Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus

Differentiation of thymocytes into mature single-positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), coreceptors (CD4 or CD8) and their appropriate major histocompatibility complex-encoded ligands. Precisely how these receptor/co-receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by coligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')₂ antibodies to mimic TCR/co-receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross-linking or targeting to Fc receptor-bearing cells. We show that TCR/CD3 co-ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co-ligation with CD8 also induces maturation of CD4T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co-ligation of TCR and CD8 appear weaker than from co-ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single-positive cell differentiation.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Separately expressed T cell receptor α and β chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4^?CD8^?TCR⁺ thymocytes and the absence of γδ cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR α chain and a transgenic TCR β chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR α chain causes thymocytes to differentiate into a CD4^?CD8^?TCR⁺ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the αβ lineage. Surprisingly, expression of the TCR α chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR β chain causes immature T cells to accelerate differentiation into the αβ lineage and thus inhibits the generation of γδ cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Molecular determinants of TCR expression and selection

The process of T cell development in the thymus is tightly regulated, being dependent on the integration of signals required for thymocyte maturation and survival. Rearrangements, expression and signaling of TCR genes play an indispensable role in this developmental program. Recent advances have provided insights into the molecular mechanisms that regulate TCR repertoire formation at the level of alphabeta versus gammadelta T cell fate and CD4(+) versus CD8(+) lineage determination.     

Lineage fate alteration of thymocytes developing in an MHC environment containing MHC/peptide ligands with antagonist properties.

A18 TCR transgenic thymocytes which are H-2E(k) restricted and normally selected into the CD4 lineage, are exclusively selected into the CD8 lineage in an H-2(q) MHC background. CD8 T cell selection in the H-2(q) background is far more efficient than default selection of A18 CD8 cells on a CD4(-/-) H-2E(k +) background. This suggests the involvement of special selecting ligands. Analogues of the cognate peptide for A18 with antagonist properties for the A18 TCR have previously been shown to effect a lineage diversion from CD4 to CD8 in fetal thymic organ cultures and intriguingly the MHC(q) background contains unidentified natural MHC class II ligands which similarly show antagonist properties for the A18 TCR. Despite the presence of these unidentified MHC class II ligands in the H-2(q) background and their potential influence on developing A18 thymocytes, however, MHC class I molecules were essential for thymic selection of A18 CD8 T cells.     

Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.     

Evidence for a selective and multi-step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules

We have characterized a prominent (15-20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4⁺8^lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4⁺8^lo population is an intermediate stage between immature CD4⁺8⁺ and end-stage CD4⁺8^- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4⁺8^lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.     

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.     

Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro

The molecular interactions provided by the thymic microenvironment that predicate T cell development remain obscure. Here, we show that a bone marrow stromal cell line ectopically expressing the Notch ligand Delta-like-1 loses its ability to support B cell lymphopoiesis, but acquires the capacity to induce the differentiation of hematopoietic progenitors into CD4 CD8 double- and single-positive T cells. Both gammadelta-TCR(+) and alphabeta-TCR(+) T cells are generated, and CD8(+) TCR(hi) cells produce gamma-interferon following CD3/TCR stimulation. These results establish that expression of Delta-like-1 on stromal cells provides key signals for the induction of T cell lineage commitment, stage-specific progenitor expansion, TCR gene rearrangement, and T cell differentiation in the absence of a thymus. Thus, it is likely that Delta-like-1/Notch interactions by the thymus underpin its unique ability to promote lineage commitment and differentiation of T cells.     

Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development

Commitment to the T and natural killer T (NKT) cell lineages is determined during alphabeta T cell receptor (TCR)-mediated interactions of common precursors with ligand-expressing cells in the thymus. Whereas mainstream thymocyte precursors recognize major histocompatibility complex (MHC) ligands expressed by stromal cells, NKT cell precursors interact with CD1d ligands expressed by cortical thymocytes. Here, we demonstrated that such homotypic T-T interactions generated "second signals" mediated by the cooperative engagement of the homophilic receptors Slamf1 (SLAM) and Slamf6 (Ly108) and the downstream recruitment of the adaptor SLAM-associated protein (SAP) and the Src kinase Fyn, which are essential for the lineage expansion and differentiation of the NKT cell lineage. These receptor interactions were required during TCR engagement and therefore only occurred when selecting ligands were presented by thymocytes rather than epithelial cells, which do not express Slamf6 or Slamf1. Thus, the topography of NKT cell ligand recognition determines the availability of a cosignaling pathway that is essential for NKT cell lineage development.     

TCR and Notch signaling in CD4 and CD8 T-cell development

Summary:  The generation of CD4 and CD8 αβ T-cell lineages from CD4⁺CD8⁺ double-positive (DP) thymocyte precursors is a complex process initiated by engagement of major histocompatibility complex (MHC) by T-cell receptor (TCR) and coreceptor. Quantitative differences in TCR signaling induced by this interaction impose an instructional bias on CD4/CD8 lineage commitment that must be reinforced by MHC recognition and TCR signaling over subsequent selection steps in order for the thymocyte to progress and mature in the adopted lineage. Our studies show that the transmembrane receptor Notch plays a role in this process by modifying TCR signal transduction in DP thymocytes. In this review, we consider the functional relationship of TCR and Notch signaling pathways in the selection and specification of CD4 and CD8 T-cell lineages.     

The Ras/MAPK cascade and the control of positive selection

Immature double positive (DP) thymocytes bearing a T cell receptor (TCR) that interacts with self‐major histocompatibility complex (MHC) molecules receive signals that induce either their differentiation (positive selection) or apoptosis (negative selection). Furthermore, those cells that are positively selected develop into two different lineages, CD4 or CD8, depending on whether their TCRs bind to MHC class II or I, respectively. Positive selection therefore involves rescue from the default fate (death), lineage commitment, and progression to the single positive (SP) stage. These are probably temporally distinct events that may require both unique and overlapping signals. Work in the past several years has started to unravel the signaling networks that control these processes. One of the first pathways identified as important for positive selection was Ras and its downstream effector, the Erk mitogen‐activated protein kinase (MAPK) cascade. In this review we examine the factors that connect the TCR to the Ras/Erk cascade in DP thymocytes, as well as what we know about the downstream effectors of the Ras/Erk cascade important for positive selection. We also consider the possible role of this cascade in CD4/CD8 lineage development, and the possible interactions of the Ras/Erk cascade with Notch during these cell fate determination processes.     

CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation.

CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8's ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8's ability to interact with class I MHC is not fixed and is developmentally regulated through the T cell's glycosylation state.     

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.     

Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire

CD4+ CD8+ thymocyte differentiation requires TCR signaling induced by self-peptide/MHC ligands. Nevertheless, the resulting mature T cells are not activated by these self-complexes, whereas foreign ligands can be potent stimuli. Here, we show that the signaling properties of TCR change during thymocyte maturation, differentially affecting responses to related peptide/MHC molecule complexes and contributing to this discrimination. Weak agonists for CD4+ CD8+ thymocytes lose potency during development, accompanied by a change in TCR-associated phosphorylation from an agonist to a partial agonist/antagonist pattern. In contrast, sensitivity to strong agonists is maintained, along with full signaling. This yields a mature T cell pool highly responsive to foreign antigen while possessing a wide margin of safety against activation by self-ligands.     

Mutually antagonistic signals regulate selection of the T cell repertoire

The sensitivity of T cells to agonist-induced death during development contrasts with their proliferative responses after agonist challenge in the periphery. The means by which TCR engagement results in these distinct outcomes is incompletely understood. It has been previously hypothesized that glucocorticoids (GC) modulate the threshold for thymocyte activation by blunting the consequences of TCR engagement. In support of this possibility, inhibition of GC production in fetal thymic organ culture was shown to result in CD4(+)CD8(+) thymocyte apoptosis. This was dependent upon MHC diversity, implying that endogenous GC might regulate antigen-specific selection. Similarly, mice expressing reduced GC receptor (GR) levels due to the presence of an antisense transgene have fewer CD4(+)CD8(+) thymocytes, which was due to an impaired transition from CD4(-)CD8(-) precursors and increased apoptosis. Here we ask how manipulating peptide diversity in the context of reduced GC signaling might affect T cell development and function. In mice with impaired GR expression there was a rescue of thymocyte cellularity and proportions as the diversity of peptides presented by self-MHC was reduced. Furthermore, whereas more CD4(+) T cells survived the selection process in mice expressing single-peptide-MHC class II complexes and reduced GR levels, these cells largely failed to recognize the same MHC molecules bound with foreign peptides. Together, these results support a role for endogenous GC in balancing TCR-mediated signals during thymic selection.