The Roles Of Chicken CD4~+CD25~+ Cells In Infectious Bursal Disease And In The Induction Of Immune Tolerance

Regulatory T cells (Tregs) are a subset of T cells which balance the immune responses and participate in many biological processes as transplant rejection, autoimmunity diseases and virus infection. Study has showed that chicken CD4+CD25+cells may have similar characteristics to those in mammals. However, there have no reports about the roles of chicken CD4+CD25+cells in chicken biological processes. In this study, we produced anti-chicken CD25 monoclonal antibody, revealed the properties of chicken CD4+CD25+cells and explored the changes of CD4+CD25+cells in infectious bursal disease virus (IBDV) infection and the induction of immune tolerance.Because there is no commercialized anti-chicken CD25 antibody, we produced anti-chicken CD25 antibody by hydrophobicity analysis of CD25 sequence, expression of chicken CD25 protein, mice immunization and cell fusion, and detected the specificity of anti-CD25 antibody with Western Blot and the specificity of RPE-conjugated anti-CD25 antibody with flow cytometry. Results showed, anti-CD25 antibody can recognized a specific band of chicken thymus and spleen protein at approximately 23.5 kDa, as expected as chicken CD25 protein molecular weight on ExPASy. The CD25 expression which the R-PE-conjugated anti-CD25 antibody recognized significantly increased on ConA stimulated CD4+ T cells. These results indicated that the anti-CD25 antibody we produced is specific and could be used for flow cytometry.To evaluate the characteristics of chicken CD4+CD25+cells, we sorted CD4+CD25+cells and CD4+ CD25- cells by flow cytometry, and determined IL-2, IL-10 and TGF-β mRNA levels by quantitative real-time PCR. Results showed that the IL-2 mRNA was not detectable in CD4+CD25+cells and the IL-10 and TGF-β mRNA levels in CD4+CD25+cells were significantly higher than those in CD4+ CD25’cells. Meanwhile, we found that CD4+CD25+cells can inhibit the proliferation of CD4+CD25-cells in vitro by co-culture test. These results suggested that chicken CD4+CD25+cells have the ability to produce suppressive cytokines and inhibit the proliferation of CD4+CD25- cells.To investigate whether chicken CD4+CD25+cells are involved in IBDV infection, SPF chickens of 4 weeks old were infected with Harbin-1 strain or Ts strain, and lymph organs thymus, spleen, bursa of Fabricius and peripheral blood lymphocytes were collected at 1-5 days after infection. The changes of CD4+CD25+cells ratio were analyzed by flow cytometry. Results showed that IBDV targeted to the bursa of Fabricius and amplified, Harbin-1 strain got its peak on the third day of infection and Ts strain got its peak on the fourth day of infection. The ratio of CD4+CD25+cells in the thymus and spleen declined rapidly at the early stage of infection. There is an interesting fluctuation of the ratio of CD4+CD25+cells in peripheral blood as it increased markedly at 1-3 days post infection and gradually returned to a normal level at 4-5 days post infection. Absence of IBDV infection, the percentage of CD4+T cells in bursa of Fabricius of SPF chickens was no more than 10%, and the CD4+CD25+cells are even not detectable; while on the fourth and fifth day after Ts infection, there had an extensive infiltration of CD4+ T lymphocytes, and CD4+CD25+cells accounted for a significant proportion. All in all, we came to the conclusion that IBDV infection can cause systemic inflammation in chickens, and CD4+CD25+ cells in thymus and spleen moved out, and migrated to the bursa of Fabricius via blood circulation to exert inhibitory effects.To assess whether chicken CD4+CD25+cells play a role in the induction and maintenance of immune tolerance, we used an immune tolerance induction chicken model our lab previously established that injection of bovine serum albumin (BSA) at a late embryonic stage via blood vessel could induce immune tolerance to BSA. First, we analyzed the percentage of CD4+CD25+cells in this process. The results showed that the ratio of CD4+CD25+cells was higher than control group along this process. Then, we attempted to decrease the CD4+CD25+cells by vascular micro-injection of anti-CD25 antibodies on embryonic incubation day (EID)16, and results showed that the ratio of CD4+CD25+cells in anti-CD25 antibody injection group was significantly lower than that in control group untile 35 days after hatched. Finally, we micro-injected anti-chicken CD25 antibody on EID16 prior to micro-injection of BSA on EID20 and then detected the immune tolerance level to BSA by enzyme-linked immunosorbent assay (ELISA). The results showed that the levels of IgY antibodies, IgM antibodies and IgA antibodies specific to BSA were weakened significantly in the CD4+CD25+cells-reduction group compared with control group (only BSA-injection group). The results indicated us that CD4+CD25+cells play an indispensable role in maintaining induced immune tolerance to BSA.Taken together, in this study we produced anti-chicken CD25 monoclonal antibody, revealed the suppressive characteristics of chicken CD4+CD25+cells and the important roles of CD4+CD25+cells in IBDV infection and induction and maintenance of immune tolerance, which provides an important basis for further researches of poultry immunology, epidemiology and immune tolerance.