Oligoclonally expanding gammadelta T lymphocytes induce IgA switching in IgA nephropathy

The aetiology of IgA nephropathy (IgAN) is closely related with abnormality of mucosal immunity. We investigated the roles of γδ T cells in the regulation of IgA production by B cells in IgAN patients. The proportion of γδ T cells in peripheral blood mononuclear cells (PBMNC) was higher in IgAN patients than in the controls and was found to be correlated with the proportion of surface IgA‐positive (sIgA+) B cells, which are precursors of IgA‐secreting plasma cells. After in vitro PWM stimulation, sIgA expression on B cells and IgA production were significantly enhanced in PBMNC obtained from IgAN patients, whereas the enhancements were abolished by removal of γδ T cells from the PBMNC. Purified γδ T cells from IgAN patients induced surface IgA expression on naïve sIgD+ B cells more effectively than did αβ T cells. Moreover, stimulated γδ T cells from IgAN patients produced a larger amount of TGF‐β1, which is one of the main cytokines that induces IgA class switching on B cells, as compared with αβ T cells and control γδ T cells. The expanded γδ T cells from IgAN patients exclusively expressed Vγ9, and the nucleotide sequences of junctional regions of Vγ9 showed very limited TCR diversities. It was therefore concluded that γδ T cells, which are expanded in response to specific antigens, enhance IgA class switching on B cells in IgAN patients.     

Circulating gamma/delta T lymphocytes from systemic sclerosis (SSc) patients display a T helper (Th) 1 polarization

Systemic sclerosis (SSc) is a connective tissue disease in which immune system activation is evidenced by high levels of different cytokines in the sera and/or in the supernatants of cultured peripheral blood mononuclear cells (PBMC) and by the presence of specific autoantibodies. gamma/delta T cells accumulate in the lung and the skin of SSc patients suggesting their potential role in the development and maintenance of the disease. The aim of this study was to assess cytokine production and cytotoxic activity of circulating gamma/delta T lymphocytes obtained from SSc patients and to evaluate their potential role during this disorder. Our results showed that both the proportion and the absolute number of IFN-gamma gamma/delta-producing cells (i.e. displaying a Th1 polarization) in SSc was significantly higher than either the proportion and the absolute number of IL-4 gamma/delta-producing cells in SSc or the proportion and the absolute number of IFN-gamma gamma/delta-producing cells in healthy controls (P < 0.05 for both groups). Furthermore, the cytotoxic activity of enriched gamma/delta T cells was significantly increased in SSc patients compared with controls. The results concerning the Vdelta1+ T cell subset paralleled those of total gamma/delta T lymphocytes. In contrast, alpha/beta T cells from SSc and control subjects displayed Th2 cytokine production. All these findings were independent of both disease subset and clinical status. Our data demonstrate that, although SSc is generally considered a Th2 autoimmune disease, Th1 polarization of gamma/delta T cells and an increase in their cytotoxic activity is observed in SSc, suggesting that gamma/delta T cells could have a relatively autonomous role in the pathogenesis in this disease.     

The cytotoxic analysis of T cell receptor V delta 1+ T cell lines derived from the synovial fluid of rheumatoid arthritis patients.

We established six human T cell lines derived from rheumatoid arthritis synovial fluid (RASF). Phenotypically, T cell receptor (TCR) gamma delta T cells occupied the majority of these lines and most of them expressed the TCR V delta 1 molecule. In contrast, V delta 2+ T cells, the majority population of peripheral blood gamma delta T cells, were rarely detected in these lines. To study the immunobiological roles of RASF V delta 1+ T cells in RA development, their cytotoxic profile was studied. The results showed that these T cells selectively lysed Daudi, but not K562 cells. The cytotoxic response was MHC-unrestricted, and was inhibited by anti-CD3 MoAb. Moreover, the cold target inhibition assay showed that the cytotoxicity was competitively inhibited by autologous and allogeneic primarily cultured RA synovial cells as well as synovial sarcoma and chondrosarcoma lines. However, PBL did not inhibit this cytotoxicity. These data suggest that V delta 1+ T cells in RASF may recognize the antigen which is commonly expressed on the surface of Daudi and the cells derived from RA synovium. We can assume that the cytotoxic V delta 1+ T cells are selectively expanded in RASF, playing a significant role for the pathogenesis of certain RA cases.     

Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation

gammadelta T cells commonly associate with mucosal and epithelial sites, fulfilling a variety of immunoregulatory functions. While lung gammadelta T cells have well-characterized pro-inflammatory activity, their potential role in the resolution of lung inflammation has yet to be explored in any detail. Indeed, given the importance of minimizing inflammation, the cellular mechanisms driving the resolution of lung inflammation are poorly understood. Using a murine model of acute Streptococcus pneumoniae-mediated lung inflammation, we now show that resolution of inflammation following bacterial clearance is associated with a > 30-fold increase in gammadelta T-cell number. Although inflammation eventually resolves in TCR delta(-/-) mice, elevated numbers of alveolar macrophages and pulmonary dendritic cells, and the appearance of well-formed granulomas in lungs of TCR delta(-/-) mice, together indicated a role for gammadelta T cells in regulating mononuclear phagocyte number. Ex vivo, both alveolar macrophages and pulmonary dendritic cells were susceptible to lung gammadelta T cell-mediated cytotoxicity, the first demonstration of such activity against a dendritic cell population. These findings support a model whereby expansion of gammadelta T cells helps restore mononuclear phagocyte numbers to homeostatic levels, protecting the lung from the consequences of inappropriate inflammation.     

Gamma/delta T cell subsets in patients with active Mycobacterium tuberculosis infection and tuberculin anergy

Earlier data suggest that gamma/delta T cells may play an important role in the immune response to Mycobacterium tuberculosis. The aim of this study was to determine the percentage of different gamma/delta subsets in peripheral blood of active tuberculosis patients with a positive or negative tuberculin reaction. Thirty-eight patients infected with M. tuberculosis and 22 healthy controls were included in the study. Venous blood was taken before starting antimycobacterial treatment. Lymphocytes were reacted with monoclonal antibodies specific for different gamma/delta V chains (Vdelta1, Vdelta2, Vgamma9 and Vgamma4). The results were analysed in the context of tuberculin reactivity and X-ray findings. Our results revealed a selective loss of Vgamma9/Vdelta2 T cells in the peripheral blood of tuberculin-negative patients with active tuberculosis compared to healthy controls, while the ratio of Vgamma9/Vdelta2 T cells in the peripheral blood of patients with a positive skin test did not differ from that of healthy controls. These findings demonstrate a relationship between the loss of the major M. tuberculosis-reactive subset of gammadelta T cells and the absence of tuberculin reactivity. The data are consistent with the hypothesis that gammadelta T cells play a role in the protective immune response to M. tuberculosis infection.     

Increase in the proportion of granulated CD56+ T cells in patients with malignancy.

Evidence is presented for the existence of a unique T cell population which expressed one of the natural killer (NK) markers, CD56 antigen, in humans. Although such CD56+ T cells were a minor population in the peripheral blood (< 10%), they were abundant in the liver (up to 50%), which was recently demonstrated to be a major organ for extrathymic T cell differentiation in mice. As in the case of extrathymic T cells in mice, these CD56+ T cells in humans contained a higher proportion of gamma delta T cells than did CD56- T cells, contained double-negative CD4-8- cells, and had the morphology of large granular lymphocytes. This unique population of CD56+ T cells tended to be elevated in the blood and among tumour-infiltrating lymphocytes in patients with colorectal cancer, especially in advanced cases. These results raise the possibility that, as in mice, CD56+ T cells with extrathymic T cell properties may also be associated with tumour immunity in humans.     

Transient inhibition of Th1-type cytokine production by CD4 T cells in hepatitis B core antigen immunized mice is mediated by regulatory T cells

The non-cytopathic hepatitis B virus (HBV) can induce chronic infections characterized by weak and limited T cell responses against the virus. The factors contributing to the failure to clear HBV and subsequent development of chronic HBV infections are not clearly understood, but a strong interferon-gamma (IFN-gamma) response by CD4+ T cells against the nucleocapsid hepatitis B core antigen (HBcAg) of the virus appears to be important for viral clearance. The present study documents depressed numbers of CD4+ T cells secreting IFN-gamma and interleukin-2 (IL-2) in enzyme-linked immunospot assay (ELISPOT) assays restimulated for 24 hr with antigen following both primary and secondary immunizations of mice with recombinant hepatitis B core antigen (rHBcAg). The kinetics of these responses showed that the depression occurred following a peak response and lasted approximately 2 weeks before returning to the previous peak levels. The depression was abrogated by depletion of CD25+ cells prior to culture in the ELISPOT assay, suggesting inhibition by regulatory T cells. This inhibition of IFN-gamma and IL-2 production was also reversed by in vitro restimulation of the test cells for 48 hr rather than 24 hr in the assay. No such transient, reversible inhibition was detected in the production of IL-5, a Th2-type cytokine. The inhibition in cytokine production did not appear to correlate with the number of antibody-secreting cells or the isotypes produced. This delay by regulatory T cells of Th1-type cytokine production could contribute to viral persistence in chronic HBV infection by interfering with the critical role IFN-gamma plays in protection against viral infections.     

T and B cell responses following immunization with tetanus toxoid in IgA nephropathy.

The B and T cell responses were investigated in IgA nephropathy before and after immunization with tetanus toxoid (TT). Both IgA and IgG anti-tetanus toxoid antibodies were elicited, but the IgA antibodies were significantly greater in patients (92.6 +/- 11.7 ELISA units) than in the controls (49.2 +/- 7.5 ELISA units). This was associated with a significantly greater proportion of IgA+ B cells in patients than controls before immunization. However, a significant increase in the proportion of IgA1 binding CD4 and CD8 cells was also found. The proportion of CD3 cells with gamma delta T cell receptors (CD3+TCR gamma delta +), was significantly greater before immunization in the IgA nephropathy patients (37.0% +/- 2.4), compared with controls (10.0% +/- 2.3; P less than 0.001). Immunization with TT further enhanced the CD3+TCR gamma delta + cells in patients to 45.8% +/- 7.2 compared with controls (16.3% +/- 4.5), with a corresponding decrease in CD3+TCR alpha beta + cells in the patients (P less than 0.001). CD3+TCR gamma delta + cells are upregulated by common microbial antigens and clinical exacerbations of IgA nephropathy are frequently associated with mucosal infections and a rise in serum IgA concentration. The increased TCR gamma delta expression may be responsible for the enhanced IgA antibody response in IgA nephropathy. The increase in IgA antibodies may than exert a controlling effect by binding to augmenting T cells and thereby inhibiting their function.     

Human T cells in hu-PBL-SCID mice proliferate in response to Daudi lymphoma and confer anti-tumour immunity.

In vitro culture of human peripheral blood lymphocytes (PBL) with Daudi (Burkitt lymphoma) cells results in selective proliferation of V gamma 9/V delta 2 T cells with high cytotoxicity against Daudi cells. After adoptive transfer into severe combined immunodeficient (SCID) mice, these cells exert specific anti-tumour activity against Daudi lymphoma. To test whether cytotoxic V gamma 9/V delta 2 T cells are induced in SCID mice, human PBL injected intraperitoneally were stimulated with irradiated Daudi cells (PBL/Daudi-SCID). After 7-14 days, PBL/Daudi-SCID had a significantly higher percentage of human gamma delta T cells in their peritoneal cavity, lymph nodes and blood than controls (PBL-SCID). DNA content analysis of T cell subsets from PBL/Daudi-SCID showed a significantly higher percentage of cells in S + G2 + M phases of the cell cycle in the TCR-gamma delta-1+ than in CD3+ cell population. Human cells recovered from PBL/Daudi-SCID showed specific cytotoxicity against Daudi cells. PBL/Daudi-SCID inoculated with a lethal dose of Daudi lymphoma survived significantly longer than controls. This protection was specific for Daudi cells and was not mediated by murine natural killer (NK) cells. Thus human peripheral blood T cells grafted in SCID mice proliferate in response to antigen and confer specific immunity.     

Increase of circulating gamma/delta T lymphocytes in the peripheral blood of patients affected by active inflammatory bowel disease.

In order to study the role of gamma/delta T cells in the pathogenesis of inflammatory bowel disease (IBD) in humans, we measured the percentage of these cells in the peripheral blood, assessed the ratio of the non-disulphide-linked (delta TCS1) type of T cell receptor (TCR) in the total gamma/delta T cells, studied the co-expression of gamma/delta TCR and accessory molecules CD8 and CD16, and compared these data with both the type and the activity of the disease. Percentage levels and absolute numbers of gamma/delta+ T cells were higher in active patients than in controls (P < 0.05), mainly as a result of an increase of V delta 1+ (delta TCS1) T cell subset (P < 0.05). This trend was strongly retained independently of disease activity and clinical picture. An increased percentage of TCR delta 1+/CD16+ cells was observed in our patients compared with controls (P < 0.05). In contrast, no difference was observed as far as the TCR delta 1+/CD8+ cells were concerned. These results suggest that IBD is associated with an expansion of gamma/delta T cells in peripheral blood, which may play a role in the pathogenesis of these disorders.     

Analysis of T cell receptors in rheumatoid arthritis: the increased expression of HLA-DR antigen on circulating gamma delta+ T cells is correlated with disease activity.

The phenotypic characteristics of peripheral blood T cells, isolated from 37 rheumatoid arthritis (RA) patients and 17 healthy controls were determined with special emphasis on gamma delta+ T cells and CD4-CD8- alpha beta+ T cells. Two- and three-colour automated flow cytometry analyses were performed using a panel of MoAbs directed against differentiation antigens and T cell receptor molecules. The results demonstrated: (i) no significant difference between the percentages of CD4-CD8- alpha beta+ T cells in patients and controls; (ii) a significant decrease of the gamma delta+ T cell level in the peripheral blood of RA patients relative to controls; (iii) phenotypic abnormalities of circulating gamma delta+ T cells in RA patients suggestive of an activation status in vivo. These abnormalities included a significant reduction in the density of the T cell differentiation antigen CD3 and an increase in the expression of HLA-DR antigen. The level of circulating HLA-DR+/gamma delta+ T cells was significantly higher in patients with active disease. HLA-DR+/gamma delta+ T cells were also present in the synovial fluid obtained from three patients with an active disease. In addition, preliminary experiments showed that the activated gamma delta+ T cells were predominantly V delta 1. Taken together, these data support the involvement of gamma delta+ T cells in the pathogenesis of RA.     

gammadelta-T cells expressing NK receptors predominate over NK cells and conventional T cells in the innate IFN-gamma response to Plasmodium falciparum malaria

Rapid production of interferon-gamma (IFN-gamma) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN-gamma-producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN-gamma-producing cells across a panel of naive human donors following 24-h exposure to live schizont-infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN-gamma-responding cells and that IFN-gamma production is unaffected by NK cell depletion. Instead, gammadelta-T cells represent the predominant source of innate IFN-gamma, with the majority of responding gammadelta-T cells expressing NK receptors. Malaria-responsive gammadelta-T cells more frequently expressed NKG2A compared to non-responding gammadelta-T cells, while non-responding gammadelta-T cells more frequently expressed CD158a/KIR2DL1. Unlike long-term gammadelta-T cell responses to iRBC, alphabeta-T cell help was not required for innate gammadelta-T cell responses. Diversity was observed among donors in total IFN-gamma output. This was positively associated with CD94 expression on IFN-gamma(+) NK-like gammadelta-T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.     

Delivery of antigen in allogeneic cells preferentially generates CD(4+) Th1 cells

We have examined the possibility of evoking antigen-specific T cell immune response by using allogeneic cells as a source of adjuvant and also as a vehicle to deliver antigen. The mice were immunized with different preparations of antigen-pulsed allogeneic and syngeneic splenocytes. It was observed during the study that the animals immunized with antigen-pulsed mitomycin C treated allogeneic cells elicited antigen specific CD(4+) Th1 cell response. Predominant release of IL-2, interferon (IFN)-gamma and IgG2a-isotype also occurred. In contrast, mice immunized with antigen-pulsed syngeneic cells chiefly enhanced the production of interleukin (IL)-4 and IgG1-isotype. Further, allogeneic macrophages induced better T cell response than B cells or splenocytes and prominently induced the expression of B7-1 and B7-2. Immunization with antigen-pulsed macrophages provided better recall responses compared to B cells. This was manifested by the high LFA-1alpha and low CD45RB expression on T cells. Because it is already known that mitomycin C-treated cells undergo apoptosis and dendritic cells engulf apoptotic cells, we therefore propose that generation of T cell response using antigen-pulsed allogeneic cells may be due to the engulfment of these cells by dendritic cells, which may then process and present antigen entrapped in allogeneic cells to activate naive CD(4+) T cells and differentiate them to Th1 cells. This study therefore provides a rational basis for manipulating antigen-specific responses by immunizing with antigen-pulsed allogeneic cells.     

Reasons why DBA/2 mice are resistant to malarial infection: expansion of CD3int B220+ gammadelta T cells with double-negative CD4- CD8- phenotype in the liver

DBA/2 (H-2(d)) mice are known to be more resistant than C57BL/6 (B6, H-2(b)) mice to the non-lethal 17XNL strain of Plasmodium yoelii. This is a very strange phenomenon because the functions of conventional T cells, especially CD8(+) T cells, are known to be somewhat lower in DBA/2 mice than in other strains of mice. We examined herein how immune responses differed between DBA/2 mice and B6 mice during malarial infection. DBA/2 mice and (DBA/2 x B6)F(1) (BDF(1), H-2(b/d)) mice were found to have milder parasitaemia and to recover more quickly from malarial infection than B6 mice. These DBA/2 and BDF(1) mice were also found to experience a marked expansion of interleukin (IL)-2Rbeta(+) CD3(int) cells and gammadelta T cells in the liver, especially in the recovery phase. The expansion of unconventional T cells (i.e. B220(+) T cells) was also marked in DBA/2 and BDF(1) mice. The majority of B220(+) T cells were gammadelta T cells and these T cells were double-negative CD4(-) CD8(-). More importantly, the production of immunoglobulin M (IgM)-type anti-DNA autoantibody was also higher in DBA/2 and BDF(1) mice than in B6 mice. In conjunction with data on cytokine production, these results indicate that primitive T and B cells, namely autoreactive extrathymic T cells and autoantibody-producing B cells, may be much more activated in DBA/2 mice and therefore resistant to the non-lethal 17XNL strain of P. yoelii.     

Regulatory gamma delta T cells in Heymann nephritis express an invariant Vgamma6/Vdelta1 with a canonical CDR3 sequence

Gammadelta T cells are expanded in human IgA nephropathy and in a rat model of adriamycin (ADR)-induced nephropathy. Despite different diseases and species, these renal gammadelta T cells use a restricted set of gammadelta T cell receptor (TCR) genes. To explore whether this phenomenon of post injury expansion of gammadelta T cells occurs in autoimmune-mediated glomerulonephritis, we studied gammadelta TCR genes in Heymann nephritis (HN). Gammadelta T cells were increased in HN kidneys (p<0.001). These gammadelta T cells predominantly expressed Vgamma6/Vdelta1 genes and used canonical matching sequences previously seen in the other models of renal injury. Gammadelta T cells from the kidneys expressed high levels of TGF-beta, IL-4 and IL-5. The gammadelta T cells from both ADR-treated and HN kidneys expressed NKG2D, the NK cell-activating receptor. These results demonstrate that the majority of gammadelta T cells in the HN kidney use a canonical Vgamma6/Vdelta1 TCR--the gammadelta TCR previously described in the rat ADR-treated kidney. The restriction in gammadelta TCR seen in two completely different models of kidney injury and the expression of an innate activating molecule NKG2D suggests that the gammadelta T cells may be responding to tissue stress from injury and producing a regulatory response.     

Evidence for the involvement of lung-specific gammadelta T cell subsets in local responses to Streptococcus pneumoniae infection

Although gammadelta T cells are involved in the response to many pathogens, the dynamics and heterogeneity of the local gammadelta T cell response remains poorly defined. We recently identified gammadelta T cells as regulators of macrophages and dendritic cells during the resolution of Streptococcus pneumoniae-mediated lung inflammation. Here, using PCR, spectratype analysis and flow cytometry, we show that multiple gammadelta T cell subsets, including those bearing Vgamma1, Vgamma4 and Vgamma6 TCR, increase in number in the lungs of infected mice, but not in associated lymphoid tissue. These gammadelta T cells displayed signs of activation, as defined by CD69 and CD25 expression. In vivo BrdU incorporation suggested that local expansion, rather than recruitment, was the principal mechanism underlying this increase in gammadelta T cells. This conclusion was supported by the finding that pulmonary gammadelta T cells, but not alphabeta T cells, isolated from mice that had resolved infection exhibited lung-homing capacity in both naive and infected recipients. Together, these data provide novel insights into the origins of the heterogeneous gammadelta T cell response that accompanies lung infection, and the first evidence that inflammation-associated gammadelta T cells may exhibit distinct tissue-homing potential.     

Gammadelta T cells assist alphabeta T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine

Para-phenylenediamine (PPD) is known to be a common sensitizer of allergic contact dermatitis and contact urticaria. To clarify the mechanism of contact hypersensitivity (CHS) to PPD, we established a mouse model of PPD-induced CHS. BALB/c mice were immunized for 3 consecutive days by painting topically a 2.5% PPD solution on their shaved abdominal skin. On days 5, 7 or 9 after the initial application, the mice were challenged by applications of a 2.5% PPD solution. Maximal ear swelling was determined at 24 h but another statistically significant and smaller ear swelling was observed 1 h after challenge with PPD in a hapten-specific manner. Adoptive cell transfer experiments demonstrated that the ear swelling of the adoptive cell transferred mice displayed an early response at 6 h and a late response from 12 h to 24 h when the recipient mice were challenged immediately after transfer. Both MoAbs and complement treatment of the transferred cells demonstrated that the phenotype of the early response cells which elicited a response at 6 h after challenge was Thy1(+), B220(+), alphabeta TCR(-), gammadelta TCR(-), CD3(-), CD4(-), CD5(+) and CD8(-). The in vitro treatment of effector cells with MoAbs against not only alphabeta TCR but also gammadelta TCR, together with complement, was found to diminish substantially the late response, elicited 12-24 h after challenge. Gammadelta T cells reconstituted the ability of alphabeta T cells to transfer 24 h CHS responsiveness. The phenotype of the gammadelta T cells that assist CHS effector alphabeta T cells was CD3(+), CD4(-) and CD8(+) and these regulatory gammadelta T cells were neither Ag-specific nor MHC-restricted. Furthermore, gammadelta T cells from normal spleen could also assist alphabeta T cells in adoptive transfer of the 24 h CHS response in a non-MHC-restricted manner. RT-PCR demonstrated that alphabeta T cells strongly expressed mRNA IFN-gamma, whereas gammadelta T cells expressed not only IFN-gamma but also IL-4 and IL-10. These data indicate that not only early response cells and alphabeta T cells but also Th2 type gammadelta T cells may play an important role in the elicitation of CHS to PPD.