Release of LL-37 by activated human Vgamma9Vdelta2 T cells: a microbicidal weapon against Brucella suis

Human Vgamma9Vdelta2 T cells play a crucial role in early immune response to intracellular pathogens. Moreover, in brucellosis, these cells are drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vgamma9Vdelta2 T cells are capable of inhibiting Brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial effects. We previously described that antibacterial factors were found in supernatants from activated Vgamma9Vdelta2 T cells. In this study, we show that Vgamma9Vdelta2 T cells express the human cathelicidin hCAP18 and its mature form, known as LL-37, is released upon activation of Vgamma9Vdelta2 T cells. We also show that LL-37 has an antibacterial effect on Brucella suis. Overall, our results demonstrate that LL-37 is a soluble factor responsible for a part of the bactericidal activity of Vgamma9Vdelta2 T cells.     

Production of TNF-alpha by human V gamma 9V delta 2 T cells via engagement of Fc gamma RIIIA, the low affinity type 3 receptor for the Fc portion of IgG, expressed upon TCR activation by nonpeptidic antigen

Human lymphocytes expressing the gammadelta TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vgamma9Vdelta2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vgamma9Vdelta2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vgamma9Vdelta2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vgamma9Vdelta2 T cells express FcgammaRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vgamma9Vdelta2 T cells. Indeed, ligation of CD16 on Vgamma9Vdelta2 T cells leads to TNF-alpha production. This TNF-alpha production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vgamma9Vdelta2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcgammaRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vgamma9Vdelta2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.     

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.     

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.     

Specific signaling pathways triggered by IL-2 in human V gamma 9V delta 2 T cells: an amalgamation of NK and alpha beta T cell signaling

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vgamma9Vdelta2 T cells represent a minor T cell subpopulation in blood (1-5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and alphabeta T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vgamma9Vdelta2 T cells behave as NK cells or as alphabeta T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in alphabeta T cells. Moreover, IL-2 induces the production of IFN-gamma in Vgamma9Vdelta2 T cells as observed in NK cells. Due to their double profiles, Vgamma9Vdelta2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.     

Innate T-cell immunity in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes

There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases. Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity. NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication. The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence. Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients. These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART). In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response. Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication. Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.     

CXCR5 identifies a subset of Vgamma9Vdelta2 T cells which secrete IL-4 and IL-10 and help B cells for antibody production

Vgamma9Vdelta2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microorganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of CXCR5 identifies a unique subset of Vgamma9Vdelta2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2, IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5+ Vgamma9Vdelta2 T cells as a distinct memory T cell subset with B cell helper function.     

In vivo gammadelta T cell priming to mycobacterial antigens by primary Mycobacterium tuberculosis infection and exposure to nonpeptidic ligands.

BACKGROUND: The recognition of phosphorylated nonpeptidic microbial metabolites by Vgamma9Vdelta2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vgamma9Vdelta2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vgamma9Vdelta2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of gammadelta T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured. RESULTS: The Vgamma9Vdelta2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vgamma9Vdelta2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of gammadelta T cell activation in vivo. The in vivo reactivity of Vgamma9Vdelta2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vgamma9Vdelta2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children. CONCLUSIONS: The increased reactivity of Vgamma9Vdelta2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vgamma9Vdelta2 responses may be useful for monitoring the efficacy of antimycobacterial therapies.     

Central memory Vgamma9Vdelta2 T lymphocytes primed and expanded by bacillus Calmette-Guérin-infected dendritic cells kill mycobacterial-infected monocytes

In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and gammadelta T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of Vgamma9Vdelta2 T cells (the major subset of gammadelta T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, Vgamma9Vdelta2 T cells increase both their expression of CD69 and CD25 and the production of TNF-alpha and IFN-gamma, in contrast to DC treated with Vgamma9Vdelta2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory Vgamma9Vdelta2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of Vgamma9Vdelta2 T cells in the control of mycobacterial infection.     

Drug-induced expansion and differentiation of V gamma 9V delta 2 T cells in vivo: the role of exogenous IL-2

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.     

TNF-alpha is a positive regulatory factor for human Vgamma2 Vdelta2 T cells

Vgamma2 Vdelta2 T cells in human peripheral blood recognize phosphoantigen and play important roles in host defense and immunoregulation. The TCR is required for Vgamma2 Vdelta2 T cell responses to phosphoantigen, but less is known about soluble or cell-associated costimulatory molecules. In this study, we show that human Vgamma2 Vdelta2 T cell responses to phosphoantigen, including activation, proliferation, cytokine production, and tumor cell cytotoxicity, require TNF-alpha binding to its receptor, with a preference for TNFR2. Because stimulated Vgamma2 Vdelta2 cells also produce TNF-alpha, this may be a positive control mechanism to sustain the response. Impaired proliferation in the presence of TNF-alpha or TNFR blocking agents was partially rescued by a TLR2 agonist, Pam(3)Cys. Our studies demonstrate that TNF-alpha plays a critical role in regulating human Vgamma2 Vdelta2 T cell immune responses.     

Differentiation of human gamma-delta T cells towards distinct memory phenotypes

Vgamma9/Vdelta2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vgamma9/Vdelta2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vgamma9/Vdelta2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-alpha, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vgamma9/Vdelta2 T cells grown with IL-21 differentiated into putative central memory CD45RO(+) T cells that did not produce TNF-alpha, IFN-gamma, or IL-4, and maintained expression of CD62L, CD81, and CCR7.     

Synthetic phosphoantigens enhance human Vgamma9Vdelta2 T lymphocytes killing of non-Hodgkin's B lymphoma.

BACKGROUND: Non-Hodgkin's B lymphomas (NHL) are often resistant to conventional treatments and, until now, immunotherapeutic approaches against NHL only aimed at inducing anti-tumor effectors. Nevertheless, human blood Vgamma9Vdelta2 T lymphocytes represent an abundant pool of cytotoxic tumor-reactive cells. Vgamma9Vdelta2 T cells are strongly activated by natural compounds, from which powerful synthetic ligands have been derived. These synthetic antigens induce efficient Vgamma9Vdelta2 T cell responses in vitro. MATERIALS AND METHODS: We set up a series of Vgamma9Vdelta2 T cell-activation experiments, including cytotoxic activity and amplification from whole blood cells. Several types of Vgamma9Vdelta2 effectors were challenged against a panel of 16 B lymphoma cell lines. These tests have been performed in the absence and presence of -specific synthetic ligands to evaluate the effect of such molecules on anti-tumor activity. RESULTS: We report here that Vgamma9Vdelta2 T cells recognize B lymphomas. This recognition is associated with the cytotoxic activity against B-lymphoma cells and/or proliferative responses, and appears to be T-cell antigen receptor (TCR)-dependent. Because few B lymphoma induce a complete set of Vgamma9Vdelta2 cell responses, a chemical ligand of Vgamma9Vdelta2 T cells was used to enhance both proliferation and cytotoxic activity of anti-B lymphoma effectors. We show that such synthetic compound improves Vgamma9Vdelta2 CTL numbers and lysis of B lymphoma lines, especially when the targets are already spontaneously recognized by these effectors. CONCLUSIONS: We report here that human Vgamma9Vdelta2 T cells anti-B lymphoma response can be improved by use of specific synthetic ligands, which enhance their cytotoxic activity and allows their rapid expansion ex vivo.     

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.     

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.     

Lack of CD27-CD45RA-V gamma 9V delta 2+ T cell effectors in immunocompromised hosts and during active pulmonary tuberculosis.

In humans, the circulating pool of mycobacteria-reactive Vgamma9Vdelta2+ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of Vgamma9Vdelta2+ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated gammadelta T cells have lost CD27 expression. In contrast, the CD27-CD45RA-Vgamma9Vdelta2+ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating Vgamma9Vdelta2+ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN-gamma-producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting gammadelta T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.     

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     

Tumor necrosis factor-alpha production is differently regulated in gamma delta and alpha beta human T lymphocytes

Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the early defense against pathogens. This cytokine is produced by several cell types including T lymphocytes expressing the alphabeta as well as the gammadelta T cell receptor (TcR). In human, the circulating gammadelta T cells, which mostly express Vgamma9Vdelta2 TcR, have been strongly suggested to play an important protective role against infectious agents. These activated cells early produce high amounts of TNF-alpha, which induce a determinant beneficial effect against development of intracellular pathogens; however, sustained production of this cytokine can result in immunopathological diseases. The signals that regulate TNF-alpha production in Vgamma9Vdelta2 T cells are totally unknown. In primary alphabeta T cells, TNF-alpha production was shown to necessitate engagement of the TcR and CD28, and to be independent of the p38 mitogen activated protein kinase pathway. We demonstrate herein that, in contrast to alphabeta T cells, TNF-alpha production in Vgamma9Vdelta2 T lymphocytes is independent of CD28 costimulation and highly dependent on TcR-induced p38 kinase and extracellular signal-regulated kinase 2 pathway activation for optimal cytokine release. Moreover, we bring elements supporting the idea that the "activation threshold" of gammadelta T cells leading to cytokine production is lower than that of alphabeta T cells.     

Functional and structural similarity of V gamma 9V delta 2 T cells in humans and Aotus monkeys, a primate infection model for Plasmodium falciparum malaria.

Gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.     

Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vgamma2Vdelta2 T cells, molecular mechanisms by which HMBPP interacts with Vgamma2Vdelta2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vgamma2Vdelta2 TCR of rhesus macaques to define HMBPP/APC interaction with Vgamma2Vdelta2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vgamma2Vdelta2 TCR tetramer. The Vgamma2Vdelta2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vgamma2Vdelta2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vgamma2Vdelta2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vgamma2Vdelta2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vgamma2Vdelta2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vgamma2Vdelta2 TCR.