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

PROBLEM: 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 grafl‐versus‐host reaction (GVH) produced by A cells injected into irradiated A × B F1s recipients. We also know by studies carried out using specific anti‐Vβ‐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 knew 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 Vβ 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). METHOD OF STUDY: 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 factor, 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‐Vβ‐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‐Vβ and were rendered specifically anergic were studied by a specific anti‐Vβ 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‐Vβ mAbs. RESULTS: The phosphorylation of the ζ chain homodimer quantitatively defective in filtrate‐treated, anti‐Vβ6‐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‐Vβ6‐stimulated cells by an unrelated anti‐Vβ (anti‐Vβ8) showed double expression of Vβ chains. The percentage of cells expressing this unrelated Vβ (Vβ 8) was normal. CONCLUSIONS: T cell anergy induced by a filtrate is linked to defective phosphorylation of the ζ‐chain homodimer. The abnormal percentage of cells expressing TcR after filtrate treatment might be due to adsorption by unstimulated cells of soluble TcR Vβ‐chain, possibly as a result of excess synthesis followed by membrane protease cleavage, allowing release i...

REVIEW ARTICLE: Tolerance to the Foetal Allograft?

“…The T-cell anergy observed in such a case is transient, requiring continuous presence of the active moiety which has been identified as being a prostaglandin (PGE2). 65 This explains the above-reported anergy 39,63,64 seen by Tafuri and others. A similar activity has been traced in the blood in the form of placental exosomes.…”

“…63,64 This can be obtained in MLR by incubating responder cells with supernatant of placental explant cultures or purified heat-resistant material. 64 Cells allostimulated in the presence of this material will not respond in a second MLR with the same stimulator MHC, whereas they will do so against a third party. The T-cell anergy observed in such a case is transient, requiring continuous presence of the active moiety which has been identified as being a prostaglandin (PGE2).…”

Regulatory T-Cells in Pregnancy: Historical Perspective, State of the Art, and Burning Questions

“…Regulatory T-cells may also be involved in pre-eclampsia (PE), together with regulatory NK T-cells (52, 61, 70, 71), as reported by several authors (62–65), except for Paeschke et al (66). Furthermore, it has been suggested that there might be an imbalance between CD4 + CD25 hi Foxp3 + and CD4 + CD25 − Foxp3 + Treg subsets in PE (61).…”

Placental secreted factors: Their role in the regulation of anti-CII antibodies and amelioration of collagen induced arthritis in rats

“…A low molecular weight factor isolated from placental cells is capable of down-regulating mouse T cell activity, inducing a transient anergy, and is linked to defective phosphorylation of TCR CD3 chain [29]. In addition, it has been demonstrated that prostaglandin E2 (PGE2) produced by chorionic villis inhibits proliferation of CTLL-2 cells dependent on IL-2 [30]; moreover, exosomes from term-delivering pregnancies suppress effector T cells by enhancing lymphocyte apoptosis and CD3-zeta chain loss [31,32].…”