Immune Defences of Teleost Fish - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 20

More recently, with the generation of monoclonal antibodies to CD8α, capable of being
used for flow cytometry, cell sorting, immunohistochemistry, western blotting and immuno-
precipitation, teleost T cell subsets have been phenotypically characterized with the ensuing
opportunity to characterize functionality. Takizawa and colleagues (2011) have been able to
characterize T cell populations and some functionality on the basis of CD8α expression. CD8α
was expressed in rainbow trout at high levels in the thymus and both intra- and subepithe-
lial T cells of the gills and intestine. In contrast, CD8α was expressed at lower levels in the
pronephros, spleen and blood. The simultaneous transcript expression analysis demonstrated
Ig and CD4 expression in the CD8α - population, that is, associated with distinct B cell and
T h subsets. Interestingly, in the case of thymocytes, CD4 was co-expressed with CD8α, sug-
gestive of the thymocyte development stage of double-positive thymocytes prior to single
positive CD4 + and CD8 + T cells. Upon cell sorting of these CD8α + and CD8α - popula-
tions, the CD8α + T cells were found to express both CD28 and CTLA-4, where the expres-
sion of the immunosuppressive receptor CTLA-4, suggestive of T reg maintenance of mucosal
homeostasis, was highly expressed in mucosal lymphocytes and much less so in non-mucosal
lymphocytes. PHA stimulation of these populations resulted in the CD8α - cells expressing
both Th 1 (IFNγ) and Th 2 (IL-4/13A) cytokines, whereas the CD8α + cells expressed the char-
acteristic CTL functional effector molecules, perforin and granulysin. Previously, functional
alloantigen-specific cytotoxicity has been demonstrated for ginbuna crucian carp ( Carassius
auratus langsdorfii) )CD8α + lymphocytes (Toda et al. 2009) and the killing mechanism was
perforin dependent (Toda et al. 2011a). Finally, although the Th 17 -associated transcription
factor RORγT homologue has not yet been characterized in teleost fish, the presence of such
aTh 17 subset has been suggested by the expression of Th 17 -associated cytokines IL-17 and
IL-22, whilst the Th 17 differentiation cytokines have also been described (see Section 2.4).
Expanding on currently available data on transcript expression analysis, by the development
of specific antibodies to T cell subsets through the recognition of subset-specific transcrip-
tion factors, specific membrane molecules in combination with functional readouts such as
effector cytokines, it will be possible to fully characterize teleost T cell subsets present and
inducible during antigenic challenge and to investigate their functional activity involved in
adaptive immune responses to pathogenic infection.
2.3.2 B cells, immunoglobulins and humoral immunity
Immunoglobulins are involved in precipitation, agglutination, neutralization, opsonization
and complement activation. As a result, they are likely to function in accord with observations
made for antibody function in higher animals/mammals, where immunoglobulins confer
humoral immune protection involved in prevention of infection, clearance and anti-pathogen
killing responses aimed at extracellular resident pathogens. Ig + cells have been described in
European sea bass ( Dicentrarchus labrax ), salmonids and cyprinids (Rombout et al. 1993;
Abelli etal. 1997). In comparison to humans, whereby functional characteristics are associated
with expression of five main antibody isotypes (IgM, IgD, IgG, IgA and IgE), until recently
teleost fish were thought to generally express IgM and IgD isotype antibodies which predom-
inate in serum and the periphery (Flajnik 2002), where IgD exhibits a restricted expression
pattern in the spleen and head kidney and IgM expression is observed in renal haematopoietic
tissue, spleen and thymus with lower levels in the skin and gill. Antibody responses in teleost
fish are generally slow, for example antibody production takes 4 to 6 weeks in salmonids,
and are of restricted diversity as a consequence of a lack of recombination by class switching