TCRβ Expression Research Articles

Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: Implications for the control of TCR-beta locus recombination.

Gene targeting studies have shown that T cell receptor (TCR)-beta gene expression and recombination are inhibited after deletion of an enhancer (Ebeta) located at the 3' end of the approximately 500-kb TCR-beta locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-beta locus, the effects of Ebeta deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Ebeta contributes to major chromatin remodeling directed to an approximately 25-kb upstream domain comprised of the Dbeta-Jbeta locus regions. Accordingly, treatment of Ebeta-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-beta gene expression and promoted recombination within the Dbeta-Jbeta loci. Unexpectedly, however, epigenetic processes at distal Vbeta genes on the 5' side of the locus and at the 3' proximal Vbeta14 gene appear to be less dependent on Ebeta, suggesting that Ebeta activity is confined to a discrete region of the TCR-beta locus. These findings have implications with respect to the developmental control of TCR-beta gene recombination, and the process of allelic exclusion at this locus.

Early T cell receptor beta gene expression is regulated by the pre-T cell receptor-CD3 complex.

We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.

TCR Gene Rearrangements and Expression of the Pre-T Cell Receptor Complex During Human T-Cell Differentiation

Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4+CD8+ through CD3−CD4+CD8− and CD3−CD4+CD8+β− intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRδ and TCRγ, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRδ rearrangements initiate in CD34+CD1a− cells preceding those in the TCRγ and TCRβ loci that commence in CD34+CD1a+ cells. Furthermore, we have examined at which cellular stage TCRβ selection occurs in humans. We analyzed expression of cytoplasmic TCRβ and cell-surface CD3 on thymocytes that lack a mature TCRβ. In addition, we overexpressed a constitutive-active mutant of p56lckF505 by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRβ selection in humans is initiated at the transition of the CD3−CD4+CD8− into the CD4+CD8+β− stage.

TCR Gene Rearrangements and Expression of the Pre-T Cell Receptor Complex During Human T-Cell Differentiation

AbstractRecent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4+CD8+ through CD3−CD4+CD8− and CD3−CD4+CD8α+β− intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRδ and TCRγ, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRδ rearrangements initiate in CD34+CD1a− cells preceding those in the TCRγ and TCRβ loci that commence in CD34+CD1a+ cells. Furthermore, we have examined at which cellular stage TCRβ selection occurs in humans. We analyzed expression of cytoplasmic TCRβ and cell-surface CD3 on thymocytes that lack a mature TCRαβ. In addition, we overexpressed a constitutive-active mutant of p56lckF505 by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRβ selection in humans is initiated at the transition of the CD3−CD4+CD8− into the CD4+CD8α+β− stage.

Expression of T-cell receptor beta-chain mRNA and protein in gamma/delta T-cells from euthymic and athymic rats: implications for T-cell lineage divergence.

The relationship between α/β and γ/δ T-cell lineages was studied in rats using RT-PCR analysis of TCRβ transcripts in γ/δ T-cell hybridomas and an intracellular staining technique to detect TCRβ protein in primary γ/δ T-cells. We report the presence of functional TCRβ transcripts in 2/9 γ/δ T-cell hybridomas. About 15 % of peripheral γ/δ T-cells and thymocytes also express TCRβ protein, giving a minimum estimate for successful Tcrb rearrangement based on ex vivo single cell analysis. In athymic rats, γ/δ T-cells expressing intracellular β protein are present but at a lower frequency than in euthymic controls, suggesting that in the thymus, more γ/δ T-cell precursors pass through a stage where functional β rearrangement has occurred than in extrathymic sites. Analysis of TCR expression in purified transitory immature CD4-8+ (iCD8SP) thymocytes and their spontaneously developing CD4+8+ (DP) progeny showed that TCRγ mRNA is expressed in iCD8SP cells but not in their immediate DP progeny that reinitiate RAG-1 transcription and commence α/βTCR expression. We conclude that rat γ/δ T cells can separate from the α/β lineage after TCRβ expression, but not after entry into the DP compartment.

Cutting Edge: The Expression In Vivo of a Second Isoform of pTα: Implications for the Mechanism of pTα Action

AbstractA second isoform of pTα, “pTαb,” is derived from the pTα locus by tissue-specific, alternative splicing. pTαb is coexpressed in the thymus with the previously characterized form of pTα (which we term pTαa) and is also expressed in peripheral cells without pTαa. While pTαa acts to retain most TCR β-chains intracellularly, pTαb permits higher levels of cell surface TCRβ expression and facilitates signaling from a CD3-TCRβ complex.

Human T cells expressing an invariant Va24-JotQ TCRα are CD4- and heterogeneous with respect to TCRβ expression

A population of mature CD4-CD8-double-negative T cells that expresses an invariant Va24-JotQ TCR has been identified in humans; the majority of these cells appear to express Vβll. A closely related invariant TCRa chain is also expressed by a population of NK1 + murine T cells, but these cells may be either CD4+ or double negative. In this report, multiple CD4+ or double-negative Va24+Vβll+ T-cell clones were isolated, and only the double-negative clones were found to express the invariant TCRa chain. Studies of TCRβ chains expressed by these cells demonstrate that a subset in some donors use Vβ genes other than Vβll. Characterization of Vβll TCRs in one donor by CDR3-length analysis was also carried out. The results indicate that multiple Vβll TCRs of differing CDR3 lengths may associate with the invariant TCRa chain.

In Vivo Infection of Human T-Cell Leukemia Virus Type I in Non-T Cells

Using three different procedures, immunomagnetic isolation, FACS and panning, each of the T4 cell, T8 cell, monocyte, and B cell population was fractionated from peripheral blood mononuclear cells (PBMC) obtained from 10 human T cell leukemia virus type I (HTLV-I)-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) cases, 3 adult T cell leukemia and lymphoma (ATLL) cases and 9 HTLV-I-infected healthy carriers. DNA from each fraction was then subjected to the quantitative polymerase chain reaction. Although T4 cells possessed the highest copy number of HTLV-I in general, we also detected a significant amount of HTLV-I provirus in T8 cells, monocytes, and B cells. To confirm virus infection of the non-T cell populations, especially the monocyte fraction, we performed further experiments and the following results were obtained: (1) HTLV-I tax RNA expression was confirmed simultaneously in the RNA of monocytes that were negative for T cell receptor β chain (TCRβ) expression in vivo. (2) We cultured these monocytes for a short period of time and detected HTLV-I antigen expression in non-specific esterase-positive monocyte/macrophage cells. Our data indicate that HTLV-I has a broad host range in vivo and that monocytes or cells of monocyte lineage such as tissue macrophages might comprise a virus reservoir in vivo.