TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.     

Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2(-/-) double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.     

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

Selective association between MHC class I-restricted T cell receptors, CDS, and activated tyrosine kinases on thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the TCR repertoire. Positive selection involves the differentiation of immature thymocytes capable of recognizing antigens complexed with self-MHC molecules to mature T cells. Besides the central role of TCR engagement by MHC in triggering selection; the interaction of CD8 and CD4 with MHC class I and class II, respectively; is thought to be important in regulating the selection process. To study potential mechanisms involved in positive selection of CD8+ cells, we have analyzed mice expressing a unique transgenic TCR. The transgenic receptor recognizes the HY male Ag in the context of the MHC class I molecule, H2-Db. We describe that CD8 and the TCR are selectively associated in thymocytes of mice expressing the restricting MHC, but not in thymocytes of mice expressing a nonrestricting MHC. pp56lck and pp59fyn, the tyrosine kinases associated with CD8 and TCR, respectively, were found to be present in this complex in an activated form. No comparable TCR-CD4 complex formation was found in thymuses undergoing positive selection to CD8+ cells. The formation of a multimolecular complex between CD8 and TCR, in which pp56lck and pp59fyn are activated, may initiate specific signaling programs involved in the maturation of CD8+ cells.     

Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice

DNA-labeling studies in alpha beta T cell receptor (TCR) transgenic mice show that the lifespan of immature CD4+8+ thymocytes is 3.5 days irrespective of whether they are selected for maturation or not. While nonselected cells die, the binding of the TCR to thymic major histocompatibility complex molecules rescues CD4+8+ cells from programmed cell death and induces first upregulation of the TCR level and then differentiation into CD4+8- or CD4-8+ cells in the absence of any cell division. When most CD4+8+ thymocytes express a selectable transgenic TCR the formation of mature cells with high TCR levels is 10-20 times as efficient as observed in normal mice, yet still only 20% of the CD4+8+ cells become mature. This is due to the limited availability of selecting 'niches': most CD4+8+ thymocytes with a selectable transgenic TCR will undergo maturation when they represent only 5% or less of all CD4+8+ cells.     

TCR-independent and caspase-independent apoptosis of murine thymocytes by CD24 cross-linking

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.     

Modeling TCR signaling complex formation in positive selection

T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4(+)8(-) or CD4(-)8(+) stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4(+)8(+) stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4(+)8(+) thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.