Novel Teleost CD4-Bearing Cell Populations Provide Insights into the Evolutionary Origins and Primordial Roles of CD4+ Lymphocytes and CD4+ Macrophages

Abstract

Tetrapods contain a single CD4 co-receptor with four immunoglobulin 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 immunoglobulin domains while CD4-2 contains only two. Since CD4-2 is reminiscent of the prototypic two-domain CD4 co-receptor, 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 monoclonal antibodies against CD4-1 and CD4-2, we identified two bona fide CD4+ T-cell populations, a predominant lymphocyte population co-expressing surface CD4-1 and CD4-2 (CD4 DP), and a minor subset expressing only CD4-2 (CD4-2 SP). While both subsets produced equivalent levels of Th1, Th17, and Treg 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 non-mammalian species. Of all myeloid subsets, we found the CD4+ population to be the most phagocytic, while 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+ monocyte/macrophages.

Gene expression analysis of isolated salmonid GALT leucocytes in response to PAMPs and recombinant cytokines

“…This may reflect fewer IgT B cells [13,44] and Th cells in the unstimulated gut, consistent with the whole gut tissue analysis (Fig. 1), although it is known that CD4-1 can also be expressed by myeloid cells in fish [45,46]. CD4 levels have also been shown to be relatively low in sea bass intestine, compared to CD8α expression [22].…”

The first functional characterization of ancient interleukin-15-like (IL-15L) reveals shared and distinct functions of the IL-2, -15 and -15L family cytokines

“…Fish thymocyte progressive development has not been studied in detail, but available knowledge of fish thymus organization, gene expression, and functions of mature T cells [e.g. (50,51,81,82), reviewed in (53)] suggest a similar development to mammals. Probably, as in mammals (80), DN thymocytes in trout importantly consist of several stages of early T cells.…”

“…S5C). Trout IL-15La and IL-15Lb containing supernatants did not detectably induce pSTAT5 in any of the investigated cell populations, except for CD8thymocytes During our studies, monoclonal antibodies against rainbow trout CD4-1 and CD4-2 became available (51); whether CD4-1 and CD4-2 have similar or different functions is not known, but in CD4-positive lymphocytes they commonly are co-expressed (51). To further investigate which thymocytes of trout were stimulated by IL-15La and IL-15Lb, thymocytes were labeled with an anti-CD8 monoclonal antibody with a different isotype (see Fig.…”

“…Three different concentrations (5,25 and 125 nM) of recombinant IL-2, IL-15-RLI and IL-15La-RLI proteins isolated from insect cells were used to stimulate CD4 + CD8 -(CD4SP [single positive]), CD4 -CD8 + (CD8SP), and CD4 -CD8 -(DN) lymphocyte fractions of thymus, intestine and spleen, while for the thymus this analysis also included the CD4 + CD8 + (DP) fraction [which is only abundant in that tissue; (51)]. Even when using high concentrations of purified cytokines, important findings obtained by using supernatants of HEK293T cells (Figs.…”

“…S3E). From previous studies it follows that, as in mammals, and although probably none of the populations was fully homogeneous, the trout CD4 + cells included helper and regulatory TCR + T cells(35,51,53), the CD8 + cells included cytotoxic TCR + T cells(50,54), the IgM + cells probably predominantly represented IgM + B cells [e.g. (55)], and the TN cells probably were a mixture of several cell populations [e.g.…”