The generation of regulatory t cells in an in vitro model of the acaid spleen

Purpose. Anterior chamber-associated immune deviation (ACAID) is composed of three distinct components: 1) eye - where antigen is processed; 2) spleen - where regulatory cells are generated; and 3) periphery - where the effector mechanisms of ACAID operate. This project utilized an in vitro model of the ACA ID spleen to study what cells and mechanisms are involved in (he generation of the efferent, CD8-4 regulatory cell population of ACAID. Methods. An in vitro model of the ACAID spleen was developed and used in combination with a previously described in vitro model of the ocular component of ACAID. Balb/c adherent peritoneal exudate cells (PEC) were collected and incubated overnight with or without 2 ng/ml TGF-β and 5 mg/ml BSA. 5 × 105 cells were washed and mixed with syngeneic whole spleen cell suspensions (5 × 107 cells/dish). In other experiments, T cells were purified by passing the spleen cells over nylon wool. B and T cells were collected by incubating the spleen cells on Primaria plates and collecting the non-adherent cells. Cell cultures were incubated for 7 days. The generation of T cells capable of suppressing the expression of DTH was tested in a local adoptive transfer assay. Briefly, equal volumes of test culture cells and positively immunized splenocytes were mixed and injected in the ear pinnae of naive Balb/c mice. Ear swelling (DTH) was measured 24 hours later. Results: PEC's incubated with, bui not without, 2 ng/ml TGF-β and BSA antigen induced the generation of regulatory T cells. PEC's incubated with splenic T cells alone, however, could not generate regulatory T cells. When splenic T and B cells were added together, the generation of regulatory T cells was restored. Ccfflcliision-4; ACAID peritoneal exudate cells incubated with whole splenocytes or splenic B and T cells alone generate T cells capable of suppressing the expression of DTH. These results suggest that ACAID antigen presenting cells do not present antigen directly to splenic T cells alone, but that splenic B cells are required.