Combined deletion of CD8 locus cis-regulatory elements affects initiation but not maintenance of CD8 expression

Developmental stage-, subset-, and lineage-specific CD8 enhancers have been identified recently by transgenic reporter analyses. Enhancer E8(II) (CIV-4,5) is active in both immature double-positive thymocytes (DP) and mature CD8 single-positive (SP) thymocytes and T cells, whereas E8(I) (CIII-1,2) directs expression only in mature cells. In mice lacking either E8(I) (CIII-1,2) or E8(II) (CIV-4,5), there was no effect on CD8 expression in DP thymocytes. However, deletion of both enhancers resulted in variegated expression of CD8, with appearance of CD4(+)CD8(-) SP thymocytes expressing surface markers characteristic of DP thymocytes. Consequently, fewer mature CD8(+) T cells developed from the reduced pool of DP cells. These results suggest that the initiation of CD8 expression is mediated by cis-regulatory elements that are distinct from any that may be involved in maintenance of expression.     

Alterations during positive selection in the thymus of nackt CD4-deficient mice

The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.     

Expression of CD21 is developmentally regulated during thymic maturation of human T lymphocytes.

CD21, the C3d/CD23/Epstein-Barr virus (EBV), receptor is expressed at low density on cells of the T lineage. Immature thymocytes express CD21 with high density. In the present study, we have analyzed the expression of CD21 during intrathymic maturation of T cells. An intense staining for CD21 was observed at the double-negative stage and at the stage of early acquisition of CD4. CD21 expression was decreased at the double-positive and single-positive stages, to then reach levels similar to those of peripheral blood T cells. Staining of thymus sections showed a bright fluorescent signal on thymocytes entering the thymus in the cortical region. Taking advantage of the immature phenotype of cells expressing high amounts of CD21 (CD21(++)), we depleted thymocyte suspensions in CD3(+) and CD8(+) cells to study the properties of CD21 on this cell subset. Triggering of CD21 with its ligands iC3b, CD23 and anti-CD21 mAb did not alter the proliferative response of thymocytes to IL-7, and did not induce the differentiation of early cells into CD4(+)CD8(+) thymocytes. Immunoprecipitation did not reveal any molecule associated with CD21 that could play a signaling role in thymocytes. Finally, EBV induced a down-regulation of CD21 and an up-regulation of CD1 in CD21(++) thymocytes. Taken together, our observations demonstrate a regulated expression of CD21 on human thymocytes and suggest that the CD21(++) subset may be a target for EBV. We further suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.     

In vitro thymocyte differentiation in MHC class I-negative Xenopus larvae

CTX is a surface antigen whose expression in larval and adult Xenopus is primarily restricted to MHC class I-negative immature cortical thymocytes. In adult Xenopus, surface expression of CTX marks a population of MHC class I(-) CD8(+) immature thymocytes that appears to be the equivalent of the mammalian CD4CD8 double positive subset. The present study reveals that transient in vitro exposure of immature CTX(+) thymocytes from MHC class I-negative tadpoles to suboptimal mitogenic concentrations of phorbol ester (PMA) plus ionomycin, induces larval cells to differentiate into more mature T-lymphoblasts that express high level of surface CD5 and CD45. These T-lymphoblasts have downregulated CTX, Rag 1 and TdT genes, whereas TCR-beta genes remain actively transcribed. Signaling induced by PMA/ionomycin modulates both class I and class II expression of MHC class I/II-negative larval thymocytes.This study also reveals that larval T-lymphoblasts are composed of two distinct subsets: CD5(high)CD8(-) and CD5 (high)CD8 (high).     

Effect of phorbol ester and calcium ionophore on human thymocytes

Positive selection of immature thymocytes is a developmental process in which TCR ligation with low avidity induces generation of mature T cells. In mouse thymocytes, CD4(+)8(+) double-positive (DP) cells which were treated with a proper combination of calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) have been reported to differentiate into CD4 single positive cells. However, in human thymocytes the effects of PMA and ionomycin have remained unclear. Here we report that DP cells that were treated with PMA and ionomycin up-regulated bcl-2 and down-regulated CD1 expression. However, CD3 expression remained low. This treatment induced prolonged CD4 down-regulation in DP cells which was an effect also seen in mature peripheral blood T cells. PMA/ionomycin-treated DP cells showed high cell proliferation and resistance to dexamethasone-induced apoptosis. These results indicate that PKC activation and calcium elevation may be part of the biochemical signals that induce positive selection of human DP cells and the system described in this paper may be a useful model to study the signals involved in the selection of human thymocytes.     

The expression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bim) in human lymphocytes

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.     

V?Gene Family Usage in Spontaneous Lymphomas of AKR Mice: Evidence for Defective Clonal Deletion

T-cell receptor (TCR) ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4⁺8⁺) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V ß gene family usage, 2 out of 14 lymphomas expressed V ß6. Normally, V ß6⁺ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V ß6⁺ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.     

Within the hemopoietic system, LAR phosphatase is a T cell lineage-specific adhesion receptor-like protein whose phosphatase activity appears dispensable for T cell development, repertoire selection and function

Expression of the receptor-type tyrosine phosphatase LAR was studied in cells of the murine hemopoietic system. The gene is expressed in all cells of the T cell lineage but not in cells of any other hemopoietic lineage and the level of expression in T cells is developmentally regulated. The CD4(-)8(-)44(+) early thymic immigrants and mature (CD4(+)8(-)/CD4(-)8(+)) thymocytes and T cells express low levels, whereas immature (CD4(-)8(-)44(-) and CD4(+)8(+)) thymocytes express high levels of LAR. Among bone marrow cells only uncommitted c-kit(+)B220(+)CD19(-) precursors, but not B cell lineage committed c-kit(+)B220(+)CD19(+) precursors, express low levels of LAR. In contrast to the c-kit(+)B220(+)CD19(+) pre-BI cells from normal mice, counterparts of pre-BI cells from PAX-5-deficient mice express LAR, indicating that PAX-5-mediated commitment to the B cell lineage results in suppression of LAR. During differentiation of PAX-5-deficient pre-BI cell line into non-T cell lineages, expression of LAR is switched off, but it is up-regulated during differentiation into thymocytes. Thus, within the hemopoietic system, LAR appears to be a T cell lineage-specific receptor-type phosphatase. However, surprisingly, truncation of its phosphatase domains has no obvious effect on T cell development, repertoire selection or function.     

Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals

Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the alphabeta T-cell lineage and plays an important role in regulating beta-selection in CD4(-)CD8(-) double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4(+)CD8(+) double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG(-/-) lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG(-/-) thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76(-/-) precursors resulted in only a minimal developmental rescue to the immature CD8(+) single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG(-/-) thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass beta-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.     

Establishment of the major compatibility complex-dependent development of CD4+ and CD8+ T cells by the Cbl family proteins

Casitas B cell lymphoma (Cbl) proteins are negative regulators for T cell antigen receptor (TCR) signaling. Their role in thymocyte development remains unclear. Here we show that simultaneous inactivation of c-Cbl and Cbl-b in thymocytes enhanced thymic negative selection and altered the ratio of CD4(+) and CD8(+) T cells. Strikingly, the mutant thymocytes developed into CD4(+)- and CD8(+)-lineage T cells independent of the major histocompatibility complex (MHC), indicating that the CD4(+)- and CD8(+)-lineage development programs are constitutively active in the absence of c-Cbl and Cbl-b. The mutant double-positive (DP) thymocytes exhibited spontaneous hyperactivation of nuclear factor-kappa B (NF-kappaB). Additionally, they failed to downregulate the pre-TCR and pre-TCR signaling. Thus, our data indicate that Cbl proteins play a critical role in establishing the MHC-dependent CD4(+) and CD8(+) T cell development programs. They likely do so by suppressing MHC-independent NF-kappaB activation, possibly through downmodulating pre-TCR signaling in DP thymocytes.     

T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential

Immature CD8-CD4- double-negative (DN) thymocytes differentiate intrathymically into CD8+CD4- and CD8-CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double-positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)alpha chain, unlike the less mature interleukin 2 receptor-positive DN thymocytes. Surface expression of the TcR-associated CD3 molecule occurs on approximately 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (approximately 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%-34% on day 1 and 48%-68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR alpha chain genes and express TcR alpha/beta heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.     

CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics

The molecular mechanisms that govern the survival, maturation and export of thymocytes are the subject of intense study, and candidates for involvement in these processes might be identified by their differential expression during thymocyte selection. One such molecule is the tetraspanin CD53, which is not expressed on most CD4(+)CD8(+) double-positive (DP) cells in the normal mouse. We have examined CD53 expression on DP from several class I- and class II-restricted TCR transgenic (Tg) mice, and have found a strong correlation between CD53 expression and positive selection. CD53 expression in DP was formally demonstrated to be dependent upon MHC recognition as evidenced by studying DP from MHC-deficient mice which totally lack expression of this molecule. This link between selection and CD53 expression was reminiscent of CD69, and indeed the majority of selected DP from normal mice that express CD53 also express CD69. We compared CD53 and CD69 induction in vitro using pre-selected thymocytes from TCR-Tg mice that were stimulated either with mAb against TCR or with antigen-presenting cells (APC) pulsed with peptides. The data shows that with either stimulus, CD69 is induced rapidly on the thymocyte surface with expression detected in as little as 2 h. CD53 induction is slower with maximal expression taking up to 20 h. We also stimulated pre-selected thymocytes from the OT-1 TCR-Tg strain with APC pulsed with peptides of varying affinities for the TCR. Here low-affinity peptides which induce CD69 expression poorly were able to induce significant levels of CD53 expression. These data demonstrate that the induction of CD53 and CD69 upon selection is not identical. Thus a combination of the CD69 and CD53 selection markers may be a powerful tool to isolate thymocytes that have either been very recently selected or have arisen from differing MHC--TCR affinity interactions during selection.     

Attenuation of cytokine responsiveness during T cell development and differentiation.

Cytokines play critical roles during T cell development; however, it is unclear to what extent development is altered by the high levels of cytokines produced during immune responses. A potential mechanism to shield developing cells from cytokine influence is attenuation of cytokine signaling. Using intracellular staining and flow cytometry to detect cytokine-induced Stat phosphorylation, we analyzed the cytokine responsiveness of developmentally defined mouse T cells. We assessed CD4(-)CD8(-) (DN), CD4(+)CD8(+) (DP), CD4(+)CD8(-) (SP4), and CD4(-)CD8(+) (SP8) in the thymus, and CD4(+)CD44(lo) (naive), CD4(+)CD44(hi) (memory), CD8(+)CD44(lo) (naive), and CD8(+)CD44(hi) (memory) in the periphery for responsiveness to interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL- 10, IL-15, interferon-alpha (IFN-alpha), and IFN-gamma. SP thymocytes responded to a wider range of cytokines than did the less mature DN and DP subpopulations. DP thymocytes were nonresponsive to all cytokines tested except for modest responses to IL-4 and IFN-alpha. Peripheral naive and memory T cells also displayed differential cytokine sensitivity. Memory T cells were less responsive to the proinflammatory cytokines IL-6 and IFN-gamma when compared with naive T cells, and the memory CD4(+) subset was less responsive to IL-4. In summary, developing thymocytes and memory T cells appear to be resistant to the influences of numerous cytokines produced during immune responses.     

Immature thymocytes that fail to express TCRbeta and/or TCRgamma delta proteins die by apoptotic cell death in the CD44(-)CD25(-) (DN4) subset.

Pre-TCR/CD3 signals are essential for survival and maturation of (CD44(-)25(+)) DN3 thymocytes via the (CD44(-)25(-)) DN4 stage to CD4(+)CD8(+) (DP) cells, a process termed beta-selection. The exact developmental stages of apoptosis resulting from lack of pre-TCR/CD3 signals have so far not been determined. Here we analyzed apoptotic cell death in relation to expression of clonotypic TCR polypeptides and to cell cycle status in immature thymocyte subpopulations of wild type (wt) mice and of several strains of mice with compromised pre-TCR/CD3 signaling complexes. In wt mice or pre-TCR/CD3-deficient mice, apoptotic cells could not be detected among DN3 cells but accumulated in a subset of DN4 expressing CD69. Apoptotic CD69(+)DN4 cells were rare in wt mice and were found among DN4 cells that were negative or low for intracellular TCRbeta and negative for TCRgamma delta polypeptide chains. Apoptotic CD69(+)DN4 cells were abundant in pre-TCR/CD3 signaling-deficient mice in which most DN4 cells failed to express clonotypic TCR polypeptides. Survival of DN4 cells, but not maturation of DN3 cells to DN4, was found to depend on the expression of clonotypic TCR polypeptides in the same cell. The results suggest that thymocytes unsuccessful in alpha beta or in gamma delta lineage development die by apoptosis in the DN4 subset.     

Development of autoreactive diabetogenic T cells in the thymus of NOD mice

Type 1 diabetes results from destruction of pancreatic beta cells by beta cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential beta cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4+CD8+ DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69+CD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4-CD8+ T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice.     

Participation of a chicken thymocyte antigen in intrathymic differentiation of T cells in vivo.

The participation of CETHB1 antigen in the intrathymic development of T cells was analysed in sublethally X-ray irradiated chickens and normal embryos which were inoculated with anti-thymocyte monoclonal antibody CETHB1. When chickens were given a sublethal X-ray irradiation on the day of hatchings immature thymo-cytes existed prominently in the thymus up to 3 weeks after irradiation. The inoculation of CETHB1 monoclonal antibody on days 14 and 18 after irradiation caused an increase of CD4(-) CD8(-) cells and a decrease of CD4 (+) CD8 (+) cells as compared to changes in control chickens. However, expression of T cell receptors CD3, alphabetaTCR, and gammadeltaTCR was not influenced. Inoculation of the monoclonal antibody on embryonic days 13 to 16 resulted in a decrease of CD4 (+) CD8(-) cells and an increase of CD4(-)CD8(-) and CD4 (+) CD8(+) cells after hatching. Thus, the binding of CETHB1 monoclonal antibody to thymocytes can inhibit the differentiation of both CD4(-)CD8(-) and CD4 (+) CD8(+) cells to CD4 (+) CD8(+) and CD4 (+) CD8(-) or CD4(-)CD8(+) cells, respectively. These results suggest that CETHB1 antigen participated in the growth and differentiation of thymocytes in the thymic microenvironment.