Delayed and restricted expression limits putative instructional opportunities of Vgamma1.1/Vgamma2 gammadelta TCR in alphabeta/gammadelta lineage choice in the thymus

Development of alphabeta and gammadelta T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRbeta and TCRgammadelta proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRgammadelta proteins or to a more rapid progression to DN4 of thymocytes expressing TCRgammadelta. The answer to this question bears on the distinction between instructive and stochastic models of alphabeta/gammadelta lineage decision. To study this question, we first monitored initial TCR protein expression in wild-type and TCR transgenic mice in reaggregate thymic organ cultures. A TCRbeta transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRgammadelta proteins. In contrast, TCRgammadelta transgenes were expressed only in a fraction of DN4 cells, did not accelerate DN3 to DN4 transition, and did not reduce the number of DN4 cells expressing TCRbeta proteins. The TCRbeta transgene partially inhibited endogenous TCRgamma rearrangements, whereas the TCRgammadelta transgenes did not inhibit endogenous TCRbeta rearrangements. Second, we analyzed frequencies of productive TCRbeta and TCRgammadelta V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRgammadelta rearrangements (Vdelta5, Vgamma1.1, and Vgamma2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding gammadeltaTCR, severely limiting their putative instructional opportunities in alphabeta/gammadelta divergence.     

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.     

Rates of recombination and chain pair biases greatly influence the primary gammadelta TCR repertoire in the thymus of adult mice

Analyses of the rearrangement status of the TCRgamma and TCRdelta chain loci in progenies of individual gammadelta thymocytes showed a hierarchy of the different Vgamma and Vdelta gene segments to participate in a recombination reaction. Moreover, individual TCRgamma chains only pair efficiently with a variable number of TCRdelta chains. Interestingly, these two parameters are inversely correlated such that the TCRgamma and TCRdelta chains that rearrange more often show a higher level of restriction in their pairing capabilities. Our data suggest that these mechanisms, together with a natural variation affecting the expected frequencies at which rearrangement of different Vgamma gene segments give raise to functional TCRgamma chains, have coevolved to maximize the diversity of the gammadelta TCR repertoire minimizing the risk that a gammadelta T cell will express more than one TCR specificity at the cell surface, despite the fact that multiple TCRgamma rearrangements take place in the same progenitor cell.     

T cell receptor-gamma allele-specific selection of V gamma 1/V delta 4 cells in the intestinal epithelium

Previous genetic analyses have shown that the relative representation of subsets of gammadelta intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRgamma, TCRdelta, and MHC loci. Here, we have analyzed V-gene use in gammadelta i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F(1) and F(2) progenies with a larger panel of Vgamma- and Vdelta-specific mAbs and have shown that the influence of TCRgamma-linked genes operates at two levels: one influencing the representation of Vgamma1 (or Vgamma7) i-IELs and other acting specifically on the Vgamma1/Vdelta4 i-IEL subset, which represents 3% and 15% of the gammadelta i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vgamma1Jgamma4Cgamma4 chain of B6 origin demonstrated that the TCRgamma-linked genes influencing the representation of the Vgamma1/Vdelta4 i-IEL subset are the structural genes of TCRgamma chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vgamma1/Vdelta4 cells bearing either parental allele in F(1) mice. The representation of Vgamma1/Vdelta4 cells among gammadelta thymocytes is similar in B6 and B10 mice, demonstrating that the Vdelta4 chain can pair well with both alleles of the Vgamma1Jgamma4Cgamma4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vgamma1-Jgamma4 junctional amino acid sequences of B6 Vgamma1/Vdelta4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vgamma1/Vdelta4 cells are the target of this cellular selection event.     

Reduced generation but efficient TCR beta-chain selection of CD4+8+ double-positive thymocytes in mice with compromised CD3 complex signaling

Maturation to the CD4+8+ double-positive (DP) stage of thymocyte development is restricted to cells that have passed TCRbeta selection, an important checkpoint at which immature CD4-8- double-negative (DN) cells that express TCRbeta polypeptide chains are selected for further maturation. The generation of DP thymocytes following TCRbeta selection is dependent on cellular survival, differentiation, and proliferation, and the entire process appears to be mediated by the pre-TCR/CD3 complex. In this study, we investigate the signaling requirements for TCRbeta selection using mice single deficient and double deficient for CD3zeta/eta and/or p56lck. While the numbers of DP cells are strongly reduced in the single-deficient mice, a further drastic reduction in the generation of DP thymocytes is seen in the double-deficient mice. The poor generation of DP cells in the mutant mice is primarily due to an impaired ability of CD25+ DN thymocytes to proliferate following expression of a TCRbeta-chain. Nevertheless, the residual DP cells in all mutant mice are strictly selected for expression of TCRbeta polypeptide chains. DN thymocytes of mutant mice expressed TCRbeta and CD3epsilon at the cell surface and contained mRNA for pre-Talpha, but not for clonotypic TCRalpha-chains, together suggesting that TCRbeta selection is mediated by pre-TCR signaling in all cases. The data suggest differential requirements of pre-TCR signaling for cell survival on the one hand, and for the proliferative burst associated with TCRbeta selection on the other.     

Mechanisms controlling termination of V-J recombination at the TCRgamma locus: implications for allelic and isotypic exclusion of TCRgamma chains

Analyses of Vgamma-Jgamma rearrangements producing the most commonly expressed TCRgamma chains in over 200 gammadelta TCR(+) thymocytes showed that assembly of TCRgamma V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRgamma and delta transgenes results in a profound inhibition of endogenous TCRgamma rearrangements in progenitor cells. The extent of TCRgamma rearrangements in these cells is best explained by a model in which initiation of TCRgamma rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional gammadelta TCR. Approximately 10% of the cells studied contained two functional TCRgamma chains involving different V and Jgamma gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRgamma and delta chains.     

Threshold of pre-T-cell-receptor surface expression is associated with alphabeta T-cell lineage commitment.

BACKGROUND: The development of immature thymocytes is regulated by the pre-T-cell receptor (pre-TCR). The pre-TCR is involved in several developmental processes including rescuing cells from programmed cell death, allelic exclusion and alphabeta versus gammadelta T-cell lineage commitment. A major issue is how the pre-TCR functions to integrate these processes in developing thymocytes. RESULTS: We have used a sensitive immunofluorescence technique to reveal the surface-expression profile of the pre-TCR on immature thymocyte subsets. We show that early pre-T cells (CD25(+)CD44(-)) can be subdivided on the basis of the level of surface pre-TCR expression. Detectable surface pre-TCR expression identified a rapidly cycling population of early pre-T cells which had successfully undergone beta-selection and been rescued from programmed cell death. Late pre-T cells (CD25(-)CD44(-)), which had traversed the beta-selection checkpoint, expressed surprisingly heterogeneous surface levels of the pre-TCR: high levels of surface pre-TCR expression were associated with commitment to the alphabeta T-cell lineage, whereas late pre-T cells with lower levels of surface pre-TCR could develop along both the alphabeta or gammadelta T-cell lineages. CONCLUSIONS: These data demonstrate that the surface expression of the pre-TCR can be used to reveal newly identified stages of T-cell development and to provide insights into alphabeta T-cell lineage commitment. They show that, although pre-TCR expression does not act as a developmental switch per se, its level of surface expression on late pre-T cells predicts their developmental potential.     

Exclusion and inclusion of TCR alpha proteins during T cell development in TCR-transgenic and normal mice

Allelic exclusion of immune receptor genes (and molecules) is incompletely understood. With regard to TCRalphabeta lineage T cells, exclusion at the tcr-b, but not tcr-a, locus seems to be strictly controlled at the locus rearrangement level. Consequently, while nearly all developing TCRalphabeta thymocytes express a single TCRbeta protein, many thymocytes rearrange and express two different TCRalpha chains and, thus, display two alphabetaTCRs on the cell surface. Of interest, the number of such dual TCR-expressing cells is appreciably lower among the mature T cells. To understand the details of TCR chain regulation at various stages of T cell development, we analyzed TCR expression in mice transgenic for two rearranged alphabetaTCR. We discovered that in such TCR double-transgenic (TCRdTg) mice peripheral T cells were functionally monospecific. Molecularly, this monospecificity was due to TCRalpha exclusion: one transgenic TCRalpha protein was selectively down-regulated from the thymocyte and T cell surface. In searching for the mechanism(s) governing this selective TCRalpha down-regulation, we present evidence for the role of protein tyrosine kinase signaling and coreceptor involvement. This mechanism may be operating in normal thymocytes.     

Distinct requirements for IL-7 in development of TCR gamma delta cells during fetal and adult life

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta cells.     

TCRbeta transmembrane tyrosines are required for pre-TCR function.

The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.     

Expression of TCR alpha beta partly rescues developmental arrest and apoptosis of alpha beta T cells in Bcl11b-/- mice

Bcl11b(-/-) mice show developmental arrest at the CD44(-)CD25(+) double-negative 3 (DN3) or immature CD8(+)single-positive stage of alphabeta T cell. We have performed detailed analysis of sorted subsets of Bcl11b(-/-) thymocytes, DN3 and CD44(-)CD25(-) double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCRalphabeta expression on the cell surface failed to promote transition from the DN3 to CD4(+)CD8(+) double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b(-/-) DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b(-/-) DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b(-/-) DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-x(L) and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRalphabeta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

Both gamma delta T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells and the induction of xenograft tolerance in mice

In murine allogeneic bone marrow transplantation recipients, treatment of the hosts with a nonmyeloablative regimen, including depleting anti-CD4 and anti-CD8 mAbs, allows establishment of long-term mixed chimerism and donor-specific tolerance. However, in the xenogeneic rat-to-mouse combination, additional anti-Thy1.2 and anti-NK1.1 mAbs are required. We have now attempted to identify the xenoresistant mouse cell populations that are targeted by anti-NK1.1 and anti-Thy1.2 mAbs. C57BL/6 (B6) wild-type, B6 TCRbeta(-/-), and B6 TCRdelta(-/-) mice received anti-CD4 and anti-CD8 mAbs, followed by 3 Gy of whole body irradiation, 7 Gy of thymic irradiation, and transplantation of T cell-depleted rat bone marrow cells. Anti-NK1.1 and anti-Thy1.2 mAbs were additionally administered to some groups. Increased rat chimerism was observed in TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-NK1.1 mAbs compared with similarly treated TCRbeta(-/-) mice. In TCRbeta(-/-) mice, but not in TCR delta(-/-) mice, donor chimerism was increased by treatment with anti-Thy1.2 mAb, indicating that CD4(-)CD8(-)TCRgammadelta(+)Thy1. 2(+)NK1.1(-) cells (gammadelta T cells) are involved in the rejection of rat marrow. In addition, chimerism was enhanced in both TCRbeta(-/-) and TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-Thy1.2 mAbs by the addition of anti-NK1.1 mAb to the conditioning regimen. Donor-specific skin graft prolongation was enhanced by anti-Thy1.2 and anti-NK1.1 mAbs in TCRdelta(-/-) mice. Therefore, in addition to CD4 and CD8 T cells, gammadelta T cells and NK cells play a role in resisting engraftment of rat marrow and the induction of xenograft tolerance in mice.     

Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer

In contrast to Ag-specific alphabeta T cells, gammadelta T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that gammadelta T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of gammadelta T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that gammadelta T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with gammadelta T cell-deficient mice (TCRdelta(-/-) mice) yielding TRAMP x TCRdelta(-/-) mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic gammadelta T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with gammadelta T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous gammadelta T cells for the treatment of prostate cancer.