Characterization of TCR Gene Rearrangements During Adult Murine T Cell Development (original) (raw)
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Characterization of T cell receptor (TCR) gene rearrangement during adult murine T cell development
The Journal of Immunology
Development of the ␣ and ␥␦ T cell lineages is dependent upon the rearrangement and expression of the TCR␣ and  or ␥ and ␦ genes, respectively. Although the timing and sequence of rearrangements of the TCR␣ and TCR loci in adult murine thymic precursors has been characterized, no similar information is available for the TCR␥ and TCR␦ loci. In this report, we show that approximately half of the total TCR␦ alleles initiate rearrangements at the CD44 high CD25 ؉ stage, whereas the TCR locus is mainly in germline configuration. In the subsequent CD44 low CD25 ؉ stage, most TCR␦ alleles are fully recombined, whereas TCR rearrangements are only complete on 10 -30% of alleles. These results indicate that rearrangement at the TCR␦ locus can precede that of TCR locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCR␦ and TCR␥ genes among CD44 high CD25 ؉ thymocytes, suggesting that functional ␥␦ TCR complexes can be formed before the rearrangement of TCR. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence ␣/␥␦ lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44 high CD25 ؉ stage of development.
Proceedings of the National Academy of Sciences, 1998
A rearranged T cell receptor (TCR) V␣ and J␣ gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic J␣ region. The introduced TCR ␣ chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR ␣ chain. This decrease is caused by a rearrangement of TCR ␣ chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR ␣ chain and the transgenic TCR  chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR ␣ chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR ␣ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR ␣ chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR ␣ chain gene play an important role in the formation of the T cell repertoire.
Blood, 1999
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(+) CD8alpha+beta- intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRdelta and TCRgamma, 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 TCRdelta rearrangements initiate in CD34(+)CD1a- cells preceding those in the TCRgamma and TCRbeta loci that commence in CD34(+)CD1a+ cells. Furthermore, we have examined at which cellular stage TCRbeta selection occurs in humans. We analyzed expression of cytoplasmic TCRbeta and cell-su...
TCRA Gene Rearrangement in Immature Thymocytes in Absence of CD3, Pre-TCR, and TCR Signaling
The Journal of Immunology, 2001
During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pT␣ and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, ␣ ؉ thymocytes do develop in pT␣-deficient mice, showing that TCR␣-chain genes are rearranged, either in CD4 ؊ CD8 ؊ or CD4 ؉ CD8 ؉ thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pT␣ and ␥c expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and ␥c signaling. However, CD3 expression was found to play a role in transcription of rearranged TCR␣-chain genes in CD4 ؊ CD8 ؊ thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.
A Molecular Map of T Cell Development
Immunity, 1998
; Fehling and von Boehmer, 1997). Early * Section of Immunology stages of thymocyte development are clearly defined Yale University School of Medicine by several molecular events that include expression of Howard Hughes Medical Institute the RAG-1 and RAG-2 gene products (Lin and Desiderio, New Haven, Connecticut 06520-8011 1993; Ferguson et al., 1994; Hoffman et al., 1996), so-† Laboratory of Immunology matic rearrangement of the T cell receptor  chain locus, National Institute of Allergy and Infectious Diseases expression of the coreceptor molecules CD4 and CD8, National Institutes of Health and, finally, recombination of the TCR ␣ locus (Dudley Bethesda, Maryland 20892 et al., 1994; Petrie et al., 1995). Productive rearrange-‡ Department of Molecular Biotechnology ment of both TCR chains allows surface expression of University of Washington School of Medicine the TCR and ensures life up to this point. Further devel-Seattle, Washington 98195-7650 opment, however, relies in part (Tourigny et al., 1997) upon signals generated by TCR interaction with MHC: self peptide complexes (von Boehmer, 1994). Most thy-Summary mocytes fail this rigorous criterion for selection and die of neglect (Janeway, 1994). Using a sensitive molecular marker for positive selec-While the early events in development can be clearly tion, the appearance of a particular functional TCR ␣ defined by molecular events, later stages of developchain sequence in cells from mice bearing a transgenic ment are typically defined by variations in the expression levels of cell surface proteins such as CD4, CD8, and  chain, we address several aspects of intrathymic T the TCR. These molecules allow for several different cell development. First, by examining specific TCR intrathymic subpopulations to be distinguished. The deprior to and after maturation, we demonstrate how a velopmental status of such populations has been, in restricted TCR repertoire is positively selected from most cases, assessed by other means including intraa highly diverse immature TCR repertoire. Second, thymic injections of genetically marked cells and reconsince this molecular marker is enriched in cells prostitution of thymocytes in irradiated mice. gressing toward the CD4 lineage and depleted in cells In this report, we identify a sensitive molecular marker progressing toward the CD8 lineage, a map of the for positive selection. This marker, which is the appeardevelopmental pathway of ␣ thymocytes can be inance or loss of a specific TCR ␣ chain sequence, is used ferred. Third, the first cells that show clear signs of to address three issues concerning the development of positive intrathymic selection are identified. TCR␣ bearing thymocytes. First, we demonstrate that the restriction of TCR usage that was apparent in the mature repertoire in "single-peptide" mice bearing a Peptide-Specific Responses of Nonimmunized TCR  Chain Transgenic Thymocytes § To whom correspondence should be addressed (e-mail: derek.
T cell migration during development: homing is not related to TCR V beta 1 repertoire selection
The EMBO Journal, 1994
at Birmingham, AL 35294, USA Communicated by J.P.Thiery T cell precursors enter the chick thymus in three waves during embryonic life. Each wave of thymocyte precursors colonizing the thymus gave rise to a similar TCR Vo repertoire in thymus, spleen and intestine both in terms of Vo1 and Jo usage as well as in the length of Vo-Do-Jo junctions. Seventeen V,1s were utilized, and a new Jo segment was found. In the progeny of the third wave, more nucleotides were deleted at the 5' end of the Jo segment, but the overall size of the CDR3 was conserved by a concomitant increase of N nucleotide addition at the V,-Do-Jo junctions during rearrangement. This CDR3 modification was observed in the spleen but not in the intestine, implying that progeny of the third wave migrate preferentially to the spleen, a possibility that was confirmed by adoptive cell transfers into congenic chickens. Very low frequencies of non-productive rearrangements in the intestine suggested that negative selection may occur in this organ. The present analysis indicates that Vo1 + T cells in spleen and intestine are primarily of thymic origin, this colonization of both organs occurs in waves and is not characterized by preselection of the TCR Vo1 repertoire.