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Immunology of Fish: Advanced Article
Immunology of Fish: Advanced Article
. Haematopoiesis
. Lymphoid Cells
. Immune Responses
. Vaccination
Innate immunity provides some form of defence against With the appearance of the jawed vertebrates, of which
pathogens in all multicellular organisms but with carti- the fish are the lowest group, came the ability to mount a
laginous and bony fish, although the lowest group of classical adaptive immune response. The fish possess the
jawed vertebrates, the addition of a classic adaptive necessary antigen receptors (immunoglobulin and T-cell
immune response becomes apparent. This provides the
receptors), antigen presentation molecules (major histo-
compatibility complex, MHC) and gene rearranging pro-
refinements of specificity for antigen recognition and
teins (rag genes). The cells mediating adaptive immunity
memory. The T- and B lymphocytes are the effector leu- are the T- and B lymphocytes. An outstanding advantage
cocytes, acquiring their antigen-specific receptors of adaptive immunity is the rapid response to a previously
(immunoglobulin for B cells and T-cell receptor for T cells) encountered and remembered pathogen. The differences in
in the anterior kidney (bone marrow being absent) and response between fish and higher vertebrates may result
thymus. The B cells are responsible for the production of from the aquatic environment and ectothermic physiology
antibodies that function as different immunoglobulin of these diverse species.
classes, whereas subsets of T lymphocytes are capable of Immunological studies of teleosts (class Osteichthyes:
killing target cells or helping with B- and other T-cell bony fish) are mainly directed at maintaining the health of
functions. Knowledge of these mechanisms is important those species that are of importance to aquaculture, such as
for the use and design of vaccines, now so essential for the
salmonids (Salmonidae), carp (Cyprinidae), catfish (Icta-
luridae), sea bass (Serranidae) and cod (Gadidae). Interest
aquaculture industry, whereas studies of fish immunology
in the origins of vertebrate immunity has led to studies on
contribute to the understanding of the evolution of the extant elasmobranchs (class Chondrichthyes: carti-
adaptive immunity. laginous fish), which include sharks, rays and skates; fish
that appear to have changed little since their earliest
appearance more than 400 Ma. The lampreys (Petromy-
zonidae) and hagfish (Myxinidae) (class Agnatha) are often
included with the fish but are outstanding in their seemingly
Introduction primitive anatomical characters, lacking both jaws and
paired fins but being immunologically advanced enough to
All fish possess innate immunity, initiated by the recog- exhibit lymphocyte-like cells expressing highly variable
nition of conserved pathogen-associated molecular pat- receptors. A form of adaptive immunity would therefore
terns (PAMPs) on microorganisms, by germline-encoded appear to have arisen independently in the agnathans.
cell receptors (pattern recognition receptors: PRRs). See also: Agnatha (Lampreys, Hagfishes, Ostracoderms);
Important among these are Toll-like receptors (TLRs); Chondrichthyes (Sharks, Rays and Chimaeras); Immune
well characterized in fish (Roach et al., 2005). The recog- Response: Evolution; Osteichthyes (Bony Fishes)
nition of PAMPs results in the induction of proin-
flammatory cytokines and the activation of antimicrobial
defence mechanisms. See also: Toll-like Receptors Lymphoid Tissues
These tissues produce and store lymphocytes and provide
ELS subject area: Immunology the sites for their interactions with antigens. In fish, such
tissues are associated with thymus, kidney, spleen and gut
How to cite: and probably, the gills where intraepithelial lymphocyte
Fletcher, Thelma C; and Secombes, Christopher J (February 2010) aggregations have been found and T cells are present
Immunology of Fish. In: Encyclopedia of Life Sciences (ELS). John Wiley
(Haugarvoli et al., 2008). The jawed fish lack bone marrow
& Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0000520.pub2
and lymph nodes and the agnathans also lack a thymus.
The latter group do not have true lymphoid organs but
ENCYCLOPEDIA OF LIFE SCIENCES & 2010, John Wiley & Sons, Ltd. www.els.net 1
Immunology of Fish
diffuse haematopoietic foci are present in the gut wall and mature in the anterior kidney and then migrate to sites of
anterior kidney of hagfish. Lymphoid cells have been activation either in the spleen or posterior kidney.
described in the kidney and intestinal lamina propria of the Lymphocytes expressing surface immunoglobulin (Ig)
lamprey and in the gut typhlosole of their larvae. See also: have been identified in the embryonic kidney of the dogfish
Lymphoid System but disappear from the kidney in the postnatal period.
A developed lymphatic system has been described in a See also: Kidney Development
flatfish and a secondary vascular system in some other
teleosts. The existence of a true lymphatic system in fish Secondary lymphoid tissues
remains equivocal.
In the secondary lymphoid tissues, the mature lymphocytes
encounter antigen and are activated to initiate the immune
Primary lymphoid tissues response.
The primary lymphoid tissues are the anatomical sites The spleen of teleosts contains a diverse population of
where T- and B lymphocytes mature and antigen-specific cells including leucocytes and antibody-producing cells
receptors are acquired by gene rearrangement. within a reticular cell framework. The lymphoid tissue may
Separate mammalian-like lymphocyte (B and T cells) be associated with melanomacrophage centres, which
functions exist in both elasmobranchs and teleosts but scavenge waste material, although antigens have been
there are still few serological reagents available to dem- observed at the surface of cells in this area. It is possible that
onstrate these distinct populations: the expression of genes they may serve as a focus for the proliferation of lympho-
involved in the development of B and T cells being more cytes and it has been suggested that they represent the
widely studied. Among the teleosts, primary lymphoid equivalent of germinal centres, which have not been
tissue in the rainbow trout has been defined by studies of described in fish. The spleen is a major lymphoid organ in
terminal deoxynucleotidyl transferase (TdT), which gen- elasmobranchs, where plasma cells are found in a supporting
erates antigen receptor diversity and serves as a develop- network of fibroblastic reticular cells. See also: Spleen
mental marker for sites of lymphopoiesis. It is highly All fish have gut-associated lymphoid tissue (GALT)
expressed in the thymus and to a lesser extent in the pro- consisting of nonencapsulated accumulations of lympho-
nephros. The recombinase-activating genes (rag) are also cytes and other leucocytes. The coexpression of both TdT
expressed within these tissues and low levels of TdT and and rag1 in the small population of intestinal lymphocytes
rag1 are also coexpressed in mesonephros, spleen and of the trout suggests that the gut may serve as an alternative
intestine but not muscle, liver or brain. Coexpression was site for lymphocyte development in teleosts. The gills are
also found in whole embryos, 20 days post-fertilization, capable of trapping antigen but although present, it is not
which correlates with the appearance of the thymus and known whether there is interaction with lymphocytes
pronephros anlages during ontogeny. It seems probable (Haugarvoli et al., 2008). See also: Lymphoid System
that the thymus and pronephros are the primary sites
involved in diversification of the teleost antigen receptors’
primary repertoire. Expression of TdT was also found to be Haematopoiesis
most prominent not only in the thymus, pro- and meso-
nephros of zebrafish but also in the spleen and intestine, Lymphopoiesis
considered to be secondary lymphoid organs (Beetz et al.,
2007). In sharks, the expression of rag1 gene has been All blood cells are derived from progenitor cells whose
reported in the thymus and in low levels in the spleen and lineage is controlled by cytokines that influence transcrip-
TdT genes have been isolated from both skates and sharks. tion factors effecting the development of specific cell types
See also: Lymphocyte Development; Lymphoid Develop- (Hanington et al., 2009). The thymus is the first organ to
ment; Spleen acquire mature lymphocytes during histogenesis. The ori-
In the Chondrichthyes, the thymus is first identified as a gins of the stem cells that colonize the thymus are not
paired organ and consists almost entirely of lymphoid cells known although advances in our knowledge of haemato-
in various stages of maturation in the cortical and medul- poiesis in fish have been rapid with the onset of the use of
lary regions, surrounded by a connective tissue capsule. the zebrafish as a model (Langenau and Zon, 2005). Con-
Organization is similar in the teleosts, where it develops as a comitant with an increase in thymocyte number is an
paired structure from the epithelial dorsal surface of the increase in the apoptotic cells in the cortex, particularly at
pharyngeal pouches. See also: The Thymic Niche and the corticomedullary junction, suggesting that negative
Thymopoiesis selection is occurring during lymphocyte development.
The teleost kidney consists of an anterior region or TdT and rag genes (rag1 and rag2) are highly expressed in
pronephros and a trunk kidney. Both are haematopoietic the thymus, confirming that it is a site where functional
but this is greater in the pronephros where renal function antigen receptors are generated. See also: The Thymic
disappears. It also functions in a secondary role where Niche and Thymopoiesis
plasma cells are recognized with immunoglobulin- The transcription factor Ikaros is a central regulator of
producing ability. Zwollo et al. (2005) suggested that B cells lymphopoiesis and in zebrafish has been shown to have a
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Immunology of Fish
role in both T- and B-cell lymphopoiesis, with evidence for Lymphoid Cells
two separate B-cell lineages in that species (Schorpp et al.,
2006). In bony fish, B cells are first seen in the anterior Lymphocytes
kidney, subsequent to the appearance of lymphoid cells, as
detected with anti-immunoglobulin sera. Weak perinuclear The presence of distinct B- and T-cell lineages is now well
localization of immunoglobulin is seen initially in large established in fish, originally by the use of monoclonal
lymphocytes, followed by the appearance of the surface Ig. antibodies and later by gene probing. Functions are in
Although firm data from thymectomy attempts in fish are many instances similar to mammals, such as the release of
limited, early thymectomy has appeared to have no effect cytokines by T cells. Such cytokines can enhance the
on lymphoid cell numbers in the kidney, suggesting that mitogenic responses of blood leucocytes and induce the
these (stem?) cells are self-generating. Shortly after Ig- activation and translocation of STAT (signal transducer
positive cells are found in kidney they become detectable in and activator of transcription)-like molecules, involved in
spleen and blood. In sharks, B cells are also first seen in the signal transduction events to leucocyte nuclei. A unique
kidney but in the Aleutian skate it is suggested that the function of teleost B cells has been their phagocytic ability
spleen may be a primary lymphoid organ for B cells. and intracellular killing of ingested microbes (Li et al.,
See also: B Lymphocytes 2006). See also: Monoclonal Antibodies
A hallmark of lymphocyte responses is their specificity,
brought about by their ability to generate a large repertoire
Myelopoiesis of antigen-specific receptors. The antigen receptors on B
and T cells are Ig and the T-cell receptor (TCR), respecti-
Granulocytes and monocytes/macrophages derived from vely, and genes for both of these molecules have been
myeloid lineage precursors develop early to provide the sequenced in cartilaginous and bony fish but not in agna-
first line of defence, with the kidney and then the spleen thans (Charlemagne, 1997). Studies of the agnathan lam-
becoming the main sites of myelopoiesis in fish, although prey and hagfish (Pancer et al., 2005) have revealed cells
granulopoiesis has also been described in the epigonal morphologically similar to mammalian lymphocytes with
organ and organ of Leydig in elasmobranchs and in the genes for variable lymphocyte receptors (VLR) that appear
typhlosole of lampreys. Long-term renal and splenic pri- specific for individual cells. These proteins have leucine-
mary cultures are haematopoietic, after the development of rich repeats and their genes are upregulated on antigen
a stromal layer, and can give rise to macrophages and stimulation of the cells. In the lamprey the response is even
dendritic cells. In addition, eosinophilic granular cells have compartmentalized in different cells (Guo et al., 2009).
been shown to differentiate from bacterially challenged gill Agnatha are able to generate a diverse repertoire of
explants. Four stages of neutrophil maturation have been lymphocyte receptors but by mechanisms different from
described in some species: myelocytes, metamyelocytes, the jawed vertebrates.
band-form granulocytes and lobed neutrophils. See also: Antibodies in the form of large oligomeric molecules
Myeloid Cell Differentiation; Neutrophils have been reported in the lamprey (Alder et al., 2008) but
A number of growth factors that may be involved in IgM is the universal Ig type produced in jawed vertebrates,
myelopoiesis in fish have been described. For example, with other classes (isotypes), IgNAR (immunoglobulin
macrophage and granulocyte colony-stimulating factor new antigen receptor) and IgW present in cartilaginous fish
(CSF) activity has been described in serum, and is enhanced and an IgD-like molecule in bony fish (Wilson et al., 1997).
post-challenge with a lipopolysaccharide (LPS) or Freund A third heavy chain class has been found in the teleosts: IgT
complete adjuvant. Similarly, macrophage supernatants (Hansen et al., 2005) and a similar IgZ in the zebrafish
have been shown to have M-CSF and G-CSF activity, and (Danilova et al., 2005). The ighz gene locus, with its specific
a goldfish macrophage cell line requires growth factors in Dz and Jz segments, is located between VH and Dm seg-
the form of conditioned media for proliferation. These ments. Although their function is unknown, if monomeric,
molecules (M-CSF and G-CSF) have now been cloned in they might reach extravascular sites more easily than tet-
fish with two forms of the M-CSF apparent (Wang et al., rameric IgM. In cartilaginous fish, the heavy chain con-
2008). The characterization of the zebrafish G-CSF and its stant regions of IgW exist in either a three- or seven-domain
receptor indicated that it functioned more widely than just form, whereas the Ig molecule termed NAR is secreted as a
in granulopoiesis (Liongue et al., 2009). See also: Haema- homodimer (i.e. without light chains) (Dooley and Flajnik,
topoietic Growth Factors 2006). The gene organization of IgM is well studied in fish
Other factors of potential importance for haematopo- (Figure 1). In cartilaginous fish there is a multicluster-type of
iesis in fish include the granulins isolated from spleen and organization of the heavy and light chain genes, with
kidney of carp, which have pleiotropic growth modulatory recombination limited to individual gene clusters. Bony
effects. They consist of 57 amino acid peptides, containing fish, on the contrary, have a translocon-type of organiza-
4 pairs of cysteine residues flanked by 2 single cysteine tion for the heavy chain genes, similar to the situation with
residues at both the N- and C-termini. An erythropoietin- mammalian Ig genes but a multicluster-type for the light
like gene has been demonstrated in trout kidney. See also: chains. It has been argued that recombination can occur
Haematopoiesis among the light chain clusters of bony fish, since they are
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Immunology of Fish
(V D1D2J C)1 (V D1D2J C)2 (V D1D2 J C)3 (V D1D2 J C)n both apoptotic and necrotic lesions in target cells, despite
an absence of azurophilic granules in their cytoplasm. The
(a) NCC have been described as the equivalent of natural killer
V1V2V3V4Vn D1D2D3D4Dn
(NK) cells but at least in catfish, there appear to be a sepa-
J1 J2 J3 J4 Jn C
rate group of large, granular cells unlike the small NCC and
these have been described as NK-like cells. Novel immune-
(b)
type receptors (NITR), containing immunoglobulin
Figure 1 Gene organization of the IgM heavy chain seen in (a) domains, have been identified in at least 13 species of tel-
cartilaginous fish and (b) bony fish. eosts and have been proposed as functional orthologues of
mammalian NK cell receptors (Yoder, 2009). Novel
relatively close together, allowing greater Ig diversity than immunoglobulin-like transcripts (NILTs), encoding
in cartilaginous fish. The multiple immunoglobulin iso- receptors with ITIMs (immunoreceptor tyrosine-based
types of fish are probably the products of distinct B-cell inhibition motifs) or ITAMs (immunoreceptor tyrosine-
lineages because there is no evidence of class switching. based activation motif), and mainly expressed in
See also: Antibody Classes; Antigens; B Lymphocytes: immunological organs, have also been described. Although
Receptors; T-cell Receptors the cell types expressing NILTs are still to be determined,
The prototypic mammalian TCR exists in two forms, they are seen to be expressed early in eggs and embryos
TCR1 composed of g and d chains, and TCR2 composed of from rainbow trout (Østergaard et al., 2009). See also:
a and b chains. The genes for TCR2, present on cytotoxic Natural Killer (NK) Cells
(TC) and helper (TH) T cells and responsible for recognizing
peptides presented in the context of self-MHC molecules,
have been sequenced in bony and cartilaginous fish. In Immune Responses
addition, genes distinct from the a and b chains, with some
identity to g and d chains, have been sequenced in carti- Antibody responses
laginous fish. Although the gene organization remains to
be elucidated, preliminary evidence in sharks suggests it is The effector functions mediated by circulating antibodies
not compatible with a multicluster organization as seen include neutralization, precipitation, agglutination and
with the Ig genes in these animals. Characterization of the opsonization of particles and the activation of the classical
complete TCRa/d locus and TCRg gene locus in Atlantic complement pathway. The pronephros and spleen are the
salmon revealed tremendous antigen receptor diversity and major sites of B cell differentiation and antibody synthesis
suggests a functional gd T cell immune component in the teleosts. On using an antiserum to dab IgM, 55% of
(Yazawa et al., 2008a, b). the kidney cells reacted with this reagent but in an ELI-
The ab TCR is unable to recognize soluble antigens, and SPOT assay (enzyme-linked immunospot assay), only
must have peptides presented by host MHC molecules. 1.5% of these cells were antibody-secreting and only 0.1%
Both MHC class I and II molecules have also been in the spleen. When fish held at 118C were immunized with
sequenced in cartilaginous and bony fish, and MHC class II killed bacteria, 1.3% of the kidney antibody-secreting cells
expression is shown to be restricted to particular cell types, produced specific antibody at 8 weeks post-immunization
including B cells and macrophages. This, in combination and there was a significant correlation between the number
with the demonstration of cytotoxic and helper cell acti- of cells and the serum agglutinating titres. See also:
vities, suggests that MHC restriction is likely to exist in fish. Antibody Function; Immune Response: Evolution
Polymorphism of fish MHC molecules can be as great as in Antibody-secreting cells have been demonstrated in the
humans. See also: Major Histocompatibility Complex intestine of trout following intraperitoneal injection with
(MHC) bacteria, but took seven weeks to appear compared with
two to three weeks in the head kidney. Conversely, anti-
Natural killer cells body-secreting cells were apparent within three weeks in
both intestinal mucosa and kidney, when immunized by the
Nonspecific cytotoxic cells (NCC) have been described in peroral route. In channel catfish mucosal antibodies are
fish. They are present in lymphoid organs, the gut, peri- produced locally from cutaneous cells rather than diffusing
toneal cavity and blood. They are able to kill a variety of from serum. Route of antigen administration is important
fish and mammalian cell lines and protozoan parasites. with immersion immunization leading to mucosal anti-
Killing begins immediately on addition of target cells and bodies appearing earlier than serum antibodies. See also:
requires direct physical contact, with cell binding being Antibodies
receptor mediated. The NCC receptor is a novel 235 amino Activation of B cells is determined by the antigen
acid (AA) protein, containing an extracellular proline-rich structure. Lipopolysaccharide antigens require an acces-
domain, a transmembrane domain (15–18 AA) and a sory cell such as a monocyte or macrophage. This appears
cytoplasmic tail and may be a Type III membrane protein. to provide interleukin-1 (IL-1) which, in the trout, is critical
Treatment of this molecule with antisense oligonucleotides for differentiation of the B lymphocyte into an antibody-
inhibits its expression and NCC cytotoxicity. NCCs inflict secreting plasma cell. Protein antigens must be processed
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Immunology of Fish
by an accessory cell (macrophages or B cells) and presented granules, form conjugates with allogeneic target cells and
to a T cell, which produces interleukins, signalling B cell kill them by inducing apoptosis via a putative perforin/
differentiation. A costimulatory pathway through mem- granzyme mechanism (Shen et al., 2002; Zhou et al., 2001).
brane-bound proteins (CD40 on B cells and its ligand In addition, some of the catfish CTL clones appear to also
CD154 on Th cells) is essential for T–B cell cooperation in use a Fas/FasL-like interaction for killing. In vivo gener-
thymus-dependent responses to protein antigens. Results ation of CTL is also possible, where, for example, per-
from zebrafish suggest that CD154 and CD40 are also ipheral blood lymphocyte (PBL) isolated from rainbow
localized on their T and B cells, respectively (Gong et al., trout DNA (deoxyribonucleic acid) vaccinated against the
2009). Three members of the B7 family of molecules G protein (guanine nucleotide-binding protein) of viral
(including the synonymous CD80/86) have been found haemorrhagic septicaemia virus (VHSV, a salmonid
expressed on the surface of fugu monocytes. These can act rhabdovirus) kill VHSV-infected, MHC class I matched
as costimulatory molecules in the activation of T cells via targets but not infectious haematopoietic necrosis virus
receptors on their surface. Genes for such CD28/CTLA4 (another salmonid rhabdovirus)-infected targets (Utke
receptors have been characterized in teleosts. Engagement et al., 2008). Interestingly, a significant killing capacity of
triggers the production of cytokine IL-2, inducing T-cell such PBL was only observed during the summer months.
proliferation and the upregulation of IL-2 was found to be When suboptimal infection with VHSV was used as a
induced in trout leucocytes, using a rainbow trout re- means to stimulate the fish, which generated similar CTL-
combinant CD80/86 (Zhang et al., 2009). See also: type responses to the above, PBL were found to have a
B Lymphocytes; Cytokines; Interleukins; Lipopolysac- higher transcriptional level of the CD8 a gene (Utke et al.,
charides; T-lymphocyte Activation 2007). See also: Graft Rejection: Mechanisms; Immunity:
Temperature is a determining factor in the immune Humoral and Cellular; T Lymphocytes: Cytotoxic
response of ectotherms and whereas immunized salmonids TH cell activity was originally indicated in fish by an
held at permissive water temperatures of 10–158C have in vivo hapten-carrier effect, where the production of anti-
shown a rise in serum antibody titres within 2–3 weeks, body to a hapten by B cells required carrier-specific
peaking at 8–12 weeks, at lower temperatures the response cooperation from putative T cells. Detailed studies in
takes longer and may even not occur. Adaptive immunity is channel catfish have confirmed their help in antibody
thought to be more temperature sensitive than innate production, in which not only hapten-specific B cells and
immunity. The immunosuppressive effects of low tem- carrier-specific T cells but also accessory cells (monocytes/
perature seem to be due to the suppression of virgin helper macrophages) are required. The regulatory role of T cells in
and cytotoxic T cells rather than memory T cells, B cells or immune responses is mediated by released cytokines, with
accessory cells. Temperatures above the ambient but the subsets of Th defined by the cytokine repertoire they
within the physiologically tolerated range can quicken the release. TH1 cells, responsible for eliciting protective
response. responses against intracellular pathogens, release IL-2,
Teleosts exhibit immunological memory with an acceler- IFNg and TNFb (tumour necrosis factor beta). TH2 cells,
ated and enhanced secondary antibody response and involved in responses to parasites, release IL-4, IL-5, IL-9
increased sensitivity to the antigen. The response differs and IL-13, whereas TH17 release IL-17 and IL-22 to com-
from that in mammals in the lack of affinity maturation bat extracellular pathogens. Although subpopulations of
(perhaps explained by the lack of germinal centres although Th have not been defined in fish, many of these cytokines
an increase in antibody affinity has been reported in trout are known and they suggest that differential regulation of
and evidence has been presented for affinity maturation in fish immune responses will occur. Thus, IL-2 and IFNg
shark IgNAR); isotype switching and an increased capacity have been cloned and the protein bioactivity studied. In the
for memory precursor cells to proliferate. Memory induc- case of rIFNg, stimulation of trout macrophages increases
tion in fish appears to be solely defined by an increase in the the expression of molecules involved in antigen presen-
number of specific antibody-producing B cells in the mem- tation to CTL (Martin et al., 2007). Although other TH1
ory pool. See also: Immunological Memory type cytokines, such as TNFb, have not been found in fish,
it is clear that additional genes are present that are related
Cell-mediated responses to IL-2 and IFNg, although their function remains to be
determined. Similarly, IL-17 and IL-22 are known in fish,
These responses are mediated by T lymphocytes, which in with isoforms of the former similar to IL-17A/F, IL-17C/E
mammals effect cytotoxic and helper functions. Evidence and IL-17D. Expression analysis indicates that these mol-
of the existence of separate cytotoxic (CD8+) and helper ecules are important in generating protective responses to
(CD4+) T cell subsets in fish is accumulating. For example, extracellular Gram-negative bacteria (Corripio-Miyar
the CD4 and CD8 (a,b) molecules have been sequenced in a et al., 2009), although studies with the recombinant pro-
variety of species, with binding sites to MHC I and II teins are still awaited. In the case of TH2 derived cytokines,
identified. In the case of functional studies, it has been to date, two genes have been discovered in different loca-
possible to successfully clone cytotoxic T lymphocytes tions within the fish genome with some homology to IL-4/
(CTL) from alloantigen-stimulated cells in channel catfish, 13, but their function is still to be determined. See also:
that do not express B- or NK cell markers but do possess Macrophages; T Lymphocytes: Helpers
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