T-cell Receptor Alpha Genes Research Articles

Cloning of self-major histocompatibility complex antigen-specific suppressor cells from adult bone marrow.

We examined if suppressor cell clones may be established from adult bone marrow that contains a population of cells capable of specifically downregulating the immune response directed toward self-major histocompatibility complex (MHC) antigens. Freshly prepared adult C3H (H-2k) marrow cells were cultured in medium containing interleukin 2 (IL-2), IL-3, or a mixture of IL-2 and IL-3. After 7-10 d, cells grown in IL-3-containing medium were screened for their capacity to suppress cytotoxic T lymphocyte (CTL) generation against self-MHC antigens in allogeneic mixed lymphocyte cultures. Cells capable of suppressing anti-C3H CTL generation were cloned by limiting dilution. Several suppressor clones were established that exhibited strong suppression of anti-H-2k, anti-H-2Kk/Ik, and anti-H-2Dk CTL generation, but failed to suppress anti-H-2d and anti-H-2b responses. When tested in a skin allograft model, intravenous injections of these bone marrow-derived anti-self suppressor cells (2.5 x 10(7) cells) together with IL-3 induced prolongation of C3H skin allografts in anti-mouse lymphocyte serum-treated B6AF1 mice. Injection of IL-3 alone had no effect on allograft survival. Moreover, these cells failed to prolong B10.AKM skin allografts on B6AF1 recipients. Northern blot analysis showed that these cells express full-length transcripts of the T cell receptor (TCR) gamma gene, but not those of TCR alpha, beta, or delta genes. However, no rearrangement of gamma gene was observed by Southern blot analysis. Flow cytometric analysis revealed that bone marrow-derived suppressor cells are strongly positive for Thy-1 antigen but negative for CD3, CD4 (L3T4), and CD8 (Lyt-2) surface markers, and express only class I MHC antigens. Suppressor cells derived from adult bone marrow may play an important role in extrathymic induction of self-tolerance.

A novel type of aberrant T cell receptor alpha-chain gene rearrangement. Implications for allelic exclusion and the V-J recombination process.

In the process of analyzing the contribution of nonproductive alpha- and beta-chain gene rearrangements to the allelic exclusion of TCR gene expression, we have found a novel type of aberrant alpha-gene rearrangement. In one alpha-allele of the mouse KB5-C20 T cell clone, a J alpha gene segment has been abutted precisely to a sequence that does not display any homology to known V and D gene segment. The appended sequence originates from within the V alpha locus and is located, in the germ-line, 1 kb upstream of a member of the V alpha 2-gene segment subfamily. No recombination signal sequences have been found contiguous to the recombination point. These observations indicate that in normal T lymphocytes, TCR alpha-genes may be affected by aberrant rearrangements similar to those that predominate in human T cell tumors containing chromosome 14 inversion or translocation. Furthermore, compilation of published data and cloning and sequencing of three additional alpha-alleles has allowed us to examine the status of alpha-loci in nine mouse T cell clones expressing functional alpha beta-heterodimers. Interestingly, in contrast to the situation observed at the beta-locus, only 1 of 18 analyzed alpha-alleles has retained a germ-line unrearranged configuration. In addition, in each T cell clone, alpha-rearrangements on homologous chromosomes were unevenly distributed over the J alpha region and shown to generally involve neighboring J alpha gene segments.

Rearrangement of the T-cell receptor alpha, beta and gamma chain genes in chronic lymphocytic leukemia

Simultaneous expression of mature B-cell and T-cell markers and subsequent abrogation of expression of the T-cell surface markers by cytotoxic chemotherapy was reported earlier in a patient (TG) with chronic lymphocytic leukemia (CLL). In addition to rearrangements of the immunoglobulin (Ig) gene loci correlating with phenotypic data, the T-cell receptor (TCR) alpha, beta and gamma chain genes also displayed clonal rearrangements in peripheral blood lymphocyte DNA of TG. The present case shows that in CLL cells not only the expression of B-cell and T-cell specific differentiation antigens but also the rearrangement of Ig as well as TCR alpha, beta and gamma genes may occur simultaneously.

IMMUNOGENETICS OF POLYMYALGIA RHEUMATICA

Evidence suggests that polymyalgia rheumatica (PMR) may be an immune-mediated disease. Therefore, the role of HLA class II genes, the switch region of immunoglobin mu and alpha 1 heavy chain as the T-cell receptor (TcR) genes were investigated by Southern blot analysis. The frequency of DR4 specificity was increased in PMR (67.4% versus 30.3%; P = 0.00017). No association was found with the DQA and DQB genes, the switch region of immunoglobulin mu and alpha 1 heavy chain genes, and the TcR alpha, beta and gamma genes. This study suggests that HLA-DR4 is an important susceptibility factor for PMR. Further studies are needed to elucidate the role of Ig and TcR genes.

LD1: a CD4−CD8− TCRαβ/CD3 + peripheral T cell line with helper function for B lymphocytes

In order to learn more about the small subset of CD4-CD8- TCR alpha beta/CD3+ peripheral T lymphocytes, we firstly characterized at the cellular and molecular levels the CD4-CD8- LD1 cell line isolated from the spleen of an MRL/lpr-lpr mouse. Secondly we studied its functional properties. LD1 cells are Thy1+ CD5+ CD4-CD8- LFA-1+ PgP-1+ and do not bind the T cell precursor-specific antibodies Joro 37-5 or Joro 75. They are negative for IgM, B-220, BP-1, J11d, Lyb8, Ia, F4/80, BP-2, and Mac-1 surface markers. LD1 cells have deleted the TCR delta locus, have rearrangements at the TCR gamma gene cluster (i.e. a V gamma 1-J gamma 1-C gamma 1 and a V gamma 4.3-J gamma 4-C gamma 4) and have two rearrangements of the TCR beta gene cluster (i.e. a D beta 1-J beta 1 and V-D-J beta 2). LD1 cells produce normal sized RNA transcripts from TCR alpha and beta genes and lower levels of gamma-mRNA. These cells bind CD3- and pan-TCR beta-specific antibodies as determined by FM analysis. We conclude that LD1 cells bear a TCR alpha beta/CD3 type of receptor complex. LD1 cells fail both in vivo and in vitro to differentiate into CD4+ or CD8+ cells. These cells help B lymphocytes to mature into antibody-secreting cells, secrete IL-3 and IL-6 but not IL-2, IL-4, or IL-5, and exert no detectable cytolytic activity. These results together with recent reports of antigen-specific CD4-CD8- TCR alpha beta/CD3+ cytotoxic T cell lines show that the CD4-CD8- TCR alpha beta/CD3+ subset comprises functionally competent helper and cytotoxic T lymphocytes and thereby argue for their potential to participate in immune responses. Our results also suggest that cells like LD1 represent terminally differentiated T lymphocytes rather than cells with precursor potential for CD4+ or CD8+ TCR alpha beta/CD3+ T lymphocytes.

Analysis of molecular events in leukemic cells arrested at an early stage of T-cell differentiation

We analyzed the rearrangement and expression of T-cell receptor (TCR) genes, including the recently identified TCR delta gene, in 21 patients with T-lineage leukemia/lymphoma. Among 8 patients with CD3-, CD4-, and CD8- (group I), 2 patients showed germline configuration of the TCR delta, gamma, beta, and alpha genes and 1 patient demonstrated only TCR delta gene rearrangement. All nine patients with CD3-, CD4+, and/or CD8+ (group II) showed concomitant rearrangements of the TCR delta, gamma, and beta genes. TCR alpha gene rearrangement was also observed in two patients. Three of four patients with CD3+ (group III) showed rearrangement of the TCR alpha gene with deletion of both alleles or of a single allele of the TCR delta gene. With Northern blot analysis, full-length transcripts of the TCR delta gene were detected in 3 of 15 examined patients. All were restricted to group I or group II. In contrast, full-length transcripts of TCR beta and alpha were observed mainly in samples from groups II and III. Based on these findings, rearrangement of the TCR delta gene may be the earliest event in T-cell differentiation, preceding rearrangements of the other TCR genes.

Analysis of Molecular Events in Leukemic Cells Arrested at an Early Stage of T-Cell Differentiation

We analyzed the rearrangement and expression of T-cell receptor (TCR) genes, including the recently identified TCR delta gene, in 21 patients with T-lineage leukemia/lymphoma. Among 8 patients with CD3-, CD4-, and CD8- (group I), 2 patients showed germline configuration of the TCR delta, gamma, beta, and alpha genes and 1 patient demonstrated only TCR delta gene rearrangement. All nine patients with CD3-, CD4+, and/or CD8+ (group II) showed concomitant rearrangements of the TCR delta, gamma, and beta genes. TCR alpha gene rearrangement was also observed in two patients. Three of four patients with CD3+ (group III) showed rearrangement of the TCR alpha gene with deletion of both alleles or of a single allele of the TCR delta gene. With Northern blot analysis, full-length transcripts of the TCR delta gene were detected in 3 of 15 examined patients. All were restricted to group I or group II. In contrast, full-length transcripts of TCR beta and alpha were observed mainly in samples from groups II and III. Based on these findings, rearrangement of the TCR delta gene may be the earliest event in T-cell differentiation, preceding rearrangements of the other TCR genes.

A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus.

A transcriptional enhancer element has been identified 4.5 kilobases 3' of C alpha (constant region alpha chain) in the human T-cell receptor (TCR) alpha-chain locus. This enhancer is active on both a TCR V alpha (variable region alpha chain) promoter and the minimal simian virus 40 promotor in TCR alpha/beta Jurkat and EL4 cells but is inactive on a V alpha promoter in human TCR gamma/delta PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and kappa B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR alpha gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR alpha/beta lineage. In addition, the TCR alpha enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR alpha locus.

Rearrangement and expression of T-cell receptor delta genes in T-cell acute lymphoblastic leukemias

We have analyzed T-cell receptor delta (TcR-delta) gene rearrangement and transcription in appropriately phenotyped mononuclear cells derived from 12 patients with T-cell acute lymphoblastic leukemia (T-ALL). The T-ALL cells were also analyzed for rearrangement and transcription of the T-cell receptor(TcR)-beta and gamma genes as well as for the presence of TcR-alpha gene transcripts. Four T-ALLs expressed TcR-gamma delta at the cell surface, while three expressed TcR-alpha beta. The other five T-ALLs did not express a TcR-CD3 complex on their cell membrane. The TcR-gamma delta + T-ALL had rearranged both TcR-delta gene alleles and contained mature 2.2 and 1.5 kb TcR-delta transcripts. In one case, immature 1.9 and 1.2 kb TcR-delta transcripts were also found. Furthermore they contained mature TcR-gamma mRNA, mature or immature TcR-beta mRNA, but no TcR-alpha mRNA. The three TcR-alpha beta + T-ALLs contained mature alpha and beta transcripts, but lacked TcR- delta transcripts as a result of deletion of both TcR-delta gene alleles. These data are in line with a mutually exclusive expression of TcR-alpha and -delta genes, which may be important to ensure the presence of only one type of TcR per T cell. One of the five CD3- T- ALLs had germline TcR-beta, gamma, and delta genes. The other four CD3- T-ALLs had rearranged their TcR-beta, gamma, and delta genes and contained immature 1.9 and 1.2 kb TcR-delta gene transcripts. Remarkably, one of these T-ALLs also contained TcR-alpha transcripts in addition to the immature TcR-delta transcripts, which was in line with the deletion of one TcR-delta gene allele and rearrangement of the other allele. This suggests that prevention of dual receptor expression may not only be regulated by the presence of germline TcR-alpha genes in TcR-gamma delta + cells or by deletion of both TcR-delta gene alleles in TcR-alpha beta + cells, but also via other regulation mechanisms. Finally, our data indicated that the combinatorial repertoire of the TcR-delta genes is limited, which has also been described for the TcR-gamma genes.

Demonstration of delta rec-pseudo J alpha rearrangement with deletion of the delta locus in a human stem-cell leukemia.

It has been hypothesized that a rearrangement between the delta recombining element (delta Rec) and a pseudo J alpha gene serves to delete the TCR-delta locus before rearrangement of the TCR-alpha genes. We have now sequenced a direct, site-specific rearrangement between the delta Rec element and a pseudo J alpha gene in a human leukemic stem-cell line. Putative "N-sequence" addition was noted at the site of recombination, suggesting that this event occurred at a time when the enzyme(s) involved in N-region addition were active in this cell. This provides support for the view that deletion of the TCR-delta locus is required before rearrangement of the TCR-alpha chain genes.

Relationship between rearrangement and transcription of the T-cell receptor alpha, beta, and gamma genes in B-precursor acute lymphoblastic leukemia

Transcription of the T-cell receptor (TCR)-gamma, beta, and alpha genes has been analyzed in 29 patients with B-precursor acute lymphoblastic leukemia (ALL) by northern blotting analysis. In addition, the configuration of the TCR-alpha gene was examined using newly developed genomic J alpha probes capable of detecting TCR-alpha gene rearrangements involving joining (J)alpha regions up to 85 kilobase (kb) from constant (C)alpha. In this study, TCR-alpha gene rearrangements were detected in 16 of the 29 patients. Thus, the frequency of TCR-alpha gene rearrangements in B precursor ALL was as high as with TCR-gamma (15 of 29) and was higher than observed for TCR- beta (nine of 29). Truncated transcripts were frequently observed in cells with rearrangements of the TCR-beta or alpha gene. In contrast, TCR-gamma transcripts were detected in only one patient despite the high incidence of TCR-gamma gene rearrangements. Results presented here suggest that although expression of TCR genes was weak in B-precursor ALL cells compared with T-lineage cells, these genes experience both transcriptional and recombinational crossover in B-precursor ALL. The results also suggest that neither transcription nor gene rearrangement, when examined alone, are sufficient to assign T or B lymphocyte lineage in lymphoblastic leukemia.

Relationship Between Rearrangement and Transcription of the T-Cell Receptor a, β, and γ Genes in B-Precursor Acute Lymphoblastic Leukemia

Transcription of the T-cell receptor (TCR)-gamma, beta, and alpha genes has been analyzed in 29 patients with B-precursor acute lymphoblastic leukemia (ALL) by northern blotting analysis. In addition, the configuration of the TCR-alpha gene was examined using newly developed genomic J alpha probes capable of detecting TCR-alpha gene rearrangements involving joining (J)alpha regions up to 85 kilobase (kb) from constant (C)alpha. In this study, TCR-alpha gene rearrangements were detected in 16 of the 29 patients. Thus, the frequency of TCR-alpha gene rearrangements in B precursor ALL was as high as with TCR-gamma (15 of 29) and was higher than observed for TCR-beta (nine of 29). Truncated transcripts were frequently observed in cells with rearrangements of the TCR-beta or alpha gene. In contrast, TCR-gamma transcripts were detected in only one patient despite the high incidence of TCR-gamma gene rearrangements. Results presented here suggest that although expression of TCR genes was weak in B-precursor ALL cells compared with T-lineage cells, these genes experience both transcriptional and recombinational crossover in B-precursor ALL. The results also suggest that neither transcription nor gene rearrangement, when examined alone, are sufficient to assign T or B lymphocyte lineage in lymphoblastic leukemia.

Developmental regulation of the extrathymic differentiation potential of the progenitor cells for T cell lineage

Interleukin-2 (IL-2) generates T lineage cell lines (IL-3B series) directly from the isolated interleukin 3 (IL-3)-induced pluripotential progenitor colonies obtained form adult spleen in vitro. The phenomenon can be reproduced by the combined IL-3/IL-2 bulk culture system. In order to compare the differentiation potential of such progenitors in various ontogenical stages, we have established a number of cell lines from fetal livers (LFD series), fetal thymus (FTD15), newborn spleens (SED series), and adult spleens (SPB series) using the IL-3/IL-2 culture system. The phenotypes of cell lines varied depending upon the developmental stages; LFD lines, Thy1+CD3-CD4-CD8-B220+, FTD15, Thy1+CD3+CD4-CD8+B220-, and SPB and IL-3B lines, Thy1+CD3+CD4-CD8+B220+. Analysis of T cell receptor (TCR) genes revealed that all of the TCR genes (alpha, beta, gamma, and delta) were in germ line configurations in LFD lines. In LFD17 and 19, from 17 and 19 gestation days (GD) respectively, however, germ line transcripts or TCR beta gene were detected, suggesting that they were in the earliest phase of T cell commitment. In LFD14 line from GD14 fetal liver, no such transcript could be detected. In contrast, FTD15 showed complete rearrangement of every TCR gene with full-length TCR alpha and beta gene mRNA. On the other hand, all of the SED, SPB, and IL-3B series of lines exhibited the rearrangement of every TCR gene with productive TCR alpha and beta gene mRNA. The results indicated that progenitors in postnatal spleen, in contrast to those in fetal liver, had the potential to rearrange and express the TCR genes in extrathymic conditions in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell receptor delta gene rearrangements in acute lymphoblastic leukemia.

Using a newly isolated cDNA clone encoding the TCR-delta gene and genomic probes, we have analyzed T cell receptor (TCR) delta gene rearrangement in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and 29 patients with B-precursor ALL. Five out of seven CD3- T-ALL and 4 of 12 CD3+ T-ALL showed bi-allelic rearrangements of the TCR-delta gene. In three CD3+ patients, a single allelic TCR-delta gene rearrangement was observed with rearrangement of the TCR-alpha gene on the other allele. In five CD3+ patients with bi-allelic rearrangements of the TCR-alpha gene, the TCR-delta gene locus was deleted. Transcription of the TCR-delta gene was also analyzed in six T-ALL. Five patients expressed TCR-delta transcripts. Only one T-ALL, presumably derived from the most immature T lineage cells, did not have TCR-delta transcripts, but expressed TCR-gamma and 1.0-kb truncated TCR-beta transcripts. In B-precursor ALL, 20 patients (69%) showed rearrangements of the TCR-delta gene. The frequency of TCR-delta gene rearrangement was higher than TCR-alpha (59%), gamma (52%), or beta (31%) genes. These findings suggest that TCR-alpha gene rearrangements may take place after rearrangements of the TCR-delta gene with concomitant deletion of rearranged TCR-delta genes in T cell differentiation. Among leukemic cells of B lineage, the TCR-delta gene is the earliest rearranging TCR gene, followed by TCR-gamma and beta gene rearrangements.

Proximity of the CTLA-1 serine esterase and Tcr alpha loci in mouse and man.

The serine esterase CTLA-1 gene was shown by in situ hybridization to map to the D segment of mouse chromosome 14, the same localization as a member of the immunoglobulin superfamily, Tcr alpha. To further demonstrate the proximity of CTLA-1 and Tcr alpha, genetic linkage was tested in mouse using restriction fragment length polymorphisms and a backcross progeny, and no recombination was observed in the 100 backcross products studied. Recombination events between Tcr alpha/CTLA-1 and the markers Gdh-X and NP-1 show that the most probable order of these loci in the mouse 14D region is NP-1-Tcr alpha/Ctla-1-Gdh-X. In man, the human homologue of CTLA-1 was shown by in situ hybridization to map on chromosome 14, at 14q11-q12, where Tcr alpha also maps. Using the human cell line SUP-T1, bearing the inversion inv(14) (q11;q32), we further demonstrated the loci order in man to be centromere-NP-1-Tcr alpha-CTLA-1. To complement the cytogenetic and genetic mapping data, we tried to determine the physical distance between the two genes by pulsed field gel electrophoresis (PFGE). DNA prepared from various cell types, both mouse and human, were digested with a panel of rare cutter enzymes and hybridized first with CTLA-1, then with Tcr alpha probes. None of the bands identified hybridized with both Tcr alpha and CTLA-1 probes for either mouse or human cells. Although the physical mapping by PFGE is inconclusive, the cytogenetic and genetic data support close linkage of the Tcr alpha and CTLA-1 genes in both mouse and man, suggesting homology between the D region of mouse chromosome 14 and the q11-q12 region of human chromosome 14, encompassing the Tcr alpha and CTLA-1 loci. These findings also provide another example of proximity of genes coding for a member of the Ig super-family and a serine esterase.

Coordinate transcriptional regulation of alpha and delta chains of the T-cell antigen receptors by phorbol esters and cyclic adenosine 5′-monophosphate in human thymocytes.

The regulation of T-cell antigen receptor (TcR) gene expression by phorbol ester and cAMP was studied in human thymocytes. Incubation of human thymocytes in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased the levels of the mRNAs of TcR-alpha and -delta while the levels of TcR-beta and -gamma mRNA were not affected. cAMP, on the other hand, caused a decrease in TcR-alpha and -delta mRNA levels but also did not affect TcR-beta and -gamma mRNA levels. The induction of TcR-alpha and -delta mRNA by 12-O-tetradecanoylphorbol-13-acetate occurred at the transcriptional level only whereas cAMP treatment decreased both TcR-alpha gene transcription and the stability of its mRNA. The similarity in the transcriptional control of TcR-alpha and -delta genes combined with their close chromosomal location raises the possibility that they share common DNA regulatory elements.

Induction of T cell receptor gamma chain transcription in murine T cells by trans-complementation.

As an initial approach to understanding the molecular basis of the developmentally regulated expression of T cell receptor (TcR) genes, we constructed hybrids among T cell clones that differ in their expression of CD4 and CD8 antigens as well as in alpha, beta and gamma mRNA levels. Here we report that the TcR gamma gene becomes transcriptionally active in hybrid cells formed between parental clones that lack TcR gamma mRNA. We also observed negative trans-regulation of TcR beta but not of TcR alpha transcription in one of these hybrids. Positive TcR gamma transcription was observed both with (CD4-CD8- X CD4-CD8-) and (CD4-CD8- X CD4+CD8+) hybrids while negative TcR beta gene regulation was only detected in (CD4-CD8- X CD4+CD8+) cells. These data suggest that the regulation of TcR alpha, beta and gamma genes is mediated by the action of specific transacting factors that become asynchronously active in the various stages of T cell development.

Sizing of the Human T Cell Receptor a Locus and Detection of a Large Deletion in the Molt-4 Cell Line

The human T cell receptor alpha (TCR-alpha) chain gene consists of discontinuous DNA segments encoding multiple variable (V), multiple joining (J), and one constant (C) region. Unlike other immunoglobulin or TCR genes, however, the TCR-alpha locus exhibits an unusual dispersal of J segments that occupy a region of greater than 50 kilobases (kb) upstream of the C region, with the exact size still unknown. We report here the study of the TCR-alpha genetic locus by using pulsed-field gel electrophoresis (PFGE), which permits the separation of large DNA fragments. Analysis of DNA prepared from normal peripheral blood mononuclear cells, human endothelial cells, and a B cell line demonstrates that both V and C sequences are contained within a single 400-kb SfiI restriction fragment. PFGE analysis of the T cell line Molt 4 suggests a greater than 600-kb deletion involving the TCR-alpha gene.

Stable T3- and T3+ subclones derived from the mosaic human T cell leukemia cell line, CEM.

An unexplained feature of the human T lymphoblastic leukemia cell line, CEM, is a heterogeneous expression of surface membrane T3-TCR complex. CEM cells constitutively maintain two distinct populations whereby a minority of cells (5 to 16%) express T3 but the remainder do not. From the parental cell line, we have derived stable subclones in which all cells express surface T3 and companion subclones that lack surface T3. Inasmuch as surface T3 expression is contingent on assembly and coexpression of the multichain T3 protein complex with the TCR, we compared the surface T3+ and T3- subclones for their rearrangement and expression of genes of the T3-TCR complex. Restricted DNA of both subclone types showed the identical monoclonal pattern of TCR gene rearrangement seen in the mosaic parental CEM line. Northern blot analysis revealed mRNA transcripts of TCR beta subunit and T3 delta in both the surface T3+ and T3- subclones. However, surface T3+ subclones transcribed abundant full length (1.5-kb) TCR alpha mRNA in contrast to the surface T3- subclones in which TCR alpha transcripts were undetectable. Lack of TCR alpha gene transcription was not attributable to either unrearranged or aberrantly rearranged TCR alpha genes because exposure of surface T3- cells to phorbol ester induced a rapid (15-min) and progressive accumulation of full length (1.5-kb) TCR alpha mRNA. This was followed by surface membrane T3 expression in 24 to 72 h. We conclude that the unusual phenotypic mosaicism of CEM cells is attributable to a reversible lack of transcription of the TCR alpha gene.

Trans-acting regulatory factors for T cell antigen receptor alpha- and gamma-chain gene expression.

BALB/c mouse thymoma-derived T cell line, CAK4.4 (Thy-1+, L3T4-, Lyt-2-), produced a large amount of TCR-gamma mRNA, a trace amount of TCR-beta mRNA but no detectable level of TCR-alpha mRNA. Another BALB/c mouse thymoma-derived T cell line, CAK1.3 (Thy-1+, L3T4+, Lyt-2+), synthesized a high level of TCR-alpha as well as TCR-beta mRNA but did not produce any amount of TCR-gamma mRNA. HAT-sensitive clones were established from the two T cell lines. Azaguanine-resistant, HPRT- CAK4.4 cells and bromodeoxyuridine-resistant, TK- CAK1.3 cells were fused by electrofusion method and the resultant hybrids were analyzed for expression of TCR genes as well as the changes of their cell surface phenotypes. Transcription of TCR-gamma gene was completely suppressed in all hybrids tested, although Southern blot analysis showed that the hybrids maintained TCR-gamma chain genes derived from both parental cells. TCR-alpha gene transcription occurred normally in one hybrid. In two other hybrids, TCR-alpha gene transcription was strongly suppressed. Treatment of the hybrid cells with 12-O-tetradecanoyl phorbol-13-acetate reversed the suppression of TCR-alpha gene transcription, but TCR-gamma gene transcription was not recovered by the same treatment. However, transcription level of TCR-beta gene was not changed in all hybrids. Our results suggested that the different trans-acting regulatory mechanisms control the transcription levels of TCR-alpha and TCR-gamma genes and that such a transcriptional control may play a crucial role in the determination of orderly appearance of TCR-gamma and TCR-alpha gene products during T cell ontogeny in the thymus.