Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

Abstract

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus)

“…In mammals, CD4 and CD8 T cells play pivotal roles in the adaptive immune system, especially in the cell-mediated immune response. However, the functional characteristics of teleost CD4 and CD8 T cells are still not well-defined due to the lack of appropriate detection tools like mAbs [ 1 , 41 ]. The function of CD4 T lymphocytes in the immune response against viral infectionswas examined using mAbs developed in previous studies [ 1 , 4 ].…”

“…In higher vertebrates, the T cells thatplayan essential role in the adaptive immune response are divided into two subsets according to their function, namely helper T cells and cytotoxic T cells [ 1 , 2 , 3 ]. Helper and cytotoxic T lymphocytes are defined by the expression of CD4 and CD8 glycoproteins on their surface, respectively [ 4 ].…”

“…The presence of CD4-like molecules, CD4-1 and CD4-2, have been investigated in several teleost species, including olive flounder ( Paralichthys olivaceus ), Atlantic salmon ( Salmo salar ), rainbow trout ( Oncorhynchus mykiss ), channel catfish ( Ictalurus punctatus ), fugu ( Takifugu rubripes ), carp ( Cyprinus carpio ) and European sea bass ( Dicentrarchus labrax ) [ 1 , 3 , 7 , 8 , 11 , 12 , 13 , 14 , 15 ]. The CD4-1 molecule possesses similar components to that of mammalian CD4, containing four Ig-like domains, a transmembrane domain and a cytoplasmic tail.…”

“…Although the expression of the CD4 and CD8 genes in teleosts appears similar to that in mammals, identification of their function in teleosts has been impeded by the lack of monoclonal antibodies (mAbs) able to detect CD4 and CD8 molecules [ 1 , 3 ]. While antibodies specific to CD4 lymphocytes and CD8 T cells are present in teleost [ 26 ], including olive flounder [ 1 , 4 , 27 , 28 ], the detailed functional activities related to CD4 and CD8 lymphocytes are still lacking at a cellular level. Our preliminary studies verified that the mAbs we produced against olive flounder CD4 lymphocytes (i.e., CD4-1 and CD4-2) were specific for these cell types, and different proportions of these CD4 T cells were found in the various tissues examined [ 1 , 4 ].…”

“…While antibodies specific to CD4 lymphocytes and CD8 T cells are present in teleost [ 26 ], including olive flounder [ 1 , 4 , 27 , 28 ], the detailed functional activities related to CD4 and CD8 lymphocytes are still lacking at a cellular level. Our preliminary studies verified that the mAbs we produced against olive flounder CD4 lymphocytes (i.e., CD4-1 and CD4-2) were specific for these cell types, and different proportions of these CD4 T cells were found in the various tissues examined [ 1 , 4 ]. The aim of the present study, was to produce mAbs able to detect CD8α and CD8β lymphocytes in olive flounder, and to examine the distribution of CD8-positive cells in various tissues of the fish.…”