A study of lymphocyte heterogeneity in the rainbow trout, Oncorhynchus mykiss

Abstract

Lymphocyte in vivo migration pathways were investigated in rainbow trout using the tracer sample method. Peripheral blood lymphocytes were separated into sig-f and sig- cells and injected back into the same fish. After 24 hours, following a passage through the thymus, the distribution of slg+ cells between the spleen and kidney was approximately equal, but more sIg- cells localised in the kidney. When unseparated lymphocytes were injected into non-histocompatible recipients 42% of the total cells were in the spleen after 72 hours. When organ specific slg+ and sig- lymphocytes were injected into non-histocompatible recipients, it was found that sig- cells (from the spleen, thymus and kidney) migrated preferentially to the spleen. It was concluded that the phenomenon of ecotaxis occurs in trout. A comparative study of rainbow trout T and B lymphocytes following their separation by nylon wool adherence and lectin agglutination techniques, was carried out. The results revealed that nylon wool separation produced an adherent and a non-adherent population of cells. The adherent population showed B cell properties, based on mitogenic responses and enzyme and immunocytochemical staining profiles, whereas the non-adherent population showed T cell properties. The lectin Soybean agglutinin was used to separate lymphocytes into an agglutinated and a non-agglutinated population. A mitogenic and immunocytochemical study of these subpopulations revealed that they were unrelated to T and B cells. In vitro studies were carried out to determine the effect of thymocyte coculture on the plaque forming cell response of spleen leukocytes from fish given a primary in vivo injection of DNP-KLH, SRBC, TNP-SRBC and TNP- LPS. Significant suppression was observed (when compared to spleen alone responses) In both allogenic and autogenic thymus-spleen cocultures from the DNP-KLH, SRBC and TNP-SRBC immunised fish, but not the TNP-LPS immunised fish. Cyclophosphamide treatment, pre-/n vitro transfer, abolished the observed suppression in the DNP-KLH, SRBC and TNP-SRBC immunised autogenic cocultures, and in some cases enhanced the PFC response as compared to the spleen alone cultures. No clear effects of NMU treatment, pre- in vitro transfer, were observed. The results showed clear evidence for a cyclophosphamide sensitive thymus derived suppressor cell population The effect of adult thymectomy on the in vivo antibody response to DNP-KLH, SRBC and A.salmonicida was examined. Two month adult thymectomy had no measurable effect on antibody response to any of the antigens, when compared to intact controls. Eight month adult thymectomy led to a significantly reduced secondary response to SRBC and DNP-KLH, which was partly restored when the fish were reconstituted with their own cryopreserved thymocytes one week before secondary immunisation. In contrast eight month adult thymectomy produced a significantly elevated secondary antibody response to A.salmonicida, which was partly returned to control levels by reconstitution of the fish’s autogenic cryopreserved thymocytes one week prior to secondary immunisation. Electrophoretic analysis of whole cell leukocytes, lysed using nonionic detergent lysis buffers, revealed organ specific protein bands in the thymus and kidney. Con A and LPS stimulation of leukocyte cultures produced clear changes in the SDS-PAGE protein patterns in silver stained and periodic acid-silver stained gels. A polyclonal antibody was produced to a 30kD thymocyte specific protein band, which stained 22% of thymocytes using the immunoperoxidase method.