Reprints Available Directly from the Publisher Photocopying Permitted by License Only Further Characterization of an Interleukin-2-1ike Cytokine Produced by Xenopus Laevis T Lymphocytes

A T-cell growth factor (TCGF) is produced by antigen-or mitogen-stimulated T lymphocytes from the South African clawed frog Xenopus laevis. This study further defines the physical and biological properties of this cytokine and demonstrates that TCGF is biochemically similar to mammalian interleukin-2 (IL-2). Biologically active TCGF eluted from SDS-PAGE displays a Mr of 16 kD and lectin-affinity chromatography indicates that the three-dimensionmal configuration of carbohydrates on TCGF and human IL-2 is similar. Secretion of TCGF is detectable 1 day after stimulation of splenocytes with the T-cell mitogen phytohemagglutinin (PHA) and peaks following 2 to 3 days of stimulation. Finally, despite the biological and physical similarities between Xenopus TCGF and mammalian IL-2, anti-human IL-2 monoclonal antibodies do not recognize Xenopus TCGF.


INTRODUCTION
Interleukin-2 (IL-2), one of numerous cytokines released during an immune response, is produced upon stimulatign of T cells with antigen (or mitogen) and interleukin-1 (IL-1). Interaction of antigen with the T-cell receptor also induces the expression of IL-2 receptors, thus ensuring antigen-specific T-cell activation (Oppenheim and Gery, 1982). Although IL-2 is primarily associated with T-cell proliferation, it can also directly or indirectly play a role in the proliferation and maturation of other cells. For example, it can induce growth of and immunoglobulin secretion by B cells, evoke superoxide production by activated macrophages, and increase the cytotoxic activity of natural killer cells (Harada et al., 1987;Wahl et al., 1987;Kehrl et al., 1988).
Human IL-2 is secreted as a single polypeptide of 133 amino acids with a molecular mass (Mr) of 15.5 kilodaltons (kD) and a single intrachain disulfide bond. The apparent molecular mass heterogeneity reported in early studies results from variable glycosylation and sialylation of the molecule (Robb et al., 1984). IL-2 genes have been *Corresponding author. cloned from a number of mammalian species including humans (Taniguchi et al., 1983), gibbon (Chen et al., 1985), mice (Yokota et al., 1985), sheep (Seow et al., 1990), pigs (Goodall et al., 1991), and cows (Cerritti et al., 1986), and the molecules they encode exhibit an overall amino acid sequence identity of 50 to 95%. Lymphocytederived culture supernatants containing IL-2-1ike activity have also been described in nonmammalian vertebrates such as fish, frogs, and birds (Cohen and Haynes, 1991).
The anuran amphibian Xenopus laevis (South African clawed frog) has an immune system that, in many ways, is comparable to that of mammals. It displays polymorphic class I and class II major histocompatibility complex (MHC) molecules that are involved in allograft rejection, mixed lymphocyte reactions, T-B-cell cooperation and restricted T-cell proliferation, and cytotoxic killing Harding et al., 1993). Additionally, Xenopus splenocytes produce an IL-2-1ike TCGF (Watkins and Cohen, 1987a). Xenopus TCGF is produced by T cells and, like mammalian IL-2, promotes growth of activated, but not resting, splenocytes and thymocytes. TCGF is also necessary for the long-term culture of Xenopus T-cell lines. There is no apparent in vitro crossreactivity between human or mouse IL-232 L. HAYNES AND N. COHEN 2 and Xenopus TCGF (Watkins and Cohen, 1987a) despite their similar biological properties. This study further defines the physical and biological properties of Xenopus TCGF and shows that it displays characteristics comparable to mammalian IL-2. Both the molecular mass of TCGF and its pattern of glycosylation are similar to mammalian IL-2. TCGF production is also similar to mammalian IL-2 in that it is only produced by activated cells (Watkins and Cohen, 1987a) and can be detected in supernatants 1 day after initial stimulation. Finally, even though Xenopus TCGF and mammalian IL-2 seem to be homologous, a panel of anti-human IL-2 monoclonal antibodies do not react with Xenopus TCGF.

Biologically Active TCGF Can Be Eluted from SDS-PAGE
To determine the Mr of the biologically active TCGF protein(s), a saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS-TCGF SN) was run on a 12% nonreducing SDS-PAGE. The gel was then cut into slices, eluted into medium, and assayed for biological activity on splenic blasts. The results of a representative experiment are shown in Fig. 1. Significant biological activity is found in the eluent from slice 9 (Mr >14 and <21 kD), which is similar to human IL-2 (14.6 to 16.2 kD for naturally glycosylated IL-2). By using this method, 40% of the activity found in the starting supernatant (SN) was recovered.
To determine the Mr of the biologically active molecule(s), eluent from a gel slice containing proteins between 14 and 21 kD was iodinated with 125I, run on a 12% reducing SDS-PAGE, and autoradiographed. As shown in Fig. 2 Representative autoradiograph of 12SI-labeled biologically active gel eluent run on a 12% reducing SDS-PAGE. 233 lus lectin (LPA) conjugated to agarose beads was performed with the SAS-TCGF SN. WGA is specific for N-acetyl glucosamine and binds human IL-2; LPA is specific for neuraminic acid and does not bind human IL-2 (Clark-Lewis and Schrader, 1982). SAS-TCGF SN was applied to each column and incubated on a rocker platform for 60 min at room temperature. Unbound protein was washed off the column and bound protein was specifically eluted with the appropriate buffer. Adherent fractions were concentrated and assayed on splenic blasts. The WGA adherent fraction exhibited biological activity on splenic blasts that was comparable to SAS-TCGF SN (Fig. 4A). The LPA adherent fraction exhibited minimal biological activity (Fig. 4B). Both nonadherent fractions exhibited biological activity (data not shown). It is not known whether this resulted from overloading of the column or the presence of growth factor(s) that cannot bind the lectin. These results with Xenopus TCGF are similar to those found with human IL-2 (Clark-Lewis and Schrader, 1982), most likely indicating a similar pattern of glycosylation.

Lectin Affinity Chromatography of SAS TCGF SN
To determine which protein(s) in the SAS TCGF SN were binding to WGA and LPA lectins, affinity chromatography was performed (as in Fig. 4) with 35S-methionine-labeled PHA SN. Both the nonadherent (non) and adherent (ad) fractions from the LPA and WGA columns were run on a 12% reducing SDS-PAGE and autoradiographed (Fig. 5 (Shoemaker et al., 1984) (data not shown). The LPA ad fraction contains the 14-kD band and very faint IgY heavy and light chains. The WGA ad fraction contains the 16-kD putative TCGF protein along with 29and 42-kD proteins.
Tunicamycin Treatment Human IL-2 has an O-linked glycosylation on threonine in amino acid position 3 (Robbet al., 1984). In order to determine if Xenopus TCGF is similarly glycosylated, the antibiotic tunicamycin (TM), which blocks the addition of N-linked but has no effect on O-linked glycosylations (Mahoney and Duskin, 1979), was used. 35S-met labeled PHA SNs were generated from freshly harvested lymphocytes incubated with PHA with or without 5/g/ml TM, run on a 12% reducing SDS-PAGE, and autoradiographed.   shows that TM has no effect on the 16-kD Xenopus TCGF is very similar in its physical and biological characterstics to human IL-2, antihuman IL-2 MoAbs (anti-HuIL-2 MoAbs) were used to try to immunoprecipitate TCGF activity from PHA-induced supernatants. Figure 7A shows that the immunoprecipitated Xenopus supernatants exhibit stimulatory activity on Xenopu s thymic blasts that is comparable to untreated supernatant; Fig. 7B reveals that the DISCUSSION Cytokines are low-molecular-weight peptides that are produced during the course of an immune response and are involved in regulating the intensity and duration of the response (Oppenheim et al., 1991). Some nonmammalian vertebrates can exhibit immune system characteristics similar to mammals (Du ), so it is not surprising that these animals also produce cytokines that are analogous to those of mammals. In fact, T-cell stimulating factors have been described in mitogen-or alloantigeninduced supernatants from fish, frog, and chicken leukocytes (Cohen and Haynes, 1991).
In this report, we have further characterized an IL-2-1ike molecule from the anuran amphibian, Xenopus laevis. Earlier experiments showed that supernatants from splenocytes stimulated with T-cell mitogens, alloantigens, or phorbol esters promoted growth of T-cell blasts and alloreactive Xenopus T-cell lines (Watkins, 1985;Watkins et al., 1988), and that TCGF was produced by T cells. TCGF was shown to promote growth of activated, but not resting, adult splenic and thymic T cells (using a monoclonal anti-T-cell antibody developed by Nagata [1985]) and activity could be absorbed by splenic or thymic lymphoblasts but not freshly harvested thymocytes or splenocytes (Watkins and Cohen, 1987b). Additionally, Xenopus PHA-induced supernatants are capable of stimulating activated Xenopus B cells to proliferate; therefore, it can also function as a B-cell growth factor (Cohen and Haynes, 1991).
The experiments described in this paper demonstrate, by SDS-PAGE analysis, that Xenopus TCGF is a secreted peptide of 16 kD. Experiments with lectin affinity chromatography also showed that TCGF binds WGA, much like human IL-2, which has variable O-linked sialylation on the threonine in position three (Robb et al., 1984). It is likely that the carbohydrates on Xenopus TCGF are O-linked because tunicamycin, which blocks the addition of N-linked glycosylations (Mahoney and Duskin, 1979), has no effect on TCGF Mr. Additionally, TCGF can be detected in radiolabeled SNs 1 day after initial stimulation; secretion peaks at day 3 and begins to decline by day 4 (data not shown).
Although Xenopus TCGF and mammalian IL-2 appear biochemically and functionally homologous, Xenopus supernatant has no activity on human lymphoblasts or murine HT2 cells and human and murine supernatants and HurIL-2 have no activity on Xenopus blasts (Watkins and Cohen, 1987a). Neither has crossreactivity been detected between fish and avian supernatants and mammalian IL-2 (Cohen and Haynes, 1991). Although TCGF preparations also contain B-cell growth-factor activity (Cohen and Haynes, 1991), we have been unable to detect functional crossreactivity between Xenopus TCGF and murine IL-4 using the IL-4-dependent cell line CT.4S (Hu-Li et al., 1989) kindly provided by Dr. W. E. Paul at the NIH (data not shown). These results are not surprising because fish, anuran amphibian and chicken immunoglobulins, and MHC molecules also exhibit structural but not functional homology to their mammalian counterparts (Hashimoto et al., 1990;Flajnik et al., 1991, Litman et al., 1991Miller, 1991). Experiments described in this report also show that several mouse anti-HuIL-2 MoAbs do not bind to Xenopus TCGF and, therefore, would not be useful in purification of the TCGF molecule.
The information compiled in this study will be useful in cloning the gene that encodes Xenopus TCGF. Because TCGF and IL-2 appear to be biochemically similar, the polymerase chain reaction (PCR) might be employed using oligonucleotide primers derived from the conserved regions of known mammalian IL-2 genes. Alternatively, primers can be derived from the TCGF amino acid sequence, once it is obtained. Attempts to sequence the biologically active gel eluent yielded very little sequence data, indicating that the N-terminal may be blocked. As was the case with mammalian interleukins, the exact nature of TCGF will not be known until the gene coding for the biologically active protein has been cloned.

Animals
Adult female Xenopus laevis were purchased from Xenopus I (Ann Arbor, MI) or the African Xenopus Facility (Noordhoek, South Africa). Juvenile Xenopus (<1 year old) were bred and maintained in our laboratory according to standardized procedures (Nieuwkoop and Faber, 1967).
Production of TCGF-Containing Supernatants Supernatants (SN) were generated as previously described (Watkins and Cohen, 1987a). Briefly, splenocytes from 50 to 70 animals were incubated at 5X10 6 cells/ml in complete medium with 0.25% bovine serum albumin (BSA) and 1/g/ml PHA-P (Sigma) or 1 to 2/g/ml PHA conjugated to agarose beads (Sigma) and the 24 hr and 48 hr supernatants were collected. Soluble PHA was removed from the supernatants by absorption with chicken erythrocytes; PHA beads were removed by centrifugation. The resulting SN was precipitated with saturated ammonium sulfate (SAS-TCGF SN), dissolved in 10% of the original volume, dialyzed (Spectra/Por membrane, Mr cut off 6,000-8,000) with amphibian PBS (APBS) and sterile filtered before use.
Assay for TCGF Activity TCGF activity was assyaed on splenic or thymic blasts in a 3-day 3H-thymidine incorporated assay. One hundred microliters of the samples to be tested were incubated with 5x104 blasts (in 100/1 complete L-15 with 1% FBS) in 96-well round-bottom plates (Costar). After 48hr, 1 /Ci/well 3H-thymidine (Amersham, Arlington Heights, IL) was added. The cultures were harvested after 72 hr and processed for liquid scintillation spectrometry. All cultures were plated in triplicate and the data are presented as the mean counts per minute (CPM)+SE.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Twelve percent reducing gels were prepared, run as described by Laemmli (1970) in a vertical gel slab unit (Hoefer Scientific, San Francisco) and stained with 0.25% Coomassie blue. To prepare autoradiographs, gels were soaked for 1 hr in Autofluor (National Diagnostics, Manville, NJ) and dried. The dried gels were exposed to X-ray film (Kodak X-Omat AR film) at-70C until the desired intensity was achieved.
Elution of Biologically Active TCGF from SDS-PAGE SAS-TCGF SN (0.5 ml) was run on a 10 or 12% nonreducing SDS-PAGE. Prestained rainbow protein molecular weight markers (Amersham) were used to eliminate the Coomassie staining step. The gel was cut into 8-mm slices and eluted into complete L15 with 0.25% BSA for 48 hr. Each sample was then dialyzed in APBS, concentrated using Centricon Microconcentrators with a Mr cutoff of 10,000, and resuspended in a final volume of 0.5 ml L15. Each fraction was assayed for TCGF activity as described before.
turer's instructions. Adherent fractions were concentrated using Centricon Microconcentrators with a Mr cutoff of 10,000, sterile filtered, and assayed for biological activity. LPA and WGA affinity chromatography was also performed using 24hr 35S-labeled PHA supernatants. Two hundred-microliter packed beads, 500-/zl binding buffer, and 100-/tl 35Slabeled PHA supernatant were incubated on a rotator overnight at room temperature. The nonadherent supernatant was collected and the beads were washed four times with the appropriate binding buffer. To collect the adherent fraction, 200/1 of elution buffer were added and the samples were rotated for 10 min at room temperature. Both the adherent and nonadherent fractions were then run on 12% reducing SDS-PAGE.

Iodination
Protein from biologically active gel eluent was iodinated with 125I (Amersham) using the Enzymobead iodination reagent (Bio-Rad, Richmond, CA) following manufacturer's instructions. After iodination, unincorporated 125I was removed by passage over an Exocellulose GF-5 desalting column (Pierce Chemical Co., Rockford, IL), and fractions containing protein were pooled.
Lectin Affinity Chromatography Five-milliliter columns were prepared with wheat germ agglutinin (WGA, from Triticum vulgaris) or lectin from Limulus polyphemus (LPA) conjugated to agarose beads (EY Laboratories, San Mateo, CA). For WGA affinity chromatography, the running buffer was APBS and the elution buffer was 0.1 M N-acetylglucosamine in APBS; for limulus lectin, the running buffer was 0.02 M Tris-HC1, 0.01 M CaC12, 0.15 M NaC1, pH 7.0, and the elution buffer was 0.02 M Na citrate, 0.15 M NaC1, pH 5.0. Approximately 2 ml of SAS-TCGF SN was applied to each column and incubated on a rocker platform for 40 to 60 min at room temperature. Unbound protein was washed off the column and bound protein was specifically eluted with the appropriate buffer. Protein concentrations were monitored using the Bio Rad protein assay reagent according to manufac-Immunoprecipitation One nanomole human recombinant IL-2 (HurIL-2) (Cetus/Perkin Elmer, Norwalk, CT) was immunoprecipitated with 100tg of an anti-HuIL-2 MoAB (17A1, 13A6 or 5A5, kindly provided by Dr. Richard Chizzonite, Hoffman-LaRoche, Nutley, NJ) overnight at 4C, rotating; 100 tl of 50% protein A-Sepharose (prA) beads (Pharmacia, Piscataway, NJ) in 1% FBS-murine PBS was added and incubated for an additional 6 hr at 4C, rotating. The resulting supernatants were sterile filtered and assayed on mouse CTLL (5X10 cells/well) in a 3-day 3H-thymidine incorporation assay. Xenopus SAS-TCGF SN was similarly immunoprecipitated except the subclass control myeloma MOPC 141 (Sigma, St. Louis) was also used. The resulting supernatants were assayed on 8-day-old Xenopus thymic lymphocytes (2104 cells/well) in a 3-day 3H-thymidine incorporation assay.