Characterization Of TIM Homologs From Zebrafish Provides Insights Into Their Essential Roles In Adaptive Humoral Immunity

T cell immunoglobulin and mucin, TIM proteins are type-I cell-surface glycoproteins. Recent studies have demonstrated that TIM family play important roles in immune system. This article focused on the identification and function of TIM family in zebrafish, emphasized in clarifying the immunological characterization of DrTIM-1L and Dr-4L in zebrafish.DrTIM-4L and DrTIM-1L, the homo logs of TIM-4 and TIM-1 respectively in mammals, were identified in zebrafish by molecular cloning. Bioinformatics analysis revealed that both DrTIM-4L and DrTIM-1L are cell-surface glycoproteins with structural characteristic in common:an N-terminal Ig domain of the V subset, followed by a heavily glycoslyated mucin domain, single transmembrane domain and an intracellular cytoplasmic tail. Subcellular localization analysis showed that DrTIM-4L was expressed primarily on cell membranes. Uniquely, DrTIM-1L was expressed originally in cytoplasm and trafficked to the cell surface after antigenic stimulation. Immunofluorescence double-staining analysis suggested that DrTIM-4L was co-localized with MHC II in APCs and DrTIM-1L was co-localized with CD4 in T cells. Stimulation of the antigen can result in up-regulation both of DrrTIM-4L+MHCⅡ+and DrrTIM-1L+CD4+double positive cells. These indicated that DrTIM-4L and DrTIM-1L might play an essential role in APCs initiated adaptive immune response. Using Abs blockade and short interfering RNA to knock down DrTIM-4L and DrTIM-1L during immunization with Ag in vivo, functional characterization demonstrated that both DrrTIM-4L and DrTIM-1L served as positive regulators in the proliferation and activation phase of adaptive immune response. Then APCs and CD4+T cells were isolated using magnetic activated cell sorting and cocltured in vitro, the result confirmed that both of DrTIM-4L and DrTIM-1L could regulate APCs initiated T cell proliferation. DrTIM-4L and DrTIM-1L not only regulated the expansion and activation of CD4+T cells, but also they were associated with KLH-induced production of IgM in humoral immunity. Immunoprotection inhibitory assay showed that DrTIM-4L and DrTIM-1L protected the fish from challenged with A. hydrophila. This strongly confirmed that DrTIM-4L and DrTIM-1L serve as costimulatory signals in humoral immunity. Furthermore, A novel soluble DrTIM-4L was discovered and can inhibit the proliferation of CD4+T cells in adaptive immune responses.These results suggested that TIM family members play a key role in humoral immunity and mechanism for the function evolution. It provided a ideal model and new scientific basis for experimental research in zebrafish.