2.3 Cellular components
The different leukocyte types derived from the lymphoid and myeloid lineages known from mammals have also been recognized in fish, including cells morphologically and functionally equivalent to mammalian B and T lymphocytes, natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, mast cells, and thrombocytes (Secombes, 1996; Whyte, 2007), as well as the recent description of dendritic-like cells in some species (Bassity and Clark, 2012; Lugo-Villarino et al., 2010).
2.3.1 Lymphocytes
The term “lymphocyte” refers to the three types of white blood cells present in mammalian blood, namely B and T lymphocytes and natural killer (NK) cells. The term was prompted because they are the main cellular components of mammalian lymph. In mammals, B and T lymphocytes mediate the specific responses of the adaptive immunity in jawed vertebrates.
Both types of lymphocytes share the unique characteristic process of somatic DNA rearrangement of their antigen-specific receptors (members of the Ig superfamily) by the random combination of the variable gene segments present in the receptor locus.
somatic 신체의
rearrangement 재배열
B cells express their antigen receptors on their cell surface as B cell receptors (BCR) and secrete them as immunoglobulin (Ig) or antibody, whereas T cell receptors (TCR) are always cell-surface bound. In both cases, one of each multiple variable (V), diversity (D), and joining (J) gene segments are randomly combined together with the constant gene segments (C) that define the type of receptor to form a mature functional VDJC antigen receptor (see Section 2.5.5).
secrete 분비하다
This process produces a vast repertoire of B or T cells bearing structurally diverse antigen receptors for specific pathogen recognition. Thus, each lymphocyte carries a unique Ig domain-containing receptor and can originate a clone of cells upon induction that will react specifically with only one antigen.
Jawless fish have a lymphoid cellular adaptive immune system based on the rearrangement of a variable lymphocyte receptor (VLR) encoded by genes of a leucine-rich repeat family of proteins not related with the Ig superfamily (Pancer et al., 2004) that does not distinguish between B-like and T-like cells.
On the other hand, in Chondrichthyes and Osteichthyes, the lymphocyte receptor system is based on the presence of variable receptors of the Ig superfamily, either BCR or TCR, and, therefore, B-like and T-like cells are present in these species, even though functional differences to their mammalian equivalents have begun to be elucidated.
The classical B cell function is the production of antibodies (or Igs) to specifically neutralize pathogens and label them to be removed by the immune system. T cells act mainly as coordinators of the responses of other immune cells and as effector cells to directly kill infected or tagged cells. Hematopoietic stem cells give rise to lymphocyte progenitors that diverge to develop into the T, B, or NK cell subsets.
2.3.1.1 B cells
In cartilaginous fish, three different Ig isotypes have been found, namely IgM, IgW, and IgNAR (Dooley and Flajnik, 2006). Chondrichthyan IgM is homologous to the IgM found in all vertebrates, while IgW has been suggested to be closely related to IgD based on phylogenetic analyses. IgNAR is a shark-specific Ig composed of heavy chains only, with no light chain association.Two different subsets of B cells have been described in sharks, expressing either IgM only or IgNAR only. There is no information available at present on IgW-expressing B cells (Flajnik, 2002; Flajnik and Kasahara, 2010; Ohta and Flajnik, 2006).
In teleosts, three different Igs have been reported, namely IgM, IgD, and IgT, the last designated as IgZ in zebrafish (Warr et al., 1979; Wilson et al., 1997; Hansen et al., 2005; Danilova et al., 2005).
While IgM and IgD seem to be essential Igs present in all teleost species, IgZ/T are only present in some of them (Fillatreau et al., 2013). Based on the expression of membrane Igs, three B cell subsets were identified in rainbow trout to date: IgM+/IgD+/IgT− (IgM+ cells), IgM−/IgD−/IgT+ (IgT+ cells), and the recently discovered IgM−/IgD+/IgT− (IgD+ cells) (Castro et al., 2014a; Hansen et al., 2005; Zhang et al., 2010).
In catfish (Ictalurus punctatus), which lack IgT, three subpopulations have been described: IgM+/IgD+, IgM+/IgD−, and IgM−/IgD+ (Edholm et al., 2010). In mammals, IgM+/IgD+, which make up the majority of peripheral B cells, down-regulate their IgD expression upon antigen binding; whether this occurs in fish and if IgM+/IgD− cells are present in all fish species is still unknown.
Concerning the IgM−/IgD+/IgT− population recently identified in trout, these cells are mainly present in the gills, and, although they still have an unknown function, they are regulated upon viral infection (Castro et al., 2014a). Also in catfish, the role in defense of the IgD+/IgM− described in peripheral blood leukocytes (PBLs) is still unknown (Edholm et al., 2010). In mammals, two types of IgD+/IgM− cells have been described.
An IgD+/IgM− population present mainly in the upper aerodigestive mucosa arises in humans after active IgM-to-IgD class switch. These plasmablast-like cells that retain IgD in the membrane secrete highly mutated mono- and polyreactive IgD, providing a layer of mucosal protection by interacting with pathogens and are either retained locally or are circulated in the blood, where they can account for up to 0.5–1% of circulating B cells (Chen and Cerutti, 2010; Chen et al., 2009).
The second type of IgD+/IgM− cells comprise up to 2.5% of circulating B cells in humans. These naïve B cells have antibody variable region genes in an unmutated configuration and are fully mature cells that are autoreactive and functionally attenuated and, therefore, have been cataloged as a new type of anergic (low reactive, tolerance-mediating) B cells (Duty et al., 2009).
In any case, four different subsets of B cells seem to be present in teleosts; three subsets with single surface expression of IgM, IgD, or IgT; and a subset co-expressing surface IgM and IgD.
Teleost B cells bearing different membrane Igs correspond to different cell linages because, in fish, there is no Ig isotype switching as in mammals (from IgM to more specific Igs). Concerning fish B cell ontogeny to date, most studies have been directed to studying the presence of IgM-bearing and IgM-secreting cells, and very little is known about the ontogeny or the tissue distribution and population dynamics of IgD and IgT/IgZ populations.
Through the study of IgM kinetics, three models of early hematopoiesis have been described in fish: in angelfish, hematopoiesis starts in the yolk sac blood islands; in zebrafish, early hematopoiesis is observed in the intraembryonic intermediate cell mass (ICM); while in trout, hematopoiesis is thought to initiate in the yolk sac for a short time before continuing in the ICM. Hematopoietic activity appears as early as 4 days post-fertilization, giving rise to erythroblasts and myeloid cells (Fillatreau et al., 2013; Salinas et al., 2011; Zapata et al., 2006).
After fertilization, B cell lymphopoiesis occurs mainly in the kidney, and detectable membrane IgM-expressing cells appear around three weeks post-fertilization (Razquin et al., 1990). Evidence for B cell development, homing, and maturation in teleost kidney is supported by the expression of conserved molecular markers and transcription factors such as Rag1 and Rag2 genes, TdT, Ikaros, EBF1, Pax5, Blimp1, or HCm, among others (Hansen, 1997; Hansen et al., 1997; Zwollo et al., 2010).
In adult fish, B cells can be found in the anterior and posterior kidney, spleen, liver, MALT tissues, and blood (Abos et al., 2013; Salinas et al., 2011).
The major functions of B cells are antibody production and antigen presentation and, in the case of teleosts, also include active phagocytic and microbicidal capacities (see Section 2.5.2) (Salinas et al., 2011). Once a B cell is activated by an antigen, part of its progeny becomes antibody-secreted cells (ASC), whereas some other cells become mid- or long-term memory cells.
The presence and characteristics of these B cell stages have been studied in a few teleost species such as rainbow trout. Different ASC populations have been defined in this species at the cellular and molecular level, including plasmablasts (replicating, low antibody secretors, bearing minimal BCR) and plasma cells (nonreplicating, terminally differentiated, high antibody secretors, bearing no BCR), further divided in short-lived plasma cells and long-lived plasma cells (Bromage et al., 2004; Zwollo et al., 2008, 2010).
The B cell development and maturation model proposed for teleost species resembles the one observed in mammalian bone marrow (see Section 2.2.1.2). On the other hand, even though affinity maturation takes place in teleosts, it is modest compared to that of mammals (Kaattari et al., 2002; Malecek et al., 2008; Yang et al., 2006; Ye et al., 2011b). Affinity maturation is the process that allows B cells to produce antibodies with increased affinity for a specific antigen during the course of an immune response.
It takes place through a double action: somatic hypermutation in the Ig genes that leads to antibodies with different binding specificities and binding affinities and the consequent clonal selection by which only the B cell progeny with the highest affinities for antigens will be selected to survive.
In those species that express IgT, such as rainbow trout, both IgM+ and IgT+ B cells are present in mucosal tissues; however, some infection models have revealed that IgT plays an important role at the mucosal level.
Despite these results, IgM also seems to react to some pathogens/antigens at a mucosal level, and recent studies have observed non-mucosal IgT responses to viral infections (Castro et al., 2013) or DNA vaccination (Castro et al., 2014b). Therefore, the exact contribution of IgT to pathogen clearance in both mucosal and systemic infections remains to be further clarified.
In any case, IgT+ cells constitute 16–28% of all trout B cells in the blood, spleen, head kidney, and peritoneal cavity, while they represent the main B cell population (54.3% of all B cells) in the gut (Zhang et al., 2010). Moreover, upon infection with an intestinal parasite, IgT responses were predominant in the gut, while IgM responses were confined to the serum (Zhang et al., 2010).
On the other hand, IgM is also modulated in different segments of the digestive tract in response to antigens (Ballesteros et al., 2013a, 2013b). Concerning the skin, again, IgT seems to be the predominant Ig isotype involved in its responsiveness to waterborne pathogens (Xu et al., 2013). However, in the gills, both IgT and IgM were discovered bound to surface structures of Ichthyophthirius multifiliis in the gills of infected rainbow trout shortly after invasion (von Gersdorff Jorgensen et al., 2011).
2.3.1.2 T cells
T cells are characterized by the presence of a T cell receptor (TCR) by which they recognize antigens. Unlike B lymphocytes, T lymphocytes fail to recognize antigens in the absence of antigen presentation, with the exception of superantigens (antigens that produce a nonspecific polyclonal T cell activation with a massive cytokine release) because the T cell receptor is restricted to recognizing antigens only when exposed in the context of an isogenic major histocompatibility complex (MHC), either class I or II, present on the cell surface (see Section 2.5.3).
A first classification of T cells can be based on the TCR chains they express, either ab or gd. ab-T cells can be cataloged as conventional T cells, whereas gd-T cells recognize unprocessed antigens in a manner similar to that of pattern recognition receptors. Consequently, in mammals, gd-T cells are more innate-like immune cells with less dependence on MHC presentation and are mostly present in epithelial and mucosal tissues, representing around 2% of the total T cell population (Bonneville et al., 2010).
On the other hand, conventional ab-T cells can be divided into T cytotoxic (Tc) or T helper (Th) cells, distinguished by the expression of the membrane-bound glycoproteins, CD8 or CD4, respectively. These molecules act as co-receptors for the TCR, stabilizing the interaction with the MHC and enhancing TCR activation through the CD3 tyrosine phosphorylation pathway.
Tc cells, expressing CD8 on the cell surface, are able to kill infected or cancerous cells after recognizing non-self or cancer-associated antigens in the context of MHC class I (Strasser et al., 2009). Th cells, on the other hand, express CD4 and produce cytokines to regulate the action of other immune cells, mainly B cells.
They can be further classified in mammals according to the expression of specific transcription factors and the secretion of representative combinations of cytokines. Although there is still some controversy as to whether these Th subsets constitute differential cell lines or cells in a different stage of activation with a certain degree of plasticity (Kleinewietfeld and Hafler, 2013), well-defined subsets in mammals include Th1, Th2, Th17, and T reg.
Upon stimulation, Th1 cells secrete effector cytokines, such as interferon g (IFN-g) and tumor necrosis factor a (TNF-a), to control intracellular infections and interleukin 2 (IL-2) to induce lymphocyte proliferation.
lymphocyte proliferation 림프구 증식
Th2 cells produce IL-4, IL-5, and IL-13 that stimulate B cells and control extracellular infections through the secretion of antibodies. Th17 cells produce IL-17 together with IL-21 and IL-22. These cells appear to be implicated in the control of extracellular bacterial infections, although their precise role is still debated.
Finally, the Th22 subset was recently discovered and secretes IL-22, but not IL-17. In mammals, Th22 cells are involved in epidermal repair.
Concerning fish, genomic studies performed in different species have identified most of the components associated with T cell function, making it possible to speculate that fish have all the different T cell subsets (Laing and Hansen, 2011); however, whether the functionalities are maintained is something that needs to be further investigated. All four TCR chains have been identified in different species of teleosts and elasmobranchs (Criscitiello et al., 2006; Flajnik and Kasahara, 2010; Nam et al., 2003), but it is still not known if gd-T cells exert roles similar to the equivalent mammalian subsets.
Mature Tc cells express a heterodimer of CD8 formed by a and b chains (Laing and Hansen, 2011). Different CD8 (both a and b chains) have been sequenced in multiple teleost species (Buonocore et al., 2006; Hansen and Strassburger, 2000; Moore et al., 2005; Patel et al., 2008; Pinto et al., 2006; Somamoto et al., 2006; Suetake et al., 2007; Sun et al., 2007). Furthermore, the combination of specific anti-CD8a antibodies, clonal fish, and MHC class I matching cell lines has permitted the verification that CD8+ Tc cells in fish also kill virus-infected cells when the TCR matches the MHC in which the antigenic peptide is exposed (Fischer et al., 2006).
CD4 orthologs have also been identified in different teleost species (Buonocore et al., 2008; Edholm et al., 2007; Laing et al., 2006; Moore et al., 2009; Patel et al., 2009; Sun et al., 2007; Wen et al., 2011); however, there is no data available to date on the functionality of CD4 cells in fish due to the lack of specific antibodies against this molecule. In some species, the presence of an additional CD4 gene has been reported (CD4L or CD 4REL) that could correspond to an ancient CD4 molecule with a different role still not elucidated (Dijkstra et al., 2006; Laing et al., 2006).
Finally, most of the cytokine representatives of the various Th populations described in mammals have been identified in teleosts (reviewed in Laing and Hansen, 2011), but again, whether they are being produced by specific Th subsets remains unknown.
T cells in fish have been reported in the thymus, spleen, head kidney, and mucosal tissues such as gills, skin, and gut (reviewed in Laing and Hansen, 2011).
They have been detected either through the use of specific CD8 antibodies or in some cases through the use of pan-T antibodies. However, most of these pan-T antibodies were developed against non-IgM expressing lymphocytes and the specific targets they are recognizing are not known (Rombout et al., 1998; Scapigliati et al., 2000).
Given the fact that we now know that B cell populations with no IgM on the cell surface are also present in fish, the specific target for these antibodies should be further characterized, despite the fact that some studies performed with some of these antibodies have rendered interesting data on T cell functionality (Abelli et al., 1999; Romano et al., 2011; Scapigliati et al., 2000).
On the other hand, some approaches focused on specific molecules have been more successful such as the development of antibodies against CD3, which is present in all T cell subpopulations. Through the use of a polyclonal antibody against the intracellular region of salmonid CD3 epsilon, high numbers of CD3+ cells were identified in the thymus, gills, and intestine, whereas lower numbers were detected in the head kidney, spleen, and peripheral blood leukocytes (Koppang et al., 2010). Surprisingly, the use of another anti-rainbow trout CD3 antibody, in this case a monoclonal, revealed high rations of CD3+ cells in thymus, skin, and posterior kidney and lower rations in head kidney, spleen, gills, and peripheral blood leukocytes (Boardman et al., 2012).
2.3.1.3 Natural killer cells (and natural cytotoxic cells)
Cell-mediated cytotoxicity is a process by which the immune system senses and kills altered, tumor, virus-infected, or foreign cells to maintain homeostasis. Natural killer (NK) cells constitute the first line of defense to carry out such processes as part of the innate immune system. NK cells are effector cells of lymphoid origin characterized by unique surface markers (CD3-CD56+CD16+) and the absence of recombined TCR or Ig genes (Vivier et al., 2008). These cells possess cytotoxic granules and their killing activity is similar to the cytotoxic T lymphocytes, except that they utilize innate nonspecific receptors to recognize antigens (Lieberman, 2003).
NK cells recognize MHC class I in the surface of the target cells through the action of three different types of receptors: killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LIRs), and receptors of the C-lectin-like family.
Depending on the receptors involved, receptor binding can lead to activation or inhibitory signals on the cell. Stimulation of the activating receptors, which associate with proteins having an intracellular ITAM motif (CD3ζ, DAP12, or DAP10), results in killing of target cells. Inhibitory signaling receptors all possess cytoplasmic ITIMs (Yoder and Litman, 2011).
For example, interaction of NK cells with normal levels of self MHC-I on a cell produces an inhibitory signal, whereas a decrease in MHC-I levels on a cell as a consequence of a tumoral process or viral infection triggers an activation signal that results in killing of the cell by the NK. This mechanism is often called the “missing self.”
On the other hand, cells infected with an intracellular pathogen display not only a unique repertoire of pathogen-derived peptides in the context of MHC-I (recognized by the TCR on Tc cells), but also peptides that can disrupt the normal assembly of MHC-I, which are then detected by NK cells.
Two types of NK cell homologs have been described in fish, mainly based on channel catfish studies: nonspecific cytotoxic cells (NCCs) and NK-like cells.
NCCs have been observed in several species, such as catfish, tilapia and damselfish, and are characterized by their ability to spontaneously kill allogeneic targets and protozoan parasites, but not autologous targets. These cells are small, agranular, and positive for the 5C6 antibody (developed against catfish NCCs). They bear a vimentin-like surface molecule and express a type III membrane protein designated as NCC receptor protein 1 (NCCRP-1) (Evans et al., 1990; Jaso-Friedmann et al., 2001; McKinney and Schmale, 1997; Yoder, 2004).
NK-like cells where inferred by the observations made on catfish from long-term alloantigen-stimulated mixed leukocyte cultures initiated with naïve PBLs.
The predominant cytotoxic cells in such a culture are large, granular cells that do not express TCR messengers, are negative for both neutrophil and macrophage markers and for reactivity with the 5C6 antibody, and are capable of killing virus-infected autologous cells (Nakanishi et al., 2011; Shen et al., 2003, 2004).
Other molecules involved in mammalian NK functions have been described in these and other fish species at the gene level, including the NK cell-enhancing factor (NKEF), immunoglobulin-like receptors, novel immune-type receptors (NITR), novel immunoglobulin-like transcripts (NILT), leukocyte immune-type receptors (LITRs), adaptor molecules like DNAX-activating proteins (DAP), perforins, NK-lysins, granulysin, granzymes, and FasL (reviewed in Fischer et al., 2013; Nakanishi et al., 2011; Yoder and Litman, 2011). Apart from these functional studies in these specific fish species, the contribution of NK cells in the context of an in vivo infection has never been evaluated.