CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection

In the thymus, phenotypically and functionally mature single positive cells are generated from immature CD4+8+ precursors by a process known as positive selection. Although this event is known to involve alphabetaTCR ligation by peptide/MHC complexes expressed on thymic stromal cells, it is clear that positive selection is a multistage process involving transition through an intermediate CD4+8+69+ phase as well as subsequent postselection phases. By analyzing the development of preselection CD4+8+69- and intermediate CD4+8+69+ thymocytes in the presence of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient thymic stromal cells, we investigated the role of MHC molecules at three distinct points during positive selection. Although the initiation of positive selection is critically dependent upon MHC interactions, we find the that later stages of maturation, involving the differentiation of CD4+8- and CD4-8+ cells from CD4+8+69+ thymocytes, occur in the absence of MHC molecules. Moreover, an analysis of the postselection proliferation of newly generated CD4+8- and CD4-8+ thymocytes shows that this also occurs independently of MHC molecules. Thus, our data provide direct evidence that, although positive selection is a multistage process initiated by TCR-MHC interactions, continuation of this process and subsequent postselection events are independent of ongoing engagement of the TCR.     

Chronic alpha1-adrenoreceptor blockade produces age-dependent changes in rat thymus structure and thymocyte differentiation

In order to examine the influence of chronic alpha1-adrenergic receptor (alpha1-AR) blockade on the thymus structure and T-cell maturation, peripubertal and adult male rats were treated with urapidil (0.20 mg/kg BW/d; s.c.) over 15 consecutive days. Thymic structure and phenotypic characteristics of the thymocytes were assessed by stereological and flow cytometry analysis, respectively. In immature rats, treatment with urapidil reduced the body weight gain and, affecting the volume of cortical compartment and its cellularity decreased the organ size and the total number of thymocytes compared to age-matched saline-injected controls. The percentage of CD4+8- single positive (SP) thymocytes was decreased, while that of CD4-8+ was increased suggesting, most likely, a disregulation in final steps of the positively selected cells maturation. However, alpha1-AR blockade in adult rats increased the thymus weight as a consequence of increase in the cortical size and cellularity. The increased percentage of most immature CD4-8- double negative (DN) cells associated with decreased percentage of immature CD4+8+ double positive (DP) thymocytes suggests a decelerated transition from DN to DP stage of T-cell development. As in immature rats, the treatment in adult rats evoked changes in the relative numbers of SP cells, but contrary to immature animals, favoring the maturation of CD4+8- over CD4-8+ thymocytes. These results demonstrate that: i) chronic blockade of alpha1-ARs affects both the thymus structure and thymocyte differentiation, ii) these effects are age-dependent, pointing out to pharmacological manipulation of alpha1-AR-mediated signaling as potential means for modulation of the intrathymic T-cell maturation.     

A role for SATB1, a nuclear matrix association region-binding protein, in the development of CD8SP thymocytes and peripheral T lymphocytes

Studies have suggested that binding of the SATB1 protein to L2a, a matrix association region located 4.5 kb 5' to the mouse CD8alpha gene, positively affects CD8 expression in T cells. Therefore, experiments were performed to determine the effect on T cell development of reduced expression of SATB1. Because homozygous SATB1-null mice do not survive to adulthood due to non-thymus autonomous defects, mice were produced that were homozygous for a T cell-specific SATB1-antisense transgene and heterozygous for a SATB1-null allele. Thymic SATB1 protein was reduced significantly in these mice, and the major cellular phenotype observed was a significant reduction in the percentage of CD8SP T cells in thymus, spleen, and lymph nodes. Mice were smaller than wild type but generally healthy, and besides a general reduction in cellularity and a slight increase in surface CD3 expression on CD8SP thymocytes, the composition of the thymus was similar to wild type. The reduction in thymic SATB1 does not lead to the variegated expression of CD8-negative single positive thymocytes seen upon deletion of several regulatory elements and suggested by others to reflect failure to activate the CD8 locus. Thus, the present results point to an essential role for SATB1 late in the development and maturation of CD8SP T cells.     

TCRαβ^＋CD^＋4CD^—8胸腺髓质细胞的表型分析

Phenotypic analysis of the medullary-type CD4+CD8- (CD4SP) thymocytes have revealed phenotypic heterogeneity within these cells. The phenotype of mature peripheral T cells is Qa-2+ HSA- CD69-, whereas in the medullary-type CD4SP thymocytes, the expression pattern of many markers were quite different, suggesting that the medullary-type CD4SP thymocytes may undergo phenotypic maturation. According to the results of two-color cytometry, seven discrete phenotypes were defined by the relative expression of Qa-2, HSA, CD69, 3G11 and 6C10: 3G11-6C10+CD69+HSAhi-->3G11+6C10+CD69+ HSAhi-->3G11+6C10-CD69+HSAint-->3G11+6C10- CD69-HSAint Qa-2(-)-->3G11+HSAlo/-Qa-2lo, at the same time, 3G11+6C10-CD69-HSAint Qa-2(-)-->3G11-HSAlo Qa-2(-)-->3G11-HSAlo/- Qa-2hi, the last two Qa-2 positive subsets could exit the thymus and home into periphery.     

A molecular marker for thymocyte-positive selection: selection of CD4 single-positive thymocytes with shorter TCRB CDR3 during T cell development

The generation of the naive T cell repertoire is a direct result of maturation and selection events in the thymus. Although maturation events are judged predominantly on the expression of surface markers, molecular markers, more intimately involved in the selection process, can be informative. We have identified a molecular marker for selection in later stages of maturation in humans. Thymocytes are selected for the expression of TCR beta-chains with shorter CDR3 at the double-positive to single-positive (SP) transition. Here we extend these studies to the mouse and show that the selection phenotype is not related to alpha-chain pairing but is a function of the MHC haplotype. Interestingly, the selection is much more apparent in CD4 SP thymocytes than in CD8 SP cells. This is in contrast to human thymocytes, where the selection is equally apparent in both lineages. The involvement of MHC in the process argues that this is a positive selection stage. The difference in the extent of this selection between the two SP lineages may indicate a class difference in the nature of the TCR-MHC interaction, the role of coreceptors in the selection process, or both.     

Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus.

We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes.     

Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.     

Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

Maturation-dependent licensing of naive T cells for rapid TNF production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4⁺ and CD8⁺ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.     

Premature expression of chemokine receptor CCR9 impairs T cell development

During thymocyte development, CCR9 is expressed on late CD4-CD8- (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.     

Effects of cyclosporine A on thymocyte differentiation in fetal thymus organ culture

During the course of differentiation in the thymus, precursor T cells are negatively selected by a self-tolerance mechanism or positively selected to acquire restriction specificity to self major histocompatibility complexes. We investigated the process of T cell differentiation and those selections using a fetal thymus organ culture with or without cyclosporine A. The agent blocked the maturation step from CD4+8+ double positive cells to mature CD4-8+ or CD4+8- single positive cells. On the other hand, the agent did not inhibit the development of CD3+4-8- T cell receptor (TCR)alpha beta- cells, which were supposed to be T cells bearing gamma delta-TCR chains. These results suggest that the development of thymocytes bearing alpha beta- or gamma delta-TCR chains differ in requirement for thymocyte-stromal cell interaction.     

Editing autoreactive TCR enables efficient positive selection

Allelic exclusion is inefficient at the TCRalpha locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4(+)CD8(-) thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, approximately 100% more CD4(+)CD8(-) thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4(+)CD8(-) thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.     

IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice

We have assessed the biologic role of IL-4 by fusing its gene to an immunoglobulin promoter/enhancer and introducing it into transgenic mice. By attenuating the transgene promoter through the insertion of E. coli lac operator sequences, we have created a series of animals that constitutively express varying amounts of IL-4. Overexpression of IL-4 results in a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion (blepharitis) with characteristic histopathologic features seen in allergic reactions. In addition, expression of the IL-4 transgene in the thymus perturbs T cell maturation, reducing the population of immature CD4+CD8+ thymocytes and peripheral T cells while increasing the population of mature CD8+ thymocytes. These results demonstrate that deregulation of a single cytokine gene in vivo can induce a complex inflammatory reaction resembling that observed in human allergic disease.     

Ephrin-B1 is critical in T-cell development

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. In this study, we investigated the role of EFNB1 and the Ephs it interacts with (collectively called EFNB1 receptors) in mouse T-cell development. In the thymus, CD8 single positive (SP) and CD4CD8 double positive (DP) cells expressed high levels of EFNB1 and EFNB1 receptors, whereas CD4 SP cells had moderate expression of both. Soluble EFNB1-Fc in fetal thymus organ culture caused significant subpopulation ratio skew, with increased CD4 SP and CD8 SP and decreased DP percentage, while the cellularity of the thymus remained constant. Moreover, in EFNB1-treated fetal thymus organ culture, CD117(+), CD25(+), DP, CD4 SP, and CD8 SP cells all had significantly enhanced proliferation history, according to bromodeoxyuridine uptake. In vitro culture of isolated thymocytes revealed that EFNB1-Fc on solid-phase protected thymocytes from anti-CD3-induced apoptosis, with concomitant augmentation of several antiapoptotic factors, particularly in CD4 SP and CD8 SP cells; on the other hand, soluble EFNB1-Fc promoted anti-CD3-induced apoptosis, as was the case in vivo. This study reveals that EFNB1 and EFNB1 receptors are critical in thymocyte development.     

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.