Contribution of α/β and γ/δ T lymphocytes to immunity against Mycobacterium bovis Bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice

Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guérin (BCG) and the relative contribution of α/β T cells and γ/δ T cells to the host immune response was assessed. Recombinase activating gene (RAG-1)^?/? mutants as well as T cell receptor (TcR) β^?/?, but not TcR-δ^?/?, mutants succumbed to M. bovis BCG infection and failed to develop granulomatous lesions. Antigen-induced IFN-γ production by spleen cells in vitro was abrogated in RAG-1^?/? mutants and markedly diminished in TcR-β^?/? and TcR-δ^?/? mice. Reconstitution experiments suggest that both α/β and γ/δ T cells are essential for antigen-specific IFN-γ secretion. Our data formally prove the crucial role of α/β T cells and reveal accessory functions of γ/δ T cells in optimum immunity against M. bovis BCG.     

Cytotoxic reactivity of gut lamina propria CD4+ αβ T cells in SCID mice with colitis

Polyclonal, mucosa-seeking memory/effector CD4⁺ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe-combined immunodeficient (SCID) mice developing colitis after CD4⁺ T cell transplantation. CD4⁺ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)-2 + IL-7. CD3 ligation enhances this cytokine-supported proliferation in CD4⁺ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine-supported proliferation in CD4⁺ T cells from the gut lamina propria in a cell density- and dose-dependent manner. Almost all CD4⁺ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4⁺ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4⁺ T cells show Fas-dependent cytotoxicity. A large fraction of gut lamina propria CD4⁺ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4⁺ T cells are apoptotic. Hence, a large fraction of colitis-inducing CD4⁺ T cells undergo activation-induced cell death in situ and can damage other cells through Fas-dependent cytotoxicity.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Activated T lymphocytes in the celiac lesion: Non-proliferative activation (CD25) of CD4+ α/β cells in the lamina propria but proliferation (Ki-67) of α/β and γ/δ cells in the epithelium

In order to identify any dominating subset of activated T cells in the celiac lesion, we examined CD3⁺, CD4⁺, CD8⁺ and T cell receptor (TcR) γ/δ⁺ Lymphocytes in jejunal cryosections from 25 patients with celiac disease and 10 controls by three-color immunofluorescence staining for expression of the nuclear proliferation marker detected by monoclonal antibody (mAb) Ki-67 and the p55 α chain of interleukin-2 receptor (CD25). mAb Ki-67⁺ intraepithelial lymphocytes (IEL) were exclusively observed in celiac patients. The median proportion of CD3⁺ IEL positive for Ki-67 increased from nil in controls to 4.5% in partly treated (range 0-19.0%; n = 10; p = 0.05) and 12.8% in untreated celiac disease (range 4.0-30.7%; n = 15; p 0.005). Only 1.5% of CD3⁺ subepithelial T cells expressed the Ki-67 marker in celiac disease (range 0-9.5%). Two- and three-color staining combining mAb to CD3 and Ki-67 with mAb to CD4, CD8 or TcR5 showed that both TcR α/β⁺ CD8⁺ and TcR γδ⁺ (but not CD4⁺) mucosal T cells proliferated in the epithelium. By contrast, CD25 were almost exclusively expressed on CD4⁺ T cells in the lamina propria. The percentage of CD25⁺ T cells increased significantly from 1.7% in controls (range 0-2.9%) to 7.5% in partly treated (range 0.8-17.8%, p 0.002), and to 14.65% in untreated celiac disease (range 3.9-21%, p 0.002). These results suggest that gluten ingestion in celiac disease induces proliferative activation of TcR α/β⁺ CD8⁺ and TcR γδ⁺ IEL but non-proliferative activation (lymphokine production?) of lamina propria CD4⁺ T cells.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Cytokine synthesis and apoptosis by intestinal intraepithelial lymphocytes: Signaling of high density αβ T cell receptor+ and γδ T cell receptor+ T cells via T cell receptor-CD3 complex results in interferon-γ and interleukin-5 production,while low density T cells undergo DNA fragmentation

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3⁺ IEL populations. Stimulation of low- or high-density CD3⁺ IEL via the T cell receptor (TCR)-CD3 complex using monoclonal anti-CD3, anti-αβ TCR or anti-γδ TCR antibodies resulted in opposing effects. In one case, a significant number of the high-density CD3⁺ T cells entered cell cycle from the resting stage (DNA replication was observed) and anti-TCR-CD3 treatment enhanced the numbers of interferon-γ and interleukin-5 spot-forming cells in this cell fraction. In contrast, when the low-density αβ TCR⁺ or γδ TCR⁺ T cells were activated via the TCR-CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR-CD3 complex resulted in their entry into the cell cycle and subsequent interferon-γ and interleukin-5 production in the high-density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low-density fraction of CD3⁺ IEL.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

Single-cell analyses of CD4+ T cells from αβ T cell receptor-transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene-specific antigen

Development of distinct CD4⁺ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double-label in situ hybridization technique was used to characterize co-expression of cytokine mRNA in antigen-specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4⁺ T cells isolated from αβ T cell receptor-transgenic mice. Interleukin (IL)-2 was the dominant cytokine expressed by antigen-stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)-γ were less frequent, and IL-4, IL-5, and IL-10 were infrequent. In contrast, cells that expressed IFN-γ or IL-10 were most frequent in the LP population, with lower frequencies of IL-2, and few IL-4- and IL-5-positive cells. Co-expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene-positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4⁺ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4⁺ T population has a distinct cytokine expression pattern with counter-regulatory properties that may be important for homeostasis in mucosal immune tissues.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

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

Germinal center formation in mice lacking αβ T cells

T cells are essential for inducing clonal B cell expansion in germinal centers during T cell-dependent antibody responses. However, class-switched antibodies are readily detectable in TCRα-deficient mice that congenitally lack αβ T cells, including those such as IgG1 that are considered to be dependent on collaboration between B cells and αβ T cells. This observation suggests that a novel form of B:T collaboration may be evident in TCRα^?/? mice. We report that germinal centers develop spontaneously in mice lacking T cell receptor α genes (TCRα^?/?), despite the absence of αβ T cells. They are not seen in TCRβ^?/? mice kept in similar conditions. Both strains of mice have γδ T cells, but it is a subset of T cells expressing TCRβ and CD4 that is dominant in the germinal centers of TCRα^?/? mice. Exceptionally, germinal centers were associated with CD4⁺ γδ T cells. The expression of CD4 seems to be important, for few extrafollicular T cells have CD4 and CD4 is largely absent from TCRβ^?/? T cells. The CD4⁺ TCRβ cells may help B cells produce autoantibodies that have been identified in TCRα^?/? mice.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

Streptozotocin-induced diabetes in mice lacking αβ T cells

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) α-chain were therefore used to assess whether TCR αβ⁺ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR αβ cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR αβ cells tipping the balance between tolerable and clinically damaging action on islet cells.     

Functional and molecular characterization of B cell-responsive Vδ1+ γδ T cells

Cells expressing the Vδ1⁺ gene segment are a minor γδ T cell population in human peripheral blood but predominate in epithelia and (inflamed) tissues. The characteristic dendritic-like morphology of these γδ T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of Vδ1⁺ γδ T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) γ chain co-expressed with the Vδ1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of Vδ1⁺ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive Vδ1⁺ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the Vδ1⁺ T cells. Finally, we investigated the diversity of the responding Vδ1⁺ γδ T cell clones by sequence analysis of the TcRδ junctional regions. No restricted V-D-J sequences were found among the LCL-responsive Vδ1⁺ T cell clones, arguing strongly against a mono- or oligoclonal Vδ1⁺ γδ T cell response to LCL. These findings may explain the presence of polyclonally activated Vδ1⁺ T cells in inflamed tissues where activated B cells are often present.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

T cells expressing the γδ T cell receptor are not required for egg granuloma formation in schistosomiasis

Immunopathology in schistosomiasis consists of a granulomatous response around parasite eggs. It has been established that granuloma formation is mediated by CD4⁺ T helper cells. However, the role of T cells bearing the γδ T cell receptor (TCR) has not been determined. In this study we utilized mutant mice that lack either αβ or γδ T cells as a result of gene targeting to investigate the relative roles of αβ and γδ T cells in the induction of immunopathology related to schistosomiasis. Mutant and control mice were infected with Schistosoma mansoni and granuloma formation as well as lymph node cell proliferative responses to egg antigens were analyzed after 8 weeks. TCR δ mutant mice (lacking γδ T cells) displayed vigorous formation of egg granulomas that were not significantly different from those observed in normal controls, both in terms of granuloma size and cellular composition. In contrast, TCR α and TCR β mutant mice (lacking αβ T cells) were unable to form granulomas. Moreover, mesenteric lymph node cells from TCR δ mutant and control mice responded strongly to egg antigens in vitro, while TCR α and β mutant mice did not. Our studies show that in schistosomiasis granuloma formation and proliferative responses to egg antigens are strictly dependent on αβ T cells. They also suggest that γδ T cells by themselves can neither mediate a granulomatous inflammation, nor significantly modify one mediated by αβ T cells.     

The role of γδ T cells in priming macrophages to produce tumor necrosis factor-α

The secretion of tumor necrosis factor (TNF)-α from macrophages is regulated by both priming and triggering signals. We found that macrophages from mice lacking γδ T cells [T cell receptor (TCR) δ^?/- mice], which lack the gene encoding the δ chain, produced only small amounts of TNF-α in response to lipopolysaccharide (LPS) and showed a reduced level of expression of CD14. Pre-incubation of macrophages from TCR δ-/- mice with γδ T cells from their TCR δ^+/- littermates restored their capacity to produce TNF-α in response to LPS. The priming activity of γδ T cells was in part inhibited by neutralizing anti-interferon (IFN)-γ monoclonal antibodies. Collectively, these results suggest that γδ T cells play a role in priming macrophages to a steady state of activation via IFN-γ secretion, which allows them to produce TNF-α when exposed to LPS.