Alfred Singer

Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.

Following successful gene rearrangement at αβ T-cell receptor (TCR) loci, developing thymocytes express both CD4 and CD8 co-receptors and undergo a life-or-death selection event, which is known as positive selection, to identify cells that express TCRs with potentially useful ligand specificities. Positively selected thymocytes must then differentiate into either CD4+ helper T cells or CD8+ cytotoxic T cells, a crucial decision known as CD4/CD8-lineage choice. In this Review, we summarize recent advances in our understanding of the cellular and molecular events involved in lineage-fate decision and discuss them in the context of the major models of CD4/CD8-lineage choice.

Asymmetric signaling requirements for thymocyte commitment to the CD4+ versus CD8+ T cell lineages: A new perspective on thymic commitment and selection

Differentiation of immature CD4+ CD8+ thymocytes into mature CD4+ CD8- and CD4-CD8+ T cells requires that synthesis of one or the other coreceptor molecule be terminated, a process referred to as lineage commitment. The present study has utilized a novel coreceptor reexpression assay to identify lineage commitment in immature thymocytes and has found that the MHC recognition requirements for CD4 commitment and CD8 commitment fundamentally differ from one another. Remarkably, we found that thymocyte commitment to the CD8+ lineage requires MHC class I-dependent instructional signals, whereas thymocyte commitment to the CD4+ lineage is MHC independent and may occur by default. In addition, an unanticipated relationship between lineage commitment and surface phenotype has been identified. These results are incompatible with current concepts and require a new perspective on lineage commitment and positive selection, which we refer to as asymmetric commitment.

Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells

Immature CD4+CD8+ (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8+ T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.

Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte‐specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta‐‐D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta‐chain locus.

Basis of CTLA-4 function in regulatory and conventional CD4+ T cells

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.

Persistence of glucose residues on core oligosaccharides prevents association of TCR alpha and TCR beta proteins with calnexin and results specifically in accelerated degradation of nascent TCR alpha proteins within the endoplasmic reticulum.

The alpha beta T‐cell antigen receptor (TCR) is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER). In this report we have examined the role of oligosaccharide processing on survival and assembly of nascent TCR proteins within the ER and their associations with molecular chaperone proteins important in TCR assembly. We found that treatment of BW5147 T cells with the glucosidase inhibitor castanospermine resulted in markedly accelerated degradation of nascent TCR alpha proteins with a half‐life of approximately 20 min. Accelerated degradation was unique to TCR alpha proteins, as the stability of nascent TCR beta and CD3 gamma,epsilon chains was unaltered. Consistent with a requirement for glucose (Glc) trimming for survival of nascent TCR alpha proteins within the ER, we found that newly synthesized TCR alpha chains were innately unstable in the glucosidase II‐deficient BW5147 mutant cell line PHAR2.7. In addition to destabilizing nascent TCR alpha proteins we found that persistence of Glc residues on core oligosaccharides markedly interfered with association of both TCR alpha and TCR beta glycoproteins with the molecular chaperone calnexin. Finally, using 2B4 T hybridoma cells in which TCR complexes are efficiently assembled, we found that rapid degradation of nascent TCR alpha proteins induced by impaired Glc trimming severely limits assembly of TCR alpha proteins with TCR beta proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of CD8β Domains in CD8 Coreceptor Function: Importance for MHC I Binding, Signaling, and Positive Selection of CD8+ T Cells in the Thymus

Abstract The contribution of the CD8β subunit to CD8 coreceptor function is poorly understood. We now demonstrate that the CD8β extracellular domain increases the avidity of CD8 binding to MHC I, and that the intracellular domain of CD8β enhances association with two intracellular molecules required for TCR signal transduction, Lck and LAT. By assessing CD8 + T cell differentiation in CD8β-deficient mice reconstituted with various transgenic CD8β chimeric molecules, we also demonstrate that the intracellular and extracellular domains of CD8β can contribute independently to CD8 + T cell development, but that both CD8β domains together are most efficient. Thus, this study identifies the molecular functions of the CD8β intracellular and extracellular domains and documents their contributions to CD8 + T cell development.

The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes.

Immature thymocytes express clonotype‐independent CD3 complexes that, when engaged by anti‐CD3 antibodies, can signal CD4‐CD8‐ thymocytes to differentiate into CD4+CD8+ cells. Clonotype‐independent CD3 complexes consist of CD3 components associated with an unknown 90 kDa surface protein. We now report the surprising finding that this 90 kDa surface protein is the molecular chaperone calnexin, an integral membrane protein previously thought to reside only in the endoplasmic reticulum (ER). We found that calnexin‐CD3 complexes escaping to the cell surface utilize interchain associations distinct from those utilized by calnexin‐CD3 complexes remaining within the ER. Specifically, we demonstrate that carbohydrate‐mediated luminal domain interactions that are necessary for formation of most internal calnexin‐CD3 complexes destined to be expressed on the cell surface, and we provide evidence that cytoplasmic domain interactions between calnexin and CD3 epsilon chains mask calnexins ER retention signal, permitting calnexin and associated proteins to escape ER retention. Thus, the present study demonstrates that partial T cell antigen receptor complexes can escape the ER of immature thymocytes in association with their molecular chaperone to be expressed at low levels on the cell surface where they may function as a signaling complex to regulate thymocyte maturation.

IL-7 Receptor Signals Inhibit Expression of Transcription Factors TCF-1, LEF-1, and RORγt Impact on Thymocyte Development

Intrathymic T cell development depends on signals transduced by both T cell receptor and cytokine receptors. Early CD4−CD8− (double negative) thymocytes require interleukin (IL)-7 receptor (IL-7R) signals for survival and proliferation, but IL-7R signals are normally extinguished by the immature single positive (ISP) stage of thymocyte development. We now demonstrate that IL-7R signals inhibit expression of transcription factors TCF-1, LEF-1, and RORγt that are required for the ISP to double positive (DP) transition in the thymus. In addition, we demonstrate that IL-7R signals also inhibit TCF-1 and LEF-1 expression in mature peripheral T cells. Thus, the present work has identified several important downstream target genes of IL-7R signaling in T cells and thymocytes that provide a molecular mechanism for the inhibitory influence of IL-7R signaling on DP thymocyte development. We conclude that IL-7R signals down-regulate transcription factors required for the ISP to DP transition and so must be terminated by the ISP stage of thymocyte development.