Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.     

Ex vivo evidence for asymmetric tyrosine phosphorylation of ZAP-70 on double-positive thymocytes in the positive selection process

Antigen stimulation via TCR in mature T cells provides rapid induction of tyrosine phosphorylation of intracellular substrates including ZAP-70. To study the potential involvement of tyrosine phosphorylation in CD4+CD8+ [double-positive (DP)] thymocytes in the positive selection process in vivo, we isolated and analyzed them in the presence of phosphatase inhibitor. DP thymocytes were obtained from TCR transgenic mice (TCR-Tg) expressing MHC class I- or class II-restricted TCR in selecting and non-selecting MHC backgrounds respectively. The phosphorylation of ZAP-70 in DP thymocytes of class I-restricted TCR-Tg was significantly higher in the positively selecting background than in the non-selecting one. However, such a phosphorylation difference between selecting and non-selecting TCR-Tg was found to be considerably less in class II-restricted TCR-Tg. A similar bias for ZAP-70 phosphorylation was also observed on selecting DP thymocytes when I-A(beta) deficient- and beta2-microglobulin-deficient mice were compared. These ex vivo studies suggest that TCR-mediated signaling on DP thymocytes induces ZAP-70 phosphorylation under a different manner of engagement of TCR to class I and class II molecules in the positive selection process.     

Preferential requirement of CD3 zeta-mediated signals for development of immature rather than mature thymocytes

Antigen recognition signals by the TCR are transduced through activation motifs present in the cytoplasmic region of CD3 chains. In vitro analysis has suggested that the CD3zeta chain mediates different signals from other CD3 chains. To analyze the in vivo function of CD3zeta-mediated signals for T cell development, mice expressing a mutant CD3zeta chain lacking all the activation motifs were generated by introducing the transgene into zeta-knockout mice. Mature CD4(+) single-positive (SP) thymocytes in these mice were greater in number than in zeta-deficient mice, and the promoted differentiation was indicated by the changes of CD69 and HSA phenotypes. We found that even in the absence of activation motifs in CD3zeta, these mature cells became functional, being able to induce Ca2+ mobilization and proliferation upon stimulation. On the other hand, CD4(-)CD8(-) double-negative (DN) thymocytes, most of which were arrested at the CD44(-)CD25(+) stage similarly to those in zeta-deficient mice, could not be promoted for differentiation into CD4(+)CD8(+) double-positive thymocytes in these mice in spite of the fact that the expression of the transgene in DN thymocytes was higher than that of zeta in wild-type mice. These results demonstrate the preferential dependence of the promotion of development and/or expansion of DN thymocytes rather than mature thymocytes upon the activation signals through the zeta chain and suggest differential requirements of TCR signaling for mature SP and immature DN thymocyte developments in vivo.     

Co-ligation of alpha4beta1 integrin and TCR rescues human thymocytes from steroid-induced apoptosis

Maturation of thymocytes represents a sequence of events during which thymocytes expressing TCR with moderate avidity for self antigen/MHC are positively selected, whereas those with high or insufficient TCR avidity die. Glucocorticoids are produced intrathymically and can contribute to apoptosis of unselected thymocytes. Thymocytes differentiate in a close contact with epithelial cells, expressing vascular adhesion molecule-1 (VCAM-1) and secreting glucocorticoids, with bone marrow-derived macrophages, and with extracellular matrix containing fibronectin (FN) and collagen. Their contact with FN is mediated by alpha4beta1 and alpha5beta1 integrins. We examined the contribution of TCR and integrin signaling to the survival of thymocytes from dexamethasone (Dex)-induced apoptosis. We demonstrate that FN and VCAM-1 (both of which bind alpha4beta1 integrin), but not collagen, considerably augment TCR-mediated protection of thymocytes from Dex-induced apoptosis. This 'survival' signal is transduced through the alphabeta1, but not through the alpha5beta1 integrin. The observed protection from Dex-induced apoptosis correlated with an increase in bcl-2 protein levels. FN-alpha4beta1 and VCAM-1-alpha4beta1 engagement induced up-regulation bcl-2 protein, while alpha5beta1 binding to FN induced a negative signal that was blocked by anti-alpha5beta1 antibody. These data suggest that alpha4beta1 integrin may contribute to protection of thymocytes with moderate avidity TCR from glucocorticoid-induced death during intrathymic maturation.     

Association of spectrin with a subcompartment of the endoplasmic reticulum in honeybee photoreceptor cells

In order to investigate the mechanisms involved in originating a diverse TCR repertoire in human peripheral blood we analyzed TCRV beta surface expression in different T cell subsets of unrelated individuals. The relative frequencies of 11 distinct V beta chains were determined for immature double positive (DP) as well as for mature CD4 single positive (4SP) and CD8 single positive (8SP) thymocytes, respectively. By comparing these data with expression in peripheral blood T lymphocytes of the same donors we were able to show that usage of TCRV beta in peripheral T cells is significantly (p < 0.001) depending on the pattern in mature SP thymocytes whereas the frequency of TCRV beta families in immature DP thymocytes has no impact (p > 0.2). No association with distinct HLA-haplotypes was observed. Preferential usage of V beta-families in either CD4- or CD8-positive peripheral T cells also correlates with the status in mature thymic precursors (p < 0.001). Altogether, this first combined study of TCR frequencies within different stages of human T cell ontogeny indicates that TCRV beta repertoire is determined mainly through selectional processes within the thymus. Since neither genomically imposed expression nor modulating events in the periphery seem to have strong influence on the relative expression of TCRV beta chains these findings have to be considered in future studies of human diseases.     

Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.     

Macrophage inflammatory protein-1beta induces migration and activation of human thymocytes

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.     

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.     

Positive selection of thymocytes expressing the same TCR by different MHC ligands results in the production of functionally distinct thymocytes distinguished by differential expression of the heat stable antigen

The issue of whether the signaling process during positive selection can affect the efficiency by which the positively selected T cells respond to antigenic stimulation has not been addressed. We approached this question by determining the consequences of positive selection of a particular transgenic TCR (2C TCR) in the H-2b and the H-2k thymus. The H-2b thymus provides a strong positive-selecting environment for the 2C TCR, whereas the H-2k thymus selects weakly for the 2C TCR. Although the positively selected CD8 thymocytes from the H-2b or H-2k thymus expressed similar levels of the CD8 coreceptor molecule, those for the H-2k thymus expressed a slightly lower level of the 2C TCR. This lower level of 2C TCR expression by H-2k CD8 thymocytes was not a result of coexpression of endogenous TCRs. Interestingly, CD8 thymocytes from H-2k mice were hyporesponsive to Ag stimulation compared with those from the H-2b mice. The functional maturity of positively selected CD8 thymocytes from the H-2b or H-2k thymus was inversely correlated with the level of heat stable Ag expressed by these cells. Furthermore, TCR-derived signals appear to be more efficiently coupled to downstream pathways leading to proliferation and cytokine production in CD8 thymocytes from H-2b 2C mice than those derived from H-2k 2C mice. These results provide the first demonstration that the intensity of the signaling process during positive selection affects the efficiency by which TCR-derived signals in positively selected thymocytes are coupled to downstream effector pathways.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2-type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross-linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.     

Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widely used in transplantation medicine. A major effect of CsA is inhibition of the differentiation of immature double-positive (DP) CD4+ CD8+ thymocytes into mature single-positive (SP) CD4+ CD8- or CD4- CD8+ thymocytes. The mechanisms underlying the changes in CD4/CD8 expression during normal differentiation of thymocytes and the way CsA interferes with this differentiation process are still unknown. Here we show that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) causes a decrease of both CD4 and CD8 expression at the cell surface level and at the mRNA level in a CD4+ CD8+ T cell line and in freshly isolated thymocytes. A PKC inhibitor, staurosporin, interferes with the differentiation from DP to SP in fetal thymus organ culture system. These data suggest that the alternation of CD4/CD8 expression from DP to SP is dependent on PKC activation. CsA blocks this decrease of CD4/CD8 expression by PMA in vitro. Moreover, this PMA effect is also blocked by treatment with cycloheximide. These results suggest that the reduction of CD4/CD8 expression requires de novo synthesis of a protein(s) induced in response to a signal conveyed by activated PKC. CsA may block the transition from DP to SP by inhibition of CD4/CD8 down-regulation induced by PKC activation.     

Intestinal epithelial cell line induction of T cell differentiation from bone marrow precursors

The mechanism whereby the intestinal microenvironment promotes T cell development in the absence of the thymus is unknown. We show that the murine intestine-derived epithelial cell line, MODE-K, can induce T cell differentiation marker expression in vitro on bone marrow (BM) T cell precursors. Three-color flow cytometry analysis of T-cell-depleted C3H BM mononuclear cells (MNC) after 4 days of coculture on monolayers of MODE-K indicated that approximately 25% of MNC expressed CD3 and TCR alpha beta. Of these CD3+ cells, 36% were CD3loCD4-CD8- double negative (DN), 34% were CD3loCD4+CD8 alpha beta+ double positive (DP), and the remainder were CD3hiCD4+CD8- or CD3hiCD4-CD8 alpha beta+ single positive (SP). In addition, the T cells which developed in coculture with MODE-K expressed the early T cell differentiation marker CD24 (heat-stable antigen). These T cells subsets did not develop when BM was cocultured with the LTA fibroblast cell line or in medium alone. Interestingly, preventing cell contact between MODE-K and BM by culturing in Transwell plates did not interfere with the development of T cells expressing the DN, DP, or SP phenotypes. Double-positive T cells did not develop if splenic MNC were cocultured with MODE-K. These results suggest that the intestinal epithelial environment can induce and support the T cell development from bone marrow precursors.     

Evidence implicating Gfi-1 and Pim-1 in pre-T-cell differentiation steps associated with beta-selection

After rearrangement of the T-cell receptor (TCR) beta-locus, early CD4(-)/CD8(-) double negative (DN) thymic T-cells undergo a process termed 'beta-selection' that allows the preferential expansion of cells with a functional TCR beta-chain. This process leads to the formation of a rapidly cycling subset of DN cells that subsequently develop into CD4(+)/CD8(+) double positive (DP) cells. Using transgenic mice that constitutively express the zinc finger protein Gfi-1 and the serine/threonine kinase Pim-1, we found that the levels of both proteins are important for the correct development of DP cells from DN precursors at the stage where 'beta-selection' occurs. Analysis of the CD25(+)/CD44(-,lo) DN subpopulation from these animals revealed that Gfi-1 inhibits and Pim-1 promotes the development of larger beta-selected cycling cells ('L subset') from smaller resting cells ('E subset') within this subpopulation. We conclude from our data that both proteins, Pim-1 and Gfi-1, participate in the regulation of beta-selection-associated pre-T-cell differentiation in opposite directions and that the ratio of both proteins is important for pre-T-cells to pass the 'E' to 'L' transition correctly during beta-selection.     

Upregulated expression of fibronectin receptors underlines the adhesive capability of thymocytes to thymic epithelial cells during the early stages of differentiation: lessons from sublethally irradiated mice

A 250-cGy whole-body gamma-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3(-)CD25(hi)CD44(+) and CD3(-)CD25(in/hi)CD44(-) cells occurred. Enhanced expression of L-selectin, alpha4, and alpha5 integrin chains (L-selhi alpha4(hi) alpha5(hi)) was also exhibited by these cells. This pattern of expression was maintained until the CD4(+)CD8(+) (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4(+) or CD8(+) single positive (SP) thymocytes (L-selin alpha4(in) alpha5(in)). In some recently generated SP cells, alpha4 expression was downregulated before the alpha5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the alpha6 integrin chain was downregulated only in maturing CD4(+) cells. Importantly, the increased expression of L-selectin and alpha4 and alpha5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the alpha4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for alpha5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the beta1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25(+) precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.     

Engagement of CD99 induces up-regulation of TCR and MHC class I and II molecules on the surface of human thymocytes

CD99 is a cell surface molecule involved in the aggregation of lymphocytes and apoptosis of immature cortical thymocytes. Despite its high level expression on immature cortical thymocytes, the functional roles of this molecule during thymic selection are only now being elucidated. Examination of the effects of CD99 engagement on the expression kinetics of the TCR and MHC class I and II molecules, which are involved primarily in thymic positive selection, revealed a marked up-regulation of these proteins on the surface of immature thymocytes. This increase was the result of accelerated mobilization of molecules stored in cytosolic compartments to the plasma membrane, rather than increased RNA and protein synthesis. Confocal microscopic analysis revealed the changes in subcellular distribution of these molecules. When CD99 was engaged, TCR and MHC class I and II molecules were concentrated at the plasma membrane, particularly at cell-cell contact sites. The TCRlow subpopulation of immature double positive thymocytes was much more responsive to CD99-mediated up-regulation than was the TCRhigh population. These findings suggest that CD99-dependent up-regulation may have possible implication in positive selection during thymocyte ontogeny.