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F Triebel

Publications and source records attributed to F Triebel.

At least 19 recordsLinked to original sources
PubMed1992

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.

Acute Disease↗
PubMed1992

Analysis of T-cell receptor alpha/beta variability in lymphocytes infiltrating a melanoma metastasis.

Multiple experimental and clinical studies have suggested that the immune system may, to some extent, control the development of melanomas. The presence of tumor-infiltrating lymphocytes could reflect an in situ immune reaction directed to the malignant cells. The characterization of T-cell receptor (TCR) expressed by tumor-infiltrating lymphocytes is one way to precisely analyze these local T-cell responses. In this study, we have assessed the TCR alpha/beta variability in tumor-infiltrating lymphocytes from a subcutaneous metastasis of a melanoma patient. Using the anchored-polymerase chain reaction 268 TCR alpha and 266 TCR beta chain transcripts have been cloned and sequenced. Their analysis shows that the T-cell infiltrate is extremely diverse, with no preferential TCR gene segment usage.

Adult↗
PubMed1992

Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

Amino Acid Sequence↗
PubMed1992

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.

Base Sequence↗
PubMed1992

Ti gamma A + delta TCS1+ T cells in human thymus. Analysis of the T cell receptor.

TcR1+ T cells in peripheral blood have been shown to express in an exclusive fashion either the Ti gamma A or the delta TCS1 epitope. Here, we characterize a subset of TcR1+ T cells in fresh thymus co-expressing the Ti gamma A and delta TCS1 epitopes. TcR1dim and TcR1bright clones can be distinguished. Biochemical and molecular studies on both types of clones generated from these thymocytes reveal a unique T cell receptor structure characterized by the use of a V gamma 9/C gamma 2-encoded 55-kDa gamma chain nondisulfide linked to a V delta 1-encoded delta chain.

Cell Line↗
PubMed1992

Studies on NK cell purification by negative selection in human peripheral blood.

We have attempted to improve negative selection procedures for the large scale purification of human CD-3 CD56+ NK cells. In a series of experiments, purifications of NK cells from 10(8) PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3+ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10(8) cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10(9) PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior to in vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.

Animals↗
PubMed1991

[Human lymphocytes carrying the T gamma/delta receptor].

The structure of the gamma and delta chains of the human T lymphocyte receptor has been studied extensively over the past three years. A directory of gamma/delta lymphocytes in different normal or pathological situations has been developed from the analysis of the different sub-populations of circulating (cytofluorometry) or tissue infiltrating (slice immunohistochemistry) gamma/delta T lymphocytes. It has been shown that the gamma and delta chains do not express a very polymorphic pattern; consequently, 2 or 3 anti-V or anti-V/J gamma or delta monoclonal antibodies are sufficient to evaluate any bias in the expression of this receptor in a given pathological situation. We present the genes coding for the gamma and delta chains, the proteins resulting from their expression, the different antibodies recognizing these proteins, the functions associated with gamma/delta + cells and finally certain pathological situations where an expression bias of the different gamma and delta chains has been described.

Antibodies, Monoclonal↗
PubMed1991

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.

Animals↗
PubMed1991

Studies on the human T cell receptor alpha/beta variable region genes. II. Identification of four additional V beta subfamilies.

The human T cell receptor (TcR) beta chain gene segment diversity has been studied using the anchored-polymerase chain reaction. Three hundred and fifty C beta-specific transcripts derived from peripheral lymphocytes were analyzed. Transcripts including V-D beta 1-J beta 2-C2 sequences were found with a high frequency (greater than 10%), suggesting that "illegitimate" joinings may constitute a cis-complementing rearrangement mechanism capable of substantially increasing the TcR beta chain combinatorial diversity. Twelve previously undescribed V beta gene segments have been identified. Five of them delineate four novel V beta subfamilies (V beta w21: two members, V beta w22, 23, 24: one member) which all have a murine homologue. The additional seven gene segments belong to the V beta 5, V beta 6, V beta 12 and V beta 13 subfamilies. In addition, the sequences of two known V beta 7 and V beta 9 gene segments have been extended. Together, the present data support the view that the contribution of the beta chain combinatorial diversity to the TcR alpha/beta variability has not yet been fully appreciated.

Base Sequence↗
PubMed1991

Molecular characterization of human T cell receptor alpha chains including a V delta 1-encoded variable segment.

Previously we have shown that a small fraction of human peripheral T cells expresses a surface receptor recognized both by the BMA031 mAb, specific for a TcR alpha/beta framework epitope, and by the A13 mAb, putatively specific for an epitope encoded by the V delta 1 gene segment. An interleukin 2-dependent polyclonal cell line (termed T2) was derived from such A13+BMA031+ circulating lymphocytes. The molecular characterization of the TcR chains expressed by T2 cells demonstrated indeed that the V delta 1 gene (one of the two major V delta genes) was transcribed with the C alpha gene segment. In the T2 polyclonal cell line, distinct V delta 1/C alpha transcripts were all found to include the same J alpha segment suggesting the existence of "hybrid" TcR alpha/delta chains encoded by unique V delta 1/J alpha rearrangements. The present study was designed to characterize further the V delta 1/J alpha rearranged genes expressed in A13+BMA031+ cells. Three additional cell lines were generated from peripheral blood of distinct adult healthy donors. Using the anchored polymerase chain reaction, it was found that 17 different J alpha segments were used in the 20 V delta 1J alpha C alpha transcripts which have been studied. Together, these data indicate that V delta 1 is a "mixed" (i.e. alpha/delta) TcR V segment which can join with most (if not all) J segments in the alpha/delta locus. In addition, it can be definitely concluded that the A13 mAb recognizes a V delta 1-encoded antigenic determinant and not a V delta 1J epitope (i.e. it can be defined and used as an anti-V delta 1 mAb, as opposed to reagents such as for example delta-TCS-1).

Adult↗
PubMed1990

T cell target 1 (TCT.1): a novel target molecule for human non-major histocompatibility complex-restricted T lymphocytes.

We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.

Antibodies, Monoclonal↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Further analysis of the T cell receptor gamma/delta+ peripheral lymphocyte subset. The V delta 1 gene segment is expressed with either C alpha or C delta.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/delta. These reagents, designated anti-A13 and anti-TiV delta 2, were found to recognize antigenic determinants encoded by the TCR V delta 1 and V delta 2 gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/delta+ cell subpopulation, the expression of V delta 2+ delta chains is largely predominant, as compared with the V delta 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV delta 2 antibodies was often greater than that detected with anti-TCR-delta 1, a reagent specific for a constant epitope of the human delta chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta dimer, these results suggested that the V delta 1 gene segment may be expressed with either C delta or C alpha. This hypothesis was confirmed using T2, an IL-2-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T2 cells were found to have productively rearranged the V delta 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V delta 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

Animals↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Phase I trial with recombinant interleukin-2 (rIL-2): immune activation by rIL-2 alone or following pretreatment with recombinant interferon-gamma.

Alterations of immunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL-2. Five-day infusions of rIL-2 at doses from 1 x 10(6) to 24 x 10(6) biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 x 10(6) U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analyzed at days 0, 8 and 11 of the rIL-2 courses. Following a leucopenia during rIL-2 infusion, a lymphocytosis was found in all patients except one. The lymphocytosis peaked at day 8 and was detected at doses of rIL-2 as low as 1 x 10(6) U/m2 per day, reaching a plateau at a dose of 16 x 10(6) U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL-2, some patients had predominant T cells (CD3+, NKH1(CD56)-), others had predominant natural killer (NK) cells (CD3-, NKH1 (CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL-2 was preceded by a course of 5 days infusion of 2 x 10(6) U/m2 per day recombinant interferon-gamma (rIFN-gamma). The infusion of rIFN-gamma prior to rIL-2 had no effect on the rIL-2-induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL-2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low-dose rIFN-gamma does not modify the immune stimulation by rIL-2.

Cytotoxicity, Immunologic↗