| Rainbow trout is a teleost fish which stands for a critical stage of the immune system evolutionary history. So it is significant to study its immune system. The distinct differentiative stages of antigen-responsive B cells in trout blood, spleen and kidney were examined over a two year period post immunization with different antigens, trinitrophenyl-lipopolysaccharides (TNP-LPS, TL, T cell independent type I antigen), trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH, TK, T cell dependent antigen) and trinitrophenyl-keyhole limpet hemocyanin-lipopolysaccharides (TNP-KLH-LPS, TKL, chimeric antigen). The kinetics of ex vivo antibody secreting cells, plasma cells and antigen inducible cells were analyzed. At the same time, rainbow trout is an important worldwide farmed fish. Now salmonid aquaculture vaccines are meanly gram negative bacterins. The protective immunity is usually used with adjuvants while this kind of formulation brings lesions on fish impacting its economic value. The most important thing to develop a more effective vaccine is to understand the mechanism of the immune system. In this thesis, the immune response induced by TI, TD and TI-TD antigen were compared. All these studies could play a critical role in developing effective vaccines for aquaculture.Bleed the trout from the four immunized group (TL, TK, TKL and control) at week 6, 16, 25, 35 and 44, three to ten fish each time from each group. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the antibody titer and antibody affinity. The result of antibody titer indicates that TL and TK antigens show rapid and robust primary response with peak during week 16 and week25. The primary response of TK antigen is slow. Response of TKL antigen is even faster and higher than TL antigen which indicates the advantage of the chimeric antigen. The result of antibody affinity demonstrates that these three antigens could induce affinity maturation. The affinity in TK and TKL group is hiher than that in TL group. TKL antigen generates highest antibody affinity and its affinity maturation is much more significant.Three or four fish in each group were dissected at week 6, 16 and 44. Blood, spleen, anterior kidney and posterior kidney were collected. Leukocytes were collected from these four organs to set up ex vivo and in vitro culture. Hydroxyurea (HU, cell cycle inhibitor) was used to distinguish plasmablast (HU sensitive) and plasma cell (HU insensitive). The in vitro inducibility of B lymphocytes was determined by adding TL into the cell culture. Then ELISPOTS assay was used to quantify the antibody secreting cells (ASCs) in the tissues. The ASCs tissue distribution was analyzed. The ELISPOTS results indicated that there is a significant number of plasma cell (greater than 90%) in the trout anterior kidney (equals the mammal bone marrow) while low number of plasma cell distribute in blood, spleen and posterior kidney. Plasmablast and inducible cells (immature B cells and memory cells) are meanly in prepherial blood (72%) and less in spleen (14%) and anterior kidney (16%). The ELISPOTS analysis of ex vivo response demonstrates that anterior kidney harbors most (86%) of the constitutive ASCs. Very few leukocytes could be isolated from the posterior kidney, so only a small amout of plasma cell, plasmablast, and antigen-responsive B cell were detected within it. The tissue distribution of three antigens induced plasmablast, plasma cells and inducible cells are approximately the same. TKL antigen shows great advantage on the number of the ASCs. Specifically, after TL priming, in vitro responsiveness to antigenic challenge increased 50-100 fold splenocytes and peripheral blood, while only 10-fold with anterior kidney leukocytes. After TD priming, responsiveness in the peripheral blood and splenocytes increased 500-fold as compared to only a 30-fold increase in the anterior kidney.At week 46, trout in TL and TK group were challenged with the same antigens. The memory response of TI-1 and TD antigen were tested. Blood, spleen, anterior kidney and posterior kidney were collected at week 16 and 46 after boost, 4 fish each time in each group. Both TI-1 and TD antigen could generate good memory response based on the result of antibody titer, affinity, and quantity of ASCs. This is different from mammals in which only TD antigen can induce memory response. In trout, the memory response to TI-1 antigen is even faster and more robust than TD antigen.Staggered introduction of HU during in vitro induction permitted resolution of the number of 1) induced ASCs undergoing proliferation (i.e. plasmablast), 2) induced plasmablasts that transition to early plasma cells (i.e. transition from HU sensitivity to HU insensitivity), 3) stable plasma cells (i.e. HU insensitive from culture inception). The result indicated that, the in vitro response of peripheral blood, regardless of when tested post-primary pr post-secondary, were composed strictly of plasmablasts. Splenocytes were able to generate short-lived plasma cells from the in vitro induced plasmablasts. In contrast, by week 16 post-primary immunizations the anterior kidney possessed a significant number of plasma cells, which appeared to be resident within the anterior kidney as they were present at culture initiation and persisted throughout the culture period.Flow cytometer were used to test the BCR affinity of the cells from immune organs at week 0, 6, 16, 44, 62, 92 post-immunization. Similar to serum antibody affinity maturation, BCR affinity also tend to experience maturation in all immune organs post immunization. |
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