Jacques Arnaud

Evolution of T cell receptor (TCR) α β heterodimer assembly with the CD3 complex

T cell antigen receptors (TCR) are composed of an antigen‐recognizing unit, the TCRα β  heterodimer, and a signal transduction ensemble, the CD3 complex. Whereas mammals possess three CD3 dimers (δ ϵ , γ ϵ , and ζ 2), birds and amphibians have only two (δ /γ ‐ϵ  and ζ 2). To understand evolutionary changes in TCR/CD3 assembly,a phylogenetic approach was employed to dissect the interaction of TCRα β  heterodimers with the CD3 components. While sheep and mouse TCRα  and TCRβ  chains could replace the corresponding human chains in mutant human T cells to restore surface TCR/CD3 expression and function, chicken TCRα , TCRβ  and CD3δ /γ  chains were unable to replace the corresponding human chains in forming a chimeric TCR/CD3 complex. The inability of chicken TCR/CD3 components to replace the human molecules in T cells was found to result from the lack of interaction between chicken TCRα β  heterodimers and the human CD3 complex. In contrast, if no CD3 molecules are present (non‐T cells), TCRα ‐TCRβ  chain pairing can take place in an apparently non‐controlled way. Thus, the TCR‐CD3 interactions have changed with the evolutionary divergence of two mammalian CD3γ  and CD3δ  genes from a single prototypic chicken δ /γ  gene. Our data suggest that the structures in mammalian TCR.C regions, which distinguish between CD3δ  and CD3γ  chains, have evolved with the appearance of two separate CD3δ  and CD3γ  functions.

TCR Dynamics in Human Mature T Lymphocytes Lacking CD3γ

The contribution of CD3γ to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3γ is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3γ-deficient (γ−) individuals. In contrast to γ− mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural γ− T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3γ. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3γ-dependent, as it was restored by retroviral transduction of CD3γ. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3γ contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.

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 αβ‐and γδ‐T cells. By means of optimal immunoprecipitation conditions with 35S‐methionine/cysteine‐ or biotin‐labelled TCR/CD3 proteins from αβ‐ or γδ‐T‐lymphoma‐cell lines, as well as TCRγδ cDNA transfectants, it was found that CD3δ chains associate less strongly with TCRγδ heterodimers compared to TCRαβ heterodimers. This preferential reactivity of CD3δ chains appears to be structural and not owing to differences in γδ‐ versus αβ‐T‐cell intracellular environments. Our results are in accordance firstly, with data from CD3δ‐deficient mice, which have γδ‐T cells but no αβ‐T cells, secondly with the suggested role of CD3δ chains in the positive selection of αβ‐T cells, a process apparently not followed by γδ‐T cells, and lastly with the differential roles of CD3δ chains versus CD3γ chains, explaining the maintenance of two CD3δ and CD3γ genes after the duplication from a CD3δ/γ gene present in avians. The impaired reactivity of CD3δ chains with TCRγδ heterodimers seems to be owing to a less efficient association with TCRγ chains. In contrast, CD3δ chains interact as strongly with TCRδ chains as do CD3γ chains with both TCRγ and TCRδ chains. These data may explain, at the molecular levels, why surface TCR/CD3 expression levels are impaired in γδ‐T cells from CD3γ‐deficient mice but not from CD3δ‐deficient mice.

Biological Function of the Extracellular Domain of the T‐Cell Receptor Constant Region

Immunoglobulin (Ig) superfamily domains are important components of transmembrane cell-cell interaction molecules, e.g. CD2, CD3, CD4, CDS. CD28, T-cell receptor (Tcr) a,0,f,S chains, Ig heavy and light chains and major histocompatibilily complex (MHC) class-I and class-II antigens etc. (see Fig 1; [1, 2]). In Ig-domains the polypeptide folds to give antiparallel /?-strands forming two ^-sheets (A,B,E and G,F,C) that are held together in a sandwich by a conserved disulfide bond and hydrophobic interactions due to in-pointing amino acids. These amino acids commonly alternate with hydrophilic residues along the /3-strands. There are two main variants of folding patterns: V-domains and Cdomains. The former domains have about 65-75 amino acid residues between the conserved disulfide bond, and there are four /3-strands in each /3-sheet plus a short /3-strand segment across the top of the domain. In C-domains, the sequence between the disulfide bond is shorter at 55 60 residues yielding sheets with four and three /3-strands; these Cdomains are designated CI-domains. Another type of Cdomain has been identified, designated C2-domain; these fold like C-domains but their sequence patterns in the region of/3-strands E and F are like those of V-domains. Among cell-ceil interaction molecules in the vertebrate immune system, V-domains and Cl-domains are evolutionarily very conserved until fish species (third domain of MHC class-1 molecules [3], second domain of MHC class-II molecules [4, 5], /32-microglobulin (/IJjm) [6-8] and Ig heavy-chain Vand C-domains [9, 10]); and C2-domains until the chicken (CD2 or CD3 domains [II, 12]). Other molecules not necessarily involved in defence systems seem to be composed at least in part by Ig-domains: Thy-1 (nervous system, [13, 14]), Fascilin II (nervous system in insects; [15]), Haemolin (nervous adhesion molecules in insects; [16]), Amalgam (nonintegrated Drosophila membrane protein; [17]), or Twitchin (an intracytoplasmic myosine-binding protein found in Coenorhabditis Elegans; [18]). The most primitive Ig superfamily domain may be Twitchin in C. Elegans (A. N. Barclay,

A highly conserved phenylalanine in the alpha, beta-T cell receptor (TCR) constant region determines the integrity of TCR/CD3 complexes.

In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr‐Cα region on Tcr‐α, β/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr‐α, β/CD3‐γɛ, δɛ complexes and ζ2 homodimers. The Tcr‐Cα region around phe195 seems together with the same region in the Tcr‐Cβ region to constitute an interaction site for ζ2 homodimers. The presence of phe195 on both Tcr‐Cα and Tcr‐Cβ causes high avidity interaction with ζ2 homodimers, whereas his195 in both Tcr‐Cγ and Tcr‐Cδ results in an apparently lower avidity interaction with ζ2 homodimers. It is suggested that the phe195 region (on β‐strand F) and eventually adjacent aromatic amino acid residues on β‐strand B region may play an important role in Tcr‐α, β/CD3 membrane expression, in Tcr‐α, β/CD3 competition with Tcr‐γ, δ/CD3 complexes for ζ2 homodimers and in the control of formation of ‘mixed’ Tcr heterodimers.

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 TCRαβ/CD3δε,γε/ζ2 structure but at the cell membrane, multimers of this minimal structure may be formed. Quantitative cytofluometry has suggested three CD3ε chains for two TCRβ (or TCRδ) chains/complex. Such data should be repeated with monoclonal antibodies (MoAb) against extracellular (EC) parts of CD3δ or CD3γ chains. In the present review, we have compared the TCR/CD3 assembly of pre‐TCR, TCRγδ and TCRαβ containing complexes, and analysed the reactivity of antibodies (Abs) against the EC part of CD3δ chains. Our data suggest an alternative assembly pathway and structure of TCR/CD3 complexes.

Clinical Pharmacokinetics of α1-Antitrypsin in Homozygous PiZ Deficient Patients

SummaryA pharmacokinetic study of α1-antitrypsin (ATT) was performed in 2 groups of homozygous PiZ-deficient patients (treated and untreated) and 1 group of healthy volunteers. The distribution of the 131I-labelled protein corresponds to a 3-compartment model. The intravenously administered protein diffused quickly to the extravascular compartment where some retention occurred. No significant difference in AAT metabolism was observed between the 3 groups.The half-life of the injected protein is slightly longer than 2.5 days. The AAT protein was not stored. These results confirm the observations collected during the clinical trials. That is, a weekly infusion is necessary to obtain stable serum AAT concentrations. Monthly infusions are unable to maintain a ‘plateau’ phase. The periodicity may be limited to every 2 weeks.

Serum cholinesterase polymorphism in France: an epidemiological survey of the deficient alleles detected by an automated micro-method.

The polymorphism of the serum cholinesterase CHE 1 was determined using an automated micro-method. The procedure developed represents an improvement of the manual method and enabled us to study more than 2400 samples obtained in 15 different geographical areas in France. Four alleles were detected: CHE1* U, A, S and F. An exceptionally high frequency of an atypical variant (CHE1*A) was observed in the central part of France (Cevennes, Limousin and Dauphiné). The populations belonging to these areas are at particular risk in case of anaesthesia.

Self-Reactive T Cell Receptor-Reactive CD8+ T Cells Inhibit T Cell Lymphoma Growth In Vivo

Syngenic C57BL/6 mice (H-2b) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8+ T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-γ and TNF-α, but little or no IL-4. By means of TCRβ-negative variant L12R4 cells, P3.3, and TCR-Vβ2 cDNA-transfected and TCR-Vβ2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vβ12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-Kb, and anti-Db mAb. Furthermore, vaccination with Vβ12 peptide p67–78, which binds to both Kb and Db MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vβ-specific, Kb-, or Db-restricted CD8+ T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.

Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision

Abstract T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR α chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated α chain does not interact with CD3 δ molecules; consequently, no TCR αβ/CD3 δεγε complexes are formed. E6.E12 cells transcribe a TCR β chain composed of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated β chain does associate with CD3 γε heterodimers, yet no TCR αβ/CD3 δεγε complexes are made. This may be due either to low assembly of TCR β/CD3 γε trimers or to lack of access of the mutated TCR β/CD3 γε trimers to the TCR α/CD3 δε compartment in the endoplasmic reticulum.