Early expression of a functional TCRbeta chain inhibits TCRgamma gene rearrangements without altering the frequency of TCRgammadelta lineage cells

To investigate the consequences of the simultaneous expression in progenitor cells of a TCRgammadelta and a pre-TCR on alphabeta/gammadelta lineage commitment, we have forced expression of functionally rearranged TCRbeta, TCRgamma, and TCRdelta chains by means of transgenes. Mice transgenic for the three TCR chains contain numbers of gammadelta thymocytes comparable to those of mice transgenic for both TCRgamma and TCRdelta chains, and numbers of alphabeta thymocytes similar to those found in mice solely transgenic for a rearranged TCRbeta chain gene. gammadelta T cells from the triple transgenic mice express the transgenic TCRbeta chain, but do not express a TCRalpha chain, and, by a number of phenotypic and molecular parameters, appear to be bona fide gammadelta thymocytes. Our results reveal a remarkable degree of independence in the generation of alphabeta and gammadelta lineage cells from progenitor cells that, in theory, could simultaneously express a TCRgammadelta and a pre-TCR.     

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.     

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.     

Role of the intracellular domain of IL-7 receptor in T cell development

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R(-/-) hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R(-/-) thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust alphabeta T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRgamma locus or development of gammadelta T cells. Thus, the IL-7R signals required in the alphabeta T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRgamma locus may require a signal that is not shared by other receptors.     

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.     

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.     

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.     

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.     

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.     

A critical role for rictor in T lymphopoiesis

Apart from a critical role for Notch and pre-TCR, the signaling pathway required for T lymphopoiesis is largely unknown. Given the potential link between Notch and mammalian target of rapamycin (mTOR) signaling in cancer cells, we used mice with conditional deletion of either Raptor or Rictor genes to determine potential contribution of the mTOR complex I and II in T lymphopoiesis. Our data demonstrated that targeted mutation of Rictor in the thymocytes drastically reduced the thymic cellularity, primarily by reducing proliferation of the immature thymocytes. Rictor deficiency caused a partial block of thymocyte development at the double-negative 3 stage. The effect of Rictor deficiency is selective for the T cell lineage, as the development of B cells, erythrocytes, and myeloid cells is largely unaffected. Analysis of bone marrow chimera generated from a mixture of wild-type and Rictor-deficient hematopoietic stem cells demonstrated that the function of Rictor is cell intrinsic. These data revealed a critical function of mTOR complex 2 in T lymphopoiesis.     

Subtle defects in pre-TCR signaling in the absence of the Tec kinase Itk

alphabeta T cell development in the thymus is dependent on signaling through the TCR. The first of these signals is mediated by the pre-TCR, which is responsible for promoting pre-T cell proliferation and the differentiation of CD4(-)8(-)3(-) (DN) thymocytes into CD4(+)8(+)3(+) (DP) cells. In many cases, T cell signaling proteins known to be essential for TCR signaling in mature T cells are also required for pre-TCR signaling in DN thymocytes. Therefore, it came as a surprise to discover that mice lacking the Tec kinases Itk and Rlk, enzymes required for efficient activation of phospholipase C-gamma1 in mature T cells, showed no obvious defects in pre-TCR-dependent selection events in the thymus. In this report, we demonstrate that DN thymocytes lacking Itk, or Itk and Rlk, are impaired in their ability to generate normal numbers of DP thymocytes, especially when placed in direct competition with WT DN thymocytes. We also show that Itk is required for maximal pre-TCR signaling in DN thymocytes. These data demonstrate that the Tec kinases Itk and Rlk are involved in, but are not essential for, pre-TCR signaling in the thymus, suggesting that there is an alternative mechanism for activating phospholipase C-gamma1 in DN thymocytes that is not operating in DP thymocytes and mature T cells.     

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.