Induction of interleukin 2 receptor beta chain expression by self- recognition in the thymus

1-2% of adult mouse thymocytes express the T cell receptor alpha/beta (TCR-alpha/beta) together with the interleukin (IL) 2R beta (p70), but not the alpha (p 55) chain. We show that the previously described alpha/beta-TCR +CD4-8- and the partially overlapping Ly6C+ thymocytes are contained within this subset. Most IL-2R beta+ alpha/beta-TCR+ cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44high, CD69+) phenotype. Overrepresentation of V beta 8.2 in both CD4-8- and CD4 and/or CD8+ IL- 2R beta+ thymocytes suggests that IL-2R beta expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2Kb-specific TCR, IL-2R beta+ cells were abundant under conditions of mainstream negative selection, i.e., in the presence of Kb, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, self-recognition by immature thymocytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R beta. IL-2- deficient mice contain normal numbers of IL-2R beta+ alpha/beta-TCR+ thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2R beta+ thymocytes is skewed towards CD4-8+, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.     

CD8 surface levels alter the fate of alpha/beta T cell receptor-expressing thymocytes in transgenic mice.

The mature T cell receptor (TCR) repertoire is established on the basis of discriminative events involving binding of the TCR alpha and beta chains and CD4 or CD8 on immature thymocytes to major histocompatibility complex (MHC)/self-peptide complexes expressed in the thymus. To ask whether the strength of the interaction between a CD8/TCR complex and a MHC/self-peptide ligand plays a pivotal role in deciding the fate of a maturing thymocyte, we generated lines of transgenic mice that express distinct and elevated levels of CD8 alpha, approximately 2, 3, and 6-10 times. These lines were then crossed to a transgenic line expressing the class I-restricted TCR, 2C. We found that thymocytes expressing the 2C TCR in combination with the highest levels of CD8 were deleted on the H-2 Kb background that is normally positively selecting for the 2C TCR. In contrast, thymocytes coexpressing the 2C TCR and moderately elevated levels of CD8 were selected for maturation. These results demonstrate directly that CD8 levels can affect the developmental fate of a maturing thymocyte and argue in support of an affinity model for thymocyte selection.     

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.     

Early molecular events induced by T cell receptor (TCR) signaling in immature CD4+ CD8+ thymocytes: increased synthesis of TCR-alpha protein is an early response to TCR signaling that compensates for TCR-alpha instability, improves TCR assembly, and parallels other indicators of positive selection

Differentiation of immature CD4+ CD8+ thymocytes into mature CD4+ or CD8+ T cells occurs within the thymus and is dependent upon expression of antigen receptor complexes (T cell receptor [TCR]) containing clonotypic alpha/beta proteins. We have recently found that CD4+ CD8+ thymocytes express low levels of surface TCR because of limitations placed on TCR assembly by the instability of nascent TCR-alpha proteins within the endoplasmic reticulum (ER) of immature thymocytes. Because TCR-alpha/beta expression increases during development, a molecular mechanism must exist for increasing the number of assembled TCR complexes present in immature CD4+ CD8+ thymocytes that have been signaled to differentiate into mature T cells, although no such mechanism has yet been described. In the current report we have examined the molecular consequences of intracellular signals generated by engagement of surface TCR complexes on immature CD4+ CD8+ thymocytes. Isolated TCR engagement generated signals that increased TCR-alpha RNA levels and increased synthesis of TCR-alpha proteins, which, in turn, significantly increased assembly of complete TCR- alpha/beta complexes in CD4+ CD8+ thymocytes. Increased TCR-alpha protein levels in TCR-signaled CD4+ CD8+ thymocytes was the result of increased synthesis and not increased stability of TCR-alpha proteins, indicating that TCR engagement compensates for, but does not correct, the inherent instability of TCR-alpha proteins in the ER of immature thymocytes. Consistent with the delivery by TCR engagement of a positive selection signal, TCR engagement also increased CD5 expression, decreased RAG-1 expression, and decreased CD4/CD8 coreceptor expression in immature CD4+ CD8+ thymocytes. These data identify amplified TCR-alpha expression as an initial response of immature CD4+ CD8+ thymocytes to TCR-mediated positive selection signals and provide a molecular basis for increased surface TCR density on developing thymocytes undergoing selection events within the thymus.     

Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28

CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self- antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.     

Restricting Zap70 expression to CD4+CD8+ thymocytes reveals a T cell receptor-dependent proofreading mechanism controlling the completion of positive selection

Although T cell receptor (TCR) signals are essential for intrathymic T cell-positive selection, it remains controversial whether they only serve to initiate this process, or whether they are required throughout to promote thymocyte differentiation and survival. To address this issue, we have devised a novel approach to interfere with thymocyte TCR signaling in a developmental stage-specific manner in vivo. We have reconstituted mice deficient for Zap70, a tyrosine kinase required for TCR signaling and normally expressed throughout T cell development, with a Zap70 transgene driven by the adenosine deaminase (ADA) gene enhancer, which is active in CD4(+)CD8(+) thymocytes but inactive in CD4(+) or CD8(+) single-positive (SP) thymocytes. In such mice, termination of Zap70 expression impaired TCR signal transduction and arrested thymocyte development after the initiation, but before the completion, of positive selection. Arrested thymocytes had terminated Rag gene expression and up-regulated TCR and Bcl-2 expression, but failed to differentiate into mature CD4 or CD8 SP thymocytes, to be rescued from death by neglect or to sustain interleukin 7R alpha expression. These observations identify a TCR-dependent proofreading mechanism that verifies thymocyte TCR specificity and differentiation choices before the completion of positive selection.     

CD4 expression is differentially required for deletion of MLS-1a- reactive T cells

Clonal deletion of thymocytes expressing potentially self-reactive T cell receptors (TCRs) occurs during thymocyte ontogeny. Mice deficient for CD4 expression provide a unique model system to study the contribution of the CD4 molecule in negative selection of T cells reactive against the major histocompatibility complex class II- associated retroviral self-superantigen, Mls-1a. In the presence of Mls- 1a determinants, mature CD8+ T cells expressing V beta 6, 8.1, and 9 were deleted in CD4-deficient mice, thus demonstrating that TCR affinity for Mls-1a is sufficient for deletion and that a signal through CD4 was not required. However, in instances where the TCR affinity for Mls-1a is low, as in the case of V beta 7+ T cells, CD4 expression was required for clonal deletion. These results demonstrate that for Mls-1a-mediated clonal deletion of T cells, the requirement for the accessory or coreceptor function of CD4 depends on the affinity of the TCR.     

In vivo and in vitro clonal deletion of double-positive thymocytes.

To study the processes of thymic development, we have established transgenic mice expressing and alpha/beta T cell antigen receptor (TCR) specific for cytochrome c associated with class II major histocompatibility complex (MHC) molecules. The transgenic TCR chains are expressed by most of the thymocytes in these mice, and these cells have been shown to efficiently mature in association with Ek- and Ab-encoded class II MHC molecules. This report describes a characterization of the negative selection of these transgenic thymocytes in vivo that is associated with the expression of As molecules. Negative selection by As molecules appears to result in the deletion of a late stage of CD4/CD8 double-positive thymocytes in that there is a virtual absence of transgenic TCR bearing CD4 single-positive thymocytes. This phenotype is accompanied by the appearance of CD4/CD8 double-negative thymocytes and peripheral T cells that are functionally antigen reactive. The process of negative selection has also been investigated using an in vitro culture system. Upon presentation of cytochrome c by Eb-expressing nonthymic antigen-presenting cells, there occurs an antigen dose-dependent deletion of the majority of CD4/CD8 double-positive thymocytes. In contrast, presentation of Staphylococcal enterotoxin A by Eb in vitro results in minimal deletion of double-positive thymocytes. In addition, we use this in vitro model to examine the effects of cyclosporin A on negative selection. In contrast to its effects on mature T cells, and the findings of others in vivo, cyclosporin A does not inhibit antigen-induced deletion of double-positive thymocytes. Finally, a comparison of the antigen dose responses for thymocyte deletion and for peripheral T cell activation indicates that double-positive thymocyte recognition is more sensitive than mature T cells to antigen recognition.     

The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post- selection intermediates leading to mature T cells

Cortical thymocytes from adult mice, separated on the basis of coexpression of CD4 and CD8 or of binding of high levels of peanut agglutinin (PNA), were subdivided according to the level of expression of the T cell receptor (TCR)-CD3 complex. The incidence of dividing cells in the resultant subpopulations was determined by DNA staining. Precursor-product relationships and the timing of TCR-CD3 acquisition were studied using continuous in vivo [3H]TdR labeling and radioautography. The extent of intrathymic selection for TCR specificity in the subpopulations was determined from the incidence of cells bearing V beta 6 or V beta 17a in different mouse strains. The majority of dividing CD4+8+ blast cells expressed extremely low levels of TCR-CD3, indicating that TCR expression and specificity selection generally occurred after division ceased. The [3H]TdR-labeling studies indicated that postdivision TCR expression was rapid, and that those nondividing cortical thymocytes which had not expressed significant levels of TCR by day 1, remained extremely low or negative for their entire 3.6-d lifespan. Small cortical thymocytes which expressed moderate levels of TCR-CD3, were predominantly an unselected population with a lifespan of 3.8 d. A small subgroup of CD4+8+ PNA+ cortical thymocytes expressing high levels of TCR-CD3 was identified as a nondividing intermediate between the small cortical thymocytes expressing moderate levels of TCR and mature medullary thymocytes. These intermediates showed a 1-d lag in [3H]TdR labeling, then a 3.4-d transit time. The cell flux through this intermediate subpopulation was approximately 10(6) cells/d, similar to the rate of turnover of mature thymocytes; thus, although only 3-4% of thymocytes progressed to this intermediate state, once reaching it most then progressed to full maturity. In accordance with this, the incidence of the V beta selection markers within the intermediate subpopulation indicated that both positive and negative selection had already occurred. Selection for TCR specificity in the systems studied appeared to take place among CD4+8+ thymocytes expressing intermediate levels of TCR.     

Mutation in fas ligand impairs maturation of thymocytes bearing moderate affinity T cell receptors

Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I- and class II-restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection.     

Autonomous maturation of alpha/beta T lineage cells in the absence of COOH-terminal Src kinase (Csk)

The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of alpha/beta T lineage cells in T cell receptor (TCR) beta-deficient or recombination activating gene (rag)-1-deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as alpha/beta T cell development is blocked in lck(-/)-fyn(-/)- csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples alpha/beta T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the alpha/beta TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I-restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR-MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.     

Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes

Differentiation of most T lymphocytes occurs within the thymus and is characterized by variable expression of CD4/CD8 coreceptor molecules, increased surface density of T cell antigen receptor (TCR) alpha beta proteins, and decreased expression of glycan chains recognized by the galactose-specific lectin peanut agglutinin (PNA). Although appreciated for several decades that PNA agglutination is useful for the physical separation of immature and mature thymocyte sub-populations, the identity of specific PNA-binding glycoproteins expressed on immature thymocytes remains to be determined. In the current report, we studied the expression of PNA-specific glycans on immature and mature T cells and used lectin affinity chromatography and immunoprecipitation techniques to characterize PNA-binding glycoproteins on thymocytes. Our data demonstrate that PNA-specific glycans are localized on a relatively small subset of thymocyte surface proteins, several of which were specifically identified, including CD43, CD45, and suprisingly, CD8 molecules. CD8 alpha and CD8 alpha' proteins bound to PNA in the absence of CD8 beta expression showing that O-glycans on CD8 beta glycoproteins are not necessary for PNA binding and that glycosylation of CD8 alpha and CD8 alpha' proteins proceeds effectively in the absence of CD8 beta. Finally, we demonstrate that PNA binding of CD8 is developmentally regulated by sialic acid addition as CD8 proteins from mature T cells bound to PNA only after sialidase treatment. These studies identify CD8 as a PNA receptor molecule on immature thymocytes and show that PNA binding of CD8 on immature and mature T cells is developmentally regulated by sialic acid modification.     

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.     

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.     

Facilitation of beta selection and modification of positive selection in the thymus of PD-1-deficient mice

PD-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine-based inhibitory motif, and disruption of the PD-1 gene results in the development of lupus-like autoimmune diseases. In this study, we examined effects of the PD-1 deficiency on the thymocyte differentiation at the clonal level using T cell receptor (TCR)-beta (Vbeta8) and TCR-alpha/beta (H-Y and 2C) transgenic mice. In these TCR transgenic lines, PD-1 expression in the thymus was variably augmented, but as in the normal mice, confined largely to the CD4(-)CD8(-) thymocytes. The transgenic mice crossed with PD-1(-/)- mice in the neutral genetic backgrounds exhibited selective increase in the CD4(+)CD8(+) (DP) population with little effect on other thymocytes subsets. Similarly, the absence of PD-1 facilitated expansion of DP thymocytes in recombination activating gene (RAG)-2(-/)- mice by anti-CD3epsilon antibody injection. On the other hand, H-Y or 2C transgenic PD-1(-/)- mice with the positively selecting background showed significantly reduced efficiency for the generation of CD8(+) single positive cells bearing the transgenic TCR-alpha/beta in spite of the increased DP population. These results collectively indicate that PD-1 negatively regulates the beta selection and modulates the positive selection, and suggest that PD-1 deficiency may lead to the significant alteration of mature T cell repertoire.     

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status

We have recently identified a dominant wave of CD4-CD8- (double- negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III- CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double- positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination- activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement- competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.     

The molecular mechanisms governing T cell development in the thymus

Intracellular signaling events required for T cell development in the thymus have been investigated using various gene deficient and transgenic mice. Here, we summarize and discuss the recent results. Immature thymocytes immigrated from bone marrow are found to be committed to lymphoid lineage. First, the lymphoid progenitor proliferates in IL-7-dependent manner, and differentiates into the cells expressing preTCR complex. The expression of preTCR is required for differentiation to CD4+ CD8+ cells. Then, CD4+ CD8+ cells express TCR alpha beta and are subjected to positive and negative selection. Essential molecules for these selection events and gene activation required for lineage commitment to CD4 or CD8 T cells are getting clear. In the near future, molecular mechanisms governing each developmental step of T cells will be clarified.     

Regulation of thymocyte development through CD3. I. Timepoint of ligation of CD3 epsilon determines clonal deletion or induction of developmental program

Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)- 2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR- beta chain homodimers and CD3. Here we show that early addition of anti- CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR- beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.