Downregulated expression of Ly-6-ThB on developing T cells marks CD4+CD8+ subset undergoing selection in the thymus

Interaction of TCRs on CD4+CD8+ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4+CD8+ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4+CD8+ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4+CD8+ thymocytes as well as in mature CD4+CD8- and CD4-CD8+ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4+CD8+ thymocytes identifies a unique subset (CD4+CD8+ThBneg-low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (approximately 20-40%) of CD4+CD8+ immature thymocytes receive these initial signals during thymic selection.     

TOX: an HMG box protein implicated in the regulation of thymocyte selection

In the thymus, pre-T cell receptor (pre-TCR)--mediated signaling and then TCR-mediated signaling initiate changes in gene expression that result in the maturation of CD4 and CD8 lineage T cells from common precursors. Using gene chip technology, we isolated a murine gene, designated Tox, that encodes a member of the HMG (high-mobility group) box family of DNA-binding proteins. TOX expression is up-regulated by both pre-TCR and TCR activation of immature thymocytes but not by TCR activation of mature na?ve T cells. Transgenic mice that express TOX show expanded CD8+ and reduced CD4+ single positive thymocyte subpopulations. We present evidence here that this phenotype results from a perturbation in lineage commitment due to reduced sensitivity to TCR-mediated signaling. This molecular marker of thymic selection events may therefore play a role in establishing the activation threshold of developing T cells and patterning changes in gene expression.     

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.     

Effects of cyclosporin A on mouse thymus: immunochemical and ultrastructural studies.

The in vivo effect of cyclosporin A (CsA) on the murine thymus was investigated by studying the ultrastructural cellular alterations, which are described for the first time, and the immunohistochemical modifications of thymocytes and thymic reticulo-epithelial cells (TREC). A marked reduction of the thymus size became apparent after 6 days of CsA treatment (10 mg/kg/day). Light microscopy studies using polyclonal antibodies (Ab) or monoclonal Ab specific to lymphoid sub-populations (anti-CD4, anti-CD5, anti-CD8), and specific to epithelial cells (anti-keratin: AKs, K8), to cortical TREC (TR4) and to subcapsular/medullary TREC (TR5, 3H9) showed that the number of CD4- 8+ or CD4+ 8- medullary thymocytes dramatically decreased and that the cortical TREC are affected, after 10 days of CsA treatment. These observations were confirmed by electron microscopy studies demonstrated that the medullary lymphoid population disappeared almost entirely. TREC, principally cortical type, presented signs of cellular lysis. A few cortical thymocytes showed some damage. At day 8 the medullary thymic tissue was disorganized, but no change was noted in the subcapsular area right up to the final day (day 10) of CsA treatment. These results suggest that CsA has a harmful effect on cortical TREC, which affects the development of immature thymocytes.     

Downregulated Expression of Ly-6-ThB on Developing T Cells Marks CD4+CD8+ Subset Undergoing Selection in the Thymus

Interaction of TCRs on CD4⁺CD8⁺ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4⁺CD8⁺ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4⁺CD8⁺ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4⁺CD8⁺ thymocytes as well as in mature CD4⁺CD8^- and CD4^-CD8⁺ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4⁺CD8⁺ thymocytes identifies a unique subset (CD4+CD8+ThB^neg–low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (Ÿ20–40%) of CD4⁺CD8⁺ immature thymocytes receive these initial signals during thymic selection.     

Analysis of recent thymic emigrants with subset- and maturity-related markers

The thymus plays an integral role in the development and production of T lymphocytes. However, thymocytes differ markedly in their phenotypic characteristics from the T cells normally found in the peripheral lymphoid organs. We have examined the phenotypic characteristics of recent thymic emigrants and compared them with both mature phenotype thymocytes (CD4+ CD8-CD3+ and CD4-CD8+ CD3+) and lymph node T cells. Recent thymic emigrants were defined as those fluorescein-positive cells found in the lymph node up to 16 h after intrathymic injection of fluorescein. Most cells emigrating from the thymus expressed CD3 and either CD4 or CD8, indicating maturity. Recent thymic emigrants, like mature phenotype thymocytes, were slightly larger on average than peripheral T cells, but this differential was lost within 24 h of emigration. Also like mature thymocytes but unlike peripheral T cells, some recent emigrants expressed heat-stable antigen. This did not change within 24 h of emigration. The antigen CD44 (Pgp-1, Ly-24) was expressed on a proportion of mature thymocytes, recent thymic emigrants, and peripheral T cells, and its expression did not show any clear relationship to maturity. The antigen CD45R also did not show marked changes associated with maturity, but our data do not parallel the published data of the expression of CD45R in the human. We conclude that recent thymic emigrants are phenotypically mature with respect to some antigens but not others. None of the antigens we investigated could have been used to uniquely distinguish recent thymic emigrants from peripheral T cells or from mature thymocytes.     

Identification of mature and immature human thymic dendritic cells that differentially express HLA-DR and interleukin-3 receptor in vivo

We have previously shown that thymic CD34+ cells have a very limited myeloid differentiation capacity and differentiate in vitro mostly into CD1a+-derived but not CD14+-derived dendritic cells (DC). Herein we characterized the human neonatal thymic DC extracted from the organ in relationship with the DC generated from CD34+ cells in situ. We show that in vivo thymic DC express E cadherin, CLA, CD4, CD38, CD40, CD44, and granulocyte-macrophage colony-stimulating factor-R (GM-CSF-R; CD116) but no CD1a. According to their morphology, functions, and surface staining they could be separated into two distinct subpopulations: mature HLA-DRhi, mostly interleukin-3-R (CD123)-negative cells, associated with thymocytes, some apoptotic, and expressed myeloid and activation markers but no lymphoid markers. In contrast, immature HLA-DR+ CD123hi CD36+ cells with monocytoid morphology lacked activation and myeloid antigens but expressed lymphoid antigens. The latter express pTalpha mRNA, which is also found in CD34+ thymocytes and in blood CD123hi DC further linking this subset to lymphoid DC. However, the DC generated from CD34+ thymic progenitors under standard conditions were pTalpha-negative. Thymic lymphoid DC showed similar phenotype and cytokine production profile as blood/tonsillar lymphoid DC but responded to GM-CSF, and at variance with them produced no or little type I interferon upon infection with viruses and did not induce a strict polarization of naive T cells into TH2 cells. Their function in the thymus remains therefore to be elucidated.     

The role of adenosine receptor engagement in murine fetal thymocyte development.

The role of adenosine receptor engagement in murine T-cell development was evaluated by culturing day 15-16 fetal thymic lobes in the presence of various concentrations of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA) or the adenosine receptor antagonist 8-phenyl-theophylline (8-PT) using the fetal thymic organ culture (FTOC) system. Before and 8 days after culture, thymocytes were isolated, counted, and analyzed for the expression of CD4 and CD8 T-cell differentiation molecules. Analysis of fresh thymocytes prior to culture showed that the majority of cells were of the CD4 single-positive or CD4+ CD8+ immature phenotype. Eight days after culture with media alone, 44% of cells were CD4+ and 23% were CD8+, and the number of viable thymocytes had increased from 1.7 x 10(5) to 2.2 x 10(5) cells per thymic lobe. A dose-dependent inhibition of maturation was observed in cultures with 8-PT, with greater than 85% inhibition at 50 microM. The double-positive thymocyte subset was most severely depleted. The number of cells obtained from cultures with NECA was also reduced, with about 65% inhibition at 50 microM, especially the CD8+ subset that was most severely affected. These results suggest that adenosine receptor engagement is required for normal T-cell differentiation and that adenosine receptor agonists and antagonists have distinct effects on thymocyte differentiation. An understanding of the cell-type-specific and developmental expression of adenosine receptors will help elucidate the mechanisms by which adenosine receptor engagement influences T-cell development.     

Age-associated decrease in proportion and antigen expression of CD8+/CD4+ thymocytes in BALB/c mice.

We studied the distribution of Thy-1.2+, CD8+ and CD4+ thymocytes in 3-, 6.5-, 12- and 18-month-old inbred BALB/c mice by staining cells with specific monoclonal antibodies (mAb) using indirect membrane immunofluorescence. The percentages of Thy-1.2+, CD8+, and CD4+ thymocytes did not vary with age until 12 months, but they exhibited at 18 months a highly significant decline. Staining thymocytes with mAb CD8 and CD4 alone or in a mixture allowed us to show that the age-related decline in proportions of CD8+ and CD4+ thymic cells is associated with the double positive (CD8+/CD4+, immature) subset. Most importantly, we observed a progressive age-related decrease in ratio of bright/dull Thy-1.2+, CD8+ and CD4+ thymocytes. The CD8+ and CD4+ thymocytes that did not adhere to peanut agglutinin (PNA-, which is supposed to represent the mature single positive subset) did not show any age-related change in the proportion of bright and dull cells. Therefore, we concluded that the double positive (CD8+/CD4+, immature, PNA+) thymocytes display reduced expression of CD8 and CD4 antigens with aging. The implications of these findings to the fundamental mechanisms of immunosenescence are discussed.     

Tyrosine kinase triggering in thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the T cell receptor (TcR) repertoire. One of these processes is positive selection. Positive selection involves the differentiation to mature T cells of thymocytes bearing TcR capable of recognizing antigens in the context of self-major histocompatibility complex (MHC) molecules. To study the potential involvement of tyrosine phosphorylation in the mechanism of positive selection, we have analyzed the activities of the tyrosine kinases pp56lck and pp59fyn in thymocytes expressing a unique TcR specific for HY antigen+ H-2 Db. Thymocytes undergoing positive selection displayed higher kinase specific activities of pp56lck and pp59fyn than nonselecting thymocytes. Furthermore, these increases in kinase activities were found only in the CD4+CD8+ subpopulation of thymocytes, where the selection process is believed to occur. These data suggest that tyrosine phosphorylation is part of the intracellular signals involved in MHC class I-driven positive selection from CD4+CD8+ immature thymocytes to CD4-CD8+ mature cells.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

An immature rat lymphocyte marker CD157: striking differences in the expression between mice and rats

We have established a novel monoclonal antibody that recognises mouse and rat CD157, and uncovered striking differences in both the level and stage of expression of this antigen in the primary lymphoid organs between these two species. Unlike mouse, the majority of rat thymocytes express CD 157. SHR and WKY rats were the exception, having unusually low levels (similar to those of the mouse) of these cells. However, in both species, a subset of CD3- CD4- CD8- thymocytes exhibited high levels of CD157. Surprisingly, these CD157high cells temporarily upregulated MHC class I molecules in both species. Furthermore, a third of CD157high rat thymocytes were CD45RC+, a marker found on immature thymocytes with regenerative capacity. Examination of the bone marrow lymphoid population shows that the expression of rat CD157 is largely observed at the CD45R+ IgM- pre-B-II cell stage, and unlike mouse, extension of expression into the IgM+ immature B cell stage was marginal. Similar to CD157high immature thymocytes, these immature B cells also expressed high levels of MHC class I. With the exception of the LEC, SHR and WKY rat strains, which have three- to four-fold less CD157+ bone marrow myeloid cells, percentages of these cells are similar between these two species. Thus, marked differences in the level and stage(s) of CD157 expression on lymphoid cells in mouse and rat indicate that CD157 may not, as previously thought, have a direct role in T or B cell differentiation.     

Effect of PGE2 on the cell surface molecule expression in PMA treated thymocytes

PGE₂ is produced by cells of the thymic microenvironment. The effects of PGE₂ are mediated by cAMP through binding to its intracellular receptor protein kinase A (PKA). Phorbol 12-myristate 13-acetate (PMA) is known to modulate CD molecule expression on thymocytes, probably through activation of protein kinase C (PKC). We have hypothesized that cross-talk between these two signalling pathways may affect modulation of the CD molecules on the cell surface of thymocytes. For this purpose, we compare the effects of PMA alone or combined with PGE₂ on CD3, CD4 and CD8 expression on mouse thymocytes by flow-cytometric analysis. PMA treatment almost completely abolished CD4 expression and slightly decreased CD3 and CD8 expression. PGE₂ alone did not change the CD3, CD4 and CD8 molecule expression. Combined with PMA, PGE₂ can overcome the decrease induced by PMA of the CD3 expression and partially reduced the disappearance of the CD4 molecule. On the other hand PGE₂ accelerated the loss of CD8 molecule expression. These events occurred only in CD4+ CD8+ immature thymocytes. An analogue of cAMP (dibutryl cAMP) mimics the effect of PGE₂, but not Br-cGMP. This differential regulation by PGE₂ of the CD molecule expression on immature thymocytes may provide additional evidence on the role of PGE₂ during the process of thymic differentiation.     

Inducible expression of a p56Lck transgene reveals a central role for Lck in the differentiation of CD4 SP thymocytes

The T lymphocyte-specific protein tyrosine kinase p56lck (Lck) is an essential component of the TCR-mediated signal transduction complex. Lck knockout mice have reduced numbers of double-positive thymocytes and very few mature single-positive cells, particularly of the CD4 lineage. Here we demonstrate the ability of a tetracycline-based tissue-specific inducible Lck transgene to restore expansion of early thymocytes and maturation of single-positive cells in Lckneg mice upon induction with doxycycline. Restoration of Lck expression is particularly important for positive selection to the CD4+ lineage but has a lesser impact on selection to the CD8+ lineage, suggesting activation of Lck is an important component of the signals involved in lineage choice during thymic differentiation.     

Two differential pathways from double-negative to double-positive thymocytes.

Murine thymocytes are divided into four major populations on the basis of expression of CD4 and CD8 antigens. The bulk of evidence favours the view that CD4-CD8- cells can develop into CD4-CD8+ and CD4+CD8- cells via the CD4+CD8+ stage in the thymus. However, CD4-CD8+ and CD4+CD8- thymocyte subsets contain not only CD3+ mature cells but also CD3- immature cells, which seem to be intermediate cells between CD4-CD8- and CD4+CD8+ cells. Here we demonstrate mouse strain differences in the proportion of immature single-positive thymocyte subsets in thymus at the steady or developing state. In C3H mice, immature CD4+CD8- is dominant in proportion over CD4-CD8+ in foetal thymus and in donor-derived thymocytes at an early stage of bone marrow transplantation. On the other hand, immature CD4-CD8+ is dominant over CD4+CD8- during T-cell development in the case of B10.BR mice. An intermediate pattern was shown in the case of F1 mice. Both of these immature single-positive subsets gave rise to double-positive cells after 24 hr culture. These results suggest that there exist two distinct differential pathways; one is from CD4-CD8- cells to CD4+CD8+ cells via CD4-CD8+ cells, and another is via CD4+CD8- cells, and that an application of the 'CD8 pathway' or 'CD4 pathway' seems to be genetically destined by BM-derived cells but not by thymic stromal cells.     

Thymic epithelial cells induce Fas-independent activation apoptosis of thymocytes

Co-cultivation of human thymocytes with homologous thymic epithelial cells (TEC) resulted in apoptosis of thymocytes and increase of CD25 expression. TEC supernatant also induced these effects. Fraction of apoptotic cells was enriched by CD69+ cells but not by CD95+ cells. Thymocytes of mice MRL-lpr/lpr bearing mutant form of gene Fas were sensitive to apoptosis induction in co-culture with TEC in the same degree as thymocytes of mice bearing Fas gene of wild type. Apoptosis of murine thymocytes can be induced by co-cultivation with both murine and human TEC.     

The expression of SV40 large T-antigen in embryonal carcinoma-SV40 transformed somatic cell hybrids

SV40 infection of most murine cell types leads to the production of only the viral early proteins, T-and t-antigens. These SV40 early proteins are not produced in murine embryonal carcinoma cells which are the stem cells of teratocarcinomas. To help elucidate the nature of the host cell range restriction to SV40 early gene expression in embryonal carcinoma cells, somatic cell hybrids were constructed between SV40 transformed cells expressing the viral T-antigen and the embryonal carcinoma cell line, F9. All four independently isolated and cloned somatic cell hybrid cell lines produced the SV40 large T-antigen. The host range restriction to SV40 in embryonal carcinoma cells thus behaves as a recessive property, lacking the required cellular functions for SV40 early gene expression.     

GSK3-mediated instability of tubulin polymers is responsible for the failure of immature CD4+CD8+ thymocytes to polarize their MTOC in response to TCR stimulation

Although mature T cells divide and differentiate when they receive strong TCR stimulation, most immature CD4+CD8+ thymocytes die. The molecular basis for this marked difference in response is not known. Observations that TCR-stimulated CD4+CD8+ thymocytes fail to polarize their microtubule-organizing center (MTOC), one of the first events that occurs upon antigen activation of mature T cells, suggests that TCR signaling routes in immature and mature T cells diverge early and upstream of MTOC polarization. To better understand the source of the divergence, we examined the molecular basis for the difference in TCR-mediated MTOC polarization. We show that unstable microtubules are a feature of immature murine CD4+CD8+ thymocytes, which also exhibit higher levels of glycogen synthase kinase 3 (GSK3) activity, a known inhibitor of microtubule stability. Importantly, CD4+CD8+ thymocytes gained the ability to polarize their MTOC in response to TCR signals when GSK3 activity was inhibited. GSK3 inhibition also abrogated TCR-mediated apoptosis of immature thymocytes. Together, our results suggest that a developmentally regulated difference in GSK3 activity has a major influence on immature CD4+CD8+ thymocyte versus mature T-cell responses to TCR stimulation.