ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck)

As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.     

p56lck Signals for Regulating Thymocyte Development Can Be Distinguished by Their Dependency on Rho Function

The tyrosine kinase p56lck regulates the differentiation and proliferative expansion of pre-T cells. However, nothing is known about other signaling molecules that operate with p56lck to mediate the pleiotropic changes that occur at this stage of thymocyte development. We used a genetic strategy to examine the requirement for the GTPase Rho in p56lck-mediated signals in the thymus. By generating mice double transgenic for a constitutively activated form of p56lck (p56lck^F505) and the Rho inhibitor C3 transferase we were able to compare thymocyte development in mice expressing active p56lck on a wild-type or Rho⁻ background. Thymocytes expressing active p56lck show enhanced proliferation of pre-T cells resulting in increased numbers of late pre-T cells, however, this dramatic effect on pre-T cell proliferation is lost when the p56lck transgene is expressed in thymocytes lacking endogenous Rho GTPase function. Expression of active p56lck also generates double positive (DP) thymocytes with low levels of CD2 antigen expression. Again, p56lck cannot prevent expression of CD2 when expressed on a Rho⁻ background. CD4⁺CD8⁺ DP cells expressing active p56lck have been shown to lack functional α/β–T cell receptor (TCR) complexes due to p56lck-mediated inhibition of TCR gene Vβ-Dβ rearrangement. This inhibition of TCR expression by active p56lck is unimpaired in the absence of Rho function. The signaling pathways that are mediated by p56lck and control thymocyte proliferation, α/β-TCR and CD2 antigen expression can thus be distinguished by their dependency on Rho function.     

Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells

Our studies have analyzed pore-forming protein (PFP) mRNA expression in resting and stimulated human peripheral blood CD3- large granular lymphocytes (LGL), CD3+ T cells, and their CD4+ or CD8+ subsets. Signals that stimulate T cells to develop cytotoxic activity (i.e., IL- 2 or OKT-3 mAb) led to the induction of PFP mRNA in T cells. The data indicated that IL-2 directly increased PFP mRNA in the CD8+ subset of T cells, in the absence of new DNA or protein synthesis. Abrogation of IL- 2-induced PFP mRNA expression and cytotoxic potential of T cells by the anti-p75 IL-2 receptor mAb suggested that low numbers of p75 IL-2 receptors on CD8+ T cells were capable of transducing signals responsible for these IL-2-induced effects. The induction of T cell PFP mRNA via CD3, using OKT-3 mAb, was less rapid but greater than that caused by IL-2; however, a combination of PMA and ionomycin, which bypasses crosslinking of the TCR/CD3 complex, could not mimic this increase in PFP mRNA levels in T cells. The role of second messenger systems in regulating PFP mRNA expression remains to be determined. In contrast, high constitutive PFP mRNA expression was observed in CD3- LGL and these mRNA levels could not be enhanced by stimulation with IL- 2. The cytotoxic potential of peripheral blood T cells and LGL induced in response to IL-2 correlated with IL-2-induced PFP mRNA levels in these cells and was consistent with PFP being one of several important molecules involved in the effector function of cytotoxic lymphocytes.     

T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture

Recent results have indicated that positive and negative repertoire selection act on the major population of CD4,8 double-positive (DP) thymocytes that express 5-10-fold less T cell receptor (TCR) than mature T cells (i.e., they are TCRlow). Since DP cells obtained ex vivo are heterogeneous with regard to their stage within thymic selection, a homogeneous population of virgin DP cells suitable for selection studies was generated in vitro from their immediate precursors, the CD8 single-positive (SP) immature blast cells. To mimic TCR-mediated selection signals, these virgin DP cells were then cultured for another 2 d in the presence of immobilized anti-TCR monoclonal antibodies with or without interleukin 2 (IL-2). Daily monitoring of recovery and phenotype showed that without TCR stimulation, the cells remained DP and became small, TCRlow cells that were lost with a half-life of 1 d, regardless of the presence of IL-2. TCR stimulation resulted in rapid downregulation of CD4 and CD8, maintenance of a larger cell size, and induction of the CD53 antigen that marks mature and CD4,8 double-negative rat thymocytes. In the absence of IL-2, viability decreased as rapidly as without TCR stimulation. Addition of IL-2 rescued TCR-stimulated virgin DP cells and prevented CD8 downregulation, so that 50-80% of input DP cells were recovered after 2 d as CD4-8+53+ cells. After release from modulation, these in vitro generated CD8 SP cells quantitatively upregulated the TCR to the TCRhigh phenotype and were readily induced to proliferate and exhibit cytotoxic T lymphocyte (CTL) activity in a polyclonal readout. Evidence is presented implicating an IL-2 receptor (IL-2R) not containing the p55 chain (i.e., most likely the p70 intermediate affinity IL-2R) in the TCR plus IL-2-driven in vitro differentiation of virgin DP cells towards the mature CD8 SP phenotype.     

The expression of IL-2 receptor subunits on various leukemic cells]

Interleukin-2 receptors are composed of at least two polypeptide chains of alpha (55KD) and beta (75KD). The IL-2R beta chain is an essential component of the functional receptor for signal transduction of IL-2. We previously reported the distribution of IL-2R subunits among peripheral blood mononuclear cells. We here present some data regarding the expression of IL-2R subunits on various hemopoietic malignant cells. Fresh leukemic cells obtained from adult T cell leukemia patients expressed both alpha and beta chains, and leukemic cells derived from some patients with T cell leukemia, B cell leukemia or myeloid leukemia expressed the alpha and/or beta chain of IL-2R. The IL-2R beta chain on these leukemic cells were demonstrated to be functional for cell growth signaling. IL-2R alpha and beta chains should be tumor markers.     

T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2- dependent differentiation pathway of CD8 alpha+/beta- T cells

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hunig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.     

Physical dissociation of the TCR-CD3 complex accompanies receptor ligation

During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2- mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes.     

Impaired development of V gamma 3 dendritic epidermal T cells in p56lck protein tyrosine kinase-deficient and CD45 protein tyrosine phosphatase- deficient mice

To determine whether p56lck protein tyrosine kinase and CD45 protein tyrosine phosphatase are involved in the signal transduction during intrathymic differentiation of gamma/delta T cells, we have examined the development of T cells expressing V gamma 3 T cell receptor (TCR) in mice deficient for either protein. The skin from both mice contained significantly reduced numbers of dendritic epidermal T cells expressing decreased levels of V gamma 3 TCR at the cell surface. Analysis of the fetal thymus from these mice suggested that maturation of V gamma 3 thymocytes was blocked at the immature stage that was characterized by the low level of V gamma 3 TCR and the high level of heat stable antigen. These results imply that both p56lck and CD45 are involved in the signal transduction during maturation of V gamma 3 T cells in the fetal thymus.     

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.     

Activation of peripheral blood T cells via the p75 interleukin 2 receptor

By using mAb and flow cytometry, a constitutive expression of the p75 IL-2R was revealed in human peripheral blood CD8+ T cells and TCR delta-1+ T cells as well as in CD16+ NK cells. Anti-p75 IL-2R mAb almost completely inhibited the induction of cytolytic activity in these T cells by brief exposure to IL-2, as estimated by anti-TCR/CD3 mAb-targeted cytotoxicity. While anti-p55 IL-2R mAb alone inhibited the response only modestly, maximal inhibition was achieved by combining both anti-p55 and anti-p75 IL-2R mAbs. These results indicate that the p75 IL-2R constitutively expressed on peripheral blood CD8+ T cells and TCR delta-1+ T cells is predominantly responsible for the direct activation of these cells by IL-2.     

Evidence for differential intracellular signaling via CD4 and CD8 molecules

Although both the CD4 and CD8 molecules enhance antigen responsiveness mediated by the T cell receptor (TCR), it is not known whether CD4 and CD8 initiate similar or different intracellular signals when they act as coreceptors. To characterize the early signals transmitted by CD4 and CD8, both CD4 and CD8 alpha were expressed in the same murine T cell hybridoma. In the double positive transfectants, CD4 and CD8 associated with equal amounts of p56lck (Lck), and both molecules enhanced interleukin 2 (IL-2) production equivalently when cross-linked with suboptimal levels of anti-TCR antibody. However, in an in vitro kinase assay, cross-linking CD4 initiated fourfold greater kinase activity compared with CD8 cross-linking. In the same assay, when CD4 or CD8 was cross-linked to the TCR, novel phosphorylated proteins were found associated with the TCR/CD4 complex but not with the TCR/CD8 complex. Consistent with this data, antiphosphotyrosine immunoblotting revealed greater tyrosine phosphorylation of intracellular substrates after TCR/CD4 cross-linking compared with TCR/CD8 cross-linking. Additionally, a specific protein kinase C inhibitor (RO318220) inhibited CD8-mediated enhancement of IL-2 production far more effectively than CD4-mediated enhancement. Thus, it appears that CD8 alpha may depend more on a protein kinase C-mediated signaling pathway, whereas CD4 may rely on greater tyrosine kinase activation. Such differential signaling via CD4 and CD8 has implications for thymic ontogeny and T cell activation.     

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.     

Defective expression of gp180, a novel CD8 ligand on intestinal epithelial cells, in inflammatory bowel disease.

Previous studies support a role for intestinal epithelial cells (IEC) as antigen-presenting cells in mucosal immune responses. T cells activated by IEC are CD8+, suppressor in function, and dependent upon CD8-associated p56lck activation. A 180-kD glycoprotein (gp180) recognized by mAbs B9 and L12 has been identified and shown to be important in CD8+ T cell activation by IEC. Since IEC derived from patients with inflammatory bowel disease (IBD) are incapable of activating CD8+ T cells, we asked whether this correlated with gp180 expression. While frozen sections of normal bowel revealed bright gp180 staining on all IEC, both inflamed and uninflamed ulcerative colitis (UC) specimens showed patchy staining. In Crohn's disease (CD), staining was faint to absent. Flow cytometry confirmed immunohistochemical data. The staining patterns correlated with the ability of IEC to activate CD8-associated p56lck. Normal IEC induced phosphorylation of p56lck in CD8alpha but not CD4+ transfectants. In contrast, both UC and CD IEC activated CD4 and, to a much lesser extent, CD8-associated p56lck. Thus, gp180 expression by IBD IEC appears to be altered, and correlates with a functional alteration of lck activation. This defect may reflect a more proximal event in the pathogenesis of IBD.     

Cell-cell adhesion mediated by CD8 and human histocompatibility leukocyte antigen G, a nonclassical major histocompatibility complex class 1 molecule on cytotrophoblasts

The lymphocyte differentiation marker CD8 acts as a coreceptor with the T cell receptor (TCR) during recognition of peptide presented by major histocompatibility complex (MHC) class I molecules. The functions of CD8 in the TCR complex are thought to be signaling through the association of CD8 with the protein tyrosine kinase p56lck and adhesion to MHC class I through the alpha 3 domain. While the ability of the CD8 alpha/alpha homodimer to bind to classical MHC class I molecules has been shown, it is unclear whether CD8 can also bind nonclassical molecules. Of particular interest is human histocompatibility leukocyte antigen (HLA)-G which is expressed on placental cytotrophoblast cells. These cells do not express HLA-A, -B and -C molecules. In this report, we demonstrate that CD8 can bind to HLA-G. It is possible, therefore, that a cell bearing CD8 may interact with HLA-G-expressing cells.     

p56lck interacts via its src homology 2 domain with the ZAP-70 kinase

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.     

Enrichment of oncolytic cells for adoptive therapy using centrifugal elutriation

We have shown that centrifugal elutriation (CE) can be used to obtain large numbers of peripheral blood lymphocytes enriched for natural killer (NK) cell activity. Lymphocytes distributed in fraction 3 (F3) were enriched 2.3 fold for large granular lymphocytes, LGL, and 1.8 fold for cells with CD16+ phenotype. Both unseparated and F3 cells proliferated and were activated following culture with interleukin-2 (IL-2), although the NK-enriched cells displayed superior cytolytic activity against tumor cell lines, as well as against fresh leukemic blasts. Comparison of the cell surface phenotype of long-term cultures indicated a significantly higher frequency of CD56+, CD3- and CD16+ NK cells, and a reduced content of CD3+ T cells in F3 populations. These results suggest that IL-2 activated NK-enriched CE-fractionated lymphocytes may be more effective than unseparated mononuclear cells for adoptive therapy purposes.     

CD3+CD16+NK1.1+B220+ large granular lymphocytes arise from both alpha- beta TCR+CD4-CD8- and gamma-delta TCR+CD4-CD8- cells

Cultivation of CD4-CD8- double negative (DN) mouse thymocytes and splenocytes with recombinant interleukin 2 (IL2) in the absence of other stimulation results in the generation of DN- CD3/TCR+CD16+NK1.1+B220+ large granular lymphocytes (LGL). Purified DN alpha-beta TCR+ thymocytes and splenocytes are CD16+IL2R alpha-IL2R beta+NK1.1+B220-CD5high. These cells are unique in that they express both CD16 and T cell receptor (TCR) which are usually mutually exclusive. In addition, they express the natural killer (NK) marker, NK1.1. Cultivation of these cells with IL2 for several days results in the generation of DN alpha-beta TCR+CD16+NK1.1+B220+CD5- LGL, suggesting that DN alpha-beta TCR+ cells in thymus and spleen are the precursors of the DN LGL reported previously. DN gamma-delta TCR+CD16- NK1.1-B220-CD5high thymocytes and splenocytes also give rise to DN gamma-delta TCR+CD16+NK1.1+B220+CD5- LGL which, as shown previously with DN alpha-beta TCR+ LGL cells, are cytotoxic against NK-sensitive YAC-1 cells. Cytotoxic activity is also induced through either CD16 or the gamma-delta TCR. DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL cells are thus similar in phenotype to TCR- NK cells. DN alpha-beta TCR+ thymocytes express low levels of the gamma subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI gamma) molecule, an essential component of CD16 expression. Fc epsilon RI gamma expression is greatly enhanced after cultivation with IL2, resulting in a higher surface expression of CD16. In contrast to DN alpha-beta TCR+ thymocytes, DN gamma-delta TCR+ thymocytes do not express detectable CD16 or Fc epsilon RI gamma mRNA but expression of both is induced by cultivation with IL2, leading to the expression of CD16 on the surface. Whereas CD16 molecules on both DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL are associated with only Fc epsilon RI gamma homodimers, the TCR on these cells are associated with an Fc epsilon RI gamma homodimer and/or CD3 zeta-Fc epsilon RI gamma heterodimers. These results demonstrate that the Fc epsilon RI gamma subunit is a component of the TCR in a fraction of T lineage cells.     

The protein tyrosine kinase p56lck regulates thymocyte development independently of its interaction with CD4 and CD8 coreceptors [corrected] [published erratum appears in J Exp Med 1993 Sep 1;178(3):1135]

The lck gene encodes a lymphocyte-specific protein tyrosine kinase of the nonreceptor type that is implicated in signal transduction pathways emanating from the CD4 and CD8 coreceptors. Previous studies also support a role for p56lck in regulating T cell receptor beta gene rearrangements and, more generally, thymocyte development. Here we report that a mutant form of p56lck, which is incapable of interacting with CD4 or CD8, behaves indistinguishably from association-competent p56lck with respect to its ability to affect thymocyte maturation. The effects of p56lck remained specific in that the closely related src- family kinase p59hck was incapable of substituting for p56lck in arresting beta locus gene rearrangements. These data support the view that src-family kinases perform highly specialized and often nonoverlapping functions in hematopoietic cells, and that p56lck acts independently of its association with CD4 and CD8 to regulate thymocyte development.     

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.     

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells

CD4(+)8(+) double positive (DP) thymocytes differentiate into CD4(+) and CD8(+) mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4(-)8(+) thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8(+) thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells.