CELLULAR PATHWAY(S) OF ANTIGEN PROCESSING IN FISH APC - EFFECT OF VARYING INVITRO TEMPERATURES ON ANTIGEN CATABOLISM

Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27-degrees-C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17-degrees-C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17-degrees-C required longer exposure to the antigen prior to fixation (i.e., greater-than-or-equal-to 8 h compared to 5 h, the optimal incubation time at 27-degrees-C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17-degrees-C to provide efficient presentation after subsequent incubation of the APC at 27-degrees-C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4-degrees-C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27-degrees-C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27-degrees-C, but not at 4-degrees-C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17-degrees-C appeared to occur at a slower rate compared to APC at 27-degrees-C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27-degrees-C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.