Differential developmental regulation and functional effects on pre-TCR surface expression of human pTalpha(a) and pTalpha(b) spliced isoforms.

Functional rearrangement at the TCRbeta locus leads to surface expression on developing pre-T cells of a pre-TCR complex composed of the TCRbeta-chain paired with the invariant pre-TCRalpha (pTalpha) chain and associated with CD3 components. Pre-TCR signaling triggers the expansion and further differentiation of pre-T cells into TCRalphabeta mature T cells, a process known as beta selection. Besides the conventional pTalpha transcript (termed pTalpha(a)), a second, alternative spliced, isoform of the pTalpha gene (pTalpha(b)) has been described, whose developmental relevance remains unknown. In this study, phenotypic, biochemical, and functional evidence is provided that only pTalpha(a) is capable of inducing surface expression of a CD3-associated pre-TCR complex, which seems spontaneously recruited into lipid rafts, while pTalpha(b) pairs with and retains TCRbeta intracellularly. In addition, by using real-time quantitative RT-PCR approaches, we show that expression of pTalpha(a) and pTalpha(b) mRNA spliced products is differentially regulated along human intrathymic development, so that pTalpha(b) transcriptional onset is developmentally delayed, but beta selection results in simultaneous shutdown of both isoforms, with a relative increase of pTalpha(b) transcripts in beta-selected vs nonselected pre-T cells in vivo. Relative increase of pTalpha(b) is also shown to occur upon pre-T cell activation in vitro. Taken together, our data illustrate that transcriptional regulation of pTalpha limits developmental expression of human pre-TCR to intrathymic stages surrounding beta selection, and are compatible with a role for pTalpha(b) in forming an intracellular TCRbeta-pTalpha(b) complex that may be responsible for limiting surface expression of a pTalpha(a)-containing pre-TCR and/or may be competent to signal from a subcellular compartment.

An endoplasmic reticulum retention function for the cytoplasmic tail of the human pre-T cell receptor (TCR) alpha chain: potential role in the regulation of cell surface pre-TCR expression levels.

The pre-T cell receptor (TCR), which consists of a TCR-beta chain paired with pre-TCR-alpha (pTalpha) and associated with CD3/zeta components, is a critical regulator of T cell development. For unknown reasons, extremely low pre-TCR levels reach the plasma membrane of pre-T cells. By transfecting chimeric TCR-alpha-pTalpha proteins into pre-T and mature T cell lines, we show here that the low surface expression of the human pre-TCR is pTalpha chain dependent. Particularly, the cytoplasmic domain of pTalpha is sufficient to reduce surface expression of a conventional TCR-alpha/beta to pre-TCR expression levels. Such reduced expression cannot be attributed to qualitative differences in the biochemical composition of the CD3/zeta modules associated with pre-TCR and TCR surface complexes. Rather, evidence is provided that the pTalpha cytoplasmic tail also causes a reduced surface expression of individual membrane molecules such as CD25 and CD4, which are shown to be retained in the endoplasmic reticulum (ER). Native pTalpha is also observed to be predominantly ER localized. Finally, sequential truncations along the pTalpha cytoplasmic domain revealed that removal of the COOH-terminal 48 residues is sufficient to release a CD4-pTalpha chimera from ER retention, and to restore native CD4 surface expression levels. As such a truncation in pTalpha also correlates with enhanced pre-TCR expression, the observed pTalpha ER retention function may contribute to the regulation of surface pre-TCR expression on pre-T cells.

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma.

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

Beta-selection is associated with the onset of CD8beta chain expression on CD4(+)CD8alphaalpha(+) pre-T cells during human intrathymic development.

T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha) chain. Such a critical developmental checkpoint, known as beta-selection, results in progression from CD4(-) CD8(-) double negative (DN) to CD4(+) CD8(+) double positive (DP) TCRalphabeta(-) thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4(+) CD8(-) intermediate stage. Here we show that the CD4(+) CD8(-) to CD4(+) CD8(+) transition involves the sequential acquisition of the alpha and beta chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha, but not CD8beta, is expressed in vivo in a minor subset of DP TCRalphabeta(-) thymocytes, referred to as CD4(+) CD8alphaalpha(+) pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta(ic)). In contrast, expression of CD8alphabeta heterodimers was selectively found on DP TCRalphabeta(-) thymocytes that express TCRbeta(ic) and are enriched for cycling cells. Interestingly, CD4(+) CD8alphaalpha(+) pre-T cells are shown to be functional intermediates between CD4(+) CD8(-) TCRbeta(ic)(-) and CD4(+) CD8alphabeta(+) TCRbeta(ic)(+) thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta(ic) expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4(+) CD8alphaalpha(+) to CD4(+) CD8alphabeta(+) transition marks the key control point of pre-TCR-mediated beta-selection in human T-cell development.

Identification of a late stage of small noncycling pTalpha- pre-T cells as immediate precursors of T cell receptor alpha/beta+ thymocytes.

During thymocyte development, progression from T cell receptor (TCR)beta to TCRalpha rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRbeta chain paired with pre-TCRalpha (pTalpha). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTalpha is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3(-) thymocytes lacking surface pTalpha, but expressing cytoplasmic TCRbeta, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early alpha transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRalpha gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTalpha+ pre-T cells and TCRalpha/beta+ thymocytes. The results support a developmental model in which pre-TCR-expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRalpha gene expression.

Enhanced green fluorescent protein as an efficient reporter gene for retroviral transduction of human multipotent lymphoid precursors.

Owing to its autofluorescence properties, green fluorescent protein (GFP) has aroused increasing interest as a marker system for many research applications. In this study we investigated the suitability of the "enhanced" GFP (EGFP), a mutant version of GFP optimized for flow cytometry and microscopy detection, as a reporter gene for retroviral transduction protocols. EGFP was shown to display a bright and stably maintained emission pattern in transfected GP+envAm12 packaging cells. Stable fluorescent emission was observed as well after transduction in NIH 3T3 fibroblasts and in the human Jurkat T cell line, in which EGFP was shown to confer no deleterious effect or growth disadvantage on the expressing cells. Moreover, EGFP expression could be detected after short-term retroviral exposure, thus allowing a rapid and quantitative retroviral titering assay, alternative to the standard colony-formation procedure. Most importantly, we showed the feasibility of EGFP as a marker gene in retroviral-mediated transduction of primary lymphoid precursors. In particular, transduction of CD34+CD1- human thymocytes by short-term cocultivation yielded up to 30% of EGFP-expressing cells, while maintaining CD34 expression levels. Finally, when cultured under multicytokine-supported conditions, such transduced intrathymic progenitors were shown to efficiently generate lymphoid-related dendritic cells, which displayed a distinct EGFP expression. Therefore, because of its rapid and easy detectability and its nontoxic characteristics, EGFP proves itself to be a valuable reporter gene by allowing the transduction of multipotential progenitors and by being compatible with the developmental programs of lymphoid lineage generation.

Identification of a common developmental pathway for thymic natural killer cells and dendritic cells.

Current data support the notion that the thymus is seeded by a yet uncommitted progenitor cell able to generate T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). We assess in this report the developmental relationship of DCs and NK cells derived from a small subset of CD34(+) human postnatal thymocytes that, like the earliest precursors in the fetal thymus, display low CD33 surface expression. Culture of these isolated CD34(+) CD33(lo) thymic progenitors with a mixture of cytokines, including interleukin-7 (IL-7), IL-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, results in predominant generation of DCs. However, the addition of IL-2 to the cytokine mixture leads to the simultaneous development of DCs and NK cells. Both developmental pathways progress through a transient population of CD34(+)CD44(bright) CD5(lo/-)CD33(+) large-sized cells, distinct from small-sized T-lineage precursors, that contain bipotential NK/DC progenitors. These data provide evidence of linked pathways of NK cell and DC development from intrathymic precursors and suggest that NK cells and DCs branch off the T lineage through a common intermediate progenitor.

Herpesvirus saimiri immortalization of alpha beta and gamma delta human T-lineage cells derived from CD34+ intrathymic precursors in vitro.

Herpesvirus saimiri (HVS), an agent that can infect many human cell types, has been shown to immortalize selectively TCR alpha beta + CD3+ T lymphocytes. Human T cell precursors defined as CD34+CD3-CD4-CD8- were isolated from thymic samples and exposed to HVS in the presence of either IL-2 or IL-7. Cultures lacking the virus were non-viable by day 15. Test cultures, in contrast, showed a sustained proliferative activity lasting > 5 months, allowing the phenotypical and molecular analysis of the cellular progeny. In the presence of IL-7, TCR alpha beta + cells with three different phenotypes (mainly CD4+CD8-, but also CD4+CD8+ and CD4-CD8+) were immortalized, whereas no TCR gamma delta + cells were recovered. Kinetic studies showed that the expansion of immortalized TCR alpha beta + cells was preceded by a gradual loss of CD34+ cells followed by a transient accumulation of two distinct cell subsets: first CD1+CD4+CD3- cells and then CD4+CD8+ thymocytes. This resembles early phenotypic changes occurring during normal intrathymic T cell development. In the presence of IL-2, in contrast, only TCR gamma delta + cells were immortalized (mainly CD4-CD8+, but also CD4-CD8-). The results show that HVS can be used to read the CD3+ cellular outcome of T cell differentiation assays, including gamma delta + CD4-CD8+, gamma delta + CD4-CD8-, alpha beta + CD4+CD8-, alpha beta + CD4-CD8+ and alpha beta + CD4+CD8+ T cells. A clear role for different cytokines (IL-2 for gamma delta + cells, IL-7 for alpha beta + cells) in early T cell commitment was also apparent.

Regulation of pre-T cell receptor (pT alpha-TCR beta) gene expression during human thymic development.

In murine T cell development, early thymocytes that productively rearrange the T cell receptor (TCR) beta locus are selected to continue maturation, before TCR alpha expression, by means of a pre-TCR alpha- (pT alpha-) TCR beta heterodimer (pre-TCR). The aim of this study was to identify equivalent stages in human thymocyte development. We show here that variable-diversity-joining region TCR beta rearrangement and the expression of full-length TCR beta transcripts have been initiated in some immature thymocytes at the TCR alpha/beta- CD4+CD8- stage, and become common in a downstream subset of TCR alpha/beta- CD4+CD8+ thymocytes that is highly enriched in large cycling cells. TCR beta chain expression was hardly detected in TCR alpha/beta- CD4+CD8- thymocytes, whereas cytoplasmic TCR beta chain was found in virtually all TCR alpha/beta- CD4+CD8+ blasts. In addition, a TCR beta complex distinct from the mature TCR alpha/beta heterodimer was immunoprecipitated only from the latter subset. cDNA derived from TCR alpha/beta- CD4+CD8+ blasts allowed us to identify and clone the gene encoding the human pT alpha chain, and to examine its expression at different stages of thymocyte development. Our results show that high pT alpha transcription occurs only in CD4+CD8- and CD4+CD8+ TCR alpha/beta- thymocytes, whereas it is weaker in earlier and later stages of development. Based on these results, we propose that the transition from TCR alpha/beta- CD4+CD8- to TCR alpha/beta- CD4+CD8+ thymocytes represents a critical developmental stage at which the successful expression of TCR beta promotes the clonal expansion and further maturation of human thymocytes, independent of TCR alpha.