Clonal anergy in self-reactive alpha/beta T cells is abrogated by heat-shock protein-reactive gamma/delta T cells in aged athymic nude mice

Although T cells proliferate and differentiate primarily in the thymus, athymic nude mice contain an appreciable level of T cell receptor alpha/beta and gamma/delta T cells, suggesting the existence of the extrathymic pathway in the development of both T cells. Recent studies with nude mice indicate that clonal deletion of self-reactive T cells does not occur extrathymically. In the present study, we have investigated the responsiveness of self-reactive T cells differentiating along an extrathymic pathway in aged BALB/c (H-2d, Mls-1b2a, I-E+, 7-8 month old) nude mice. Consistent with recent reports, T cells bearing V beta 3 or V beta 11, which are important for recognizing proteins encoded by the Mls-2a or the I-E allele, respectively, are readily detected in age nude mice. The V beta 3- or V beta 11-bearing T cells, however, do not proliferate in response to staphylococcal enterotoxin A which specifically stimulates V beta 3- or V beta 11-bearing T cells. When exogenous recombinant interleukin 2 was added to the culture, the V beta 3-bearing T cells in aged nude mice significantly proliferated in response to staphylococcal enterotoxin A. Aged nude mice also contained a substantial level of gamma/delta T cells which account for 15.6% of all Thy-1.2+ cells. The gamma/delta T cells proliferated and produced a significant level of interleukin 2 in response to the 65-kDa mycobacterial heat-shock protein, which is highly homologous to its eukaryotic counterpart. These results suggest that unresponsiveness of self-reactive T cells may be reversed by T cells responding to stress proteins expressed by the invading microbes and/or the stressed autologous cells.     

gamma delta T cells play a crucial role in the expression of 65,000 MW heat-shock protein in mice immunized with Toxoplasma antigen.

Toxoplasma gondii is an obligate intracellular protozoan parasite and cellular immunity plays a crucial role in protection against infection with this pathogen. When mice are immunized with Toxoplasma homogenate, they readily acquire resistance against infection with a lethal dose of a low virulence Beverley strain of T. gondii. We have reported previously that expression of 65,000 MW heat-shock protein (hsp 65) in host macrophages closely correlates with protective potentials of hosts, while this protein is not expressed in Toxoplasma themselves. In this study, we examined the mechanism of expression of hsp 65 in mice immunized with Toxoplasma homogenate. Heat-shock protein was detected in peritoneal macrophages of BALB/c mice immunized 7 days previously by electroblot assay with a specific monoclonal antibody (mAb) for microbial hsp 65. Furthermore, an immunogold ultracytochemistry assay demonstrated that this protein was expressed on the cell surface of peritoneal macrophages in immune mice. This expression was not induced in those of immune athymic nude mice and SCID mice. Treatment of BALB/c mice with anti-Thy-1.2 mAb 1 day before immunization led to an almost complete loss of the expression of hsp 65. To determine the subsets of T cells responsible for induction of this protein, mice were depleted of gamma delta T cells, alpha beta T cells, CD4+ T cells or CD8+ T cells by treating with corresponding antibodies before immunization. From these experiments, gamma delta T cells were shown to be essential for the expression of hsp 65, although CD4+ alpha beta T cells also contributed to some extent. Thus, gamma delta T cells appear to play an important role in protective immunity against infection with T. gondii through mediating the expression of hsp 65 in host macrophages.     

Contribution of extrathymic gamma delta T cells to the expression of heat-shock protein and to protective immunity in mice infected with Toxoplasma gondii.

We demonstrated that gamma delta T cells contribute to protective immunity against Toxoplasma gondii by inducing the expression of a 65,000 MW heat-shock protein (hsp 65) in host macrophages. Here we examined the role of extrathymic and intrathymic gamma delta T cells in protective immunity and hsp 65 expression in mice infected with T. gondii. Intrathymic gamma delta T cells were obtained from severe combined immunodeficiency (SCID) mice grafted with syngeneic fetal thymus (TG-SCID), in which only T cells derived from the donor thymus developed, whereas extrathymic gamma delta T cells were obtained from nude mice that lack thymus. Extrathymic gamma delta T cells from T. gondii-infected nude mice differed from intrathymic gamma delta T cells of infected TG-SCID mice, in terms of Thy1.2 expression and V-region gene usage of T-cell receptor (TCR) gamma delta. Extrathymic gamma delta T cells expressed extremely high levels of Thy1.2, and had V gamma 7 repertoire but lacked V gamma 5,6 and V delta 1,5. On the other hand, intrathymic gamma delta T cells express intermediate and low levels of Thy1,2. These cells possessed V gamma 5,6 and V delta 1,5 but failed to rearrange the V gamma 7 gene. Peritoneal macrophages from infected nude mice contained hsp 65, whereas this protein was scarcely expressed in those of infected TG-SCID mice. Transfer of extrathymic, but not of intrathymic gamma delta T cells to SCID mice enabled their macrophages to express hsp 65. Athymic nude mice were significantly resistant to the infection compared with SCID mice which lack gamma delta T as well as alpha beta T cells. The resistance was dependent upon extrathymic gamma delta T cells, since nude mice depleted of gamma delta T cells using a corresponding monoclonal antibody became extremely susceptible. These results indicated that extrathymic rather than intrathymic gamma delta T cells play some crucial roles in protection against T. gondii and in hsp 65 expression.     

Chagas' disease is attenuated in mice lacking gamma delta T cells.

The role of gamma delta T cells in the immunopathology of Chagas' disease is evaluated by monitoring the course of Trypanosoma cruzi infection in mice lacking gamma delta T cells after disruption of the T-cell receptor C delta locus. Levels of parasitemia, states of lymphocyte activation, and levels of lymphokine production as well as tissue pathology are compared in delta knockout mice and their littermates in acute and chronic phases of infection. Although the levels of circulating parasites do not significantly differ in the two groups, mortality scores and numbers of inflammatory lesions of skeletal and cardiac muscles are lower in gamma delta T cell-deficient m ice than in littermate controls. Furthermore, polyclonal lymphocyte activation, as measured by proliferative activities and numbers of B- and T-cell blasts in the spleen, are reduced in deficient mice in the acute and chronic phases of infection. Levels of gamma interferon mRNA obtained from total spleen cells, known to be a critical lymphokine in resistance to T. cruzi infection, are significantly higher in uninfected gamma delta T cell-deficient mice than in control animals and slightly above levels for littermates in the course of acute infection. Interestingly, however, in chronic phases, the levels of this lymphokine are not statistically different between the two groups of mice. These results indicate that gamma delta T cells do not play a crucial role in parasite clearance during the acute phase of the disease but contribute to the mechanisms leading to tissue damage and pathology.     

Kinetics of accumulation of gamma delta receptor-bearing T lymphocytes in mice infected with live mycobacteria.

The kinetics of accumulation of T cells bearing the gamma delta heterodimer form of the T-cell receptor in mice infected with live Mycobacterium bovis BCG or M. tuberculosis was studied. Substantial numbers of gamma delta T cells accumulated in mice given primary mycobacterial infections, although this accumulation was in parallel to, but not preferential to, that of alpha beta receptor-bearing T cells. In contrast, no accumulation of gamma delta cells was observed in memory immune mice upon rechallenge, thus suggesting that gamma delta cells play no role in the anamnestic response. The results of the study show, further, that large accumulations of gamma delta T cells can also be induced by inoculation with oil adjuvant vehicles containing heat-killed mycobacteria, although not by inoculation of the heat-killed bacteria alone.     

In vivo induction of gamma/delta T cells with highly potent and selective anti-tumor cytotoxicity.

High frequencies of CD5+TcR alpha/beta- T cells were induced in the peritoneal cavity of rats immunized with syngeneic W439 lymphoma cells. These TcR alpha/beta- cells expressed TcR delta mRNA as analyzed by the polymerase chain reaction technique. The delta + (TcR gamma/delta +) T cells were of the CD2+, CD3+, CD4-, CD8+, CD45RB+ phenotype and showed stronger anti-tumor cytotoxicity compared to the TcR alpha/beta + T cells. The cytotoxic effects of both alpha/beta and gamma/delta T cells were selective for the W439 lymphoma cells and were not directed to other syngeneic tumors, natural killer targets and syngeneic or allogeneic normal cells. T cells, including both alpha/beta and gamma/delta cells, were induced when WF rats were immunized with allogeneic BN spleen cells. In this case the gamma/delta T cells showed allo-selective cytotoxicity, although weaker compared to the TcR alpha/beta + T cells. The gamma/delta T cells, induced by immunization with either W439 cells or BN spleen cells, were selective for the immunogen used and had no effect on irrelevant target cells, indicating that these effector cells were not activated by a shared gamma/delta T cell-related superantigen. Since highly potent tumor-selective gamma/delta cytotoxic T lymphocytes could be induced by syngeneic lymphoma cells, we suggest a role for gamma/delta T cells in the defense against certain types of tumors.     

Increase of gamma/delta T cells in hospital workers who are in close contact with tuberculosis patients.

gamma/delta T cells are likely to participate in the immune response to tuberculous infection in humans. In this study, we carried out an investigation to characterize the responsiveness of gamma/delta T cells from tuberculous patients and healthy individuals to mycobacterial stimulation in vitro. Healthy subjects were assigned to the following two groups: those who had been exposed to tuberculosis (contacts) and those who had not been exposed (noncontacts). The percent gamma/delta T cells in fresh peripheral blood obtained from health care workers who were tuberculin skin test positive and who had constant contact with patients with active tuberculosis (healthy contacts) was significantly higher, whereas healthy noncontacts showed the normal range of gamma/delta T cells. Patients with active pulmonary tuberculosis also had low levels of gamma/delta T cells. HLA-DR antigen-bearing activated gamma/delta T cells were observed in higher percentages among healthy contacts than among healthy noncontacts or patients with pulmonary tuberculosis. In healthy contacts, gamma/delta T cells increased as a percentage of peripheral blood mononuclear cells after in vitro stimulation with purified protein derivative (PPD) tuberculin compared with the percentage of fresh peripheral blood mononuclear cells that they made up, whereas no such increase was observed in patients with tuberculosis or in healthy noncontacts. Phenotypic analysis of the gamma/delta T cells in healthy contacts, which increased in number in vitro in response to PPD, revealed the preferential outgrowth of CD4+ V gamma 2+ gamma/delta T cells. This expansion of gamma/delta T cells by PPD required accessory cells, and it was inhibited by the addition of an antibody against HLA-DR in culture. Proteolytic digestion of PPD showed that gamma/delta T cells increased in number in response to peptide, but not nonpeptide, components of PPD. These findings suggest that gamma/delta T cells, especially CD4+ V gamma 2+ gamma/delta T cells, may participate in the immune surveillance of tuberculous infections in humans.     

Functionally distinct subsets of human gamma/delta T cells.

To determine if effector subsets exist among human gamma/delta T cells, we examined the cytokine production and cytotoxic activity of gamma/delta T cell clones with different accessory molecule phenotypes, V delta and V gamma gene expression, and J gamma rearrangements. T cell clones bearing gamma/delta T cell receptor produce an array of cytokines like alpha/beta T cell clones. Individual gamma/delta T cell clones produced a characteristic array of cytokines without correlation with V delta or V gamma gene expression. However, when phenotypic subsets were considered, CD4+ gamma/delta clones produced significantly higher levels of interleukin 2 and granulocyte-monocyte colony-stimulating factor compared with CD4-CD8- and CD8+ gamma/delta clones. Similarly, when cytotoxic potential was assessed, CD4+ gamma/delta clones exhibited minimal activity when compared with CD4-CD8- and CD8+ adult peripheral blood gamma/delta clones. We conclude that functionally distinct gamma/delta T cell subsets exist and suggest that these subsets may correlate with expression of the CD4 accessory molecule.     

Biology of murine gamma delta T cells.

Murine lymphocytes express either a T-cell receptor alpha beta or a gamma delta heterodimer. The function of alpha beta cells are well characterized, while gamma delta cells remain an enigmatic population. In the mouse, gamma delta cells appear in significant proportions in the epithelia of various nonlymphoid tissues such as the skin, intestine, tongue, lung, and reproductive organs. While gamma delta T-cell subsets with distinct antigen receptor repertoires are associated with certain organs, diversified populations of gamma delta cells showing heterogeneous TCR phenotypes, as a result of junctional region diversification and usage of different V chains, can be found in the lymphoid organs and in the intestinal epithelia. Recent evidence has shown that gamma delta cells might recognize heat shock proteins, possibly in association with classical and nonpolymorphic MHC molecules. Together with their tissue distribution, gamma delta cells may represent the first line of defense of the immune system. gamma delta Cells are the first T cells to colonize the thymus. Intriguingly, there is more evidence to support the hypothesis that they might also affect the development of alpha beta cells and other hematopoietic stem cells.     

Development and selection of gamma delta T cells.

The gamma delta T-cell population, a subpopulation of T cells formed through cell lineages that are independent of the alpha beta T-cell lineage, consists of multiple subsets with distinct receptor repertoires and homing properties. While the cell sublineage is a critical factor in the determination of homing specificity, both cell sublineage and receptor-dependent selection are instrumental in the determination of the functional repertoire.     

Predominant appearance of gamma/delta T lymphocytes in the liver of mice after birth

gamma/delta T lymphocytes residing in the liver of mice were systematically characterized with respect to their age-related variation, phenotype and V gene segment usage of gamma/delta T cell receptor (TcR). Previous human and murine studies have shown that a high proportion of gamma/delta T cells reside in the liver and that such liver gamma/delta T cells have lymphoblastic morphology and can spontaneously proliferate in vitro. In the present study, a predominant appearance of gamma/delta T cells (up to 23% among CD3+ cells) in the liver was confirmed in 4-week old mice of various strains. gamma/delta T cells in the liver preferentially co-expressed CD8 antigens, whereas the vast majority of gamma/delta T cells in the spleen lacked the CD8 antigens. The identification of gamma/delta T cells in various lymphoid and non-lymphoid organs also revealed the liver to be one of the organs where gamma/delta T cell are most abundant. The level of such liver gamma/delta T cells showed a clear age-related variation. In the fetal stage and just after birth, gamma/delta T cells were not detectable in the liver (less than 0.2%). However, a significantly higher percentage of gamma/delta T cells among both the total population of mononuclear cells and CD3+ cells was detected in the liver of young 2- to 8-week-old mice; this percentage subsequently declined. As the total number of liver mononuclear cells increased in aged mice, the absolute number of liver gamma/delta T cells also increased as a function of age. V gene segment usage analysis by the polymerase chain reaction method demonstrated that V gamma 1 or V gamma 2/V delta 6 were preferentially used by liver gamma/delta T cells. The age-related increase of gamma/delta T cells was more prominent in the liver of athymic nude mice, and such gamma/delta T cells highly co-expressed the CD8 antigens and also utilized the V gamma 1 or V gamma 2/V delta 6 for gamma/delta Tcr. The predominant appearance of unique gamma/delta T cells in the liver, which was inversely related to the existence of the thymus, indicates that these gamma/delta T cells may differentiate extrathymically in the liver.     

The role of gamma/delta T cells in the feto-maternal relationship.

Polymorphic MHC is absent from the trophoblast, therefore, it resists NK as well as CTL-mediated lysis in vitro. Activated gamma / delta TCR positive cells are significantly enriched in the decidua as well as in peripheral blood of healthy pregnant women. Human peripheral gamma / delta lymphocytes preferentially express the V gamma 9/V delta 2 TCR, whereas those of the decidua use the V delta 1 chain. These subpopulations are functionally polarized, the former being Th1, the latter Th2. Potentially cytotoxic V delta 2+ lymphocytes recognize HLA-E on the trophoblast via the CD94/NKG2A receptor, which induces an inhibitory signal, thus potentially inhibiting Th1 type cytokine production.     

Characterization of coaggregation between Bacteroides gingivalis T22 and Fusobacterium nucleatum T18.

Bacterial adherence is a key factor in the colonization of the oral ecosystem, yet little is known about the mechanisms by which the pathogen Bacteroides gingivalis adheres in the periodontal environment. We examined the ability of strains of B. gingivalis to coaggregate with selected microorganisms isolated from the subgingival microbiota of the cynomolgus monkey. A strong interaction was demonstrated between strains of B. gingivalis and Fusobacterium nucleatum, whereas less pronounced or no interaction was observed with other oral isolates. Electron microscopic examination of coaggregates revealed large masses of bacteria, in which the fusiform F. nucleatum T18 and coccobacillary B. gingivalis T22 cells formed a woven pattern. To investigate this interaction and the nature of the bacterial cell surface molecules involved, we used a microcoaggregation assay. Galactose and galactose-related sugars blocked coaggregation, in contrast with the lack of effect of glucose or glucose-related sugars. The ability of F. nucleatum T18 cells to coaggregate was diminished by pretreatment with pronase. Pretreatment of B. gingivalis T22 cells with pronase resulted in an inhibition of coaggregation, whereas pretreatment with sodium metaperiodate completely abolished coaggregation. These data suggest that the coaggregation between B. gingivalis T22 and F. nucleatum T18 represents a carbohydrate-lectin interaction, mediated by a galactose-containing carbohydrate on B. gingivalis T22 and a protein on F. nucleatum T18.     

Virus-induced demyelination in nude mice is mediated by gamma delta T cells

Infection of mice with mouse hepatitis virus (MHV), strain JHM, results in acute and chronic demyelination with many similarities to the human disease multiple sclerosis. This pathological process is primarily T cell-mediated and MHV infection of mice lacking B and T cells does not result in demyelination. In apparent contradiction to these results, robust demyelination is detected in MHV-infected young nude (athymic) mice. Herein, we show that demyelination in nude mice was mediated by gamma delta T cells. These cells, but not conventional CD4 or CD8 alpha beta T cells, were detected in the central nervous system of MHV-infected nude mice and their depletion with neutralizing antibody resulted in an 80% reduction in demyelination. These results show, for the first time, that gamma delta T cells can substitute for alpha beta T cells in a virus model of demyelination and further support a pathological role for gamma delta T cells in patients with multiple sclerosis.     

Analysis of gamma delta T cells in the chicken.

T cell development in birds and mammals is remarkably similar. One hallmark of these similarities is the division of T cells into two sublineages characterized by different T cell receptor (TCR) molecules--the gamma delta and alpha beta TCRs. These two sublineages differ in several ways besides the molecular form of the TCR, such as the mechanisms involved in TCR repertoire selection and their pattern of tissue localization. The extensive similarity between the avian and mammalian gamma delta T cells suggest that the separation of these two lineages is relatively ancient evolutionarily. The advantages of the avian system as a developmental model have allowed information to be obtained on the gamma delta T cells which may be of general relevance.     

Specificity of gamma delta receptor bearing T cells.

T cells expressing the gamma delta receptor heterodimer can recognize a broad array of different antigens, including classical and non-classical major histocompatibility complex (MHC) proteins, MHC-like CD1 antigens, and bacterial antigens such as the mycobacterial heat shock proteins and staphylococcal enterotoxins. Reactivity to self antigens including autologous stress proteins implicates TCR gamma delta T cells in autoimmune disease. It is as yet unclear whether the responses of gamma delta T cells specific for soluble proteins are restricted by conventional or non-classical MHC molecules. Correlations of TCR gamma delta usage with specificity suggest that, like TCR alpha beta, sequences encoded within both the V regions and the V(D)J junctions are important in determining receptor specificity.     

The role of gamma/delta T cell receptor positive cells in pregnancy.

PROBLEM: Due to the lack of classical HLA antigens on the trophoblast, fetal antigens are possibly presented in a non major histocompatibility complex (MHC) restricted way. Decidual gammadelta T cells, which significantly increase in number during pregnancy, might play a role in recognition of fetal antigens and also in determining the quality of the response to these antigens. Our study was aimed at investigating the role of this cell population in progesterone-dependent immunomodulation. METHOD OF STUDY: Peripheral lymphocytes from healthy pregnant women and from habitual aborters were tested by immunocytochemistry for the presence of gamma/delta T cell receptor (TCR) and progesterone receptor. To investigate the effect of treatment with a pan anti gamma/delta antibody, lymphocytes were incubated for 3 hr with the antibody, and then interleukin (IL)-10, IL-12 and progesterone-induced blocking factor (PIBF) expression (by immuno-cytochemistry) as well as natural killer (NK) cell activity were determined. RESULTS: In peripheral blood of healthy pregnant women the percentage of gamma/delta TCR+ cells was significantly higher (P < 0.001) than in that of recurrent aborters or of non-pregnant individuals. Ninety-seven percent of gamma/delta TCR+ pregnancy lymphocytes expressed progesterone receptor. Binding of a specific antibody to the gamma/delta TCR inhibited PIBF- as well as IL-10 production, whereas it increased NK activity and IL-12 expression. CONCLUSIONS: These data suggest the role of gamma/delta TCR-bearing lymphocytes in progesterone-dependent immunomodulation.     

Differences in the induction of tumorspecific T suppressor lymphocytes among the peritoneal exudate cells in histocompatible BALB/cAnPt and BALB/cJ mouse sublines.

The BALB/c inbred strain of mice exists in several sublines, two of which are of special interest to tumorimmunologists. BALB/cAnPt mice develop plasmacytomas upon intraperitoneal injection of mineral oil in 61% of the animals. BALB/cJ mice on the other hand are relatively resistant and only a few animals develop tumors. The cellular immune response of BALB/cAnPt mice towards the transplantable plasmacytoma ADJ-PC-5 is characterized by the dominance of specific T suppressor (Ts) lymphocytes, when an immunization protocol is used which mimics some aspects of early stages of tumorigenesis by increasing exponentially the antigenic tumor load. Application of this in vivo induction protocol for Ts cells reveals that ADJ-PC-5-specific Ts cells capable of suppressing the generation of an in vitro cytotoxic response can be induced in BALB/cAnPt but not in BALB/cJ mice. No subline difference could be found when specific Ts cells were induced in vitro. The data point towards a subline difference of in vivo cell interactions rather than T cell repertoire composition.     

Role of gamma delta T cells in inflammatory bowel disease.

gammadelta T cells have previously been shown to play a protective role in various animal models of chronic inflammation (e.g., experimental autoimmune encephalomyelitis, collagen-induced arthritis, and non-obese diabetes). This immunoregulatory potential is exerted by synthesizing various anti-inflammatory cytokines and growth factors (e.g., transforming growth factor-beta). As the normal balance between inflammatory and regulatory cytokines is perturbed in inflammatory bowel disease (IBD) a protective effect of gammadelta T cells seems likely. This notion is supported by our finding of increased mortality of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis following gammadelta T cell depletion. In contrast, no effect was observed after depletion of gammadelta T cells in a Crohn's disease animal model with terminal ileitis (TNF(DeltaARE) mice). Therefore, future studies must further define where in the intestinal immune system gammadelta T cells exert their protective function and how this can be used in the treatment of IBD.     

Involvement of gamma delta T cells in immunity to trypanosomiasis.

In this study the involvement of peripheral gamma delta T cells, prepared by flow cytometry, in the immune response of cattle to primary infection with Trypanosoma congolense was assessed. Negligible in vitro proliferative responses were observed in gamma delta T cells isolated from trypanosusceptible Boran (Bos indicus) cattle at all stages examined post-infection when stimulated in vitro with parasite antigens. In contrast, both CD8+ T cells and gamma delta T cells from trypanotolerant N'Dama (Bos taurus) cattle proliferated markedly when stimulated in vitro with a complex of invariant trypanosome antigens with MW between 100,000 and 140,000 (100,000 MW complex). Neither species of cattle exhibited significant T-cell recognition of trypanosome variable surface glycoprotein (VSG). To study further the functional and phenotypic characteristics of the gamma delta T-cell response, four T-cell lines were established from infected N'Dama cattle. These cell lines were comprised of up to 96% gamma delta (WC1+) T cells, the remainder being CD8+ T cells. Two of these gamma delta T-cell lines exhibited 100,000 MW complex antigen specificity which was not major histocompatibility complex (MHC) restricted in one line.