Xenopus laevis Research Resource for Immunobiology

Xenopus laevis
Research Resource for Immunobiology funded by NIAID

Background Information

This Research Resource is directed by:

Jacques Robert
Associate Professor
Microbiology & Immunology
585.275.5359

Email:
jacques_robert@urmc.rochester.edu

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.
(see Robert and Ohta 2009 for a recent review)

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
Classical MHC class Ia	Absent on the cell surface	Present
Nonclassical MHC class Ib mRNAs	Present	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
Viral immunity	Weak	Stronger

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.
XL-1	All leukocytes from early larval stage, ventral blood island stage 35
XL-2	All leukocytes from early embryonic stage 24. [135 Kd]
*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), a thymocyte specific marker CTX recognized by X71 and 1S9.2 mAbs (Chretien et al., 1996a; Robert et al., 1997) 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 (http://xlaevis.cpsc.ucalgary.ca/common/index.do)

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) Evans 2007. 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 cold shock or hydrostatic pressure during the last meiotic division (Kobel, al., 1975 (not 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. laevis	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	+	+	+	+
β2-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 β2-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 (15/0, 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, 1984b) 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.)

Generation of transgenic clones

Image of X. laevis clone with "Sleeping Beauty" transposase

Fig 7: Generation of transgenic isogenetic X. laevis clone with the "Sleeping Beauty" transposase. Dejellied LG-6 eggs were co-injected with 10² ng of transposase mRNA and 15 ng in 10mL volume of vector containing a GFP reporter transgene under the control of the X. laevis Ef-1α promoter. Live larvae were screened for GFP expression with a fluorescence stereomicroscope at a pre-metamorphic stage (st 56, 1 month old). Arrows: thymus

We are adapting transgenic (Tg) technology to our X. laevis clones (e.g., LG-6, LG-15) for immunologoical studies (reviewed in Robert et al., 2009). The advantage of using these clones is that they are MHC defined. Furthermore, since progeny from clonerd Tg founders are produced by gynogenesis (UV-irradiated spermatozoa are used to activate diploid eggs and do not contribute any genetic material to the offspring), no time consuming screening is required. Three different Tg techniques that allow the direct insertion of transgenes into X. laevis genomic DNA are currently used in our lab: the φC31 integrase (Allen & Weeks,2005), the I-Sce meganuclease (Pan et al., 2006; Ogino et al.., 2006), and the transpossae "Sleeping Beauty" (Sinzelle et al., 2006). These techniques are used to express reporter GFP genes under the regulation of immunologically relevant genes, over-expressing or knocking down by siRNA immunologically relevant genes.

Skin graft rejection & whole-mount histology

Skin graft rejection is a well-established technique in X. laevis to determine the segregation of both major and minor H-antigen (Ag) differences (Du Pasquier & Bernard, 1980; Ramanayake et al., 2007). Briefly, a piece of ventral skin (5x5mm) from a donor is inserted under the dorsal skin of a recipient and 24 hrs later the overlying host skin is removed. The time for complete skin rejection is determined by the time required for the destruction of all silvery iridophore pigment cells in the grafted skin (Fig. 8). At 21-22ºC, a recipient rejects donor skin displaying a 1 or 2 MHC haplotype mismatch subacutely within 18-22 days in X. laevis. In contrast, skin grafts from MHC identical donors that differ only by minor H-Ags are rejected more slowly (from 30 to 200 or more days). No rejection at all occurs when donor and recipient are genetically identical (e.g., fully inbred). This technique, therefore, is very powerful for both characterizing MHC combination and the degree of inbreeding, with the advantage of keeping both the donor and the recipient alive. It is also a very convenient method to assess T cell responses. For example, priming LG-6 with the heat-shock protein gp96 derived from minor H-Ag-disparate LG-15 tissue induces an accelerated rejection of LG-15 skin graft as compare to control recipient injected with cognate LG-15-derived gp96 or unprimed control (Robert et al., 2004). Whereas adult MHC-disparate skin grafts are rejected by larvae, minor H-Ag-disparate skin grafts are fully accepted (Fig. 6D), and remain permanently in the adult recipient after metamorphosis. This allotolerance to adult skin graft from minor H-Ags mismatched donor is Ag-specific since it induces permanent survival of a second-set skin graft from the same H-Ag mismatched donor that is grafted after metamorphosis but not a third-set skin graft from a different minor H-Ag-disparate donor (reviewed in Robert and Ohta, 2009 )

Fig 6: Analysis of skin graft rejection 12 days post-transplantation by stereomicroscopy.
X. laevis adult recipient MHC-homozygous inbred of the J strain received a skin graft from either a MHC-identical (A, B) or a MHC-disparate outbred donor (C). Note the disappearance of the silvery pigments in the MHC-disparate rejected, but not MHC identical skin graft (Arrow). (D) Absence of rejection of adult LG-6 skin graft (arrows) by a pre-metamorphic LG-15 larva that differs at multiple minor histocompatibility loci.

To better define the effector cells involved in the immune response to skin allo-antigens of X. laevis, we have adapted a whole-mount immunohistology procedure used in mice that enables us to visualize leukocyte infiltration into unfixed transplanted skin tissues using fluorescent antibodies (Ramanayake et al., 2007). This is a very convenient, powerful and simple technique. It does not require fixation, clearing or embedding, and, therefore, it preserves inherent tissue architecture and protein conformation, including antigenic determinants. The skin graft surgically removed at different time following grafting, allows one to monitor in detail different leukocyte populations by direct incubation with X. laevis-specific mAbs, such as anti-CD8 mAb (Fig. 9).

Image collage of Leukocyte infiltration in skin graft.

Fig 9: Leukocyte infiltration in skin graft by whole-mount immuno-histology.

A) Leukocyte infiltration in the vicinity of blood vessels (*) of MHC-disparate X. laevis skin undergoing rejection, 7 days post-transplantation stained with anti-class II mAb. Scale bar = 200 microns. (B) Magnification of the boxed area. Scale bar = 50 microns
(adapted from Ramanayake et al., 2007)

(B, C) CD8 T cell infiltration in minor H-Ag-disparate skin undergoing rejection. Whole-mount immunohistology of LG-6 skin tissues 12 days posttransplantation stained for mouse IgM isotype control (B) or X. laevis-specific anti-CD8 mAb AM22 followed by a PE-conjugated mouse mu-specific Ab (C). LG-6 skins were grafted onto LG-15 recipients 1 wk after adoptive transfer of LG-15 PLs (500,000) that were pulsed 1 h with 800 ng of LG-6-derived gp96. Whole-mount immunohistology of each transplant was performed 12 days postgrafting (30-35% rejection). Scale bar, 50 microns
(adapted from Robert et al. 2008)

Thymus/lymphocyte embryonic chimeras

Fig. 8: 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

Our animal resource facility include four newly renovated rooms in the vivarium that are designated for maintaining experimentally manipulated amphibians and for maintaining and breeding stocks of outbred, partially inbred, and cloned lines of Xenopus. Rooms are controlled for light cycle and temperature and have filtered and declorinated water "on tap". Room one (A) houses two four-tier racks; each rack holds holding 24 aquariums (each measuring 23" x 15" x 8.5" (length x width x depth)). A temperature-controlled continuous flow water system supplies temperature-adjusted water drip-wise, via a stopcock, to each of these aquariums. The flow system is equipped with thermal sensors that automatically shut off the flow of water if the temperature deviates from preset values. Room two (B) houses 10 50-gallon aquariums; a photo of some of these large aquariums is shown below. Room three houses mouse cage racks with standard shoebox "cages" for keeping individual or small numbers of frogs. Room four (C, D) is mainly devoted in rising larvae in large tanks. A small area with a table and stereomicroscope in this will allow the selection of diploid eggs after gynogenesis, and minor animal surgery. A satellite facility, outside the main vivarium, is devoted to studying an emerging infectious disease caused by ranaviruses (Iridoviridae). This facility can house up to 500 animals in containment.

All animal facilities and programs are responsible to the vivarium and Division of Laboratory Animal Medicine of the School of Medicine and Dentistry and are fully accredited by the American Association for Accreditation of Laboratory Animal Care and in compliance with state law, federal statute and Federal Policy.