title: Induction of transient murine T cell anergy by a low molecular weight compound obtained from supernatants of human placental cultures is linked to defective phosphorylation of TCR CD3 chain.
abstract: OBJECTIVE
  Allopregnancy induces specific transient tolerance to paternal grafts, and we know that a low molecular weight material ("filtrate") present in a human placental supernatant can do so in vitro (specific unresponsiveness) as well as in vivo, such as when preventing graft-versus-host reaction (GVH) produced by A cells injected into irradiated A x B F1s recipient. We also know by studies carried out using specific anti-V beta-specific stimulation as well as secondary and primary mixed lymphocyte reaction in major histocompatibility complex (MHC) only incompatible combinations that the material acts by inducing T cell anergy rather than clonal deletion. We explored the mechanism of such an anergy, which we know was not dependent on calcium fluxes, cyclic adenosine monophosphate (cAMP) levels, or PkC by studies of protein phosphorylation. Having observed in previous studies that expression of T cell reactivity (TcR) in anergic cells was enhanced, but that the numbers of cells expressing a given reactivity (TcR) V beta after specific stimulation in the presence of a filtrate was much higher than it should be, we monitored the receptor expression by fluorescence-activated cell sorter (FACS).
  
  
  METHODS
  We used short-term stimulation of the T-cell-derived Jurkat E6-1 cells by anti-CD3 monoclonal antibody (mAb) or phorbol myristite acetate plus calcium ionophore in the presence or absence of human placental low molecular weight suppressor factors, followed by Western blotting. Transfer on nitrocellulose filters so as to allow the revelation of the phosphorylations was realized by means of a specific antiphosphotyrosin mAb. The final revelation was obtained by chemiluminescence. Similar experiments were performed on anti-V beta-stimulated BALB/c splenocytes, as well as cyproflaxin-treated cells, which are hyper-responsive in cell proliferation assays in the presence of the filtrate. In parallel, cells that were stimulated by a specific anti-V beta and were rendered specifically anergic were studied by a specific anti-V beta and were rendered specifically anergic were studied for other TcR expression using an FACS and both fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-labelled, related and unrelated anti-V beta mAbs.
  
  
  RESULTS
  The phosphorylation of the zeta chain homodimer quantitatively defective in filtrate-treated, anti-V beta 6-stimulated splenocytes as well as in Jurkatt cells. In parallel, cells from cyproflaxin-treated Jurkatt cells were showing enhanced phosphorylation of all bands. The labelling of filtrate-treated anti-V beta 6-stimulated cells by an unrelated anti-V beta (anti-V beta 8) showed double expression of V beta chains. The percentage of cells expressing this unrelated V beta (V beta 8) was normal.
  
  
  CONCLUSIONS
  T cell anergy induced by a filtrate is linked to defective phosphorylation of the zeta-chain homodimer. The abnormal percentage of the cells expressing TcR after filtrate treatment might be due to adsorption by unstimulated cells of soluble TcR V beta-chain, possibly as a result of excess synthesis followed by membrane protease cleavage, allowing release in a soluble form of TcR V beta-chain nonspecifically captured by other cells.
authors.affiliation: Université Paris Sud/Assistance Publique, Hôpital Antione Béclère, Clamart, France.
authors.forename: J L
authors.initials: JL
authors.lastname: Voluménie
authors.forename: B
authors.initials: B
authors.lastname: Mognetti
authors.forename: D
authors.initials: D
authors.lastname: de Smedt
authors.forename: E
authors.initials: E
authors.lastname: Menu
authors.forename: G
authors.initials: G
authors.lastname: Chaouat
mesh_terms: D000818:Animals; D017252:CD3 Complex / Q000378:metabolism*; D019281:Dimerization; D005260:Female; D006801:Humans; D007108:Immune Tolerance; D066298:In Vitro Techniques; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000737:chemistry / Q000276:immunology*; D011247:Pregnancy; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D017252:CD3 Complex; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; C054979:antigen T cell receptor, zeta chain