A keratinocyte-responsive gamma delta TCR is necessary for dendritic epidermal T cell activation by damaged keratinocytes and maintenance in the epidermis

A unique population of T lymphocytes, designated dendritic epidermal T cells (DETC), homes to the murine epidermis during fetal development. DETC express a canonical gammadelta TCR, Vgamma3/Vdelta1, which recognizes Ag expressed on damaged, stressed, or transformed keratinocytes. Recently, DETC were shown to play a key role in the complex process of wound repair. To examine the role of the DETC TCR in DETC localization to the epidermis, maintenance in the skin, and activation in vivo, we analyzed DETC in the TCRdelta(-/-) mouse. Unlike previous reports in which the TCRdelta(-/-) skin was found to be devoid of any DETC, we discovered that TCRdelta(-/-) mice have alphabeta TCR-expressing DETC with a polyclonal Vbeta chain repertoire. The alphabeta DETC are not retained over the life of the animal, suggesting that the gammadelta TCR is critical for the maintenance of DETC in the skin. Although the alphabeta DETC can be activated in response to direct stimulation, they do not respond to keratinocyte damage. Our results suggest that a keratinocyte-responsive TCR is necessary for DETC activation in response to keratinocyte damage and for DETC maintenance in the epidermis.     

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.     

Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers

We previously reported that resident gammadelta T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing gammadelta T cells did not produce IFN-gamma or IL-4, similar to Th17 cells. IL-17-producing gammadelta T cells specifically express CD25 but not CD122, whereas CD122(+) gammadelta T cells produced IFN-gamma. IL-17-producing gammadelta T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-gamma-producing CD122(+) gammadelta T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing gammadelta T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing gammadelta T cells. The number of thymic IL-17-producing gammadelta T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vgamma6(+) Vdelta1(+) gammadelta T cells. The number of IL-17-producing gammadelta T cells was decreased in fetal thymus of Vdelta1-deficient mice, whereas Vgamma5(+) fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vgamma6(+) Vdelta1(+) T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.     

Conservation of nonpeptide antigen recognition by rhesus monkey V gamma 2V delta 2 T cells

We have previously found that monkey Vgamma2Vdelta2(+) T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guérin infections. We have now analyzed rhesus monkey gammadelta T cell responses to nonpeptide Ags and superantigens. Like human Vgamma2Vdelta2(+) T cells, rhesus monkey gammadelta T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags. Responsiveness was limited to gammadelta T cells expressing Vgamma2Vdelta2 TCRs. Rhesus monkey Vgamma2Vdelta2(+) T cells also responded to the superantigen, staphyloccocal enterotoxin A. Sequencing of the rhesus monkey Vgamma2Vdelta2 TCR revealed a strong sequence homology to human Vgamma2Vdelta2 TCR that preserves important sequence motifs. Moreover, chimeric TCRs that pair human Vgamma2 with monkey Vdelta2 and monkey Vgamma2 with human Vdelta2 retain reactivity to nonpeptide Ags and B cell lymphomas. A molecular model of the rhesus monkey Vgamma2Vdelta2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vgamma2Vdelta2 TCR and preserves the topology of the complementarity-determining region loops. Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human gammadelta T cell Ag responses.     

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.     

Exacerbation of collagen-induced arthritis by oligoclonal, IL-17-producing gamma delta T cells

Murine gammadelta T cell subsets, defined by their Vgamma chain usage, have been shown in various disease models to have distinct functional roles. In this study, we examined the responses of the two main peripheral gammadelta T cell subsets, Vgamma1(+) and Vgamma4(+) cells, during collagen-induced arthritis (CIA), a mouse model that shares many hallmarks with human rheumatoid arthritis. We found that whereas both subsets increased in number, only the Vgamma4(+) cells became activated. Surprisingly, these Vgamma4(+) cells appeared to be Ag selected, based on preferential Vgamma4/Vdelta4 pairing and very limited TCR junctions. Furthermore, in both the draining lymph node and the joints, the vast majority of the Vgamma4/Vdelta4(+) cells produced IL-17, a cytokine that appears to be key in the development of CIA. In fact, the number of IL-17-producing Vgamma4(+) gammadelta T cells in the draining lymph nodes was found to be equivalent to the number of CD4(+)alphabeta(+) Th-17 cells. When mice were depleted of Vgamma4(+) cells, clinical disease scores were significantly reduced and the incidence of disease was lowered. A decrease in total IgG and IgG2a anti-collagen Abs was also seen. These results suggest that Vgamma4/Vdelta4(+) gammadelta T cells exacerbate CIA through their production of IL-17.     

Predominance of Vgamma9/Vdelta2 T lymphocytes in the cerebrospinal fluid of children with tuberculous meningitis: reversal after chemotherapy.

BACKGROUND: We analyzed the gammadelta T cell composition and responses in the peripheral blood and cerebrospinal fluid (CSF) of children affected by tuberculous meningitis (TBM) and in control children. MATERIALS AND METHODS: Peripheral blood and CSF samples were stimulated with different phosphoantigens and IL-2, and expansion of Vgamma9/Vdelta2 T cells assessed by FACS analysis. Vgamma9/Vdelta2 lines were obtained by culturing CSF or peripheral blood mononuclear cells (PBMC) in vitro with phosphoantigens and IL-2 for 2 months, and tested for proliferation and cytokine production in response to phosphoantigens. Vdelta2(D)Jdelta junctional sequence length was assessed by PCR. RESULTS: The repertoire of gammadelta T cells from the CSF of TBM patients was characterized by the predominance of Vgamma9/Vdelta2 T lymphocytes, which accounted for >80% of gammadelta T cells. Vgamma9/Vdelta2 cells from the CSF of TBM children responded to different synthetic and natural (mycobacterial) phosphoantigens and produced discrete amounts of IFN-gamma and TNF-alpha. The in vitro expansion of Vgamma9/Vdelta2 T cells from CSF and peripheral blood of TBM patients prominently decreased following chemotherapy, and similarly, the proportion of ex vivo unstimulated Vgamma9/Vdelta2 T cells in CSF of TBM patients decreased to levels detected in the CSF of control subjects. Vdelta2 CDR3 TCR analysis showed that the remaining Vdelta2 cells in the CSF of TBM patients were still polyclonal. CONCLUSIONS: These findings are consistent with an involvement of Vgamma9/Vdelta2 T cells in TBM. http://link. springer-ny.com/link/service/journals/00020/bibs/5n5p301. html     

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.     

An autoreactive gamma delta TCR derived from a polymyositis lesion

To investigate the role of gammadelta T cells in human autoimmune disease we expressed and characterized a gammadelta TCR from an autoimmune tissue lesion. The TCR was first identified in a rare form of polymyositis characterized by a monoclonal infiltrate of gammadelta T cells which invaded and destroyed skeletal muscle fibers. The Vgamma1.3-Jgamma1-Cgamma1/Vdelta2-Jdelta3 TCR cDNA of the original muscle invasive gammadelta T cell clone was reconstructed from unrelated cDNA and transfected into the mouse hybridoma BW58alpha(-)beta(-). Appropriate anti-human gammadelta TCR Abs stimulated the TCR transfectants to produce IL-2, thus demonstrating that the human gammadelta TCR functionally interacted with murine signaling components. The transfected Vgamma1.3/Vdelta2 TCR recognized a cytosolic protein expressed in cultured human myoblasts and TE671 rhabdomyosarcoma cells. The Ag was recognized in the absence of presenting cells. Using a panel of control gammadelta TCR transfectants with defined exchanges in different positions of both TCR chains, we showed that the gammadelta TCR recognized its Ag in a TCR complementarity-determining region 3-dependent way. To our knowledge, this is the first example of a molecularly defined gammadelta TCR directly derived from an autoimmune tissue lesion. The strategy used in this study may be applicable to other autoimmune diseases.     

Phosphostim-activated gamma delta T cells kill autologous metastatic renal cell carcinoma

Metastatic renal cell carcinoma, inherently resistant to conventional treatments, is considered immunogenic. Indeed, partial responses are obtained after treatment with cytokines such as IL-2 or IFN-alpha, suggesting that the immune system may control the tumor growth. In this study, we have investigated the ability of the main subset of peripheral gammadelta lymphocytes, the Vgamma9Vdelta2-TCR T lymphocytes, to induce an effective cytotoxic response against autologous primary renal cell carcinoma lines. These gammadelta T cells were expanded ex vivo using a Vgamma9Vdelta2 agonist, a synthetic phosphoantigen called Phosphostim. From 11 of 15 patients, the peripheral Vgamma9Vdelta2 T cells were amplified in vitro by stimulating PBMCs with IL-2 and Phosphostim molecule. These expanded Vgamma9Vdelta2 T cells express activation markers and exhibit an effector/memory phenotype. They display a selective lytic potential toward autologous primary renal tumor cells and not against renal NC. The lytic activity involves the perforin-granzyme pathway and is mainly TCR and NKG2D receptor dependent. Furthermore, an increased expression of MHC class I-related molecule A or B proteins, known ligands of NKG2D, are detected on primary renal tumor cells. Interestingly, from 2 of the 11 positive cultures in response to Phosphostim, expanded-Vgamma9Vdelta2 T cells present an expression of killer cell Ig-like receptors, suggesting their prior recruitment in vivo. Unexpectedly, on serial frozen sections from three tumors, we observe a gammadelta lymphocyte infiltrate that was mainly composed of Vgamma9Vdelta2 T cells. These results outline that Vgamma9Vdelta2-TCR effectors may represent a promising approach for the treatment of metastatic renal cell carcinoma.     

Mechanisms of Vdelta1 gammadelta T cell activation by microbial components

There are two major subsets of gammadelta T cell in humans. Vgamma2Vdelta2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases. Ags identified for the Vdelta2 T cells are nonpeptide phosphate, amine, and aminobisphosphonate compounds. In contrast, Vdelta1-encoded TCRs account for the vast majority of gammadelta T cells in tissues such as intestine and spleen. Some of these T cells recognize CD1c and MHC class I-related chain (MICA/B) molecules [correction]. These T cells are cytotoxic and use both perforin- and Fas-mediated cytotoxicity. A fundamental question is how these gammadelta T cells are activated during microbial exposure to carry out effector functions. In this study, we provide evidence for a mechanism by which Vdelta1 gammadelta T cells are activated by inflammatory cytokines in the context of the Vdelta1 TCR. Dendritic cells are necessary as accessory cells for microbial Ag-mediated Vdelta1 gammadelta T cell activation. Cytokine (IL-12), adhesion (LFA3/CD2, LFA1/ICAM1) and costimulatory (MHC class I-related chain (MICA/B) molecules/NK-activating receptor G2D) molecules play a significant role along with Vdelta1 TCR in this activation.     

Human V gamma 2V delta 2 T cells augment migration-inhibitory factor secretion and counteract the inhibitory effect of glucocorticoids on IL-1 beta and TNF-alpha production

In immune cells, proinflammatory cytokine gene expression is regulated by glucocorticoids, whereas migration-inhibitory factor (MIF), a pleiotropic cytokine, has the unique property of counteracting the inhibitory effect of glucocorticoids on TNF-alpha and IL-1beta secretion. A few lines of evidence suggest that gammadelta T cells play an important role in immunoregulation. However, it is unknown whether human gammadelta T cells participate in regulating MIF secretion, and how gammadelta T cells, glucocorticoids, and cytokines converge to give a unified physiological response. In this study, we demonstrate that human Vgamma2Vdelta2 T cells augment MIF secretion. Remarkably, these Vgamma2Vdelta2 T cells, functioning similarly to MIF in part, counteracted inhibition of dexamethasone on production of IL-1beta and TNF-alpha. SCID mice reconstituted with human PBMC that were mock depleted of Vdelta2 T cells and repeatedly infected with lethal dose of Escherichia coli had shorter survival time than those reconstituted with PBMC that were depleted of Vdelta2 T cells. Thus, human Vgamma2Vdelta2 T cells are likely to play broad-spectrum roles in immunoregulation and immunopathology by influencing MIF secretion and the immunomodulatory function of glucocorticoids.     

gammadelta T cells contribute to control of chronic parasitemia in Plasmodium chabaudi infections in mice

During a primary infection of mice with Plasmodium chabaudi, gammadelta T cells are stimulated and their expansion coincides with recovery from the acute phase of infection in normal mice or with chronic infections in B cell-deficient mice (mu-MT). To determine whether the large gammadelta T cell pool observed in female B cell-deficient mice is responsible for controlling the chronic infection, studies were done using double-knockout mice deficient in both B and gammadelta cells (mu-MT x delta-/-TCR) and in gammadelta T cell-depleted mu-MT mice. In both types of gammadelta T cell-deficient mice, the early parasitemia following the peak of infection was exacerbated, and the chronic parasitemia was maintained at significantly higher levels in the absence of gammadelta T cells. The majority of gammadelta T cells in C57BL/6 and mu-MT mice responding to infection belonged predominantly to a single family of gammadelta T cells with TCR composed of Vgamma2Vdelta4 chains and which produced IFN-gamma rather than IL-4.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.     

Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.     

Targeting of tumor cells for human gammadelta T cells by nonpeptide antigens.

Human Vgamma2/Vdelta2(+) gammadelta T cells respond to low molecular-mass nonpeptide Ags in a gammadelta TCR-dependent manner. Although requirements of Ag presentation have remained controversial, we have indicated that specific responses of the primary gammadelta T cells to pamidronate were dependent on monocytic adherent cells for Ag presentation. Here, we show that human tumor cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to gammadelta T cells, inducing specific proliferation and IFN-gamma production. gammadelta TCR dependency of the response to Ag-pulsed tumor cells was confirmed by using a Jurkat line transfected with a Vgamma2/Vdelta2 gammadelta TCR. Furthermore, gammadelta T cells exhibited markedly enhanced cytotoxicity against the Ag-pulsed tumor cells as compared with untreated tumor cells. Survey of a number of human tumor cell lines of different origins revealed that the majority of them became susceptible for gammadelta T cell-mediated cytotoxicity following the Ag pulsing except for breast cancer lines so far examined, while normal PHA blast cells remained resistant. The results not only imply a unique mode of nonpeptide Ag recognition by human gammadelta T cells but also may provide a novel strategic clue for immunotherapy of human malignancy.     

Lymphocyctes Tgammadelta in clinically normal skin and peripheral blood of patients with systemic lupus erythematosus and their correlation with disease activity

Human Tgammadelta lymphocytes constitute from 1 to 15% of all peripheral blood lymphocytes. Recent work has demonstrated that this population plays a major role in the pathogenesis of infectious and immune diseases. Increased numbers of gammadelta T cells have been found in affected skin from systemic sclerosis and chronic cutaneous lupus erythematosus patients. In our study, we have determined the numbers of Tgammadelta lymphocytes and their subpopulations in peripheral blood from 29 patients with systemic lupus erythematosus (SLE) and in 19 healthy volunteers using flow cytometry and specific monoclonal antibodies. The same cells in uninvolved skin from SLE patients and human controls using immunohistochemical analysis were estimated. T-Cell receptor (TCR) delta chain gene rearrangement was identified with primers for Vdelta1, Vdelta2 and Vdelta3 by the polymerase chain reaction. Statistical analysis showed a significantly decreased number of gammadelta T cells in SLE patients (26.4+/-16.9/microl) compared with the control group (55.3+/-20.6/microl (p < 0.001). The number of Vdelta2 TCR+ and Vgamma9 TCR+ subpopulations was also lower in SLE patients than in healthy persons. No statistical correlation between disease activity and the number of gammadelta T cells was demonstrated. The percentage of Tgammadelta lymphocytes in clinically normal skin from SLE patients was twice (22.0+/-9.4%) that found in the skin from healthy persons (11.1+/-5.5%) (p < 0.002). Higher percentages of the Vdelta2 TCR+ and Vgamma9 TCR+ subpopulation of lymphocytes were found in the skin from SLE patients. We have also found positive correlation between the percentage of Tgammadelta lymphocytes in skin and the activity of SLE (r=0.594, p < 0.001), and between subpopulation Vdelta3 TCR+ and disease activity (r=0.659, p< 0.001). In conclusion, the results of our studies demonstrate that, in patients with SLE, accumulation of Tgammadelta lymphocytes can be seen in clinically normal skin, and the percentage of these cells correlates with the activity of the disease.     

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.     

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.