Channel catfish leukocyte immune-type receptor mediated inhibition of cellular cytotoxicity is facilitated by SHP-1-dependent and -independent mechanisms

Abstract

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins belonging to the immunoglobulin superfamily that likely play an important role in the regulation of teleost immune cell effector responses. IpLITRs are expressed by myeloid and lymphoid subsets and based on their structural features can be classified as either putative stimulatory or inhibitory forms. We have recently demonstrated at the biochemical and functional levels that stimulatory IpLITR-types induced intracellular signaling cascades resulting in immune cell activation. Alternatively, we have shown that putative inhibitory IpLITRs may abrogate immune cell responses by recruiting teleost Src homology 2 (SH2) domain-containing cytoplasmic phosphatases (SHP) to their tyrosine-containing cytoplasmic tails. In the present study, we used vaccinia virus to express recombinant chimeric proteins encoding the extracellular and transmembrane regions of human KIR2DL3 fused with the cytoplasmic tails of two putative inhibitory IpLITRs (i.e. IpLITR1.2a and IpLITR1.1b) in mouse spleen-derived cytotoxic lymphocytes. This approach allowed us to study the specific effects of IpLITR-induced signaling on lymphocyte killing of B cell targets (e.g. 721.221 cells) using a standard chromium release assay. Our results suggest that both IpLITR1.2a and IpLITR1.1b are potent inhibitors of lymphocyte-mediated cellular cytotoxicity. Furthermore, using a catalytically inactive SHP-1 mutant in combination with site-directed mutagenesis and co-immunoprecipitations, we also demonstrate that the IpLITR1.2a-mediated functional inhibitory response is SHP-1-dependent. Alternatively, IpLITR1.1b-mediated inhibition of cellular cytotoxicity is facilitated by both SHP-1-dependent and independent mechanisms, possibly involving the C-terminal Src kinase (Csk). The involvement of this inhibitory kinase requires binding to a tyrosine residue encoded in the unique membrane proximal cytoplasmic tail region of IpLITR1.1b. Overall, this represents the first functional information for inhibitory IpLITR-types and reveals that catfish LITRs engage SHP-dependent and -independent inhibitory signaling pathways to abrogate lymphocyte-mediated killing.