INDUCTION OF TRANSIENT MURINE T-CELL ANERGY BY A LOW-MOLECULAR-WEIGHTCOMPOUND OBTAINED FROM SUPERNATANTS OF HUMAN PLACENTAL CULTURES IS LINKED TO DEFECTIVE PHOSPHORYLATION OF TCR CD3 CHAIN

PROBLEM: Allopregnancy induces specific transient tolerance to paterna l grafts, and we know that a low molecular weight material (''filtrate '') present in a human placental supernatant can do so in vitro (speci fic unresponsiveness) as well as in vivo, such as when preventing graf t-versus-host reaction (GVH) produced by A cells injected into irradia ted A x B F1s recipients. We also know by studies carried out using sp ecific anti-V beta-specific stimulation as well as secondary and prima ry mixed lymphocyte reaction in major histocompatibility complex (MHC) only incompatible combinations that the material acts by inducing T c ell anergy rather than clonal deletion. We explored the mechanism of s uch an anergy, which we knew was not dependent on calcium fluxes, cycl ic 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 V beta after specific stimulation in the presence of a filtrate was much higher than it should be, we m onitored the receptor expression by fluorescence-activated cell sorter (FAGS). METHOD OF STUDY: We used short-term stimulation of the T-cell -derived Jurkat E6-1 cells by anti-CD3 monoclonal antibody (mAb) or ph orbol myristite acetate plus calcium ionophore in the presence or abse nce of human placental low molecular weight suppressor factor, followe d by Western blotting. Transfer on nitrocellulose filters so as to all ow the revelation of the phosphorylations was realized by means of a s pecific antiphosphotyrosin mAb. The final revelation was obtained by c hemiluminescence. Similar experiments were performed on anti-V beta-st imulated BALB/c splenocytes, as well as cyproflaxin-treated cells, whi ch are hyper-responsive in cell proliferation assays in the presence o f the filtrate. In parallel, cells that were stimulated by a specific anti-V beta and were rendered specifically anergic were studied by a s pecific anti-V beta and were rendered specifically anergic were studie d for other TcR expression using an FAGS and both fluorescein isothioc yanate (FITC) and phycoerythrin (PE)-labelled, related and unrelated a nti-V beta mAbs. RESULTS: The phosphorylation of the zeta chain homodi mer quantitatively defective in filtrate-treated, anti-V beta 6-stimul ated splenocytes as well as in Jurkatt cells. In parallel, cells from cyproflaxin-treated Jurkatt cells were showing enhanced phosphorylatio n of all bands. The labelling of filtrate-treated anti-V beta 6-stimul ated cells by an unrelated anti-V beta (anti-V beta 8) showed double e xpression of V beta chains. The percentage of cells expressing this un related V beta (V beta 8) was normal. CONCLUSIONS: T cell anergy induc ed by a filtrate is linked to defective phosphorylation of the zeta-ch ain homodimer. The abnormal percentage of cells expressing TcR after f iltrate treatment might be due to adsorption by unstimulated cells of soluble TcR V beta-chain, possibly as a result of excess synthesis fol lowed by membrane protease cleavage, allowing release in a soluble for m of TcR V beta-chain nonspecifically captured by other cells.