Poster Sessions: Part II

The lymphocytes from six human donors produced interferon (IFN) in response to supernatants derived from M. arthritidis broth cultures (MAS). The IFN was characterized as IFN-y. Monoclonal antibodies to HLA.DR determinants suppressed the IFN response. Introduction: Previous work has established that mycoplasmas are inducers of a interferon [1,4,9]. Our recent findings on the presence of a T-cell mitogen in M. arthritidis supernatants (MAS), active for both mouse [5] and human [6,7] lymphocytes, prompted us to evaluate its ability to induce IFN. Material and Methods: Supernatants collected from M. arthritidis broth cultures (MAS) were filtered to remove intact organisms, dialyzed, and frozen at 70°C 15]. Lymphocyte suspensions prepared by Ficoll-Hypaque gradient centrifugation 12] were adjusted to 2.5 x 105/ml in RPMI 1640 medium containing 10 percent fetal calf serum and were exposed to various dilutions of MAS, phytohemagglutinin (PHA), or Newcastle Disease virus (NDV). Supernatants, collected after one to four days, were assayed for IFN using the microtiter plate procedure of Green et al. [8] with human WISH cells and encephalomyocarditis virus. Titers consisted of the reciprocal of the highest dilution calculated to cause 50 percent reduction in viral plaques. Monoclonal antibodies to HLA.DR determinants were generously supplied by Dr. Noel Warner (Clone L243, Becton Dickinson) and by Ortho Laboratories (OKIa). Results and Discussion: The lymphocytes from six healthy human donors all produced IFN in response to MAS, NDV, or PHA. MAS-induced IFN peaked after three to four days, giving maximal levels of 281 to 2,187 units. The IFN activity was inactivated with actinomycin D and IFN was destroyed by trypsin, treatment at pH 2.0 for 24 hours and at 56°C for 30 minutes. MAS-induced IFN was inactivated by anti-IFN-y antibody but not by anti-IFNa antibody. We conclude that MAS induces IFN-y. Since previous work indicated that murine [5] and human [7] T-cell proliferative responses to MAS were dependent on Ia or HLA.DR antigen-bearing cells, respectively, we studied the effect of monoclonal antibodies to HLA.DR determinants on IFN responses to MAS. Both monoclonals markedly inhibited IFN induced by MAS but were effective for NDVand PHAinduced IFNs only at the higher concentrations. The significance of these findings is that both lymphocyte proliferation and IFN induction require the presence of HLA.DR antigens or HLA.DR-bearing cells. This may relate to other work in the mouse system which suggests that an Ia antigen may be the receptor site for the mitogen [3]. The mitogen system provides an excellent model to elucidate the nature of the control exercised by products of the HLA gene complex on lymphocyte functions. REFERENCES 1. Beck J, Brunner H, Marcucci F, et al: J Interferon Res 2:31-36, 1982 2. Boyum A: Scand J Clin Lab Invest 21 (Suppl 97):77-89, 1968 3. Cole BC, Daynes RA, Ward JR: J Immunol, In press 4. Cole BC, Overall JC Jr, Lombardi PS, Glasgow LA: Infect Immun 14:88-94, 1970 5. Cole BC, Sullivan GJ, Daynes RA, et al: J Immunol 128:2013-2018, 1982 6. Cole BC, Washburn LR, Sullivan GJ, et al: Infect Immun 36:662-666, 1982 7. Daynes RA, Novak JM, Cole BC: J Immunol, In press 8. Green JA, Yeh T-J, Overall JC Jr: J Clin Microbiol 12:433-438, 1980 9. Rinaldo CR, Cole BC, Overall JC Jr, et al: Infect Immun 10:1296-1301, 1974 Some Characteristics of Protective Antigen(s) in Mycoplasma hyopneumoniae. R.F. Ross, B.J. Zimmermann, T.F. Young. Veterinary Medical Research Institute, Iowa State University, Ames, IO Immunity to Mycoplasma hyopneumoniae disease has been demonstrated in convalescent swine and in vaccinated swine challenged experimentally with the organism. However, protection against experimental challenge induced by vaccination has been only partial and, in field outbreaks, has been negligible. In view of these inadequacies, we attempted to determine some of the characteristics of protective activity of M. hyopneumoniae. M. hyopneumoniae strains J and 11 were grown in Friis medium and harvested by centrifugation after two to three days' incubation, color changing units (ccu) were determined, and the cells were stored at 70°C. Thawed cells, extract, or other preparations were diluted to the appropriate ccu and 0.15 percent formalin was added. Heat treatment, when used, consisted of 80°C for 60 minutes. Freeze-thaw-saline extracts were prepared from strain J as described for M. pneumoniae by Chandler and Barile [19801. Sixto ten-week-old Yorkshire, Hampshire, or cross-bred swine were obtained from SPF herds. Vaccines were given intramuscularly in two doses 14 days apart, and challenge, consisting of lung homogenate containing M. hyopneumoniae strain 11, was administered endotracheally two to three weeks later. Protection was assessed at necropsy 28 to 45 days after challenge by macroscopic evaluation of pigs' lungs. Results obtained in one trial indicated that vaccine containing 109 ccu was comparable to that obtained with 1010 or 1011 ccu/ml. The heat stability of protective activity in whole cells and the immunogenicity of supernate from cultures are given in Table 1. The effect of a freezethaw-saline extraction on protective activity is given in Table 2. TABLE 1 Protective Activity of Whole Cells and Supernate from M. hyopneumoniae