The common cytokine receptor gamma chain controls survival of gamma/delta T cells

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.     

MHC-independent presentation of mycobacteria to human gamma delta T cells

The majority of human peripheral gamma delta T cells express antigen receptors using the V gamma 9 and V delta 2 gene products. Cells of this subset have been previously shown to uniformly recognize mycobacteria regardless of their V-(D)-J junctional sequences in an MHC-unrestricted manner. This reactivity superficially resembles activation of alpha beta cells by bacterial superantigens, which are thought to be presented by monomorphic regions of MHC class II molecules. It is not known whether presentation of the mycobacterial antigen to V gamma 9/V delta 2 T cells is also mediated by class II MHC molecules. In order to examine the similarity between presentation of bacterial superantigens to alpha beta T cells and the presentation of mycobacteria to gamma delta T cells we have studied the role of class II MHC molecules in presentation of the mycobacterial antigen AP-MT to V gamma 9/V delta 2 clones. Activation of gamma delta T cells by AP-MT required direct contact with antigen presenting cells, indicating that an interaction with cell surface molecules on antigen presenting cells is required. Class II MHC molecules were neither sufficient nor necessary for effective presentation of AP-MT to the gamma delta T cells, as transfectants expressing class II MHC molecules were unable to present, whereas cell lines lacking expression of MHC class II molecules could present this mycobacterial antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early preferential stimulation of gamma delta T cells by TNF-alpha

Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS. However, gamma delta T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state. As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.     

The gamma delta T lymphocytes of the perinatal murine thymus

The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of methoxytrimethylbenzenesulphonyl (Mtr) and pentamethylchromansulphonyl (Pmc) as arginine side-chain protecting groups in the presence of tryptophan without lateral protection or protected with t-Boc is discussed. The synthetic VP3 peptide has been administered to mice in different forms: (i) free, (ii) coupled to keyhole limpet hemocyanin, (iii) encapsulated in multilamellar (MLV) liposomes, and (iv) incorporated to a tetrameric branched lysine core. The immune response induced by these preparation is reported.     

Crucial role of TCR gamma chain junctional region in prenyl pyrophosphate antigen recognition by gamma delta T cells

Hysteresis of the nasal airway pressure-flow relationship (PFR) is seen during hyperpnea, with lower nasal resistance during increasing inspiratory flow than during decreasing flow. We hypothesized that the nasal PFR hysteresis arose in the nasal vestibule airway because of progressive collapse during the inspiration. We measured the inspiratory transnasal and transvestibular PFR for one nasal passage in five normal subjects breathing via a nasal mask during voluntary hyperventilation, both with voluntary nostril flaring and without flaring. The inspiratory hysteresis (IH) was quantified as the ratio of the areas under the descending and ascending pressure-flow curves. Flaring reduced the vestibular IH from 1.96 +/- 0.06 to 1.15 +/- 0.06 and the nasal IH from 2.05 +/- 0.13 to 1.28 +/- 0.06 (both P < 0.01). Our results demonstrate that hysteresis arises in the compliant vestibule segment of the nasal airway, likely because of progressive collapse of the nasal vestibule during inspiration. The findings suggest that hysteresis is prevented by voluntary nostril flaring maintained throughout inspiration.     

Differential effects of type I IFNs on the growth of WC1- CD8+ gamma delta T cells and WC1+ CD8- gamma delta T cells in vitro

Type I IFNs have a broad array of immunoregulatory functions that include up-regulation of type 1 immune responses through enhancing differentiation and activation of CD8+ T cells and CD4+ Th1 cells. Ovine trophoblast IFN-tau is a recently described type I IFN with the potential for therapeutic use, based on its potent antiviral activity yet low toxicity. Studies were designed to determine the immunoregulatory effects of IFN-tau on Ag-stimulated T cells, and a novel effect of type I IFNs on gammadelta T cells was observed. In cultures of parasite Ag-stimulated bovine T cells that contained a mixture of alphabeta and gammadelta T cells, both IFN-tau and IFN-alpha suppressed the expansion of WC1+ CD2- CD6- CD8- gammadelta T cells, yet stimulated the growth of WC1- CD2+ CD6+ CD8+ gammadelta T cells and CD8+ alphabeta T cells. The CD8+ gammadelta T cell subset expressed high levels of the IL-2R alpha-chain. Furthermore, we showed that type I IFN enhanced IL-2 production by these Ag-stimulated T cell lines. In short term cultures of PBMC, IL-2 stimulated an expansion of WC1- CD6+ CD8+ gammadelta T cells, which was significantly increased by IFN-tau, even though IFN-tau alone did not support cell survival. These studies demonstrate for the first time that type I IFNs differentially modulate the proliferation of different subsets of gammadelta T cells, which appears to act in part via IL-2.     

T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.     

Abscess formation in Listeria monocytogenes-infected gamma delta T cell deficient mouse mutants involves alpha beta T cells

To evaluate the usefulness of the transgenic Muta mouse for investigating radiation-induced mutations in vivo, we have examined the effects of whole-body X irradiation and compared them to the effects of ultraviolet light. The spontaneous mutation frequencies in young adults were about 7 x 10(-5) in the spleen, liver and skin. The mutation frequencies 1 week after a lethal dose of X radiation (8 Gy) were 3.2, 2.6 and 2.7 times the spontaneous levels in the spleen, liver and skin, respectively. When the skin was irradiated with 10 kJ m-2 of UVB, the mutation frequency increased about 6 times. The mutation frequencies induced by an acute dose of 4 Gy or by a fractionated dose of 11.7 Gy (0.15 Gy x 78 times, 3 times/week) in the spleen and liver were less than 2-fold the spontaneous levels at 16 weeks after irradiation. A comparison of X and UV radiation was also conducted with cultured cells derived from the mouse embryo. UVC of 5 J m-2 raised the mutation frequency to 15 times that of unirradiated cells, while 10 Gy X rays raised it 2.6 times. The findings indicate that the Muta mouse is less sensitive to X-ray-induced mutation than UV-induced mutation.     

gamma/delta T cell/endothelial cell interactions

Ruminant gamma/delta T cells exhibit unique patterns of tissue- and inflammation-specific recruitment. Studies of other cells, such as alpha/beta T cells and even neutrophils, have clearly shown that interactions with the vascular endothelium regulate the entry of these cells into tissues. The leukocyte/endothelial cell interaction is a dynamic event that occurs under considerable shear force associated with blood flow, and it involves a variety of adhesion molecules expressed by both the leukocyte and endothelium. We have begun a systematic analysis of gamma/delta T cell interactions with endothelial and other cells in novel in vitro assays that reflect blood flow to gain insight into the molecular basis of the selective recruitment of these lymphocytes to epithelial-associated tissues and sites of inflammation. We have found that bovine gamma/delta T cells in newborns predominantly use the selectin family of leukocyte and vascular adhesion proteins to interact with endothelial cells. This is in direct contrast to other lymphocytes, such as alpha/beta T cells, which predominantly interact with selectins only after conversion to a memory cell phenotype. Our analyses of gamma/delta T cell-selectin interactions will be summarized.     

Lethal murine graft-versus-host disease induced by donor gamma/delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens

Although T-cell receptor (TCR) alpha/beta expressing cells have a well-known role in graft-versus-host disease (GVHD) generation, the role of TCR gamma/delta expressing cells in this process has remained unclear. To elucidate the potential function of TCR gamma/delta cells in GVHD, we have used transgenic (Tg) H-2d mice (termed G8) that express gamma/delta heterodimers on a high proportion of peripheral T cells. In vitro, G8 Tg gamma/delta T cells proliferate to and kill C57BL/6 (B6) (H-2b) which express gene products (T10b and T22b) from the nonclassical major histocompatibility complex (MHC) class Ib H-2T region. The infusion of G8 Tg (H-2Td) TCR gamma/delta cells into lethally irradiated [900 cGy total body irradiation (TBI)] B6 (H-2b) mice resulted in the generation of lethal GVHD characterized histologically by destruction of the spleen, liver, lung, and colon. Lethal GVHD was prevented by the injection of anti-TCR gamma/delta monoclonal antibodies. Immunohistochemical analysis of B6 recipients post-bone marrow transplantation (BMT) confirmed that G8 Tg TCR gamma/delta cells infiltrated GVHD target tissues (skin, liver, colon, and lung) and were absent in recipients treated with anti-TCR gamma/delta monoclonal antibodies (MoAbs) but not anti-CD4 plus anti-CD8 MoAbs. In contrast, injection of TCR gamma/delta+ cells into irradiated (900 cGy TBI) B6.A-TIaa BoyEg mice that do not express either T10b or T22b did not induce lethal GVHD. Similarly, in a different GVHD system in which sublethal irradiation without bone marrow (BM) rescue was used, B6 but not B6.A-TIaa/BoyEg mice were found to be susceptible to TCR gamma delta+ cell mediated GVHD-induced lethality characterized by an aplasia syndrome. These results demonstrate that TCR gamma/delta cells have the capacity to cause acute lethal GVHD in mice and suggest that nonclassical MHC class Ib gene products expressed on GVHD target organs are responsible for G8 Tg TCR gamma/delta+ cell mediated lethality.     

Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, V gamma 9+/V delta 2+ T cells in their blood and lungs. Here we have determined whether this gamma deltaT loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ alpha beta or gamma delta T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of V gamma 9+V delta 2+ peripheral blood T cells from healthy subjects (30-45%) and TB patients (55-68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in gamma delta T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by gamma delta T cells, and in the presence of metalloproteinase-inhibitors >70% of gamma delta T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated gamma delta T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive gamma delta T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of gamma delta T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.     

Intraepithelial gamma delta T cells are closely associated with apoptotic enterocytes in the bovine intestine

The T cell population in the intestinal epithelium, comparable in size to the T cell pool in the spleen, is characterized by the predominant distribution of T cells bearing gamma delta T cell receptors. To determine the functional significance of the intraepithelial lymphocytes, we observed gamma delta T cells present in the jejunal epithelium in cattle, in which there is predominance of gamma delta T cells. Immunohistochemistry of frozen sections demonstrated that gamma delta T lymphocytes were densely distributed in the villous epithelium but there were fewer in the lamina propria and they were not present in the crypt epithelium. Ultrastructurally, intraepithelial gamma delta T cells were characterized by possessing electron-dense granules and interdigitating with enterocyte cytoplasm. Enterocytes, which were inserted by processes of intraepithelial lymphocytes or contacted by their cell bodies, showed morphologic changes seen in apoptotic cell death, such as elevated electron density of the cytoplasm and condensation of the chromatin. Apoptotic cells and cell debris were found in macrophages, which gathered in the subepithelial region of villus tips. These findings suggest that in the small intestine of cattle, gamma delta T cells are involved in the renewal of epithelial cells by inducing apoptosis of epithelial cells.     

Lyme arthritis synovial gamma delta T cells respond to Borrelia burgdorferi lipoproteins and lipidated hexapeptides

Lyme arthritis synovial fluid contains a large proportion of gamma delta T cells that proliferates upon stimulation with the causative spirochete, Borrelia burgdorferi. A panel of Borrelia-reactive gamma delta T cell clones was derived from synovial fluid of two patients with Lyme arthritis. Each of six gamma delta clones from one patient used the V delta 1 TCR segment but had otherwise unique CDR3 sequences and diverse V gamma segment usage. Stimulation of the V delta 1 clones was optimal in the presence of Borrelia, dendritic cells, and exogenous IL-2, which was reflected by proliferation, TCR down-modulation, as well as induction of CD25 and Fas ligand expression. Stimulation by B. burgdorferi-pulsed dendritic cells withstood chemical fixation and was not restricted to class I or class II MHC, CD1a, CD1b, or CD1c. In contrast, anti-gamma delta antibody potently inhibited proliferation. Extraction of B. burgdorferi lipoproteins with Triton X-114 enriched for the stimulatory component. This was confirmed using lipidated vs nonlipidated hexapeptides of Borrelia outer surface proteins. These observations suggest that synovial V delta 1 T cells may mediate an innate immune response to common lipoprotein products of spirochetes.     

Chemokine expression by intraepithelial gamma delta T cells. Implications for the recruitment of inflammatory cells to damaged epithelia

T cells expressing gamma delta TCR may have evolved to recognize Ag in a different manner as well as perform a broader set of functions than T cells with alpha beta TCR. In this study, we tested the hypothesis that dendritic epidermal T cells (DETC) bearing the invariant V gamma 3V delta 1 TCR may be able to signal the migration of peripheral alpha beta T cells to the epidermis by secreting specific chemokines. Expression of macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES, and lymphotactin was inducible in DETC 7-17 cells, whereas mRNA for monocyte chemoattractant protein (MCP)-1 could not be detected. Strikingly, lymphotactin was the most abundant chemokine produced by activated DETC 7-17 cells. Activated primary DETC cultures also produced copious amounts of lymphotactin mRNA. Similarly, freshly isolated and activated intestinal intraepithelial T cells (i-IEL) with gamma delta TCR expressed high levels of lymphotactin mRNA. In contrast, lymphotactin mRNA was present in activated spleen gamma delta T cells at low basal levels. Migration of CD8+ T cells induced by culture supernatants from stimulated DETC 7-17 cells was strongly reduced in the presence of a neutralizing anti-lymphotactin antiserum and to a lesser extent by neutralizing anti-MIP-1 alpha, anti-MIP-1 beta, or anti-RANTES antiserum. The presence of lymphotactin in supernatants from activated DETC 7-17 cultures was directly demonstrated by Western blot analysis. These observations are consistent with a model in which gamma delta IEL play an active multi-faceted role in the maintenance of epithelia homeostasis.     

The response of gamma delta T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules

Peripheral blood gamma delta T cells from non-exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum, in vitro. This response, largely caused by T cells bearing the V gamma 9+ chain of the T-cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of V gamma 9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti-MHC class II antibodies can be overcome by the addition of interleukin-2 (IL-2) to the cultures, suggesting that gamma delta T cells themselves do not recognize MHC class II molecules but require an MHC class II-dependent response taking place in the culture. In contrast, the inhibition by anti-class I antibodies cannot be reversed by addition of IL-2. Since an accompanying CD4+ T-cell response occurred in peripheral blood mononuclear cells cultured with P falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of V gamma 9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched gamma delta T cells to P falciparum schizont extract by addition of purified CD4+ T cells.     

Local expansion of allergen-specific CD30+Th2-type gamma delta T cells in bronchial asthma

BACKGROUND: T lymphocytes infiltrating airways during the allergic immune response play a fundamental role in recruiting other specialized cells, such as eosinophils, by secreting interleukin 5 (IL-5), and promoting local and systemic IgE synthesis by producing IL-4. Whether these presumed allergen-specific T cells are of mucosal or systemic origin is still a matter of conjecture. MATERIALS AND METHODS: Immunophenotype, IL-4 production, and in vitro proliferative response to specific or unrelated allergens were analyzed in the bronchoalveolar lavage (BAL) fluid lymphocyte suspensions obtained from untreated patients with allergic asthma. Healthy subjects and patients affected by pulmonary sarcoidosis, a granulomatous lung disease characterized by infiltrating Th1 CD4+ lymphocytes, served as controls. RESULTS: The proportions of gamma delta T lymphocytes, mostly CD4+ or CD4- (-)CD8-, was higher in asthmatic subjects than in controls (p < 0.05). Most BAL gamma delta CD4+ lymphocytes of asthmatic patients displayed the T cell receptor (TCR)-gamma delta V delta 1 chain. While CD30 antigen coexpression on the surface of BAL alpha beta(+) T lymphocytes was low (ranging from 5 to 12%), about half of pulmonary gamma delta T cells coexpressed it. These cells produced IL-4 and negligible amounts of interferon-gamma (IFN gamma), and proliferated in vitro in response to purified specific but not unrelated allergens. In contrast, control or sarcoidosis gamma delta T cells never displayed the CD30 surface molecule or produced significant quantities of IL-4. CONCLUSIONS: These findings not only confirm our previous hypothesis that the allergen-specific Th2-type lymphocytes found in the lungs of asthmatic patients are gamma delta T cells belonging to airway mucosal immunocytes, but also strongly support the notion that asthma is a local rather than a systemic disease.