Strength of TCR signal from self‐peptide modulates autoreactive thymocyte deletion and Foxp3+ Treg‐cell formation

Autoreactive CD4⁺CD8^? (CD4SP) thymocytes can be subjected to deletion when they encounter self‐peptide during their development, but they can also undergo selection to become CD4SPFoxp3⁺ Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP‐76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3⁺ thymocytes. CD4SP thymocytes expressing TCR Vβ‐chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3⁺ thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg‐cell formation in response to a defined self‐peptide, SLP‐76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3⁺ Treg‐cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg‐cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg‐cell formation.     

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.     

Reduced CD4 and CD8 Expression in Human Thymuses Treated with Soluble CD4

Recent data suggest that accessory molecules like CD4 and CD8 act as co-receptors in intrathymic T-cell development. Soluble CD4 (sCD4) molecules offer a novel experimental approach to investigate the relevance of CD4 interaction with its putative intrathymic receptor for T-cell maturation. We attempted to inhibit binding of surface CD4 on thymocytes to its intrathymic receptor competitively by introduction of human sCD4 into human thymus tissue cultures. Our results demonstrate that sCD4, while not affecting peripheral T-cell responses as shown in control experiments, significantly affects intrathymic development of T lymphocytes. Immature CD4CD8 double positive (DP) thymocytes responded with reduced expression of both CD4 and CD8 molecules. This phenomenon could be followed up to the stage of single positive (SP) thymocytes: density of CD4 molecules on CD4 SP thymocytes and, even more interestingly, CD8 expression on CD8 SP cells, were reduced, indicating that the effect observed in immature DP thymocytes persists during their further development. Beyond that, analysis of T-cell receptor (TCR) expression in the low density CD4CD8 DP population revealed a slight decrease of alpha beta-TCR surface expression, suggesting a possible role of CD4 engagement in the generation of TCR in man. Since sCD4 is considered a therapeutical agent in HIV infections, these findings are not only of basic but also of clinical interest.     

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.     

The Src-like adaptor protein downregulates the T cell receptor on CD4+CD8+ thymocytes and regulates positive selection

In this report, we show that the Src-like adaptor protein (SLAP) plays an important role in thymocyte development. SLAP expression is developmentally regulated; it is low in CD4-CD8- thymocytes, it peaks in the CD4+CD8+ subset, and it decreases to low levels in more mature cells. Disruption of the SLAP gene leads to a marked upregulation of TCR and CD5 expression at the CD4+CD8+ stage. The absence of SLAP was also developmentally significant because it enhanced positive selection in mice expressing the DO11.10 transgenic T cell receptor. Moreover, SLAP deletion at least partially rescued the development of ZAP-70-deficient thymocytes. These results demonstrate that SLAP participates in a novel mechanism of TCR downregulation at the CD4+CD8+ stage and regulates positive selection.     

CD4-CD8- thymocytes that express the T cell receptor may have previously expressed CD8

Amongst CD4-CD8- (double negative) thymocytes there is a sizeable population (variable from strain to strain) of cells expressing surface T cell receptor (TCR). These TCR+ double negatives are predominantly non-cycling, have very little precursor activity, and, unlike the TCR-CD4-CD8- thymocytes, appear not to be part of the mainstream of thymocyte development. A unique feature of this population is the biased V beta-gene region usage. In CBA mice, 60-70% of TCR+ CD4-CD8- cells express receptors that utilize V beta 8 gene products, compared with peripheral T cells from the same strain which are only 20-30% V beta 8+. This suggests that the high V beta 8 usage may be the result of some selective process. A growing body of experimental data suggests that TCR specificity selection occurs at the CD4+CD8+ stage of thymocyte development. In order to gain some insight into the previous history of the TCR+ double negatives, in particular whether or not they have previously expressed CD8 and therefore been eligible for selection, we have determined the methylation state of the CD8 gene and compared it to other thymocyte populations. We show that the TCR+ CD4-CD8- thymocytes are demethylated at some sites in the CD8 gene, consistent with previous CD8 expression. However, the demethylation pattern is distinct from that seen on typical peripheral T cells or on mature thymocytes, suggesting that the TCR+ CD4-CD8- thymocytes are not derived from mature thymocytes or peripheral T cells which have returned to the thymus and downregulated CD8 expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4(+)CD8(+)TCR(low) thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid-induced apoptosis

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4(+)CD8(+) thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cells within subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four-color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4(-)CD8(-)TCR(-) thymocytes, and decreased during development via the CD4(-)CD8(+)TCR(-) subpopulation into the CD4(+)CD8(+)TCR(low) subset. Interestingly, the latter population, although expressing less than half the GR density of CD4(-)CD8(-)TCR(-) cells, is the most sensitive subset to GC-induced apoptosis. Up-regulation of TCR expression by the CD4(+)CD8(+)TCR(low) subset to CD4(+)CD8(+)TCR(high) cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR-mediated signals at a constant rate relative to TCR expression.     

Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes

To understand how CD8 expression is regulated during the transition process from CD4+8+ (CD4 and CD8 double positive, DP) to CD4-8+ (CD8 single positive, CD8SP) cells in the thymus, the involvement of Runx proteins in the alteration of chromatin configuration was investigated. Using the chromatin immunoprecipitation assay, we first demonstrated that Runx proteins bind to the stage-specific CD8 enhancer, as well as the CD4 silencer, in CD8SP thymocytes. Among Runx family members, Runx3 expression was initiated in DP thymocytes receiving a positive selection signal and increased in concert with differentiation to the CD8SP stage. Furthermore, reactivation of the CD8 gene, as well as CD4 silencing, was suppressed in positively selected thymocytes of Runx dominant-negative transgenic mice. These results suggest that Runx proteins, especially Runx3, are involved in lineage specification of CD8 T cells and provide important information for understanding the mechanism for the mutually exclusive expression of coreceptors in mature thymocytes.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus

Differentiation of thymocytes into mature single-positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), coreceptors (CD4 or CD8) and their appropriate major histocompatibility complex-encoded ligands. Precisely how these receptor/co-receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by coligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')₂ antibodies to mimic TCR/co-receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross-linking or targeting to Fc receptor-bearing cells. We show that TCR/CD3 co-ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co-ligation with CD8 also induces maturation of CD4T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co-ligation of TCR and CD8 appear weaker than from co-ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single-positive cell differentiation.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected. We found that sorted CD3medium CD4+CD8- thymocytes from adult mice up-regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3high counterparts, CD3medium CD4+CD8- thymocytes were unable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3medium thymocytes of both CD4+CD8- and CD4-CD8+ subsets were also considerably more responsive than CD3high SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO-1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vbeta+ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3medium subpopulations than in CD3high subpopulations. These findings argue that the CD3medium SP thymocyte subpopulations contain apoptosis-susceptible precursor cells of CD3high SP cells and are subject to negatively selecting pressures.     

Differentiation of CD4highCD8low coreceptor-skewed thymocytes into mature CD8 single-positive cells independent of MHC class I recognition

Thymocytes with a CD4^hiCD8^lo coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4^hiCD8^lo cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2^bm13 background, generate a large number of CD4^hiCD8^lo CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4^hiCD8^lo population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4^hiCD8^lo CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.     

Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD⁺8⁺ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

Exogenous Thy-1.1 expression as a marker for thymocyte maturation in transgenic mice.

We established a line of transgenic mice carrying the exogenous mouse Thy-1.1 gene (8.2 Kb Eco RI genomic DNA fragment). In these mice, Thy-1.1 was expressed on thymocytes but not on peripheral T cells, presumably due to the lack of cis-acting element(s) on the microinjected genomic DNA. Even in the thymus, however, while most of the CD3/TCR- thymocytes were positive for the transgenic Thy-1.1 gene expression, the CD4+ or CD8+ single positive (SP) thymocytes were composed of two groups, one Thy-1.1+ and one Thy-1.1-, suggesting that the former belongs to cells at premature stages of terminal T cell differentiation. There was no difference in the amount of CD3/TCR complexes expressed on two such SP thymocyte subsets. In the double negative (DN) thymocytes, all the CD3/TCR+ cells were Thy-1.1-. These results suggest that the maturational process of CD3+ DN thymocytes differs from that of SP thymocytes. The unique distribution of Thy-1.1+ population among CD3+ thymocytes suggests that the transgenic Thy-1.1 gene expression can serve as a useful marker to examine terminal maturation processes of CD3+ thymocytes.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.