Biomedical subjects
G Gorochov
Publications and source records attributed to G Gorochov.
Expression of V beta gene segments by Sezary cells.
The T-cell receptor V beta repertoire expressed by Sezary cells was determined in a series of 16 patients whose samples have been shown to contain a majority of tumor cells. By using anti-V beta monoclonal antibodies, polymerase chain reaction analysis of expressed V beta, and, in selected cases, nucleotide sequencing, we have shown that the expressed V beta segments belong to five V beta families (V beta 5, V beta 6, V beta 8, V beta 13, and V beta 18), which contain a large fraction of the T-cell receptor V beta repertoire and do not share significant similarities in complementary determining region 4. V beta segments from these five families were also found to be strongly expressed by CD4 + CD7- peripheral blood cells obtained by fluorescence-activated cell sorting from two healthy donors. The diversity of the V beta repertoire expressed by Sezary cells appears to be similar to that expressed by circulating non-neoplastic T cells. These data do not support the hypothesis that a common superantigen is involved in the initiation of this form of cutaneous T-cell lymphoma.
More about the T-cell receptor V beta repertoire usage in mycosis fungoides/Sézary syndrome.
Explore the source record for details and available documents.
Oligoclonal expansion of CD8+ CD57+ T cells with restricted T-cell receptor beta chain variability after bone marrow transplantation.
A major expansion of CD8+57+ lymphocytes expressing an alpha-beta T-cell receptor (TCR) is frequent after bone marrow transplantation (BMT). We examined the clonality of the TCR beta gene repertoire in these expanded CD8+57+ cells after allogeneic or autologous BMT. We performed a polymerase chain reaction (PCR) analysis of the V beta chain usage in CD8+57+ cells purified from nine BMT recipients with a series of oligonucleotides specific for 20 V beta gene families. PCR products from selected TCR beta gene rearrangements were sequenced. The CD8+57+ cells from eight of nine patients used a restricted set of V beta families, with a marked predominance of two to three V beta gene families per patient, whereas the control autologous CD57- subset expressed the whole 20 V beta families. A direct sequencing analysis confirmed the V beta 16 and V beta 17 clonality in six patients, showing a striking homology in the CDR3 sequences of the V beta 16 products. The CD8+57+ cells, but not the CD57- cells, displayed an oligoclonal pattern of TCR rearrangements as shown by PCR analysis of TCR gamma gene rearrangements. Such an oligoclonal expansion of CD8+57+ cells, using a restricted set of the V beta gene families, may result from a specific TCR stimulation of a limited number of T-cell clones in BMT recipients.
Anti-leucocyte function-associated antigen-1 antibodies inhibit T-cell activation following low-avidity and adhesion-independent interactions.
Anti-leucocyte function-associated antigen-1 (LFA-1) antibodies can provide either stimulatory or inhibitory signals to T cells, depending on the epitope they recognize, type and stage of activation of the T cells, and nature of the activation stimulus. Because of the low affinity of interaction between the T-cell receptor (TcR) and the antigen/major histocompatibility complex (MHC), it was proposed that the LFA-1 molecule strengthens the adhesion between the interacting cells, thus contributing in an additive manner to TcR-specific interactions. To check if high-avidity, TcR-specific interactions still require the accessory function of the adhesion molecule, we studied the effect of anti-LFA-1 antibodies on T-cell triggering mediated through chimeric receptors composed of an Fv of an antibody and a constant region of the TcR. Such chimeric TcR (cTcR) confer on T cells antibody-type specificity and affinity. We made use of transfected T-cell hybridomas expressing various amounts of either one cTcR chain (composed of VH linked to C beta) or double-chain cTcR (VHC beta + VLC alpha). When such transfectants were stimulated with hapten-modified cells, anti-LFA-1 antibodies inhibited activation predominantly mediated through cTcR composed of a single chimeric chain and did not inhibit stimulation of the double-chain transfectants. Moreover, these anti-LFA-1 antibodies blocked antigen-specific T-cell activation regardless of whether the stimulus was adhesion dependent or not, such as in the case of stimulation by immobilized hapten-protein conjugates. These studies show that the 'off-signal' provided by anti-LFA-1 antibodies is adhesion independent and affects mainly low-avidity TcR-antigen interactions.
In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.
We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.
CD8 lymphocytosis and pseudotumoral splenomegaly in HIV infection.
Three patients infected with human immunodeficiency virus (HIV) presented with pseudotumoral splenomegaly, CD8 lymphocytosis (3.5-5.1 x 10(9)/l), and hypergammaglobulinaemia. Spleen and bone marrow showed diffuse CD8 lymphocyte and plasma-cell infiltration. Amplification of the T-cell-receptor gamma chain gene did not reveal any clonal T-cell population. Phenotypic analysis showed a predominance of CD8/CD57 suppressor T cells with expression of activation markers (DR and CD38). No cytotoxic T lymphocytes specific for HIV could be detected. The three patients shared the HLA haplotype A1, B8, DR3. The association with this haplotype suggests a genetically determined host immune response to HIV.
Functional assembly of chimeric T-cell receptor chains.
We have generated cytotoxic T-cell hybridomas expressing chimeric T-cell receptors (cTCR) with an antibody-type specificity for the TNP hapten. Transfectants expressing the cTCR genes could mediate specific lysis of haptenated tumor cell lines of various types and secrete IL-2 upon stimulation with TNP modified cells. In a previous report, we showed that double-gene transfectants expressing either VHC alpha and VLC beta or VHC beta and VLC alpha could be activated by TNP-modified stimulator cells or TNP proteins immobilized on plastic. Single-chain transfectants (expressing VHC alpha or VHC beta alone) could be mainly activated by TNP-cells. We now report that transfection of chimeric VHC alpha gene into an alpha-chain-defective mutant restores the surface expression of the TCR/CD3 complex. In parallel, such transfectants regained the ability to respond to mitogen and anti-CD3 antibodies and responded weakly to TNP cells. Double gene transfectants, bearing 2 complementary chimeric chains, expressed high amounts of cTCR on their surface, sufficient to acquire sound anti-TNP reactivity. Cells expressing the VHC beta gene only were not functional and had no detectable surface TCR chains. Taken together, our results suggest that chimeric VHC alpha chains can pair with endogenous V beta C beta chains, but that there is preferential association between complementary chimeric chains, resulting in higher functional expression of the chimeric TCR.
Phenotype and function of peripheral blood and bone marrow T-cell colonies: identification of DC3-, 4-, 8-autoreactive T cells.
The existence of immature autoreactive T cells has been examined in peripheral blood (PB) and bone marrow (BM) derived T-lymphocyte colonies (TLC) that have previously been shown to be potentially generated from CD3-negative BM-T cells. An extensive phenotypical analysis of total and T-depleted TLC showed that both PB- and BM-derived TLC contained a mean of 5% immature T lymphocytes (ITL), which were negative for the CD3, CD4, and CD8 antigens but displayed the CD2 and CD7 antigens. The only detectable immune functions of these isolated ITL were an allo- and an autoreactivity without cytolytic activity. The self-reactivity of ITL was not detected in bulk non T-depleted TLC cells and seemed to be actively suppressed by autologous mature T cells. In addition, the auto-MLR of ITL was totally inhibited by anti-HLA class II but not by anti-class I monoclonal antibody (MoAb) and only partially by anti-CD4 moAb, whereas the anti-CD3 and anti-CD2 MoAbs gave no inhibition. Once activated, ITL could acquire in culture a mature T cell CD3 + CD4 + phenotype. The CD3-, 4-, 8-auto-reactive T cells present in T colonies could represent pre- or post-thymic cells that have not yet undergo or that have escaped the thymic selection.
Generation of effector T cells expressing chimeric T cell receptor with antibody type-specificity.
Explore the source record for details and available documents.