Some hints concerning the shape of T-cell receptor structures.

Several models are proposed for T-cell antigen receptor (TCR) assembly and structure. However, there is little experimental data favouring directly either one or the other(s). The minimal complex appears to be composed of a TCRalphabeta/CD3deltaepsilon,gammaepsilon/zeta2 structure but at the cell membrane, multimers of this minimal structure may be formed. Quantitative cytofluometry has suggested three CD3epsilon chains for two TCRbeta (or TCRdelta) chains/complex. Such data should be repeated with monoclonal antibodies (MoAb) against extracellular (EC) parts of CD3delta or CD3gamma chains. In the present review, we have compared the TCR/CD3 assembly of pre-TCR, TCRgammadelta and TCRalphabeta containing complexes, and analysed the reactivity of antibodies (Abs) against the EC part of CD3delta chains. Our data suggest an alternative assembly pathway and structure of TCR/CD3 complexes.

On the role of CD3delta chains in TCRgammadelta/CD3 complexes during assembly and membrane expression.

The present study was performed in order to analyze whether T-cell receptor (TCR)/CD3 assembly, intracellular transport and surface expression are carried in a similar way in alphabeta-and gammadelta-T cells. By means of optimal immunoprecipitation conditions with 35S-methionine/cysteine- or biotin-labelled TCR/CD3 proteins from alphabeta- or gammadelta-T-lymphoma-cell lines, as well as TCRgammadelta cDNA transfectants, it was found that CD3delta chains associate less strongly with TCRgammadelta heterodimers compared to TCRalphabeta heterodimers. This preferential reactivity of CD3delta chains appears to be structural and not owing to differences in gammadelta- versus alphabeta-T-cell intracellular environments. Our results are in accordance firstly, with data from CD3delta-deficient mice, which have gammadelta-T cells but no alphabeta-T cells, secondly with the suggested role of CD3delta chains in the positive selection of alphabeta-T cells, a process apparently not followed by gammadelta-T cells, and lastly with the differential roles of CD3delta chains versus CD3gamma chains, explaining the maintenance of two CD3delta and CD3gamma genes after the duplication from a CD3delta/gamma gene present in avians. The impaired reactivity of CD3delta chains with TCRgammadelta heterodimers seems to be owing to a less efficient association with TCRgamma chains. In contrast, CD3delta chains interact as strongly with TCRdelta chains as do CD3gamma chains with both TCRgamma and TCRdelta chains. These data may explain, at the molecular levels, why surface TCR/CD3 expression levels are impaired in gammadelta-T cells from CD3gamma-deficient mice but not from CD3delta-deficient mice.

A new monoclonal anti-CD7 antibody reactive on paraffin sections.

CBC.37 monoclonal antibody (mAb) was generated using balb/c mice immunized with CEM T cell line. It was selected because of its strong reactivity on T lymphocytes on paraffin tissue sections. The anti-CD7 specificity of CBC.37 mAb was assessed by immunohistochemistry, cross-blocking, and cross-immunoprecipitation experiments using CBC.37 and the anti-CD7 mAb DK24. CBC.37 mAb immunoprecipitated a 40-kDa protein. Cross-blocking and cross-immunoprecipitation experiments demonstrated that the two antibodies recognized the same molecule. Immunostaining of a large number of reactive lymph nodes and B and T cell lymphomas confirms that CBC.37 mAb was directed against T cells. As expected, on reactive lymph nodes the staining pattern was comparable to that of CD3. Among the 110 T cell lymphomas examined, all T lymphoblastic lymphomas were positive (15+/15; 100%). As a result of the frequent loss of CD7 antigen, only 25+/95 (26%) of peripheral T cell neoplasms were found to be positive for CBC.37. A marked reduction in the number of CBC.37-positive T cells was observed in 7 of the 60 cases of benign inflammatory dermatoses studied (approximately 12%). CBC.37 was unreactive with all healthy and neoplastic non-lymphoid samples examined. Because the lack of CD7 expression in T cell lymphomas is of diagnostic value, CBC.37 mAb in association with other anti-T cell antibodies working on paraffin sections could be of particular value in asserting the diagnosis of T cell lymphomas in routine histopathology.

A new monoclonal anti-CD3epsilon antibody reactive on paraffin sections.

We generated a new monoclonal antibody (MAb), F7.2.38, by immunizing mice with CD3varepsilongammadelta/CD3omega complexes purified from human T-cells by OKT3 MAb-Sepharose affinity chromatography. Immunoprecipitation experiments and Western blotting analysis showed that MAb F7.2.38 recognized the CD3varepsilon chain in CD3varepsilon cDNA-transfected FOX B-cells and in various T-cell lines. Using flow cytometry on permeabilized or intact cells, the epitope was found to be located in the cytoplasmic tail of the CD3varepsilon chain. Immunohistochemical staining on paraffin-embedded sections showed that the reactivity of MAb F7.2.38 was comparable to that of the commercially available anti-CD3varepsilon polyclonal antibody. Of the 52 well-characterized T-cell lymphomas, 41 were positive for F7. 2.38 (79%), whereas all 37 B-cell lymphomas and 69 non-lymphoid tumors were unreactive. This new anti-CD3varepsilon antibody would be particularly useful for phenotyping T-cell lymphomas on routinely processed paraffin-embedded tissue sections.

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma.

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

Molecular mechanisms in the TCR (TCR alpha beta-CD3 delta epsilon, gamma epsilon) interaction with zeta 2 homodimers: clues from a 'phenotypic revertant' clone.

The association between the TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers in the endoplasmic reticulum (ER) constitutes a key step in TCR assembly and export to the T cell surface. Incompletely assembled TCR-CD3 complexes are degraded in the ER or the lysosomes. A previously described Jurkat variant (J79) has a mutation at position 195 on the TCR Calpha domain causing a phenylalanine to valine exchange. This results in a lack of association between TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers. Two main hypotheses could explain this phenomenon in J79 cells: TCR-CD3 hexamers may be incapable of interacting with zeta2 due to a structural change in the TCR Calpha region; alternatively, TCR-CD3 hexamers may be incapable of interacting with zeta2 due to factors unrelated to either molecular complex. In order to assess these two possibilities, the TCR-CD3 membrane-negative J79 cells were treated with ethylmethylsulfonate and clones positive for TCR membrane expression were isolated. The characterization of the J79r58 phenotypic revertant cell line is the subject of this study. The main question was to assess the reason for the TCR re-expression. The TCR on J79r58 cells appears qualitatively and functionally equivalent to wild-type TCR complexes. Nucleotide sequence analysis confirmed the presence of the original mutation in the TCR Calpha region but failed to detect compensatory mutations in alpha, beta, gamma, delta, epsilon or zeta chains. Thus, mutated J79-TCR-CD3 complexes can interact with zeta2 homodimers. Possible mechanisms for the unsuccessful TCR-CD3 interaction with zeta2 homodimers are presented and discussed.

An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination.

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction. In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.

Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3.

Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV)-1 genomes. The immortalized cell line was assigned to the T-lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T-cell receptor (TCR) alpha-chains but not beta-chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin-2 (IL-2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL-2 was secreted. This new human immortalized T-lymphocytic cell line, which is cell-surface TCR/CD3-negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.