Xenopus laevis Research Resource for Immunobiology

Background Information

Overview:

The University of Rochester is home to the world's most comprehensive resource specializing in the use of the amphibian Xenopus laevis for biomedical and immunological research. Several genetically-defined inbred strains and clones are available for study. The facility also maintains and develops research tools such as transgenic animals, monoclonal antibodies, cell lines, DNA libraries and molecular probes. The resource includes a satellite facility devoted to study infectious diseases caused by iridovirus. Technical assistance, education and training are also provided. The resource is supported by the National Institute of Allergy and Infectious Diseases (NIH/NIAID).

The frog X. laevis is a unique comparative model for immunobiology

Ever since it was discovered that X. laevis could easily be induced to breed in the laboratory by injecting human gonadotrophin, this anuran amphibian has become the model of choice for research in all sorts of biological fields ranging from early developmental biology and metamorphosis to behavior and immunity. In particular, evolutionary distance of X. laevis from mammals permits distinguishing species-specific adaptation from more conserved features of the immune system.

Model to study ontogeny of the immune system

Fig. 1: Two developmentally and physiologically distinct immune systems coexist in the same species

Unlike mammals, the Xenopus immune system undergoes striking developmental changes twice during its life (Fig 1): first during embryogenesis, and then again during the metamorphosis (Flajnik al., 1987). The thymus, first colonized by embryonic stem cells a few days after fertilization, undergoes a second wave of stem cell immigration after losing about 90% of its lymphocytes during metamorphosis. The embryonic and larval periods of thymocyte differentiation take place in different environments, since during metamorphosis the whole organism is remodeled and many new proteins are expressed that could be considered antigenic by the larval immune system. The emerging adult lymphocytes, therefore, are likely to be subjected to a new education by the adult "self," resulting in a new balance of self-tolerance. Despite the drastic remodeling, a long lasting immunological memory persists through metamorphosis for B and T cell antigens.

Table 1: Comparative overview of the immune system capacity of larval and adult *X. laevis*
Immune System Characteristics	Larva (Ancestral-like system)	Adult (Mammalian-like system)
Thymus-dependent functions
MLR	poor	Better
CTL	Not demonstrated	Yes (MHC-restricted)
IgM to IgY switch	Poor	Yes
Rejection of MHC identical but minor H-antigen disparate skin grafts	Incomplete (tolerance)	Acute
MHC classical class I	Absent (cell surface and mRNA)	Present
Non-classical class Ib	No mRNA detected	Present
CD8+ T-cell	Present	Present
NK cell and NK activity	Present at late stages	Present
Tumor Immunity
Tumor Ag recognition	Yes	Yes
Anti-tumor effector	Weak	Stronger
Hsp immunogenicity	Yes but not peptide specific	Yes, peptide-specific

X. laevis adults display an efficient immune system (Table 1) very similar to mammals (e.g. rearranging TCR and Ig genes, as well as MHC class I- and class II-restricted T cell recognition; Du Pasquier et al., 1989), whereas the larval presents some deficiencies such as a poor switch from IgM to IgY (Xenopus IgG functional equivalent), a specific tolerogenic rather than allodestructive response to minor histocompatibility antigens, and an inefficient anti-tumor effector system. Both MHC class I and class II genes are differentially regulated during metamorphosis. Consistent expression of class I does not occur until metamorphosis; class II expression is restricted in larvae to thymic epithelium, peripheral B and accessory cells. After metamorphosis, class II antigens are expressed constitutively on virtually all thymocytes and mature peripheral T- as well as B-cells. The MHC class I-deficient larva constitutes a naturally ocurring "knockout".

Reagents (Monoclonal antibodies, DNA probes, cDNA and genomc libraries)

Table 2: Expression pattern of *X. laevis* surface markers detectable with currently available mAbs.
Markers (mAbs)	Expression pattern
CD8 (AM22, F17)	Larval and adult thymocytes (70-80%) and T-cells (about 20% of splenocytes). All lymphoid tumor lines.
CTX (X71, 1S9.2)	Larval and adult thymocytes (60-70%); no consistent expression in peripheral lymphocytes. All lymphoid tumor lines, gut epithelial tissue.
XT1 (XT1)	Most, but not all, larval and adult T-cells; earliest marker of thymocytes. All lymphoid tumor lines
MHC class I (TB17)	Ubiquitous in adult. Not consistent expression until metamorphosis.
MHC class II (AM20, 14A2)	Thymocytes, B and T-cells (99% of spleen lymphocytes), only B-cells in larvae.
CD5 (2B1)	Thymocytes (>95%), T-cells and some activated IgM+ B cells. All lymphoid tumor lines.
CD45 (CL21)	T and B cells. All lymphoid tumor lines.
NK-like (1F8)	Non-B and non-T, peripheral lymphoid cells.
RC47	Leukocyte lineage from very early stage. Thymic cortex and medulla (>90% of total thymocytes).
IgM (10A9, 6.16)	Larval and adult B cells.
IgY (11D5)	Some larval and adult B cells.
IgX (410D9)	Some larval and adult B cells, especially in the gut.
Light chain (1E9, 13B2)	Larval and adult B cells.
*No mAbs specific for CD4 or TCR have been described so far.

MAbs (Table 2) allowing the phenotypic characterization of various leukocyte populations include: markers of thymocytes and peripheral T-cell subpopulations such as the pan T cell XT-1 mAb (Nagata, 1985); two mAbs against a CD8-equivalent T cell co-receptor (Flajnik et al., 1991; Ibrahim et al, 1991); a mAb that recognizes a CD5 epitope (Jürgens et al., 1995), and another that detects a CD45-like molecule (Barritt & Turpen, 1995). All these mAb reagents are available in our laboratory, as are mAbs that detect MHC class I and class II molecules (Flajnik et al., 1990; 1991).

The mAb 1F8 identifies a surface protein specifically expressed by a NK-like lymphoid population (Horton et al., 2000) that remains present in T-cell deficient frogs (thymectomized) and that kill MHC class I-negative tumor targets. NK cells emerge as a small population in the spleen of metamorphosing larvae one week after the first appearance of surface MHC class Ia (Horton et al., 2003).

Fig. 2: Immunochemistry microscopy on frozen spleen section of at low magnification from a Xenopus juvenile double stained for IgM+ (blue) and proliferating BrdU+ (brown) cells. (From Du Pasquier et al., 2000)

Three immunoglobulin isotypes have been described in X. laevis (Hsu, 1998; Du Pasquier et al., 2000): IgM, IgY and IgX. X. laevis IgM is clearly homologous to mammalian IgM. IgY is believed to be a modern equivalent of an ancient protein that gave rise to mammalian IgE and IgG. IgX is functionally analogous to mammalian IgA but structurally is more similar to X. laevis IgM (Mussmann et al., 1996). IgM and IgX appear to be thymus independent isotypes, whereas IgY is produced during T-dependent immune responses. Mabs specific for the three different heavy chains as well as for Light chain isotypes have been characterized (Table 1, Bleicher & Cohen, 1981; Hsu et al., 1984). The various X. laevis cDNA libraries available include: those from adult, metamorphic, and larval total splenocytes (from naïve and immunized animals), those form purified adult NK and CD8 T cell population, and those from the 15/0 and ff-2 lymphoid tumors. Finally, there is available a whole variety of cDNA clones of immunologically relevant genes useful to generate molecular probes. EST and BAC libraries are also available (ref)

Inbred MHC-defined strains, and isogenetic clones (gynogenesis)

Table 3: List of *X. laevis* strains and isogenetic clones
	Name (MHC genotypes)
Partially Inbred, MHC homozygous strains	F, J, A8(r/r), K, G
Isogenetic X. laevis/gilli (LG) clones with identical heterozygous (a/c) MHC but different minor H genotype	LG-6 LG- 7 LG-15
MHC-disparate LG isogenetic clones	LG-3 (b/d), LG-5 (b/c)
Isogenetic X. laevis/mulleri (LM) clones	LM3 (w/y)

Our laboratory maintains several inbred X. laevis with different MHC homozygous genotype, as well as several different MHC-defined isogenetic clones (Table 3). These clones were generated by gynogenesis between X. laevis and X. gilli or X. mueleri (Kobel & Du Pasquier, 1975). These clones and inbred strains permit classic adoptive transfer and transplantation manipulations as in mice (Robert et al., 2004). Unlike mice, they also permit transfer of tissues and cells between larva and adult (Robert et al., 2004).

Natural and artificial polyploidy (2n, 3n, 4n, 5n, 8n, 12n)

The Xenopus genus includes various tetra-, octo- and dodecaploid species (Table 4) that were generated times over a period ranging from 80 to 10 millions years ago by interspecies hybridization through allopolyploidization (Evans et al., 2004) [11, 12]. These species offer a unique model to study the consequences of whole genome duplication (i.e., study the fate of duplicated genes). In addition, polyploidy animals can be generated easily in laboratory providing an ideal genetic marker. X. laevis tetraploid are produced artificially by subjecting diploid egg from isogenetic clones to hydrostatic pressure during the last meiotic division (Kobel, al., 1970; Chrétien al., 1996). Tetraploid cells can be distinguished from diploid cells by differences in their DNA content as well as by the number of nucleolar organizers. After surface staining with various mAbs and an APC-conjugated secondary reagent, cells are fixed and stained with propidium iodide to detect by flow cytometry differences in DNA amounts (Chrétien al., 1996; Turpen 2004). By using isogenetic clones rather than inbred strains to generate tetraploids, the progeny can be directly maintained by gynogenesis

Table 4: List of Existing Xenopus Species

2N (20)	4N (36)	8N (72)	12N (108)
Xenopus (Silurana) tropicalis	X. mulleri X. borealis X. clivii X. fraseri x. gilli¹	X. vestitus X. amieti X. andrei² X. vitei²	X. ruwenzoriensis X. longipes²
1 Species almost extinct, a few individuals protected in South Africa (Capetown) 2 Species rare, a few live specimens are in Geneva, Switzerland

Embryonic, fibroblast cell lines and lymphoid tumor cell lines

X. laevis is the only amphibian where true lymphoid tumors have been discovered and cell lines have been obtained opening new avenues for tumor biology and tumor immunity. These lymphoid tumor cell lines have been extensively characterized (Table 1; Robert al., 1994). They display a unique dual T/B phenotype, which, in mammals, is encountered only in rare lymphocytic leukemias.

Table 5: Characteristics of four different X. laevis cloned thymic tumor cell lines

Name of tumor cell line	BB7	ff-2	15/0	15/40
Genetic background of initial tumor-bearing host	Partially inbred MHC homozygous ff strain		LG-15 (MHC a/c) isogenetic clone
T-cell surface markers (CD8, CD5, XT-1 pan T-cell) and Thymocyte surface marker CTX	+++	+++	+++	+++
Ig mRNA	-	+	+	+
Ig protein	-	-	-	-
TdT, Rag 1 and 2 Expression	+	+	+	+
MHC class I mRNA and Protein	-	+	-	+
MHC class II mRNA and protein	-	-	-	+
Non-classical class Ib mRNAs	+	+	+	+
b2-microglobulin mRNA	+	+	+	+
Tumorigenicity in syngeneic Larvae Adult	- -	+ -	++ ++	+ ++

All these cell lines express specific T-cell lineage markers (CD8, CD5 and XT-1 pan T-cell), and immature thymocytes CTX marker. They also express Rag 1, Rag 2, and TdT that are involved in Ig and TCR rearrangements, and as such, are features of undifferentiated lymphocytes. Except for the B3B3 line that has undergone unproductive gene rearrangement, all other lines express IgH and L mRNAs but do not synthesize any Ig protein (Robert et al., 1994). B3B7, 15/0 do not express MHC class I and class II mRNA or protein. However, all these different cell lines do express non-classical MHC class Ib (XNC) and b2-microglobulin mRNA.

Fig.3: X. laevis LG15 cloned adult injected sc with 10,000 15/0 tumor cells. A solid tumor developed at the site of injection after 1 month (red arrow). The frog was also grafted with white ventral skin from a syngeneic donor (black arrow).

Some of these tumor lines (15/50, 15/40 and ff-2) remain tumorigenic when transplanted in MHC compatible hosts. The 15/0 tumor line derived from the LG-15 clone (MHC a/c haplotype), grows well in LG-15 (Fig. 3) and other LG sharing the same MHC (LG6 and 7). In contrast the ff-2 tumor line grows well in F larvae but is rejected by adults. This rejection results from a thymus-dependent immune response that develops during metamorphosis.

Other types of cell lines are also available in X. laevis including: fibroblasts lines of the different strains and LG clones, and a kidney cells line (A6).

Thymectomy, generation of T-cell deficient frogs

Fig. 4: Absence of thymus (T) in thymectomized (Tx) larvae. Kindly provided by J. Horton (U.K.)

T cell-deficient adult X. laevis can be generated by thymectomy (Tx) at an early larval stage before stem cell immigration (review in Horton et al., 1998b). Tx adults have dramatically impaired anti-tumor responses (Robert et al., 1997) and skin allograft immunity (Horton et al., 1998b). In Tx animals, peripheral T cells (PBL and splenocytes) are absent whereas the percentage of B cells (Hsu & Du Pasquier, 1984) and NK cells (Horton et al. 2000) are increased; in vitro responses of Tx splenocytes to phytomitogens or alloantigens are also abrogated (Horton et al., 1998). Tx adult frogs are instrumental in assessing the importance of T cells in X. laevis anti-tumor and anti-viral immunity

Aneuploidy and in situ hybridization techniques

Fig. 5: Triple stain with the class II a and b and the class Ib (non-classical XNC class I genes) on the B3B7 tumor cell line chromosomes. From Courtet et al., (2001)

X laevis is the only species where aneuploid animal can be generated for studying the segregation of functions linked to a specific chromosome (Kobel et al., 1979). In situ hybridization techniques are now available both for chromosome (Courtet et al., 2001) and for whole-mount embryos (Pollet et al., 2003).

Current transgenic technology

Fig. 6: “Plucky” a X. laevis transgenic albino expressing the green fluorescent protein (GFP) in her eye. Kindly provided by D. Papermmaster http://www.uchc.edu/dsp/plucky.html)

Known as restriction enzyme-mediated insertion (REMI), this method involves mixing transgene DNA with purified and permeabilized sperm, along with a small amount of restriction enzyme and a high-speed interphase egg extract. This mixture is then injected into unfertilized dejellied eggs (Kroll & Amaya, 1996). The extract partially decondenses the sperm chromatin but does not promote replication and the restriction enzyme stimulates recombination and integration by creating double stranded breaks in the sperm chromatin. This technique permits large-scale transgenesis in X. laevis embryos. Unlike embryos injected with plasmids, the transgenic embryos show correct spatial and temporal regulation of integrated promoter constructs. It is generally considered that one of the great advantages of this system in X. laevis over transgenesis in mice or zebrafish is that the transgene is usually integrated into the male genome prior to fertilization. Therefore, the resulting embryos are not chimeric and breeding of animals is not required. However, exchange of genetic material between male and female genomes as well as multiple gene insertions have been observed (Kobel & Du Pasquier, pers, comm.)

Thymus/lymphocyte embryonic chimeras

Fig. 7: Chimeras made by exchanging the anterior and posterior regions of two embryos at 24 hrs after fertilization. One embryo was from an A8 albino (white skin and red eyes) strain and the other embryo was from a J (green) strain. From Basel Institute for Immunology Annual Report 1982

Another unique advantage of X. laevis for experimental immunology is the possibility to manipulate frog embryos very early in development to generate chimeras. For example, the anterior one third of a 24-h-old tail bud embryos containing the thymus anlagen can be fused by microsurgery to the posterior two-thirds of a MHC-mismatched embryos that contains the enlagen of all hematopoietic cells (Flajnik et al., 1985). Chimeras can also be made between embryos of different ploidy to follow the fate of a particular precursor population during development (Turpen, 1998). In summary, chimeras provide an excellent in vivo experimental system to study hematopoiesis, thymic education, tolerance and MHC restriction.

Vivarium

Photos of one room with bank of tanks (left) and another room with 50 gallons tanks for larger number of animals (lower right). Each room is equipped with temperature-controlled continuous flow water system that supplies temperature-adjusted water drip-wise, via a stopcock, to each of these aquariums