Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.     

Imbalances in T cell subpopulations in multiple sclerosis patients.

Abnormal proportions of a distinct T cell subpopulation able to bind IgG immune complexes (T.G cells) were found in peripheral blood samples from patients with MS. About 50% of the patients examined had an overabundance of T.G cells. The possible role of these cells in the pathogenesis of MS is considered.     

Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes

In this study, we demonstrate that malignant mature CD4(+) T lymphocytes derived from cutaneous T cell lymphomas (CTCL) variably display some aspects of the T regulatory phenotype. Whereas seven cell lines representing a spectrum of primary cutaneous T cell lymphoproliferative disorders expressed CD25 and TGF-beta, the expression of FOXP3 and, to a lesser degree, IL-10 was restricted to two CTCL cell lines that are dependent on exogenous IL-2. IL-2, IL-15, and IL-21, all of which signals through receptors containing the common gamma chain, induced expression of IL-10 in the IL-2-dependent cell lines as well as primary leukemic CTCL cells. However, only IL-2 and IL-15, but not IL-21, induced expression of FOXP3. The IL-2-triggered induction of IL-10 and FOXP3 expression occurred by signaling through STAT3 and STAT5, respectively. Immunohistochemical analysis of the CTCL tissues revealed that FOXP3-expressing cells were common among the CD7-negative enlarged atypical and small lymphocytes at the early skin patch and plaque stages. Their frequency was profoundly diminished at the tumor stage and in the CTCL lymph node lesions with or without large cell transformation. These results indicate that the T regulatory cell features are induced in CTCL T cells by common gamma chain signaling cytokines such as IL-2 and do not represent a fully predetermined, constitutive phenotype independent of the local environmental stimuli to which these malignant mature CD4(+) T cells become exposed.     

Alpha genes of the T cell receptor: a possible implication in genetic susceptibility to multiple sclerosis

Multiple sclerosis (MS) is a neurological disease in which 60% of patients are DR2 (versus 20% in controls). Restriction fragment length polymorphism (RFLP) associated with T cell receptor alpha-chain and beta-chain genes have been analysed in a sample of 46 MS patients and compared with those of 142 controls. The alpha-chain gene polymorphism is localized to the V-J region and consists of 3 Bgl II alleles (alpha a = 3.2 kb; alpha b = 2.9 kb; alpha c = 2.8 kb). A significant difference was found in the distribution of these three alleles since 97% of DR2 patients versus 60% in DR2 non-MS individuals were found to be homozygotes alpha a/alpha a. These results suggest the influence of T cell antigen receptor germ line repertoire on the etiopathology of this disease.     

Questionable relevance of gamma delta T lymphocytes in renal cell carcinoma

Adoptive gammadelta T cell immunotherapy has moved briskly into clinical trials prompted by several small studies suggesting abundant accumulation of gammadelta T cells within renal cell carcinoma (RCC). In this study, we re-examined levels of gammadelta T cells within RCC tumors and correlated levels of these cells with pathologic features and outcome associated with this form of cancer. Tissues from 248 consecutive clear cell RCC tumors obtained from 2000 to 2003 were stained and quantified for total CD3+ and gammadelta T cells per mm2. Wilcoxon rank sum and Kruskal-Wallis tests were used to evaluate associations between T cell amounts and prognostic factors (age, gender, tumor size, stage, grade, tumor necrosis). Cox models were used to assess associations with RCC-specific death. Median numbers of total CD3+ and gammadelta T cells were 281/mm2 (interquartile range (IQR): 149-536) and 2.6/mm2 (IQR: 1.3-4.6), respectively. The median percentage of CD3+ T cells that were gammadelta T cells was 1.0% (IQR: 0.4-1.9). This low percentage of intratumoral gammadelta T cells was diluted even further with rising CD3+ T cell infiltration. Percentages of gammadelta T cells were not associated with even one single clinicopathologic feature examined. Median follow-up for this study was 3.1 years (48 patients died of RCC) and Cox analysis failed to demonstrate that gammadelta T cells (hazard ratio=1.02, p=0.25) were predictive of RCC-specific death. gammadelta T cells are rare and not recruited nor expanded within RCC tumors. Percentages of gammadelta T cells fail to correlate with any prognostic features of RCC nor specific death. As such, the role of gammadelta T cells in RCC immunobiology remains questionable.     

Gamma/delta T cell receptor positive T cells in the inflammatory joint: lack of association with response to soluble antigens

In patients with inflammatory synovitis, the proliferative response by lymphocytes from synovial fluid to soluble mycobacterial antigens is enhanced relative to those from peripheral blood. Earlier studies suggested that gamma/delta T cell receptor positive (TCR+) T lymphocytes may significantly contribute to the mycobacterial-specific synovial fluid response. We therefore examined the relationship of the T cell proliferative response to Mycobacterium tuberculosis antigens and the presence of gamma/delta TCR+ T cells employing several monoclonal antibodies. No consistent increase of gamma/delta TCR+ T cells was noted in inflammatory synovial fluids or tissues. Nonetheless, lymphocytes from the majority of the synovial fluids proliferated vigorously in response to water-soluble M. tuberculosis antigens. There was no relationship between the percentage of gamma/delta TCR+ T lymphocytes and the intensity of the proliferative response. In contrast, stimulation with whole mycobacterial organisms was capable of enriching the gamma/delta TCR+ cell population obtained from the peripheral blood of tuberculosis skin test positive normal controls and from some inflammatory synovial fluids. These observations do not support a role for mycobacteria reactive gamma/delta TCR+ synovial T lymphocytes in response to soluble mycobacterial antigens or in the local pathogenesis of inflammatory synovitis.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

A susceptibility locus for multiple sclerosis is linked to the T cell receptor beta chain complex

Inheritance of T cell receptor beta chain (TCR beta) genes was analyzed in families of 40 sibling pairs concordant for the relapsing-remitting form of multiple sclerosis (MS). TCR beta haplotypes were determined by segregation analysis of polymorphic markers within the TCR beta complex. The mean proportion of TCR beta haplotypes identical by descent (IBD) inherited by MS sibling pairs was significantly increased compared with expected values (means test, p less than 0.004), whereas the distribution of haplotype sharing was random when MS patients were compared with their unaffected siblings. Furthermore, one allelic form of a TCR beta variable region gene segment was overrepresented on MS chromosomes compared with those parental chromosomes not transmitted to MS offspring both in the MS sibling pair families and in a second group of families containing only one individual affected with MS. These results demonstrate that a gene within the TCR beta complex or a closely linked locus influences susceptibility to MS.     

Stimulation of a major subset of lymphocytes expressing T cell receptor gamma delta by an antigen derived from Mycobacterium tuberculosis

To investigate the possible function(s) of T cell receptor (TcR) gamma delta expressing lymphocytes, we generated a series of gamma delta TcR surface positive hybridomas. Spontaneous producers of IL-2 were quite common among these hybridomas, particularly those expressing a certain V delta gene or gene family (V delta M23). Several other experiments indicated that IL-2 production in these hybridomas is triggered via TcR gamma delta. Surprisingly, every spontaneously reactive gamma delta+ hybridoma was further stimulated by purified protein derivative (PPD) of Mycobacterium tuberculosis, perhaps due to crossreaction with a bacterial antigen homologous to certain eukaryotic heat shock proteins. The finding of an antigen recognized by a gamma delta TcR could aid in understanding the functional role of the gamma delta TcR+ lymphocytes.     

Quantitative analysis of the role of receptor recycling in T cell polarization

Activation of T cells of the immune system involves recognition of the antigen by the T cell receptor and subsequent internalization and recycling of this receptor. We present a numerical model for this process that accounts for the polarity of the intracellular traffic determined by the polarization of the microtubule-organizing center to the immunological synapse. Unexpectedly, the model explains the observed accumulation of receptors at the immunological synapse mainly as dynamic maintenance of the receptor density there, while the surface receptors everywhere else are depleted, even though the internalization occurs primarily at the synapse. In the case of an unsuccessful polarization of the microtubule-organizing center, which alters the polarity of the receptor trafficking, the model explains the absence of receptor accumulation as a dynamic downregulation at the synapse. The experiment shows that in this case the interaction of the T cell with its target is aborted. Disruption of recycling leads in the experiment to accumulation of the incompletely polarized cells. We propose that receptor recycling is a mechanism whereby the cell can sense its internal structure and detect polarity errors, analogous to checkpoint signaling mechanisms that ensure fidelity of cell division.     

Idiotype-specific T lymphocytes responsible for the selection of somatic variants of a B cell hybrid

A B cell hybrid (2C3E1), which when cultivated in vitro stably expresses a serologically defined private idiotype on the cell surface, undergoes a somatic variation which culminates in the generation of idiotype-negative variants when propagated as a tumor in syngeneic BALB/c mice. Lyt-1+ and Lyt-2- BALB/c T cells derived from appropriately primed spleen cells when co-cultured with 2C3E1 tumor cells in vitro are responsible for the generation or selection of the idiotype-negative tumor variants. The idiotype-specific effector T cells responsible for the variant production in vitro and in vivo are triggered either by viable or irradiated 2C3E1 tumor cells, but not by soluble idiotype-positive monoclonal antibody secreted by the tumor. The idiotype-positive antibody did stimulate the production of the effector T cells when the soluble protein was covalently linked to the plasma membrane of BALB/c spleen cells, but not when the idiotype-positive protein was presented on allogeneic C57BL/6 spleen cells. A single exposure of spleen cells to 2C3E1 cells in vivo was sufficient to prime the idiotype-specific T cells, but the frequency and reproducibility of variant selection increased when the in vivo primed effector T cells were restimulated by irradiated 2C3E1 cells in vitro prior to co-cultivation with the wild-type 2C3E1 cells. The initial variant B cell lines could be segregated into several different phenotypes. However, after extensive cultivation either in vitro or after repeated transfer in vivo, all of the variant cell lines acquired a single stable phenotype that was characterized by the loss of both the surface and secreted idiotype marker and antigen-binding activity associated with the wild-type 2C3E1 cells.     

Sequence diversity, natural selection and linkage disequilibrium in the human T cell receptor alpha/delta locus

T cell receptors (TR), through their interaction with the major histocompatibility complex, play a central role in immune responsiveness and potentially immune-related disorders. We resequenced all 57 variable (V) genes in the human T cell receptor alpha and delta (TRA/TRD) locus in 40 individuals of Northern European, Mexican, African-American and Chinese descent. Two hundred and eighty-four single nucleotide polymorphisms (SNPs) were identified. The distribution of SNPs between V genes was heterogeneous, with an average of five SNPs per gene and a range of zero to 15. We describe the patterns of linkage disequilibrium for these newly discovered SNPs and compare these patterns with other emerging large-scale datasets (e.g. Perlegen and HapMap projects) to place our findings into a framework for future analysis of genotype–phenotype associations across this locus. Furthermore, we explore signatures of natural selection across V genes. We find evidence of strong directional selection at this locus as evidenced by unusually high values of F _st Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Gamma delta T cell responses associated with the development of tuberculosis in health care workers

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

Critical role for BIM in T cell receptor restimulation-induced death

Background

Upon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals.

Results

Upon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption.

Conclusion

Our data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis.

Reviewers

This article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr.     

Biochemical analysis of heat-resistant mouse tumor cell strains: a new member of the HSP70 family.

A series of heat-resistant mutants selected from a murine tumor cell line, RIF-1, display a markedly increased and stable resistance to heat shock. The mutant cell lines were analyzed for differences that may explain their increased resistance. Membrane lipid analysis showed no change in cholesterol content but an increase in the proportion of saturated fatty acids in the phospholipid fraction. Two-dimensional gel analysis revealed a generally increased constitutive synthesis of several major heat shock proteins (HSP), including HSP90, 68, 60, and 28. In addition, a new protein in the 70-kilodalton region is present in the resistant lines. The new protein has a lower isoelectric point than the constitutive HSP70 does, is only weakly induced by heat shock, and is immunologically cross-reactive with other members of the HSP70 family. After heat shock, the mutants display increases in HSP similar to those seen in the wild-type cells and they develop further transient tolerance to heat. Analysis of these mutants may help in understanding the function of HSP, both in normal growth and after heat shock.     

Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining

Nucleotide sequences of a large number of V-(D)-J junctions of T cell receptor (TCR) gamma and delta genes show that most fetal thymocytes express on their surface one of just two gamma delta TCRs known to be expressed by epidermal gamma delta T cells (s-IEL) or intraepithelial gamma delta T cells associated with female reproductive organs (r-IEL). In contrast, gamma delta TCRs expressed on adult thymocytes are highly diverse as a result of multiple combinations of gene segments as well as junctional deletions and insertions, indicating that developmental time-and cell lineage-dependent mechanisms exist that control the extent of gamma delta TCR diversity. In addition, this study revealed a new type of junctional insertion (P nucleotides), which led to a new model of V-(D)-J joining generally applicable to immunoglobulin and TCR genes.