A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4−CD8−double negative to the CD4+CD8+ double positive differentiation stage in vitro

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.     

Thymic Nurse Cells Support CD4^-CD8^＋ Thymocytes to Differentiate into CD4^＋CD8^＋ Cells

Thymic nurse cells （TNCs） represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4^-CD8^＋ cells differentiated into CD4^＋CD8^＋ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4^-CD8^＋ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4^＋CD8^＋ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4^-CD8^＋ thymocytes to differentiate into CD4^＋CD8^＋ T cells by direct interaction.     

'Radioresistant' CD4-CD8- intrathymic T cell precursors differentiate into mature CD4+CD8- and CD4-CD8+ T cells. Development of 'radioresistant' CD4-CD8- intrathymic T cell precursors

Using lectin (PNA) and monoclonal antibodies for Pgp-1, IL-2R, H-2k, CD3, and F23.1 (T cell receptor V beta 8), we characterized the 'radioresistant' CD4-CD8- double negative thymocytes at an early stage after 800 rad irradiation. Most of the CD4-CD8- cells on day 8 after irradiation expressed a high level of Thy-1, H-2k, and PNA, while a small proportion of these cells were CD3+ and/or F23.1+. The appearance of Pgp-1 and IL-2R on the 'radioresistant' double negative precursors was also sequentially examined from day 5 to day 9 after irradiation. The double negative thymocytes at day 5 expressed the highest level of Pgp-1 antigens and these cells gradually decreased in number from day 7 to day 9. By contrast, IL-2R was transiently expressed on the double negative cells on the day 7 and 8 after irradiation. These results indicate that progression of thymocyte development occurred within the CD4-CD8- thymocytes after irradiation. We further examined the homing ability of the double negative 'radioresistant' intrathymic T cell precursors to the periphery by intrathymic cell transplantation method. The double negative thymocytes proliferate and differentiate into CD4+CD8+ cells and CD4+CD8- cells but few CD4-CD8+ cells in the thymus, while only CD4-CD8+ cells were detected in the peripheral lymphoid organs 14 days after intrathymic transplantation of the double negative cells in the H-2 compatible Thy-1 congenic mice. These results suggest that the 'radioresistant' intrathymic precursors differentiate and mature in the thymus and migrate to the periphery.     

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.     

CD45 can act as a negative regulator for the transition from early to late CD4+ CD8+ thymocytes

The differentiation process from CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) stage is accompanied by vigorous proliferation. The resulting DP cells contain a sizable proportion of large cycling cells, but most DP cells are small resting cells. To explore the molecular mechanisms which regulate cell proliferation of DP thymocytes prior to further development, we used TCR-transgenic (Tg) mice with non-selecting MHC (Tg-Neut), which contain almost exclusively DP thymocytes that are not subject to either positive or negative selection. In Tg-Neut, the thymus contained DP cells of relatively large size, which showed higher extracellular signal-regulated kinase activity and enhanced responsiveness to mitogen compared to small DP cells. This indicates that all the large DP cells in the thymus are not positively selected and that they possess proliferative potential. When Tg-Neut mice were backcrossed with CD45 knockout mice (CD454-/- Tg-Neut), the thymus showed an increase of large DP cells and cycling cells, but a decrease of apoptotic cells. Furthermore, Bcl-2 expression and Jun N-terminal kinase activity, which are associated with resistance to apoptosis, were enhanced. These observations suggest that thymocyte proliferation in the DP stage is suppressed by a CD45-related process with regulation of mitogen-activated protein kinase and Bcl-2 unless DP cells receive TCR-mediated signals.     

Deletion of CD4 and CD8 coreceptors permits generation of alphabetaT cells that recognize antigens independently of the MHC

The thymus generates major histocompatibility complex (MHC)-restricted alphabetaT cells that only recognize antigenic ligands in association with MHC or MHC-like molecules. We hypothesized that MHC specificity might be imposed on a broader alphabetaTCR repertoire during thymic selection by CD4 and CD8 coreceptors that bind and effectively sequester the tyrosine kinase Lck, thereby preventing T cell receptor (TCR) signaling by non-MHC ligands that do not engage either coreceptor. This hypothesis predicts that, in coreceptor-deficient mice, alphabeta thymocytes would be signaled by non-MHC ligands to differentiate into alphabetaT cells lacking MHC specificity. We now report that MHC-independent alphabetaT cells were indeed generated in mice deficient in both coreceptors as well as MHC ("quad-deficient" mice) and that such mice contained a diverse alphabetaT cell repertoire whose MHC independence was confirmed at the clonal level. We conclude that CD4 and CD8 coreceptors impose MHC specificity on a broader alphabetaTCR repertoire during thymic selection by preventing thymocytes from being signaled by non-MHC ligands.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

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.     

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.     

Lck activity controls CD4/CD8 T cell lineage commitment

Thymocytes carrying MHC class I-restricted TCRs differentiate into CD8 T cells, while those recognizing MHC class II become CD4 T cells. The mechanisms underlying how MHC class recognition, coreceptor expression, and effector function are coordinated are not well understood. Since the tyrosine kinase Lck binds with more affinity to CD4 than CD8, it has been proposed as a candidate to mediate this process. By using transgenic mice with altered Lck activity, we show that thymocytes carrying a class II-restricted TCR develop into functional CD8 T cells when Lck activity is reduced. Conversely, thymocytes carrying a class I-restricted TCR develop into functional CD4 T cells when Lck activity is increased. These results directly show that quantitative differences in the Lck signal control the CD4/CD8 lineage decision.     

Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes

The present study has assessed the impact of the intracellular domains of CD4 and CD8 on positive selection and lineage direction of MHC class I-restricted thymocytes. Contrary to current presumption, we found that the CD4 tail promotes the generation of both CD4+ and CD8+ T cells without preference for the CD4+ T cell lineage. We also found that the identity of the coreceptor tail and hence the strength of coreceptor signaling determine the number of thymocytes undergoing positive selection but not their ultimate CD4/CD8 phenotype. These findings demonstrate that the strength of coreceptor signaling has a significant quantitative but not qualitative impact on positive selection and provide a simple explanation for the greater numbers of CD4+ than CD8+ T cells selected in the normal thymus.     

CD4+CD8+ thymocytes develop into CD4 or CD8 single-positive cells in athymic nude mice

A differentiation pathway from CD4+CD8+ cells to CD4+CD8- or CD4-CD8+ cells was investigated in athymic nude mice. Using fluorescence-activated cell sorter, CD4+CD8+ cells were sorted out from AKR thymocytes (H-2k, Thy-1.1) stained with two monoclonal antibodies against CD4 and CD8 (anti-L3T4 and anti-Ly-2). These CD4+CD8+ AKR thymocytes were injected i.v. into CBA or C3H nude mice (H-2k, Thy-1.2) which had received 650 rads and had been reconstituted with syngeneic nude bone marrow cells. The lymph node cells of the nude recipients at 4 wks post-thymocyte transfer were shown to contain 50% AKR-derived Thy-1.1+ cells. The majority of the Thy-1.1+ cells were found to express either CD4 or CD8 alone but not to express both CD4 and CD8. These findings indicate that CD4+CD8+ thymocytes can develop into CD4+CD8- and CD4-CD8+ single-positive cells in extrathymic tissues.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

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.     

Ontogeny of T cell maturation in LEC mutant rats which bear a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- thymocytes.

LEC rats bear a congenital deficiency in CD4+CD8- thymocytes and peripheral CD4+ T cells, and consequently a deficiency in Th cell functions. Ontogeny of T cell maturation in normal and LEC mutant rats was, therefore, investigated. Prenatal development of thymocytes in normal rat strains, with respect to the expression of CD4/CD8 and TcR antigens, was similar to that of mice except that its kinetics was delayed by approximately 24 h. The kinetics of T cell maturation in LEC rats was comparable with that of normal rats up to day 19 of gestation, at which stage double-negative thymocytes (CD4-CD8-) developed into double positives (CD4+CD8+) through immature CD4-CD8+ subset. At day 19 of gestation in LEC as well as normal rats, double positives occupied approximately 80% of the total thymocytes, half of which were TcR-dull positive, indicating that TcR was normally rearranged and then expressed in LEC rat thymocytes. These data indicate that double negatives normally mature into at least double positives in LEC rats. Both single positives appeared after day 19 of gestation in normal rats, while in LEC rats CD4+CD8- cells did not appear, suggesting that the deficiency in CD4+CD8- cells is due to a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- cells, but not due to a postnatal deletion.     

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.     

Phenotypic changes accompanying positive selection of CD4+CD8+ thymocytes.

We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the alpha beta T cell receptor (TcRhigh) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co-receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I-restricted transgenic TcR develop in vitro exclusively into CD4-CD8+ cells while small CD4+CD8+ TcRhigh cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR-MHC ligand interactions in the generation of mature alpha beta T cells.