Suppression of Lymphocyte Proliferation by a >30 000 Molecular Weight Factor in Horse Conceptus-Conditioned Medium1

Abstract

In this experiment we have identified and partially characterized the immunosuppressive activity of preimplantation horse conceptus-conditioned medium (HCCM). Horse conceptuses were nonsurgically flushed from mares at Days 9-10 (n = 6), 15-16 (n = 3), and 25-26 (n = 3). After incubating the conceptuses for 24 h in RPMI-1640 supplemented with 15% fetal calf serum (FCS) and 1% penicillin/streptomycin, HCCM was obtained from cultures and tested for immunosuppressive activity in lymphocyte proliferation assays. Peripheral blood lymphocytes obtained from randomly selected mares were stimulated with mitogens (pokeweed mitogen [PWM], concanavalin A [Con A], and phytohemagglutinin [PHA]) in cultures supplemented with 0%, 25%, or 50% HCCM. HCCM from all cultures suppressed lymphocyte proliferation induced by all three mitogens (p less than 0.001). After being subjected to various treatments (heating, freeze-thawing, and nitrocellulose filtration), HCCM maintained its full biological suppressor activity. Amicon microconcentrators with 10,000 and 30,000 molecular weight (MW) exclusion filter membranes were used to fractionate HCCM by molecular weight. The suppressor factor was found to be in the greater than 30,000 MW fraction. HCCM was further tested interspecifically on donkey and goat lymphocytes stimulated with PWM. HCCM did suppress proliferation of interspecific lymphocytes (p less than 0.01); however, the suppressive capacity of HCCM in caprine lymphocyte cultures was less (p less than 0.05) than that observed in equine cultures. These data support the hypothesis that the horse conceptus produces an immunoregulatory factor. This factor is extremely stabile and appears to exhibit some degree of species-specificity. The production and immunosuppressive effectiveness of such a factor may play an important role in maintaining the fetal allograft throughout gestation.

Involvement of interleukin 2 receptors in conceptus-derived suppression of T and B cell proliferation in horses

“…We reported the production of an immunosuppressive factor produced by day 10 to day 26 horse conceptuses (Roth et al, 1990). In that study, the conceptus cultures were performed as described here for obtaining HCS 1.…”

“…This noncytotoxic suppressor factor is effective only if present during the early stages of lymphocyte acti¬ vation and its effect on cells is reversible. HCS suppression is highly dependent on its presence during the early stages of cell activation and could explain why the suppressive factors are produced as early as day 10 of pregnancy (Roth et al, 1990). Our results suggest that the factor(s) suppress cell proliferation by preventing IL-2R activation.…”

“…A 30 000 kDa factor in medium conditioned by horse conceptuses has shown immunosuppressive activity (Roth et al, 1990). Roth et al (1991) have shown that this suppressor factor, horse conceptus-derived immunosuppressive factor (HCS) has M, > 100 000.…”