IL-4/13 expressing CD3γ/δ+ T cells regulate mucosal immunity in response to Flavobacterium columnare infection in grass carp.

Interleukin (IL) 4 and 13 are signature cytokines orchestrating Th2 immune response. Teleost fish have two homologs, termed IL-4/13A and IL-4/13B, and have been functionally characterized. However, what cells express IL-4/13A and IL-4/13B has not been investigated in fish. In this work, the recombinant IL-4/13A and IL-4/13B proteins of grass carp (Ctenopharyngodon idella) were produced in the Escherichia coli (E. coli) cells and purified. Monoclonal antibodies (mAbs) against the recombinant CiIL-4/13A and CiIL-4/13B proteins were prepared and characterized. Western blotting analysis showed that the CiIL-4/13A and CiIL-4/13B mAbs could specifically recognize the recombinant proteins expressed in the E. coli cells and HEK293T cells and did not cross-react with each other. Confocal microscopy revealed that the CiIL-4/13A+ and CiIL-4/13B+ cells were present in the gills, intestine and spleen and could be upregulated in fish infected with Flavobacterium columnare (F. columnare). Interestingly, the cells expressing CiIL-4/13A and CiIL-4/13B were mostly CD3γ/δ+ cells. The CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells were significantly upregulated in the gill filaments and the intestinal mucosa after F. columnare infection. Our results imply that the CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells are important for homeostasis and the regulation of mucosal immunity.