a characteristic and selective feature of circulating Vdelta2 gammadelta T cells, which is in line with their suspected function as Th1 effector T cells.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

Activation of V gamma 9V delta 2 T cells by NKG2D

Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.     

In vivo immunomanipulation of V gamma 9V delta 2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model

Vgamma9Vdelta2(+) cells represent the major population of gammadelta T cells in primate blood and react in an MHC-unrestricted fashion to a set of low m.w. nonpeptide phosphoantigens. Two types of structurally related agonists have been discovered so far: the natural phosphoantigens (hydroxydimethyl allyl-pyrophosphate or isopentenyl-pyrophosphate (IPP)) acting directly on Vgamma9Vdelta2(+) TCR and aminobisphosphonates, which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vgamma9Vdelta2(+) cells. We demonstrate in the cynomolgus monkey that Vgamma9Vdelta2 can be manipulated in vivo with bromohydrin pyrophosphate (BrHPP)/Phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vgamma9Vdelta2, inducing production of a high level of Th1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vgamma9Vdelta2 in blood in a dose-dependant manner. This transient response returns to baseline within 10-15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic Th1 cytokines and massive expansion of gammadelta T cell subset with antitumor and anti-infectious properties.     

Transcriptional profiling of gamma delta T cells identifies a role for vitamin D in the immunoregulation of the V gamma 9V delta 2 response to phosphate-containing ligands

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vdelta2Vgamma9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1alpha,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced gammadelta T cell expansion, IFN-gamma production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of gammadelta T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.     

T cells in cryptopatch aggregates share TCR gamma variable region junctional sequences with gamma delta T cells in the small intestinal epithelium of mice

The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.