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.     

Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains

Immature thymocytes express a pre-T cell receptor (pre-TCR) composed of the TCRbeta chain paired with pre-Talpha. Signals from this receptor are essential for passage of thymocytes through a key developmental checkpoint in the thymus. These signals were efficiently delivered in vivo by a truncated form of the murine pre-TCR that lacked all of its extracellular immunoglobulin domains. De novo expression of the truncated pre-TCR or an intact alphabetaTCR was sufficient to activate characteristic TCR signaling pathways in a T cell line. These findings support the view that recognition of an extracellular ligand is not required for pre-TCR function.     

Proliferation kinetics associated with T cell receptor-beta chain selection of fetal murine thymocytes

After productive rearrangement of a TCR beta chain gene, CD4-8- double negative (DN) thymocytes express TCR beta polypeptide chains on the cell surface together with pre-T alpha and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCR beta + DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas DN cells that fail to generate a productive TCR beta gene rearrangement do not continue in development. This process is termed TCR beta chain selection. Although it is likely that differences between proliferation dynamics of TCR beta + and TCR beta-cells may play a role, the exact mechanisms of TCR beta chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR beta + and TCR beta-thymocytes during fetal development, i.e., when TCR beta chain selection takes place for the first time. We analyzed in situ accumulation of TCR beta + thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR beta + and TCR beta-populations by flow cytometry. About 600 TCR beta + cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5, and 15.5. As of dg 14.5, most TCR beta + cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR beta + cells has slowed down to less than 1/d. About three quarters of TCR beta-cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCR beta-cells, but not TCR beta + cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCR beta-cells. Positive selection of fetal TCR beta + cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.     

Requirement for p56lck tyrosine kinase activation in T cell receptor-mediated thymic selection

The nonreceptor protein tyrosine kinase p56lck (Lck) serves as a fundamental regulator of thymocyte development by delivering signals from the pre-T cell receptor (pre-TCR) that permit subsequent maturation. However, considerable evidence supports the view that Lck also participates in signal transduction from the mature TCR. We have tested this conjecture by expressing a dominant-negative form of Lck under the control of a promoter element (the distal lck promoter) that directs high expression in CD4+CD8+ thymocytes, mature thymocytes, and peripheral T cells, thereby avoiding, complications that result from the well-documented ability of dominant-negative Lck to block very early events in thymocyte maturation. Here we report that expression of the catalytically inactive Lck protein at twice normal concentrations inhibits thymocyte positive selection by as much as 80%, while leaving other aspects of cell maturation intact. This effect was studied in more detail in mice simultaneously bearing the male-specific H-Y alpha/beta TCR transgene and ovalbumin-specific DO10 alpha/beta TCR transgene, where even equimolar expression of the dominant-negative Lck protein substantially vitiated the positive selection process. Although deletion of H-Y alpha/beta thymocytes proceeded normally in male mice despite the presence of catalytically inactive Lck, modest inhibition of superantigen-mediated deletion was in some cases observed. These data further implicate Lck in the propagation of all TCR-derived signals, and indicate that even very modest deficiencies in the representation of functional Lck molecules could in humans, profoundly alter the character of the peripheral TCR repertoire.     

Distinct effects of Jak3 signaling on alphabeta and gammadelta thymocyte development

Janus kinase 3 (Jak3) plays a central role in the transduction of signals mediated by the IL-2 family of cytokine receptors. Targeted deletion of the murine Jak3 gene results in severe reduction of alphabeta and complete elimination of gammadelta lineage thymocytes and NK cells. The developmental blockade appears to be imposed on early thymocyte differentiation and/or expansion. In this study, we show that bcl-2 expression and in vivo survival of immature thymocytes are greatly compromised in Jak3-/- mice. There is no gross deficiency in rearrangements of the TCRdelta and certain gamma loci in pre-T cells, and a functional gammadelta TCR transgene cannot rescue gammadelta lineage differentiation in Jak3-/- mice. In contrast, a TCRbeta transgene is partially able to restore alphabeta thymocyte development. These data suggest that the signals mediated by Jak3 are critical for survival of all thymocyte precursors particularly during TCRbeta-chain gene rearrangement, and are continuously required in the gammadelta lineage. The results also emphasize the fundamentally different requirements for differentiation of the alphabeta and gammadelta T cell lineages.     

Exit of the pre-TCR from the ER/cis-Golgi is necessary for signaling differentiation, proliferation, and allelic exclusion in immature thymocytes

A major issue is whether surface expression of the pre-TCR is necessary for signaling the development of immature thymocytes. To address this question, we generated transgenic mice expressing a TCRbeta chain that had a strong endoplasmic reticulum (ER) retrieval signal (TCRbetaER) and that was expressed intracellularly but failed to reach the cell surface. In TCRbetaER transgenic mice, there was a failure of allelic exclusion. Also, the transgene failed to rescue the developmental defects observed in TCRbeta-null mice. In contrast, TCRbeta transgenes with a mutant ER retrieval sequence or lacking this sequence signaled efficient allelic exclusion and suppressed the TCRbeta-/- defect. These data show that exit of the pre-TCR from the ER/cis-Golgi is required for progression through the double-negative thymocyte checkpoint.     

Lineage relationships of the fetal thymocyte subset that expresses the beta chain of the interleukin-2 receptor

The beta chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R beta+ thymocytes are gamma delta cells. The precursor-progeny relationships of fetal IL-2R beta+ thymocytes to the alpha beta T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2R beta is expressed on a subset of very immature cells, along with high amounts of Pgp1 and Fc gamma RII/III, partially preceding the expression of intracellular CD3 epsilon. IL-2-R beta disappears before expression of IL-2R alpha. IL-2R beta+ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR) alpha beta+, TCR gamma delta+, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2R alpha+ day 15 fetal thymocytes. These data suggest that IL-2R beta expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.     

CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4(-)CD8(-) (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56(lck). At the CD4(+)CD8(+) (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR-major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4(+) or CD8(+) (single positive, SP) thymocytes is induced by engagement of the alpha/beta-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR-MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.     

Calnexin association is not sufficient to protect T cell receptor alpha proteins from rapid degradation in CD4+CD8+ thymocytes

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRalpha molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRalpha instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRalpha proteins in all other T cell types examined. Here, we compared the processing of TCRalpha glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRalpha glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRalpha proteins stably associated with calnexin in both cell types. Interestingly, however, TCRalpha association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+ thymocytes compared with splenic T cells. Finally, TCRalpha degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin.TCRalpha complexes. These data demonstrate that calnexin association is not sufficient to protect TCRalpha proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRalpha glycoproteins.     

Separately expressed T cell receptor alpha and beta chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4-CD8-TCR+ thymocytes and the absence of gamma delta cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR alpha chain and a transgenic TCR beta chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR alpha chain causes thymocytes to differentiate into a CD4-CD8-TCR+ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the alpha beta lineage. Surprisingly, expression of the TCR alpha chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR beta chain causes immature T cells to accelerate differentiation into the alpha beta lineage and thus inhibits the generation of gamma delta cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.     

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.     

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.