Influence of human leukocyte antigen genes on TCR V gene segment frequencies.

Human leukocyte antigen (HLA)-dependent selection mechanisms exerted during thymic maturation are supposed to be main contributing factors to the genetic predetermination of the TCR repertoire and may have a detectable effect on adult peripheral blood lymphocyte V segment frequencies. Here, we analyzed whether polymorphic or non-polymorphic HLA determinants are associated with selected expression of some V gene segment specificities. We first examined the reactivity of 17 V segment specific mAb on purified CD4+ and CD8+ cell fractions in 10 unrelated people. We found a significant overexpression of only three V segment products (V beta 2, V beta 5.1 and V beta 6.7) in CD4+ and none in CD8+ cell fractions in most individuals. Skewing of certain V beta segments by non-polymorphic HLA determinants (i.e. class II for CD4+ and class I for CD8+ cells) is therefore more limited (3/17) than previously thought. Considering the effects of polymorphic HLA determinants, we compared TCR V segment frequencies in HLA-identical siblings to sibling pairs who differ at one or both HLA haplotypes, using 13 V beta specific mAb. In pairwise comparisons, we found that the HLA complex had no detectable effect on TCR repertoire in five large families with multiple siblings. Together, these observations suggest that HLA-predicted selection mechanisms exerted during thymic maturation might not have a predominant influence shaping the TCR repertoire of normal adults.

TCR gene segments from at least one third of V alpha subfamilies rearrange at the delta locus.

Using PCR and an experimentally validated V alpha subfamily-specific oligonucleotide panel (V alpha 1-w29), we have investigated whether the TCR delta chain may increase its combinatorial diversity by using V genes considered as alpha chain-specific. We show that at least 10 distinct human V alpha segments rearrange at the J delta locus, leading to scrambling of the two V gene repertoires. Fifty-five per cent of the V alpha/J delta transcripts characterized here were in frame. The 17 V alpha/C delta chains analysed included an extended CDR3 region with up to 18 aa encoded by the junctional region. In addition, a new J delta segment (J delta 4) has been characterized. Together, these findings demonstrate that combinatorial diversity in the human delta locus is larger than previously thought.

Limited T-cell receptor diversity in liver-infiltrating lymphocytes from patients with primary biliary cirrhosis.

Primary biliary cirrhosis is associated with the presence of high-titer anti-mitochondrial autoantibodies as well as T-cell infiltration of the liver, suggesting the involvement of autoimmune mechanisms. We have studied here the sequences of T-cell receptor alpha and beta chains expressed by T-cell clones derived from liver-infiltrating lymphocytes of two patients with primary biliary cirrhosis. Among the eight clones studied from the first patient, four expressed the same member of the V beta 6 subfamily, associated with either V alpha 4 (three clones) or V alpha 21 (one clone) gene segment. Two other clones expressed an identical V beta 12 transcript, and two in-frame alpha chain transcripts, involving V alpha 2 and V alpha 7 gene segments. From the second patient, eight out of the nine clones were found to rearrange V beta 17-J beta 2.1 and V alpha 3 gene segments. The remaining clone expressed distinct T-cell receptor chains, involving V beta 9 and V alpha 11 gene segments. As deduced from the analysis of their junctional regions, the eight T-cell clones expressing V beta 17/V alpha 3 gene segments derived from only three different T cells. Furthermore, conserved amino acid motifs were found to be encoded in both the alpha and the beta-chain junctional regions. Together, these data show a local amplification of unique T lymphocytes in both patients. The use of identical V beta J beta and V alpha gene segments with similar junctional sequences by three different cells, evidenced in one of the two cases, strengthens the view that liver-infiltrating T lymphocytes are selected locally by autoantigens in PBC.

The use of anchored polymerase chain reaction for the study of large numbers of human T-cell receptor transcripts.

Anchored-PCR (A-PCR) is an approach designed to amplify and clone sequences with unknown 5' or 3' extremities. A-PCR is therefore appropriate for studying variable region of T-cell receptors (TCRs) expressed in polyclonal T-cell populations since it does not prejudge which variable gene segments are actually being used. We report here some critical modifications in the initial procedure to make it easy to clone and sequence large series of TCR transcripts. They have been introduced to improve both the yield and specificity of TCR amplified products and include re-amplification, size selections of the material combined with the successive use of nested TCR constant region specific primers. This procedure has been successfully applied to the study of the repertoire of both TCR alpha/beta+ and gamma/delta+ human T-cells. The efficiency of the present A-PCR protocol will help to precisely analyze TCR usage in normal and pathological situations.

Influence of interleukin-2 administration on the expression of T-cell receptor V gene segments in patients with renal-cell carcinoma.

Tumor regression induced by IL-2 in a fraction of patients with metastatic renal-cell carcinoma (MRCC) could not be predicted by immunological monitoring. In addition, the general mechanisms leading to tumor regression or even the distinct cell subsets (e.g., T vs. NK cells) involved are poorly identified. To evaluate the influence of IL-2 administration on circulating T-cell subpopulations, TCR V alpha and V beta gene segment usage was analysed by PCR in 7 MRCC patients using a panel of V gene segment subfamily-specific oligonucleotide primers (V alpha I-w29/V beta I-w24). Three samples were examined in each patient: (i) peripheral blood cells (PBMCs) before therapy (day I); (ii) PBMC 2 days after the interruption of IL-2, at day 36 (i.e., at the lymphocytic rebound), (iii) the CD25-enriched cell fraction at day 36. Virtually all V alpha and V beta subfamily specificities were found in pre- as well as in post-treatment PBMCs and CD25+ cell fractions. These results support the view that circulating T-cell subpopulations are highly polyclonal after IL-2 therapy without any major alteration in the TCR V alpha and V beta repertoire. In addition, the results of quantificative densitometric analysis of V alpha and V beta amplified products suggest that a unique V beta 18-expressing T-cell subpopulation may be expanded in the CD25+ cell fraction after IL-2 therapy. Further characterization of these T cells may contribute to a better understanding of in vivo effects of IL-2 on renal-cell carcinoma metastases.

Fine specificity of monoclonal antibodies directed at human T cell receptor variable regions: comparison with oligonucleotide-driven amplification for evaluation of V beta expression.

Seven distinct anti-human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR V beta genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide-driven amplification and cDNA sequencing. Four mAb were found to delineate the V beta 3, V beta 8, V beta 17 and V beta 19 subfamilies, respectively. The remaining reagents recognize subsets within the V beta 2, V beta 5 and V beta 13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide-driven amplification for evaluation of V beta 3, V beta 8, V beta 17 and V beta 19 subfamily expression in the peripheral blood. Although the V gene subfamily-specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR beta chain. The present data confirm the necessity to establish a complete set of well-characterized monoclonal reagents to study human T cell responses.

Analysis of T-cell-receptor variable gene segment usage in peripheral-blood lymphocytes of advanced cancer patients.

Advanced cancer patients generally display impaired T-cell immune functions. The underlying mechanisms are not well understood. The aim of this study was to analyze whether major alterations of TCR variable gene segment usage could be detected in the blood of these patients. Seventeen individuals with various malignancies were tested using PCR and a panel of V-gene-segment-sub-family-specific (V alpha 1-w29/V beta 1-w24) oligonucleotide primers. The results indicate that these cancer patient lymphocytes expressed most V alpha and V beta sub-family specificities, similarly to the lymphocytes of healthy donors (n = 10). This suggests that immunodepression in advanced cancer patients is not related to major deletions in their T-cell repertoires. We also compared the mean relative expression of each V-sub-family specificity of patients and healthy donors by quantitative densitometric analysis. We found significant differences in 4 V beta specificities (and no V alpha). Our analysis identified unique T-cell sub-sets putatively involved in the mechanisms leading to immunodepression in advanced cancer patients. Alternatively, the observed differences in terms of V beta specificity expression may reflect the host response against the tumor.

Analysis of T cell receptor variability in tumor-infiltrating lymphocytes from a human regressive melanoma. Evidence for in situ T cell clonal expansion.

Malignant melanomas are often infiltrated by T lymphocytes. It is postulated that the presence of tumor-infiltrating lymphocytes (TIL) reflects ongoing immune responses against transformed cells. Such "responses" appear generally inefficient with the potential exception of infrequent clinical situations characterized by spontaneous tumor regression. We have characterized here the molecular structure of the T cell receptor beta chain expressed by TILs in a case of regressive melanoma. Advantage was taken of the PCR technology to study T lymphocytes directly without cell culture. Experimentally validated V beta subfamily specific primers were used to evaluate the V beta usage in TILs and control samples. Our results reveal that clonal T cell populations, precisely defined by their V-D-J junctional sequences, are amplified at the tumor site. The existence of such local antigen-driven selections support the hypothesis that antitumor responses may indeed take place in regressive melanoma.

Analysis of T-cell receptor variability in transplanted patients with acute graft-versus-host disease.

T lymphocytes play a pivotal role in graft-versus-host disease (GVHD) and largely contribute to the graft-versus-leukemia (GVL) effect. Most mature T lymphocytes specifically recognize antigens through the alpha/beta T-cell receptor (TCR). Each alpha/beta TCR chain includes a constant region and a variable region, the latter being encoded by V-J alpha or V-D-J beta rearranged gene segments. To better characterize T cells involved in GVHD, V alpha and V beta gene segment usage was analyzed, after cDNA amplification, in peripheral blood mononuclear cells (PBMC) and skin samples from three patients with grade II cutaneous GVHD. At time of GVHD diagnosis (days 11, 22, and 25), when first signs of engraftment were detectable, virtually all V alpha and V beta subfamilies were represented in PBMC RNAs of the three recipients. These results suggest that diversified TCR gene segment expression is observed early after allogenic bone marrow transplantation (alloBMT). Lymphocytes infiltrating GVHD skin also expressed a large series of V alpha and V beta subfamily specificities. However, analysis of the V alpha and V beta amplified products showed substantial differences between PBMC and the skin lymphocyte RNAs. These observations indicate that a large variety of T lymphocytes are present at the disease site, while some of them may be specifically amplified or decreased in response to minor histocompatibility antigens (miHA). Further characterization of the latter T-cell subpopulations should lead to a better understanding of human in vivo responses directed at miHA.

An experimentally validated panel of subfamily-specific oligonucleotide primers (V alpha 1-w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.

We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.

Studies on the human T cell receptor alpha/beta variable region genes. I. Identification of 7 additional V alpha subfamilies and 14 J alpha gene segments.

The anchored-polymerase chain reaction has been used to study further the diversity of the human T cell receptor alpha chain. The analysis of 308 cDNA transcripts from human peripheral lymphocytes hybridizing with a C alpha probe led to the identification of a series of additional V alpha and J alpha gene segments. The sequences of seven V alpha gene segments which individually define a novel V alpha subfamily (termed V alpha w23 to V alpha w29) are reported. The sequences of some previously described V alpha 1, V alpha 2, V alpha 5, V alpha 7 and V alpha 22 gene segments are also extended. In addition, we report 14 novel J alpha gene segment sequences. Taken together, these data indicate that the contribution of the alpha chain combinatorial diversity to the human T cell receptor alpha/beta variability has not yet been fully appreciated.

Cloning and expression of a lymphocyte activation gene (LAG-1).

Using subtractive hybridization of a cDNA library we have identified a human gene, termed LAG-1 (for "Lymphocyte Activation Gene-1"). This cDNA codes for a 69 amino-acid polypeptide which belongs to a new class of recently described proteins secreted by activated lymphocytes and/or monocytes. The LAG-1 gene was cloned, sequenced and its chromosomal location assigned to chromosome 17 (q21 band). The promoter region of the LAG-1 gene was shown to include a GM-CSF-like decanucleotide sequence. Using a baculovirus vector expression system, we found that a 10 kDa recombinant LAG-1 protein is secreted by AcNPV infected SF9 cells, as determined in Western blot experiments by the reactivity of specific anti-peptidic heteroantibodies. Finally the natural LAG-1 protein was precipitated from the supernatant of internally labeled activated Nk cells and shown to migrate as a single entity of 14 kDa in SDS-PAGE analysis.

LAG-3, a novel lymphocyte activation gene closely related to CD4.

We have identified a novel human gene of the Ig superfamily, designated LAG-3. Expression of this gene is undetectable in resting PBL, while it is found (a 2-kb message) in activated T and NK cells. The LAG-3 gene includes eight exons; the corresponding cDNA encodes a 498-amino acid membrane protein with four extracellular IgSF domains. The first one belongs to the V-SET; it is particular since it includes an extra loop in the middle of the domain and an unusual intrachain disulphide bridge. The three other domains belong to the C2-SET. Strong internal homologies are found in the LAG-3 molecule between domains 1 and 3, as well as between domains 2 and 4. It is therefore likely that LAG-3 has evolved by duplication of a pre-existing gene encoding a two IgSF-domain structure. The compared analysis of LAG-3 and CD4, with respect to both their peptidic sequence as well as their exon/intron organization, indicated that the two molecules are closely related. This point is strengthened by the finding that both genes are located on the distal part of the short arm of chromosome 12.

The T-cell receptor V delta genes predominantly used by human peripheral gamma/delta+ T lymphocytes are not rearranged in CD3- natural killer cells.

We have analyzed, in 19 CD3- natural killer cell clones, the genomic organization of the T-cell receptor delta locus with two distinct V delta probes, V delta 1 and V delta 2. These two V delta genes code for surface proteins expressed in more than 90% of peripheral blood T-cell receptor gamma/delta+ lymphocytes, as shown by double color immunofluorescence analysis with anti-TCR delta 1, anti-BB3, and anti-delta TCS1 monoclonal antibodies. The V delta 1 and V delta 2 genes were found to be in germline position in all these clones, which are distinct phenotypically and represent a variety of the corresponding peripheral natural killer cell populations. We also studied in these cloned cell lines the transcriptional activity of the T-cell receptor delta locus with a C delta probe: short transcripts (1.7 and 0.8 kb) were found exclusively. These experiments further suggest that CD3- natural killer peripheral cells are likely to constitute a unique lineage distinct from T lymphocytes.

cDNA cloning of functional T cell receptor gamma/delta chains expressed in human peripheral blood lymphocytes.

We have identified in earlier studies two V delta rearrangements corresponding to a 4.5-kb Eco RI fragment detected with a V delta1 probe and to a 7-kb Eco RI band detected with a V delta2 probe. These rearrangements have been found in two human T cell clones, F6C7 and G6, displaying surface phenotypes unfrequent in human peripheral blood, namely Ti gamma A+ BB3- (F6C7) and Ti gamma A- BB3+ (G6). Herein, we report the sequences of the functional transcripts encoded by these rearranged genes and show that the 4.5- and the 7-kb Eco RI fragments correspond to V1/D3/J delta 3 and to V2/D3/J delta 3 recombinations, respectively. In addition, we have sequenced the V2/D3/J1/C delta transcripts expressed in two clones, AB12 and VTC, which have a Ti gamma A+ BB3+ surface phenotype corresponding to that of most gamma/delta peripheral lymphocytes. Analyses of the delta transcripts expressed by these four cells further strengthen the hypothesis that anti-BB3 and anti-delta-TCS-1 monoclonal antibodies recognize a V delta 2- and a V1/(D)/J delta 1-encoded epitope, respectively. Sequence of the gamma transcripts expressed by AB12 and F6C7 cells shows that they encode a V9/JP/C gamma 1 chain. Finally, we confirm that non-combinatorial diversity in the gamma and delta proteins is generated by both junctional flexibility and N-region addition without any somatic mutation.

A novel human V delta gene expressed predominantly in the Ti gamma A fraction of gamma/delta+ peripheral lymphocytes.

We have characterized a functional T cell receptor (TcR) delta transcript in a Ti gamma A+ human cloned cell line derived from peripheral blood. This cDNA includes a novel V gene (V-AB12), whose expression was initially studied in a series of TcR gamma/delta+ clones. Nine Ti gamma A+ clones derived independently from distinct donors have been tested: each of them was found to possess a unique V-AB12/J-IDP2 5.5-kb Eco RI rearrangement, which was constantly transcribed. Surface expression of the protein encoded by this unique rearranged gene was demonstrated by immunoprecipitations performed on three Ti gamma A+ polyclonal cell lines using a specific rabbit heteroantiserum. Further analysis strongly suggested that a monoclonal antibody (mAb), designated anti-BB3, detects a V-AB12-encoded antigenic determinant on the cell surface. Double-color immunofluorescence analysis of peripheral blood lymphocytes from ten donors indicated that most BB3+ cells are recognized by anti-Ti gamma A mAb. In previous studies, we have shown that a majority of TcR gamma/delta+ peripheral T cells expresses a gamma chain including V9 (Ti gamma A) and most frequently JP-encoded peptides. Given the present results on the delta chain, it can be concluded that, in many individuals, a predominant fraction (V gamma 9+/V-AB12+) of circulating CD3+ TcR alpha/beta- T lymphocytes expresses a receptor with little, if any, combinatorial diversity.