Gabriela Hernandez-Hoyos

GATA-3 Expression Is Controlled by TCR Signals and Regulates CD4/CD8 Differentiation

GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 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.

Constitutive Expression of PU.1 in Fetal Hematopoietic Progenitors Blocks T Cell Development at the Pro-T Cell Stage

The essential hematopoietic transcription factor PU.1 is expressed in multipotent thymic precursors but downregulated during T lineage commitment. The significance of PU.1 downregulation was tested using retroviral vectors to force hematopoietic precursors to maintain PU.1 expression during differentiation in fetal thymic organ culture. PU.1 reduced thymocyte expansion and blocked development at the pro-T cell stage. PU.1-expressing cells could be rescued by switching to conditions permissive for macrophage development; thus, the inhibition depends on both lineage and developmental stage. An intact DNA binding domain was required for these effects. PU.1 expression can downregulate pre-Talpha, Rag-1, and Rag-2 in a dose-dependent manner, and higher PU.1 levels induce Mac-1 and Id-2. Thus, downregulation of PU.1 is specifically required for progression in the T cell lineage.

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.

A New Regulatory Region of the IL-2 Locus That Confers Position-Independent Transgene Expression

Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endogenous IL-2 expression. In contrast, a 2-kb promoter green fluorescent protein transgene, lacking the new hypersensitive sites, is expressed in only a few founder lines, and expression is dysregulated in CD8+ cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells.

Analysis of T-Cell Development by Using Short Interfering RNA to Knock Down Protein Expression

We have applied RNA interference (RNAi) technology to the analysis of genes involved in T-cell development, combining a reaggregate fetal thymic organ culture (rFTOC) system with retroviral delivery of short interfering RNA (siRNA) hairpins. The process involves the isolation of murine fetal liver or fetal thymocytes, infection with retroviral particles carrying the construct of interest, followed by reaggregation of the transduced precursors with fetal thymic stroma into lobes. Subsequently, individual lobes are harvested and analyzed for development at various time points. These reaggregate cultures recapitulate most features of T-cell development in vivo, including pre-TCR selection and expansion, positive selection of CD4 and CD8 T cells, and negative selection. In our hands, the combination of retroviral delivery of RNAi and rFTOCs is a quick alternative to conventional knockouts for the analysis of gene function during T-cell development. This chapter describes the methods we have developed to knock down gene expression in T-cell precursors, using retroviral delivery of siRNA hairpins.

A Notch so simple influence on T cell development.

T cell precursors undergo a series of developmental choices that progressively narrow their ability to give rise to different cell lineages. Evidence accumulated in the last few years suggests that Notch occupies a central place among the signal transduction pathways that regulate many of these choices, including the T/B, alphabeta/gammadelta and CD4/CD8 lineage decisions. Nevertheless the mechanisms by which Notch exerts its effects are not well understood, and in some cases the physiologic role is unclear. In this review we try to present succinctly the experiments and highlight the areas of controversy.

Regulation of GATA-3 Expression during CD4 Lineage Differentiation

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.