1.1. Innate Immune System (NK Cells, Leukocytes, Complement, Antimicrobial Peptides)
Based on morphology and Giemsa staining pattern, typical leukocyte types such as neutrophils, basophils, eosinohils, polymorphonuclear (PMN) cells, monocyte and macrophage-like cells, and smaller lymphocytes can be observed in the blood and the peritoneal fluid (Du Pasquier et al.,1985; Hadji-Azimi et al.,1987). Although mAbs recognizing markers specific for particular leukocyte subsets are not available, there are several mAbs identifying surface markers of all leukocytes (Table 1) such as F1F6 (Flajnik et al.,1988), and even leukocytes at early developmental stages such as CD45 (CL-21 mAb; Smith and Turpen,1991; Barritt and Turpen,1995), Xl-1 (Ohinata et al.,1989), Xl-2 (Miyanaga et al.,1998), and RC47 (Du Pasquier and Flajnik,1990).
Table 1. Expression Pattern of Xenopus Lymphocyte Surface Markers Detectable with Currently Available mAbs
| ||Xenopus markers (mAbs) *||Expression pattern [Molecular weights]||Ref.|
|B cells||IgM (10A9, 6.16)||Larval and adult B cells. [73 Kd]||1, 2|
| ||IgY (11D5)||Some larval and adult B cells. [66 Kd].||1|
| ||IgX (410D9)||Some larval and adult B cells, especially in the gut. [80 Kd]||3, 4|
| ||IgL (1E9, 13B2, 409B8)||Some larval and adult B cells. [30-35 Kd]||5|
|T cells, thymocytes||CD3 epsilon (CD3-2) anti-human||Cross-reacts with Xenopus CD3 epsilon, and coprecipitates the TCR-CD3 complex of T-cells and lymphoid tumor lines.||6|
| ||CD5 (2B1)||Thymocytes (>95%), T-cells and some PMA-activated IgM+ B cells.||7|
| ||CD8 (AM22, F17)||Larval and adult thymocytes (70-80%) and T-cells (about 20% of splenocytes). [35 Kd]||8-10|
| ||CTX (X71, 1S9.2)||Larval and adult thymocytes (60-70%); no consistent expression in peripheral lymphocytes. Also expressed in gut epithelial tissue.||11, 12|
| ||T-cells (XT1)||Most, but not all, larval and adult T-cells; earliest marker of thymocytes.||13|
| ||T cells (AM15)||Subpopulation of thymocytes and peripheral T cells. [18 Kd]||8|
|MHC||MHC class I (TB17)||Ubiquitous in adult; all lymphopoietic lineages. Not expressed until metamorphosis. [44-45 Kd]||14|
| ||MHC class II (AM20, 14A2)||Thymocytes, B and T-cells (99% of spleen lymphocytes), only B-cells in larvae. [30 Kd]||8|
|Leukocytes||CD45 (CL21)||All leukocytes from early embryonic stage 28. Different size variants expressed by thymocytes and B cells. [180-200 Kd]||15|
| ||NK cells (1F8)||Non-B and non-T, peripheral lymphoid cells [55 Kd].||16|
| ||F1F6||All mature leukocytes and erythrocytes, but not immature cells. [13 Kd]||17|
| ||RC47||Leukocyte lineage from very early stage (day 6 post-fertilization). Thymic cortex and medulla (>90% of total thymocytes).||9|
| ||XL-1||All leukocytes from early larval stage, ventral blood island stage 35.||18|
| ||XL-2||All leukocytes from early embryonic stage 24. [135 Kd]||19|
Many of the genes known to be involved in mammalian innate immunity have been identified in X. laevis and S. tropicalis (Table 2). Among them, toll-like receptors (TLR) are one of the innate receptors that recognize PAMPs on pathogens that initiate innate as well as adaptive immune responses (Janeway,1992). Of interest, in contrast to mammals that have 10 TLRs, a total of 20 different TLR genes, as well as some adaptor proteins, have been identified in the S. tropicalis genome (Fitzgerald et al.,2001; Inoue et al.,2005). All these TLR genes are constitutively expressed in tadpoles and adults (Ishii et al.,2007; Roach et al.,2005), suggesting that the innate immune response through TLR signaling is active throughout life. While most TLRs are evolutionarily conserved due to the strong selection for maintenance of specific PAMP recognition, Xenopus TLR4 (i.e., the receptor responsible of response to the endotoxin lipopolysaccharide [LPS] in mammals; reviewed in Janeway,1992) seems to be divergent. In this regard, it is interesting to note that Xenopus is poorly responsive to purified LPS (e.g., adult can receive up to 1 mg of LPS without any sign of inflammation or other untoward effects; Bleicher et al.,1983; Marr et al.,2005). Thus, Xenopus carry all the human orthologs and some TLR family members that are expanded in a Xenopus-specific manner (e.g., TLR14).
Table 2. Some Relevant Mammalian Immune Gene Orthologs Identified* in the S. tropicalis Genome
| || ||Types of molecules||Genes||Ref.|
|Innate immunity||Leukocyte receptor||NK receptor||NKp30, KIRs,||1|
| || ||Toll-like receptor||TLR1, 2, 3, 4, 5, 6, 7, 8, 9|
| || ||Fc receptor||FcR-like, FcRγ||1, 3|
| || ||C-type lectin||CLEC||31|
| || ||Others||SIGLEC,||31|
| ||Signaling molecules|| ||NFKB, IKBB, MyD88 TNFα, IL-6 DAP10, DAP12||4 OR|
| ||Effector molecules||Cytokines||IL-1β, LTα, LTβ, TNFα, IL-6, IFNα||5, 6|
| || ||Cytotoxic killing||iNOS, granzyme, perforin||31|
| || ||Anti-bacterial peptide||magainin, xenopsin, caerulein||31|
| || ||Complement||C1∼9, MASP, Bf||7-10|
|Adaptive immunity||Antigen receptors||Immunoglobulins||IgH (M, D, X, Y, F), IgL (λ, σ, κ)||11- 17|
| || ||T cell receptors||TCRα, β, γ, δ||18, 19|
| || ||GOD||Rag 1, 2, AID, TdT||20-22|
| ||Antigen presentation||MHC||Class I, Class II, DM, β2M||6, 23-26|
| || ||Immunoproteasome||Psmb8, 9,10||6|
| || ||Transporters||Tap1, Tap2||6, 27|
| || ||Cathepsins||Cathepsin L||31|
| || ||Others||Tapasin, calreticulin, calnexin||6 (Tapasin), 31|
| ||Accessory molecules|| ||CD4, CD8α, β, BTLA||28|
| || ||B7 family||CD274, CD276, VTCN1||28|
| || ||B7 receptors||CD28, CTLA4||28|
| || ||TNFSF family||CD40LG, HVEM||28|
| || ||TNFRSF family||CD40||31|
| || ||Adhesion molecules||MCAM, JAM, ICAM, CD2||31|
| || ||Cytokines||IL-2, 3, 4, 5, 7, IFNγ||29 (IFNγ), 31|
| ||Effector molecules|| ||IL-6, 17, 21, 23||31|
| ||Signaling molecules|| ||LCK, Fyn, Igα, CD3ϵ, CD3ζ||3, 30|
As many other amphibians, the granular glands (also called serous glands) in the dermal layer of the Xenopus skin produce potent antimicrobial peptides (e.g., magainin, xenopsin, caerulein precursor fragments) effective against bacteria and fungi (reviewed in Simmaco et al.,1998; Zasloff,2002; Rollins-Smith and Conlon,2005). The variety and remarkable molecular heterogeneity of these antimicrobial peptides that can be secreted in large amount under stress suggest that they provide an important protection of the host against a wide range of pathogens.
NK cells are leukocytes of the innate immune system that can kill targets by cell-mediated lysis, especially virus-infected cells and tumors that have down-regulated their surface expression of MHC class I molecules. In addition, through production of cytokines such as interferon-γ, NK cells are involved in the orchestration of both innate and the adaptive immune responses. NK cells have been characterized in X. laevis using a mAb, 1F8 (Horton et al.,2000) that recognizes a 55-kDa surface protein (Horton et al.,2003). 1F8 recognizes a population of large granular non-B, non-T leukocytes that remain present in larvally thymectomized (Tx) T-cell–deficient Xenopus and that kill MHC class I-negative tumor but not MHC-expressing autologous lymphoblast targets (Horton et al.,2000).
In mammals, receptors on NK cells are encoded in two different genetic regions unlinked to the MHC; however these regions are suggested to be the paralogs of the MHC (Du Pasquier,2000; Teng et al.,2002). Indeed, the linkage of these NK receptors to the MHC has been identified in nonmammalian species such as birds and marsupials; thus, their close linkage is proposed to be primordial (Kaufman et al.,1999; Belov et al.,2006). In the Xenopus MHC, there is a cluster of putative membrane-bound molecules that might function as candidate primordial NK receptors that may have co-evolved with particular MHC class I alleles (Ohta et al,. 2006). There are other NK receptors in the S. tropicalis genome that are not linked to the MHC; these may be orthologous to the mammalian NK receptors (Ohta, unpublished observation). In fact, recent studies of the S. tropicalis genome reveal that receptor systems regulating function of leukocytes and NK cells are rather complex. Indeed, the diploid S. tropicalis genome contains at least 75 genes encoding paired Fc-related receptors (FcR) designated XFLs (Guselnikov et al.,2008). Many homologous genes that are primarily expressed in lymphoid tissues are also found in the allotetraploid X. laevis, and two signaling adapter receptors, FcRγ and TCRζ, have been characterized (Guselnikov et al.,2003). The function of these genes is currently unknown, but their extraordinary diversity and their subdivision into two signaling class of receptors: inhibitors and activators, suggests that they are involved in the regulation of immune function.
The three pathways of the complement system (classical, the lectin, and the alternative) are present in Xenopus (reviewed in Fujita et al.,2004). This system, consisting of more than 30 proteins and receptors, bridges the innate and adaptive immune system and is critical for a potent humoral response. Most genes of the complement are present in the S. tropicalis genome, and many of them have been characterized in X. laevis (Alsenz et al.,1992; Kato et al.,1994; Mo et al.,1996; Endo et al.,1998; reviewed in Fujita et al.,2004) including serum proteins involved in the complement lectin pathways such as ficolins (Kakinuma et al.,2003). Similarly, proinflammatory cytokines (e.g., IL-1, 6, TNFα), chemokines, and the respective receptors genes involved in the activation of innate effector responses are present in the genome of S. tropicalis. However, to date there are very few cytokine/chemokine expression and functional studies in Xenopus. An IL-1β-like factor produced by stimulated peritoneal leukocyte has been reported in X. laevis (Watkins and Cohen,1987), and more recently, up-regulated expression of IL-1β message upon inflammatory signal such as LPS has been shown (Zou et al., 2008; Marr et al.,2005). Some studies suggest that TGF-β inhibit proliferation of Xenopus splenic blasts induced by a supernatant containing a T-cell growth factor (Haynes and Cohen,1993a).
1.2. Adaptive Immune System (B cell, T cells, Ag Presenting Cells)
The mammalian adaptive immune system consists of a network of specialized B and T cells expressing surface Ag receptors generated by combinatorial somatic rearrangements that can recognize foreign antigens. The B-cell receptors (BCR) can recognize and bind directly to a particular motif or epitope in a large molecule, whereas TCRs only recognize short peptides that are processed and complexed with MHC molecules on the surface of the specialized or professional antigen presenting cells (APCs) such as dendritic cells, monocytes, and macrophages (Fig. 2). Surface class II molecules loaded with 10 to 30 amino acid-long antigenic peptides are recognized by TCRs on the CD4 helper T cells, whereas surface class I molecules complexed with β2-microglobulin (β2-m) and 8 to 11 amino acid-long antigenic peptides are recognized by the TCR on the surface of cytotoxic CD8 T cells. In addition to TCR signaling, activation of both naive CD4 and CD8 T cells require a second signal from co-stimulatory molecules such as B7 ligands and CD40 provided by APCs interacting with CD28 and CD40L on T cells, respectively. As such APCs bridge the innate and adaptive immune system: through PRRs such as TLRs, APCs initiate innate immune responses by up-regulating co-stimulatory receptors and releasing proinflammatory cytokines, and through Ag presentation APCs trigger adaptive immune responses (Fig. 2). After their activation, T-cell clones expand and differentiate into cytotoxic CD8 T-cell effectors (CTLs) or CD4 T helper cells. CTLs can kill any cell targets expressing the class I/peptide complex that activated them without costimulation. Various CD4 helper T-cell effector types (e.g., Th1, Th2) are generated that secrete different cytokines critical to help various other immune cells including B cells.
Figure 2. Schematic overview of a typical mammalian adaptive immune response. Productive T-cell activation requires two signals from an APC (e.g., dendritic cells, macrophages): the first signal is due to the recognition by the TCR of the MHC molecules complexed with the antigenic peptide (CD4 T cells interact with class II molecules, CD8 T cells with class I molecules), the second signal is provided by costimulatory molecules (e.g., B-7, CD40) that are up-regulated following APC activation by pathogen products or PAMPs binding to PRRs such as TLRs. Activated T cells proliferate and differentiate into cell effectors: CTLs able to kill target expressing the same Ags-class I complex and CD4 T helper cells producing various cytokines that act on the pathogen as well as on other immune cells including CD8 T and B cells. Most T cells die from apoptosis after the response (contraction phase), except a long-lived minor population of memory T cells able to respond faster to a second pathogen exposure. For abbreviations, see list.
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Although the Xenopus adaptive immune system is less well characterized than that of mammals, its general pattern is very similar. However, in contrast to mammals where BM is the main site for lymphopoiesis and a reservoir for antibody-secreting B cells, adult Xenopus B cells differentiate mostly in the liver and the spleen (reviewed in Du Pasquier et al.,2000). Furthermore, no accumulation of antibody-secreting B cells in adult Xenopus BM was detected during immune responses to bacteria and virus (Marr et al.,2007). Therefore, even though some lymphopoietic activity is suggested by the expression of RAG (Greenhalgh et al.,1993), the Xenopus BM does not appear to be a major lymphoid organ. The surface BCR is recognized by several mAbs including anti-Ig heavy chain isotypes μ, ν, and χ, as well as anti-Ig light chain mAbs (Hsu and Du Pasquier,1984a; Schwager et al.,1991b; Hsu et al.,1991; Table 1). Expression of IgM and IgX are thymus-independent, whereas the production of IgY is thymus-dependent (Turner and Manning,1974) and requires collaboration between B and T cells (Blomberg et al.,1980). Majorities of B cells express the IgM isotype and are widely distributed in most tissues. IgX-expressing B cells are a minor population of mostly secretory plasma cells found mainly in the gut epithelium (where they may be involved in mucosal immunity), and to a lesser extent in the spleen and the liver (Mussmann et al.,1996). IgY-producing B cells are found in the liver, spleen, and blood circulation, but not in the intestine (Mussmann et al.,1996). Other B-cell–specific markers are currently lacking although B-cell–specific gene orthologs are present in the S. tropicalis genome (e.g., CD19, CD138, CD180; Otha and Robert, unpublished observations). The Ig gene loci in Xenopus and mammals have a similar organization and usage with somatic combinatorial joining of V, D, and J elements (reviewed in Du Pasquier et al.,2000) that are RAG-dependent (Greenhalgh et al.,1993) and involve terminal deoxynucleotidyl transferase (TdT; Lee and Hsu,1994) for generation of diversity. Allelic exclusion, which prevents the expression of more than one Ig heavy chain loci, also operates in Xenopus B cells (Du Pasquier and Hsu,1983; reviewed in Hsu,1998). Thus Xenopus Ig heavy and Ig light chain repertoires are as diverse as those of mammals.
For a long time, Xenopus was considered to have only the three Ig heavy chain isotypes: IgM, IgX, and IgY (Schwager et al.,1988; Haire et al.,1989; Amemiya et al.,1989). Recently, however, two new isotypes, IgD and IgF, were discovered by in silico analysis of the S. tropicalis genome (Zhao et al.,2006; Ohta et al.,2006). Because of the evolutionary transitional position of Xenopus, the discovery of IgD in Xenopus tied the connection of mammalian IgD to the previously unknown immunoglobulin isotype, IgW in lungfish (Ota et al.,2003) and IgW/X in cartilaginous fish (Kobayashi et al.,1984; Harding et al.,1990; Bernstein et al.,1996; Greenberg et al.,1996), thereby demonstrating that IgD is as old as IgM. Without the genomic database, the discovery of IgD would have not been possible, because of its low level of expression only by naive B cells and its large molecular weight. IgF is the shortest Ig isotype having a hinge exon. The two constant domains of IgF are very similar to those of first and fourth constant domains of IgY, suggesting that IgF was generated by tandem duplication of IgY followed by a loss of internal constant domains. IgX (Mussman et al.,1996) and IgY (Warr et al.,1995) are homologs to the mammalian IgA and IgG (also IgE) isotypes, respectively. Two mammalian orthologous Ig light chain isotypes, λ, κ, (Bengtén et al.,2000; Criscitiello and Flajnik,2007) have been identified as well as the σ-isotype that is specific to the nonmammalian species, and their genomic organization is similar to that of mammals (Qin et al.,2008).
Class switch recombination (CSR) from IgM to IgY, and somatic hypermutation (SHM) at the Ig locus, occurs during the course of an antibody response in Xenopus (reviewed in Hsu,1998, Du Pasquier et al.,2000). These two processes typical of affinity maturation involve AID (Ichikawa et al.,2006), but in contrast to mammals, SHM is targeted to G/C nucleotide in Xenopus (Wilson et al.,1992). Although the sequences of the genomic region that serve as substrate for the switch (S region) are divergent between mammals and Xenopus (i.e., Xenopus Sμ is rich in AT and not prone to form R-loops), a Xenopus S region can functionally replace mouse equivalents to mediate CSR in vivo (Zarrin et al.,2004), and Xenopus AID (XlAID) can induce CSR in AID-deficient murine B cells (Ichikawa et al.,2006). Despite the absence of germinal centers in X. laevis, in situ hybridization has detected XlAID mRNA mainly in the spleen, preferentially distributed in follicular B-cell zones. XlAID is markedly up-regulated with different kinetics upon bacterial stimulation and viral infection (Marr et al.,2007). Additionally, during secondary anti-viral response XlAID is also noticeably expressed by PBLs, suggesting that XlAID remains active in a subset of circulating B cells and plasma cells.
Despite these fundamental similarities, affinity maturation in Xenopus is poor when compared with mammals. For example, the affinity of IgY antibody against dinitrophenol (DNP) increases less than 10 times during a humoral response in contrast to more than a 10,000 fold affinity increase in mammals (reviewed in Hsu,1998). Sequence analysis of the variable domain of Ig heavy chain VH1 gene family during antibody response to DNP has revealed that the rate of somatic mutations is not very different between Xenopus and mice (Wilson et al.,1992). However, the relatively low ratio of replacement to silent mutation in the CDRs (Wilson et al.,1992) suggests that, in Xenopus, the selection mechanism is not optimal. This could be related to the simple organization of the lymphoid organs in Xenopus that lack lymph nodes and germinal centers (Marr et al.,2007; reviewed in Du Pasquier et al.,2000), an observation that correlates with poor affinity maturation. It is important, however, to mention that protective neutralizing antibodies are generated following a secondary infection with the iridovirus Frog virus 3 (FV3), and that immunological memory to FV3 lasts at least 15 months (Maniero et al.,2006). Therefore, when examined in a physiological context involving a natural viral pathogen, antibodies generated by Xenopus appear to provide protective defense against subsequent viral infection even though they are of a weaker affinity than mammalian antibodies.
In contrast to mammalian mature B cells that are generally not phagocytic, peripheral differentiated B cells from teleost fish species and X. laevis are phagocytic and capable of killing ingested microbes. This finding suggests that evolutionarily, B cells and macrophages may share a common origin. That such a primordial phagocytic function has also been demonstrated for Xenopus B cells, offers additional support of this frog as an excellent model of transitional evolution (Li et al.,2006).
T cells are the second main type of lymphocytes. All four types of TCR chains are found in the Xenopus genome, suggesting the presence of γδT cells as well as the conventional αβT cells. Other signaling components of the Xenopus TCR have also been described, including CD3γ and δ (Dzialo and Cooper,1997; Göbel et al.,2000) and TCRζ (Guselnikov et al.,2003). Co-stimulatory (CD28) and co-inhibitory receptors (CTLA4) that regulate T-cell activation as well as other receptors that include BTLA and ICOS have been identified in Xenopus (Bernard et al.,2007). Most of the B7 family members are also present in the Xenopus (Ohta, manuscript in preparation), suggesting that a similar co-signaling mechanism for T-cell activation by APCs was present before the divergence of amphibian species. So far, the X. laevis TCR repertoire has only been studied for TCRβ (Chretien et al.,1997), α, and γ (Haire et al.,2002).
In mammals, αβT-cell differentiation occurs in the thymus, and is characterized by the successive expression of certain cell surface molecules, including the coreceptors CD4 and CD8 (Anderson et al.,1996). Briefly, thymocyte maturation can be divided into three broad categories based on coreceptor surface expression: (1) an early double negative (DN) CD4−CD8− stage; (2) a predominant double positive (DP) CD4+CD8+ stage; and (3) a mature CD4+ or CD8+ single positive (SP) cell stage (Krangel et al.,2004). Immature DN thymocytes up-regulate the coreceptors following TCRβ locus rearrangement. TCRα chain rearrangement is initiated and TCR αβ heterodimers are expressed on the cell surface at the DP stage. At this point, these thymocytes become eligible for both positive and negative selection. Positive selection ensures that these T cells are able to recognize cognate MHC and hence, is termed the “MHC restriction” phase, whereas negative selection, or “tolerance,” ensures that self-reactive T cells are eliminated. Thus, the mature T-cell repertoire is established in association with self peptide-MHC molecules.
Multiple lines of evidence suggest that thymic education is fundamentally similar in mammals and Xenopus. First, several key factors involved in the regulation of T-cell development are highly conserved and expressed in the thymus in Xenopus (reviewed in Hansen and Zapata,1998; Rothenberg and Taghon,2005). These include GATA3 (Zon et al.,1991; Du Pasquier et al.,1995) and Ikaros (Hansen et al.,1997). The thymus dependency of T cells is also conserved between frogs and humans as evidenced by the severe T-cell deficiency that results from thymectomy (Tx) at an early larval stage before stem cell immigration (reviewed in Horton et al.,1998). T-cell–deficient adult X. laevis generated by early larval Tx have dramatically impaired anti-tumor responses (Robert et al.,1997) and skin allograft immunity (reviewed in Horton et al.,1998). In Tx animals, peripheral T cells are absent, whereas the percentage of B cells (Hsu and Du Pasquier,1984b) and NK cells (Horton et al.,2000) is increased; in vitro responses of Tx splenocytes to phytomitogens or alloantigens are also abrogated (reviewed in Horton et al.,1998). The active role of the thymus in T-cell selection is also supported by studies with chimeras made at embryonic stages between LG clones of different MHC haplotypes. For example, young adults generated by microsurgical fusion of the anterior one-third of a 24-hr-old tail bud embryo containing the thymus anlage with the posterior two-thirds of a MHC-mismatched embryo that contains the anlage of all hematopoietic cells, do not reject skin grafts of the thymus MHC haplotype (Flajnik et al.,1985). Although this tolerance of the thymus haplotype is not absolute (see next section), these data do suggest that there is thymic selection of the T-cell repertoire in Xenopus. In addition, the transcription factor autoimmune regulator (AIRE), that in mammals is required for expression of tissue-specific self-Ags by medullary thymic epithelial cells (reviewed in Mathis and Benoist,2007), has recently been characterized in X. laevis and S. tropicalis (Saltis et al.,2008). Finally, by using mAbs that recognizes CTX, a thymoctye differentiation pathway has been characterized in vitro in Xenopus. CTX, a homodimeric surface glycoprotein, is a marker of an immature stage that appears equivalent to the mammalian thymocyte double positive stage (Chrétien et al.,1996; Robert et al.,1997; Robert and Cohen,1998a). In this Xenopus thymocyte developmental series, cells transition from CTX+, CD8+, CD5low, CD45low to more mature CTXneg, CD5bright, CD45bright cells that can be further subdivided into CD8bright and CD8neg (Robert and Cohen,1999). The CTX gene, first identified in X. laevis, is conserved in S. tropicalis (Du Pasquier et al.,1999), and has subsequently been characterized in other vertebrates (Kong et al.,1998; Chrétien et al.,1998).
At the cellular level, the availability of mAb recognizing the CD8 co-receptor has provided an excellent opportunity to characterize this differentiated T-cell subset in more detail. CD8 T cells co-express XT-1, a pan-T-cell marker (Nagata,1985), a high level of CD5 (a T-cell marker in Xenopus; Jürgens et al.,1995) and CD45 (Barritt and Turpen,1995); these markers are not detectable in Tx frogs (Gravenor et al.,1995). Interestingly, a minor population of CD8 T cells co-express the NK cell-associated surface molecules recognized by 1F8 mAb (Rau et al.,2002), suggesting the occurrence in X. laevis as in mammals of CD8 NK-CTLs (Moretta et al.,2003). Biochemical analysis of the TCR/CD3 complex (Göbel et al.,2000) and characterization of TCRβ genes in Xenopus (Chrétien et al.,1997) also revealed features that are similar to mammals. For example, cell-mediated MHC-restricted cytotoxicity against major and minor H-Ags, characterized in vitro with whole splenocytes (Bernard et al.,1979; Watkins et al.,1988), was formally shown to be due to CD8 T cells (Robert et al.,2002). Using immunization against minor H-Ags, cytotoxicity of primed CD8 T cells purified with anti-CD8 mAb was Ag-specific and MHC-restricted, whereas only NK-like killing (e.g., killing of class I-negative tumor but not class I-expressing lymphoblast targets) was detected in CD8-depleted splenocytes. Effector capacity of CD8 T cells was further studied in vivo by developing an adoptive cell transfer in isogenetic clones of X. laevis (Maniero and Robert,2004). Splenocytes from immunized donors of the same clone but immunized against minor H-Ags were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify transferred cells as well as the extent of their proliferation in response to exposure of the recipient to the same Ags (reviewed in Robert et al.,2004). Primed anti-LG-15, but not naive CFSE+ T cells accumulated and divided in the spleen of allografted recipients to a greater extent than in those of autografted recipients. Similar accumulation and division occurred when CD8 T cells primed against 15/0 tumor Ags were transferred to an isogenetic recipient bearing the same MHC class I-negative tumor. Furthermore, the transfer of such primed anti-tumor splenocytes into naïve recipients before tumor challenge delayed the appearance of tumors.
With respect to CD8 T-cell effector function, the crucial role of CTLs in Xenopus resistance to viral infection has been demonstrated using FV3, a ranavirus implicated in diseases and mortality of wild and captive amphibian populations (reviewed in Chinchar,2002). Depletion of CD8 T cells by mAb treatment markedly increases adult susceptibility to FV3 infection (Robert et al.,2005). The anti-FV3 CD8 T cell response was further investigated in vivo using bromodeoxy-uridine (BrdU) incorporation to assess lymphocyte proliferative responses by flow cytometry (Morales and Robert,2007; reviewed in Morales and Robert,2008a). After primary infection, CD8 T cells significantly proliferated in the spleen and accumulated in infected kidneys (the main site of FV3 infection), from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide evidence of protective Ag-dependent CD8 T cell proliferation, recognition, and memory against a natural pathogen in Xenopus.
Recently, a nonconventional CD8 T-cell subset has been characterized in Xenopus. This subset recognizes and kills thymic lymphoid tumor that do not express classical MHC class I (class Ia). Therefore, by definition, this killing is not MHC classical class Ia-restricted (Goyos et. al.,2004). These CTLs (or CCU-CTL as they have been called) interact with nonclassical MHC class Ib gene products (Goyos et al.,2007). Although the 15/0 lymphoid tumor is class Ia-negative, mRNAs for both class Ib (class Ib; Robert et al.,1994, Salter-Cid et al.,1998) and β2m (Stewart et al.,2005; Goyos et al.,2007) are expressed Stable 15/0 tumor transfectants with impaired class Ib expression induced by RNA interference targeting β2m or directly class Ib are more resistant to CD8 T-cell killing and more tumorigenic (Goyos et al.,2007). This finding suggests that class Ib molecules are indeed involved in the recognition of tumor cells by nontypical CD8 cytotoxic T cells. X. laevis possesses at least 20 Xenopusnonclassical class Ib molecules (XNC) genes that have been grouped in 9 subfamilies based on sequence similarity (Flajnik et al.,1993). Two additional novel subfamilies, XNC10 and XNC11, have recently been characterized that are preferentially expressed in thymic lymphoid tumors, thymocytes and T cells (Goyos et al.,2009).
Due to the lack of specific mAbs, little is still known about CD4 T cells other than that a CD4 gene ortholog is present in the S. tropicalis genome, as are cytokines and transcription factors critical for differentiation of Th1 (e.g., T-bet; IFNγ), Th2 (e.g., GATA3; IL-4, 5, 6, 10), Treg (FOXP3; CTLA4), and Th17 (e.g., RORγ; IL-17, 6, 21, 23, TGFβ) cells (Ohta and Robert, unpublished observations). However, typical helper T-cell functions have been identified in X. laevis adults. These include T cell collaboration for B-cell response that is MHC restricted (Blomberg et al.,1980; Flajnik et al.,1985) and proliferative responses in MHC-disparate mixed lymphocyte reactions (MLR; Du Pasquier et al.,1985; Watkins et al.,1988) that are inhibited by anti-class II mAbs (Harding et al.,1993).
Very few studies exist on Xenopus cytokines and chemokines involved in T-cell function. An IL-2–like T-cell growth factors have been functionally characterized in X. laevis (Watkins et al.,1987; Watkins and Cohen,1987; Haynes and Cohen,1993b), but IL-2 awaits identification at the molecular level. Recently, the interferon γ gene has been characterized in S. tropicalis, and its expression has been shown to be induced by stimulation of splenocytes with LPS and synthetic double-stranded poly(I:C) (Qi and Nie,2008).
The Xenopus MHC was identified many years ago (Flajnik and Du Pasquier,1990; Shum et al.,1993; Sato et al.,1993), and has been extensively studied for functions and polymorphism. The MHC region is also known as the most gene-dense region; in the human genome there are ∼250 genes spanning a ∼4 Mbp region. A search of the S. tropicalis genome for MHC genes has revealed a total of 122 genes found in 8 genomic scaffolds encompassing ∼3.65Mbp. The overall genomic organization is remarkably similar to that of human MHC with some block inversions (Ohta et al.,2006). Yet, the Xenopus MHC demonstrates unique features of which some may be primordial: (1) a single MHC class I gene is tightly linked to the class I processing genes (transporter and immunoproteasome), thus defining the true “class I region,” present in all nonmammalian jawed vertebrates (Nonaka et al.,1997); (2) a strong co-evolution of class I and three other class I processing genes (proteasome subunit β type 8, transporter associated with antigen processing 1 and 2; Namikawa et al.,1995; Ohta et al.,2003; Bos and Waldman,2006a,b) that can be separated into two ancient (separated 80–100 MYA) allelic lineages. No other species demonstrates such extensive co-evolution in the class I system; (3) maintenance of the two lineages among various Xenopus species; (4) all nonclassical class Ib genes are found outside the MHC region (Courtet et al.,2001) as seen in other nonmammalian species (Juul-Madsen et al.,2000; Belov et al.,2006; Afanassieff et al.,2001); and (5) a cluster of NK-like receptors found in the class III region that may have co-evolved with MHC class I as receptor/ligand relationship (Ohta et al.,2006).
Based on morphological criteria and some markers (e.g., formalin-resistant ATPase activity, MHC class II Ag, and vimentin), putative dendritic cells and Langerhans cells have been described in Xenopus adult skin (Mescher et al.,2007). Peritoneal leukocytes (PLs) also contain putative APCs. Large numbers of PLs are obtained by peritoneal lavage of frogs 3–4 days after elicitation of a cellular exudate by injection with heat-killed E. coli (Marr et al.,2005). These elicited PLs are 50–60% monocucleated cells with multiple pseudopods typical of macrophages. Compared with PLs from untreated frogs, bacteria-elicited PLs display an increased size (i.e., high forward scatter), are enriched for nonlymphocytic leukocytes (e.g., low signal for B- and T-cell markers), and still express, albeit at a lower level, surface MHC class II, and class I. Notably, the Ag-presentation ability of these cells has been demonstrated using the minor H-Ag system provided by LG clones. Adoptive transfer of PLs pulsed with minor H-Ags complexed to the heat shock protein gp96 induces potent CD8 T-cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts through an active process involving an endocytic receptor (CD91) expressed at the APCs surface (Robert et al.,2008).
1.3. Immune Responses to Pathogens and Tumors
X. laevis and S. tropicalis have become an important and useful model to study host pathogen interaction (reviewed in Morales and Robert,2008b). The prevalence of infections and die offs caused by two pathogens: the chytrid fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses (RV, family Iridoviridae) have markedly increased over the past decade. These pathogens are postulated to be among the major causal factors associated with the world-wide amphibian decline (reviewed in Carey et al.,1999; Daszak et al.,2000; Lips et al.,2008). Xenopus is providing a realistic alternative to study anti-fugal and anti-viral defenses of natural populations of endangered amphibians (reviewed in Morales and Robert,2008b). Study of the X. laevis immune response to RVs using FV3 is already well established (Gantress et al.,2003; Robert et al.,2005). Critical involvement of CD8 T cells (Morales and Robert,2007), and antibodies (Gantress et al.,2003; Maniero et al.,2006) have been well documented. Several amphibian antimicrobial peptides in the skin are reported to inhibit Bd growth (reviewed in Rollins-Smith and Conlon,2005) as well as frog viruses including FV3 (Chinchar et al.,2004). Studies of immune responses against Bd using microarrays in S. tropicalis, a species that unlike X. laevis is susceptible to this pathogen, suggest a prominent role of innate immune responses including antimicrobial peptides (Ribas and Fischer, unpublished observations); more studies are needed to evaluate the role of adaptive immune responses in controlling Bd infection. In this regard, it could be informative to evaluate the contribution of adaptive immunity to the resistance of X. laevis by depleting B and T cells (e.g., sublethal γ-irradiation, antibody treatments).
A peculiar feature of Xenopus is that like other ectothermic vertebrates, its immune system is affected by temperature (reviewed in Hsu,1998; Meier,2003). For example, helminth clearance in X. laevis is slower at 15°C than 25°C (Jackson and Tinsley,2002). Similarly, minor H-disparate skin grafts are rejected faster at 27°C than at 21°C (Robert et al.,1995) and more slowly than MHC-disparate skin graft at 19°C (Du Pasquier et al.,1975). In addition, switch from IgM to IgY is prevented at 19°C (Wabl and Du Pasquier,1976). Likewise, in vitro T-cell proliferation in MLR or induced by mitogen (reviewed in Meier,2003), and of lymphoid thymic tumor cell lines (Robert et al.,1994), occur faster at 27°C (optimum) than at lower temperature (18–25°C). Collectively, these data suggest a selective inhibitory effect of low temperature on T-cell function in Xenopus, and as such could provide a useful model to further explore the influence of temperature on T-cell responses and particularly on MHC presentation and Ag recognition.
X. laevis has also been instrumental in revealing the conservation of immune responses to tumors. It is the only amphibian in which series of true lymphoid tumors (e.g., metastatic proliferation of genetically altered cells, and not other types of deregulated proliferation resulting from wound repair or infection) have been discovered, and cell lines that are transplantable and tumorigenic have been obtained. These lymphoid tumors have opened up new avenues for comparative tumor biology and tumor immunity (reviewed in Robert and Cohen,1998b; Goyos and Robert,2009). Four lymphoid tumor lines (e.g., B3B7, ff-2, 15/0 and 15/40) were initially derived from spontaneous thymic lymphoid tumors (Du Pasquier and Robert,1992; Robert et al.,1994); a similar spontaneous thymic tumor was also reported later independently (Earley et al., 1995). All these tumor cell lines display a unique dual immature T/B phenotype, which, in mammals, is featured only by rare lymphocytic leukemias. All Xenopus lymphoid tumor cell lines express T-cell lineage markers (CD5, CD8, XT-1) and the immature thymocyte marker CTX. They also express RAG1, RAG2, and TdT that are involved in Ig rearrangements, and have undergone extensive Ig heavy and Ig light gene rearrangements, but do not synthesize any Ig protein (Du Pasquier et al.,1995). Interestingly, ff-2 and 15/40 but not B3B7 and 15/0 cell lines express MHC class Ia molecules, whereas they all express nonclassical MHC class Ib and β2m molecules (Robert et al.,1994; Goyos et al.,2007;2009).
The tumor cell lines differ in their tumorigenicity. The ff-2 tumor cell line that was derived from the partially inbred frog strain homozygous for the f MHC haplotype, is transplantable and tumorigenic in larvae that share at least one MHC haplotype with the tumor, whereas it is rejected by adults of any genetic background (Robert et al.,1995; Robert and Cohen,1998b). Tumor rejection in adults critically depends on thymic-derived T cells, because it manifests just after metamorphosis in parallel with the T-cell renewal in the thymus and the recovery of T cell effector functions (see section 3.1), it is abrogated by sublethal γ-irradiation that preferentially deplete thymocytes (Robert et al.,1995), and it is impaired in T-cell–deficient Tx animals (Robert et al.,1997). Immune responses to tumor have been further characterized using the 15/0 tumor line, derived from the LG-15 clone, that is very tumorigenic and grows both in larval and adult histocompatible (a/c) hosts. Involvement of NK cells in antitumor response is suggested by the accelerated growth of 15/0 tumor transplanted into animals pretreated with anti-NK cell mAb (Rau et al.,2002) and by NK cell cytotoxicity toward 15/0 tumor targets in vitro (Rau et al.,2002; Goyos et al.,2004). However, evidence strongly supports the fundamental and prominent role of T cells in antitumor responses: First, transplanted 15/0 tumors can develop in T-cell deficient Tx outbreds (Goyos and Robert,2009), and growth of transplanted 15/0 tumor is accelerated in histocompatible host sublethally γ-irradiated or depleted of CD8 T cells by mAb treatment (Rau et al.,2001); Second, immunization with 15/0 tumor-derived heat shock proteins (e.g., gp96, hsp70) generates potent T-cell responses against 15/0 tumor in vivo (Robert et al.,2001a) that critically involves CD8 T cells (Maniero and Robert,2004). Gp96 and hsp70 have been shown to chaperone antigenic peptides and to elicit CD8 T cell responses against these Ags as efficiently in Xenopus as in mammals (Robert et al.,2001a,2002). Because 15/0 tumor is perfectly transplantable in LG-6 recipients that share the same MHC but differ at minor H loci with LG-15 clone (Fig. 1), this system permitted to increase T-cell response by immunizing against both tumor and minor H-Ags (Goyos et al.,2004). Further studies have revealed that T cells involved in antitumor responses in Xenopus include conventional CD8 T cells and the previously discussed unconventional CD8 T cells (CCU-CTLs) that interact with nonclassical MHC class Ib molecules (Goyos et al.,2004,2007). Parenthetically, these tumor cell lines have also been used to reveal the conserved ability of certain heat shock proteins (gp96, hsp70) to generate potent antitumor responses (Robert et al.,2001a; Maniero and Robert,2004).