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.     

Inefficient cell spreading and cytoskeletal polarization by CD4+CD8+ thymocytes: regulation by the thymic environment

CD4(+)CD8(+) double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4(+)) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

The quantity of TCR signal determines positive selection and lineage commitment of T cells

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.     

CD5 costimulation up-regulates the signaling to extracellular signal-regulated kinase activation in CD4+CD8+ thymocytes and supports their differentiation to the CD4 lineage

CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-gamma (PLC-gamma) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-gamma, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8-CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8- cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.     

Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos, and RasGRP1

Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.     

Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.     

Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions

Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection.     

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.     

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.     

Analyzing expression of perforin, Runx3, and Thpok genes during positive selection reveals activation of CD8-differentiation programs by MHC II-signaled thymocytes

Intrathymic positive selection matches CD4-CD8 lineage differentiation to MHC specificity. However, it is unclear whether MHC signals induce lineage choice or simply select thymocytes of the appropriate lineage. To investigate this issue, we assessed thymocytes undergoing positive selection for expression of the CD8 lineage markers perforin and Runx3. Using both population-based and single-cell RT-PCR analyses, we found large subsets of MHC class II (MHC-II)-signaled thymocytes expressing these genes within the CD4+ 8+ and CD4+ 8(int), but not the CD4+ 8- populations of signaling competent mice. This indicates that MHC-II signals normally fail to impose CD4 differentiation and further implies that the number of mature CD8 single-positive (SP) thymocytes greatly underestimates CD8 lineage choice. We next examined whether MHC-II-restricted CD4+ 8- thymocytes remain competent to initiate CD8 lineage gene expression. In mice in which expression of the tyrosine kinase Zap70 and thereby TCR signaling were impaired selectively in SP thymocytes, MHC-II-signaled CD4+ 8- thymocytes expressed perforin and Runx3 and failed to up-regulate the CD4 marker Thpok. This indicated that impairing TCR signals at the CD4 SP stage switched gene expression patterns from CD4- to CD8-lineage specific. We conclude from these findings that MHC-II-signaled thymocytes remain competent to initiate CD8-specific gene expression even after CD8 down-regulation and that CD4 lineage differentiation is not fixed before the CD4 SP stage.     

Activated p56lck directs maturation of both CD4 and CD8 single-positive thymocytes

p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.     

Tonic ubiquitylation controls T‐cell receptor:CD3 complex expression during T‐cell development

Expression of the T‐cell receptor (TCR):CD3 complex is tightly regulated during T‐cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ε proline‐rich sequence, Lck, c‐Cbl, and SLAP, which collectively trigger the dynamin‐dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ‐monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T‐cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T‐cell development.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

The generation of mature, single-positive thymocytes in vivo is dysregulated by CD69 blockade or overexpression.

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.     

Cross-positive selection of thymocytes expressing a single TCR by multiple major histocompatibility complex molecules of both classes: implications for CD4+ versus CD8+ lineage commitment

This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8+ CTL clone. The Idhigh+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, Dq or Lq molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2q background, mature Idhigh+ T cells appeared among both CD4+ and CD8+ subsets in periphery, even in the absence of RAG-2 gene. When examined on beta2-microglobulin-/- background, CD4+, but not CD8+, Idhigh+ T cells developed, suggesting that maturation of CD8+ and CD4+ Idhigh+ cells was MHC class I (Dq/Lq) and class II (I-Aq) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4+/CD8+ lineage commitment are discussed.     

The novel murine CD4+CD8+ thymocyte cell line exhibits lineage commitment into both CD4+ and CD8+ T cells by altering the intensity and the duration of anti-CD3 stimulation in vitro

A CD4(+)CD8(+) double-positive thymocyte cell line, 257-20-109 was established from BALB/c mice thymocytes and used to analyze the requirements to induce CD4 or CD8 single-positive (SP) T cells. CD4SP cells were induced from 257-20-109 cells by anti-CD3 stimulation in the presence of the FcR-positive macrophage cell line, P388D1. During stimulation, maturation events, such as the down-regulation of CD24 and the up-regulation of CD69, H-2D(d), CD5, and Bcl-2, were recognized. Furthermore, these CD4SP cells appeared to be functional because the cells produced IL-2 and IL-4 when activated with phorbol ester and calcium ionophore. In contrast, CD8SP cells could be induced by stimulation with fixed anti-CD3 after removal of stimulation. To investigate the extent of signals required for CD4SP and CD8SP, the cells stimulated under either condition for 2 days were sorted and transferred to different culture conditions. These results suggested that the fate of lineage commitment was determined within 2 days, and that CD4 lineage commitment required longer activation. Furthermore, the experiments with subclones of 257-20-109 demonstrated that the lower density of CD3 did not shift the cells from CD4SP to CD8SP, but only reduced the amount of CD4SP cells. In contrast, when the 257-20-109 cells were stimulated by the combination of fixed anti-CD3 and anti-CD28, the majority of the cells shifted to CD4SP, with an enhancement of extracellular signal-regulated kinase 1 phosphorylation. Our results indicate that the signals via TCR/CD3 alone shifted the double-positive cells to CD8SP cells, but the reinforced signals via TCR/CD3 and costimulator could commit the cells to CD4SP.