The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.
We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.
1. Animals
X. laevis x X. gilli hybrids LG-6 and LG-15 isogenetic clones 1 are from our breeding colony at the University of Rochester (http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). LG-6 and LG-15 share the same heterozygous MHC haplotype (a/c) but differ at minor histocompatibility (H) loci. Progeny from these clones are produced by gynogenesis, in which diploid eggs produced by the female are activated by UV-irradiated sperm (no DNA contribution to the progeny).
The use of gloves is facultative. Some people prefer to not wear them because it is more difficult to handle the frogs (slippery) and in fact it appears to make the frogs uncomfortable.
2. Purification of gp96 from 15/0 Tumor (Expresses Both Tumor and Minor H-Ags)
Gp96 purification has been previously described 2, 3. Briefly, gp96 is purified by 50-70% ammonium sulfate fractionation, followed by conA-sepharose and DEAE chromatography. About 20-50 μg of protein can be obtained per 1 mL of tumor tissue. Purity of the preparation is determined by SDS-PAGE and sliver staining.
3. Elicitation and Harvest of Peritoneal Leukocytes (PLs) from Minor H-Ag-disparate LG-6 Frogs
4. Pulsing and Adoptive Transfer of Lg-6 Pls Into Naíve Lg-6 Recipients
5. Skin Graft Assay
Skin graft rejection is a well-established assay in Xenopus 4, 5, 6. In Xenopus, a recipient rejects donor skin displaying 1 or 2 MHC haplotype mismatches within 18-22 days at 21-22°C. In contrast, skin grafts between LG-6 and LG-15 clones that differ only by minor H-Ags are rejected more slowly (more that 30 days). However, this rejection is accelerated in recipients that have been primed against donor minor H-Ags either by a previous skin graft or by immunization with gp96 purified from the donor. No rejection at all occurs when the donor and the recipient are genetically identical (e.g., cloned or fully inbred animals). Therefore, this simple technique is very powerful for characterizing in vivo immune responses elicited by gp96.
6. Whole-mount Immunohistology of Transplanted Skin
Frog whole-mount immunohistology has been previously described 6. Briefly, the frog is anesthetized and placed under the microscope on a sterile paper towel pre-wet with frog water (the working area is also aseptically prepared). The transplanted skin is then harvested together with a small amount of surrounding host skin and it is stained with antibodies similarly to cell staining for flow cytometry analysis 6. Autoclaved instruments and sterile buffers need to be used. After staining the skin is placed on a microscope slide and gently pressed with a cover slip. At this point the skin can be visualized using a fluorescent microscope for different cell populations that have infiltrated the graft. After the procedure the frog is kept in water with antibiotics as described in section 5.5. and returned in normal water. The small wound left where the skin was removed heals within a week.
7. Characterization of Blood Leukocytes
8. Tumor Transplantation Assay
9. Reagents Needed:
10. Representative Results
Figure 1. Stereomicroscopic analysis of skin graft rejection 12 days post-transplantation. LG-6 cloned frog received a skin graft from either (A) a MHC-identical LG-6 (shows no rejection) or (B) a MHC-disparate outbred donor (80% rejection). Arrows shows silvery iridophore pigmented cells marking healthy (non-rejected) grafted tissue. (*) Mark of forceps not due to rejection.
Figure 2. Gp96 facilitates cross-presentation of tumor antigens in Xenopus. LG-15 PLs (5 x 105) were pulsed for 1 hr on ice either with APBS (negative control), 1 μg of recombinant gp96 purified from an E. Coli culture, or 1 or 0.5 μg of gp96 purified from 15/0 tumor tissue. After 3 washes, cells were adoptively transferred into LG-15 adult recipients (1 x 106/individual). Three days later, live 15/0 tumor cells (5 x 105) were transplanted by s.c. injection. Each curve represents the kinetics of tumor growth in one frog. Days post challenge when tumors first appeared were monitored, and tumor size was determined periodically with a caliper (length x width x thickness).
The amphibian Xenopus is a unique versatile non-mammalian model to study immunity. Its extensive use in biomedical and immunological research has yielded in many important research tools such as the MHC defined clones LG-6 and LG-15 as well as different cell lines and monoclonal antibodies. Using these tools we have established different in vitro and in vivo assays to study the ability of heat shock proteins such as gp96 to mediate potent Ag-specific anti-minor H-Ag and anti-tumor T cell responses 7. This model system allows us to further investigate the immunological properties gp96 during the priming and effector cell phases.
Concerning the priming phase, initial studies of these responses used subcutaneous immunization with purified gp96 that required two injections of 10 μg gp96 at two week intervals before in vivo assays such as skin grafting or tumor transplantation 2, 8. In comparison, the cross-presentation method we have developed 7 and are currently using is more convenient and efficient. The multiple advantages of this priming strategy include time and amount of protein needed for each experiment. For instance in order to immunize an animal it takes 4 weeks and 20 μg of gp96, while with PL priming we need only 3 days and 0.5 to 1 μg of protein. Additionally there is less variability because the priming consists of only one injection of PLs pulsed with gp96. This is a critical step because if the needle is pulled out too quickly during one of the immunization by s.c. injection, a significant fraction of protein may be lost therefore causing a greater individual variability. Importantly, this cross-presentation assay provides a way to further investigate the mechanisms of gp96-mediated immune responses. For example, we can also modulate the expression of certain molecules such as class Ia on the surface of PLs before pulsing with gp96 to investigate their role in gp96 mediated immune responses and T cell priming. The process of gp96 internalization can also be studied by pre-incubating PLs with antibodies or competitors interfering with endocytic receptors 7.
Concerning the effector phase, the Xenopus model is not only suitable to characterize immune cell effectors in vitro by flow cytometry, killing and proliferation assays 8, 9, but also provides powerful in vivo assays such as minor H-Ag-disparate skin grafting and tumor transplantation assays. Both of these assays are well established in the Xenopus model however there are a few critical steps that must be followed. For instance in the skin grafting assay special attention must be paid when handling the graft especially when cutting out the window of overlaying host skin. The window has to be slightly smaller than the graft itself so that the graft will not fall out. Furthermore, during tumor transplantation it is important to first inject half of the control animals followed by the experimental ones and than finish with the second half of the control frogs. This will ensure that the tumor is viable during the injection process and will produce more consistent data. The fact that this model system relies on cloned animals further allows to extend studies of effector cells stimulated by hsps using adoptive cell transfer 10.
In summary, the methods presented here highlight the frog Xenopus as an exceptional non-mammalian model system to study hsps such as gp96 role in immune surveillance and immune responses.
The authors have nothing to disclose.
The expert animal husbandry provided by Tina Martin and David Albright is gratefully appreciated. This research was supported by grants T32-AI-07285 (H. N.), NIH R25 2GM064133 (T.C.L), 1R03-HD061671-01, R24-AI-059830-06 from the NIH.
Material Name | Typ | Company | Catalogue Number | Comment |
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Reagents needed: | ||||
Amphibian Phosphate Buffered Saline (APBS): | ||||
NaCl, 1.15 g/L | ||||
Na2HPO4, 0.2 g/L | ||||
KH2PO | ||||
10N NaOH | ||||
Tricaine Methane Sulfonate (TMS, MS-222) | Crescent Research Chemicals | CAS#886-86-2 | ||
Sodium bicarbonate | Fisher Scientific | S-233-500 | ||
Histopaque-1077 | Sigma-Aldrich | 10771 | 100ml | |
Heparin Sodium Salt | Sigma-Aldrich | H3149-50KU | ||
Culture medium for Xenopus 15/0 tumors [see 3 for more details]: | ||||
Iscove DMEM basal medium | Gibco-Invitrogen | 11965 | ||
Insulin | ||||
Non-essential amino acids | ||||
Penicillin-streptomycin | ||||
Kanamycin | ||||
Primatone | Sheffield Products Division | |||
β2-mercaptoethanol | ||||
NaHCO3 | ||||
30% double distilled water | ||||
5% featal bovine serum | ||||
20% superantant from a Xenopus kidney cell line A6 | ||||
0.25% of normal Xenopus serum | ||||
Materials and Equipment: | ||||
50 and 15 ml conical centrifuge tubes (sterile) | ||||
25 gauge 5/8 Precision Glide sterile needles | BD | |||
18 gauge 1½ Precision Glide sterile needles | BD | |||
1 ml Tuberculin Slip Tip Syringe sterile | BD | |||
10 ml Slip Tip Syringe sterile | BD | |||
1.5 ml MicroCentrifuge tubes (sterile) | ||||
60 X 15 mm Polystyrene Petri Dishes sterile | Falcon | |||
Razor blades | ||||
25 X 75 mm X 1 mm Premium Microscope Slides | Fisher Scientific | |||
10 cm glass petri dishes | ||||
9″ Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable | VWR | |||
Tygon tubing | ||||
Two # 5 Swiss Jeweler’s Forceps | Miltex Inc. | |||
Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm | Roboz | |||
Helios calipers | ||||
Dissecting microscope | ||||
High intensity illuminator | ||||
Hemacytometer | ||||
Un-bunsen burner | ||||
37°C shaking incubator | ||||
Centrifuge |