Human alpha beta and gamma delta thymocyte development: TCR gene rearrangements, intracellular TCR beta expression, and gamma delta developmental potential--differences between men and mice

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development, molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells, indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame, suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast, in alphabeta cells, the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were, however, depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell, suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway, an instructional role for the TCR in the alphabeta/gammadelta lineage choice, and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci, the timing of beta selection, and maintenance of gammadelta potential through the early double positive stage of development.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

Early TCR alpha beta expression promotes maturation of T cells expressing Fc epsilon RI gamma containing TCR/CD3 complexes

In a previous study we presented data indicating that the expanded population of CD4(-)CD8(-) (DN) alphabeta T cells in TCRalpha-chain-transgenic mice was partially if not entirely derived from gammadelta T cell lineage cells. The development of both gammadelta T cells and DN alphabeta T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN alphabeta T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRalpha-chain is expressed by precursor thymocytes already at the CD3(-)CD4(-)CD8(-) (triple negative, TN) CD44(+)CD25(-) stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRbeta-chains matured into DN alphabeta T cells at several stages of development. The genes encoding the gamma-chain of the high affinity receptor for IgE (FcepsilonRIgamma) and the CD3zeta protein were found to be reciprocally expressed in TN thymocytes such that during development the FcepsilonRIgamma expression decreased whereas CD3zeta expression increased. Furthermore, in a fraction of the transgene-induced DN alphabeta T cells the FcepsilonRIgamma protein colocalized with the TCR/CD3 complex. These data suggest that similarly to gammadelta T cells and NKT cells, precursors expressing the TCR early in the common alphabetagammadelta developmental pathway may use the FcepsilonRIgamma protein as a signaling component of the TCR/CD3 complex.     

Biochemical evidence for the presence of a single CD3delta and CD3gamma chain in the surface T cell receptor/CD3 complex

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.     

Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Leptospira interrogans activation of human peripheral blood mononuclear cells: preferential expansion of TCR gamma delta+ T cells vs TCR alpha beta+ T cells

Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-gamma, IL-12, and TNF-alpha. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6-8 days, and the majority of cells contained in these cultures were gamma/delta T cells. These cultures showed a 10- to 50-fold expansion of gamma/delta T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce gammadelta T cell or NK cell expansion, but induced significant alphabeta T cell expansion. Vgamma9/Vdelta2 were expressed on all gamma/delta T cells expanded by exposure of PBMC to Leptorspira: Leptospira stimulation of purified TCRgammadelta(+) T cells, obtained from 8-day cultures of Leptospira-stimulated PBMC, induced high levels of IFN-gamma production, but no cell proliferation, suggesting that such stimulation of gammadelta T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCRgammadelta(+) T cells. These results indicate that Leptospira can activate gammadelta T cells and alphabeta T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

Regulation of T cell-dependent autoantibody production by a gammadelta T cell line derived from lupus-prone mice

Lupus-prone (MRLxC57BL/6) F(1) mice lacking gammadelta T cells show more severe lupus than their T cell-intact counterparts, suggesting that gammadelta T cells down-modulate murine lupus. To determine the mechanisms for this effect, we assessed the capacity of gammadelta T cell lines derived from spleens of alphabeta T cell-deficient MRL/Mp-Fas(lpr) (MRL/Fas(lpr)) mice to down-regulate anti-dsDNA production generated by CD4(+)alphabeta T helper cell lines and activated B cells from wild-type MRL/Fas(lpr) mice. One line, GD12 (gd TCR(+), CD4(-)CD8(-)), had the capacity to reduce anti-dsDNA production in a contact-dependent manner. GD12 also killed activated MRL/Fas(lpr) (H-2(k)) B cells, with less cytolysis of resting B cells than that generated by in comparison to cytokine-matched gammadelta T cell lines. In addition, GD12 also killed activated B cells derived from C57BL/6-Fas(lpr) (H-2(b)) or beta(2)-microglobulin (beta(2) M)-deficient MRL/Fas(lpr) mice, suggesting cytolysis was neither MHC- nor CD1-restricted. Killing by GD12 was inhibited by anti-TNFalpha and anti-TNF-R1, and partially blocked by anti-gd TCR Fab fragments, but not by anti-FasL, anti-TNF-R2 (p75) or concanamycin A. IL-10 produced by GD12 also partially inhibited alphabeta Th1-dependent but not alphabeta Th2-dependent autoantibody production. These findings prove that we have identtified a gammadelta T cell line that suppresses autoantibody synthesis by alphabeta T-B cell collaboration in vitro.     

Activation and control of self-reactive gammadelta T cells

Mammalian and avian CD3+ T cells can be separated into two lymphocyte subsets bearing heterodimeric T-cell receptors (TCR) composed of either alphabeta or gammadelta chains. Although it is now widely accepted that gammadelta and alphabeta T cells fulfill mandatory and nonredundant roles, the generality of this assumption and the exact functions played by gammadelta T cells remain uncertain. While an early protective role of gammadelta T cells has long been suspected, recent observations drawn in particular from transgenic models suggest their implication in the homeostatic control of immune and nonimmune processes. This hypothesis is also supported by the existence of several self-reactive gammadelta T-cell subsets in rodents and humans, whose specificity and effector properties will be detailed and discussed here. The present review will also describe several mechanisms that could allow efficient control of these self-reactive subsets while permitting expression of their regulatory and/or protective properties.     

Tissue localization and CD8 accessory molecule expression of T gamma delta cells in humans

In this study, we used TCR isotype-specific antibodies to examine the frequency, phenotype, and histologic localization pattern of T gamma delta cells in humans. The TCR delta 1+ cells comprised an average of 15% of the splenic CD3+ cells and 7% of circulating T cells. The T gamma delta cells in these human tissues, like their avian counterparts, were often not "double-negative" for the CD4 and CD8 accessory molecules. Approximately 50% of the splenic delta+ cells expressed CD8, and 30% of the delta+ cells in blood were CD8+. T cells of both gamma delta and alpha beta TCR isotypes were exceedingly rare in the skin. The T gamma delta cells exhibited preferential homing to the sinusoidal areas (red pulp) of the spleen and into the epithelial layer of the intestine in humans, as had been previously noted in chickens. Although 80% of the T gamma delta cells in the human intestinal mucosa were localized in the epithelial layer, these cells represented only 5 to 10% of all the CD3+ T cells in this microenvironment. We conclude that T gamma delta cells represent a sizeable subpopulation of the T cells in human peripheral tissues. The phylogenetic conservation of the CD8 expression by peripheral T gamma delta cells and of their preferential homing pattern suggests a special role in bodily defense for this T cell subpopulation.     

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.     

TCR and CD3 antibody cross-reactivity in 44 species.

The production of monoclonal antibodies is very costly, and antibodies are only available for a limited number of species. Until a more cost effective method of antibody production is found, identification of cross-reactive antibodies is an alternative approach that can provide investigators studying immunity in minor species with valuable antibody reagents. Flow cytometry was used to test 21 monoclonal antibodies (mAb), raised against alphabeta and gammadelta T cell receptors and CD3 from human and five animal species, for cross-reactivity in 44 different species including 16 species of nonhuman primates, marsupials, carnivores, lagomorphs, rodents, ruminants, swine, cetacean, horse, birds, a reptile, and fish. Fifteen of the mAbs cross-reacted with orthologous molecules in one or more species. Two antibodies, anti-human TCR gammadelta (B1.1), and anti-human CD3 (SP34) were found to costain in 13 species of nonhuman primates. This study has identified valuable new reagents for studying T cell populations in different animal species and for the first time characterized antibodies useful for studying gammadelta T cell populations in many species of primates. These antibodies may be used for further immunity research in species with less well-characterized immune systems.