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The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Tetrapods contain a single CD4 coreceptor with four Ig domains that likely arose from a primordial two-domain ancestor. Notably, teleost fish contain two CD4 genes. Like tetrapod CD4, CD4-1 of rainbow trout includes four Ig domains, whereas CD4-2 contains only two. Because CD4-2 is reminiscent of the prototypic two-domain CD4 coreceptor, we hypothesized that by characterizing the cell types bearing CD4-1 and CD4-2, we would shed light into the evolution and primordial roles of CD4-bearing cells. Using newly established mAbs against CD4-1 and CD4-2, we identified two bona-fide CD4+ T cell populations: a predominant lymphocyte population coexpressing surface CD4-1 and CD4-2 (CD4 double-positive [DP]), and a minor subset expressing only CD4-2 (CD4-2 single-positive [SP]). Although both subsets produced equivalent levels of Th1, Th17, and regulatory T cell cytokines upon bacterial infection, CD4-2 SP lymphocytes were less proliferative and displayed a more restricted TCRβ repertoire. These data suggest that CD4-2 SP cells represent a functionally distinct population and may embody a vestigial CD4+ T cell subset, the roles of which reflect those of primeval CD4+ T cells. Importantly, we also describe the first CD4+ monocyte/macrophage population in a nonmammalian species. Of all myeloid subsets, we found the CD4+ population to be the most phagocytic, whereas CD4+ lymphocytes lacked this capacity. This study fills in an important gap in the knowledge of teleost CD4-bearing leukocytes, thus revealing critical insights into the evolutionary origins and primordial roles of CD4+ lymphocytes and CD4+ monocytes/macrophages.

Our previous work has shown the existence of distinct subsets of B cells in vertebrates that are highly phagocytic. To understand further the involvement of B cells in other potential innate immune functions, we performed the first comparative transcriptome analysis on FACS-sorted IgT+ and IgM+ B cells in rainbow trout, a primitive teleost fish species. To our surprise the gene that showed the highest differential expression between these two B cell subsets was perforin, a molecule that is not typically associated with B cells. The results obtained by transcriptome were expanded by further RT-PCR analysis confirming that unlike IgM+ B cells, IgT+ B cells expressed high transcript levels of several perforin isoforms, including, prf1-like-B, prf1-like-C and prf1-like-D. We also confirmed the unique expression of these perforin genes in IgT+ B cell by single cell transcriptome analysis. Moreover, we produced antibodies against these perforin molecules and demonstrated by immunohistochemistry the presence of several of these perforin isoforms in IgT+ B cells. Since perforin is a cytolytic protein we hypothesized that IgT+ B cells could possess cytotoxic activity similar to that of other perforin-expressing cells. To confirm this hypothesis, we tested the potential cytotoxic capacity of IgT+ B cells towards several mammalian cell lines, such as HL-60. Our results show that the killing activity of IgT+ B cells was significantly greater than that of IgM+ B cells. As IgT+ B cells and the IgT antibodies they produce play key roles in fish mucosal surfaces, our results strongly suggest a cytotoxic role for mucosal B cells in these species. Overall, our findings demonstrate a previously unrecognized new function for vertebrate B cells in immunity.

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.

A homologue of complement component C3 (SpC3) has been cloned and sequenced from the purple sea urchin, Strongylocentrotus purpuratus. The preprocessed, deduced protein size is estimated to be 186 kDa with a short leader and two chains, α and β. There are cysteines in conserved positions for interchain disulfide bonding, and there is a conserved thioester site in the α-chain with an associated histidine. There are five consensus N-linked glycosylation sites, and putative cleavage sites for factor I and C3 convertase. Partially purified SpC3 on protein gels shows a nonreduced size of 210 kDa and, under reducing conditions, reveals an α-chain of 130 kDa and a β-chain of 80 kDa. These sizes are larger than the deduced sizes, suggesting that the protein has carbohydrates added to most of the consensus N-linked glycosylation sites. Phylogenetic analysis of SpC3 compared with other members of the thioester protein family, which includes C3, C4, C5, and α2-macroglobulin, shows that SpC3 is the first divergent complement protein, falling at the base of the complement protein clade. Transcripts from the SpC3 gene (Sp064) are 9 kb, and the gene is expressed specifically in coelomocytes, which are the immunocytes in the sea urchin. Genome blots suggest that SpC3 is encoded by a single copy gene per haploid genome. This is the first identification of a complement component in an invertebrate, and suggests homology of the innate immune system within the deuterostome lineage of animals.

In mammals, interaction of CD28 with CD80 or CD86 molecules provides costimulatory signals for T cell activation. Such signals lead to increased gene and protein expression of IL-2 by activated T-cells. CD80 and CD86 have only been cloned in mammals and birds thus far, and their functional characterization is basically limited to their study in mammals. Thus, in the present study we show for the first time the cloning of a rainbow trout molecule (rtCD80/86) that shows a high degree of homology to CD80 and CD86 molecules. In addition, we have identified and cloned trout IL-2 and we have shown that recombinantly produced trout CD80/86 upregulates the expression of IL-2 in trout blood leukocytes. This finding indicates that this capacity to modulate IL-2 expression is a primordial function that has been conserved both in fish and mammalian CD80/CD86 molecules throughout evolutionary time.