Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions

Abstract

Introduction: Previously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model.

Methods: The IL2RG X^KOY pigs used in this study were obtained by mating IL2RG X^KOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity.

Results: Tumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed.

Conclusions: IL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Keywords: DPF, designated pathogen-free; IL, Interleukin; IL2RG, interleukin-2 receptor gamma; Interleukin-2 receptor gamma; KO, knock out pigs; NK cells, natural killer cells; OIDP, operational immunodeficient pig; PCR, polymerase chain reaction; Pig; SCID; SCID, Severe combined immunodeficiency; SCNT, somatic cell nuclear transfer; SD, standard deviation; U-iR, uterectomy-isolated rearing; WT, wild-type pigs; XLGD, X-linked genetic diseases; Xenotransplantation; ZFN, Zinc finger nuclease; tdTomato, tandem dimer Tomato.

Fig. 1

Transplantation of HeLa S3 cells into subcutis of the ear in IL2RG KO and WT pigs. (A) Body weight gain of pigs over time. No differences were observed in the growth curves between the two groups. Blue line: IL2RG KO pigs (n = 7); Green line: WT pigs (n = 6). (B, C) Transplanted cells were monitored for tumor development. Photographs show an external view of tumor growth in the ear. (B) IL2RG KO pigs. Tumors derived from transplanted cells were identifiable in pig ears and their size increased with time. (C) WT pigs. Palpable tumors were observed in the ears of pigs 1 week after transplantation; however, no obvious tumors were observed at the transplantation site from 2 weeks after transplantation. (D) Broad photograph of the dissected tumor derived from an IL2RG KO pig. For the donor cells in this study, we selected the HeLa S3 cell line, which was modified to express tdTomato. The tumor was inspected with a fluorescent lamp and filter to verify whether the tumor emitted tdTomato. (D, left) Bright-field stereomicroscopic images of dissected tumors at 3 weeks after transplantation. (D, right) Fluorescence-stereomicroscopic image (left panel). The tumor exhibited a strong red signal. Scale bar: 5 mm. (E) Tumor volume in both groups. Two IL2RG KO pigs and three WT pigs received cell transplants in both ears, while all others received them in one ear only. Blue line: IL2RG KO pigs (n = 9); Green line: WT pigs (n = 9). The time after birth is indicated as weeks. Quantitative data are presented as mean ± SD.

Fig. 2

Histological evaluation of tumors within the subcutis of the ear of pigs at 1 week after transplantation. (A, A′) IL2RG KO pigs. (B, B′) WT pigs. (A) HE staining. Tumors were visible, well demarcated, and formed nests in the ears (A, left). The transplanted cells expanded clonally at the transplantation site (A, right). (A′) CD45 staining. Scarce CD45+ cells were detected in the tumor. (B) HE staining. Within the subcutis of the ears of WT pigs, there were moderately demarcated tumors (B, left); however, they were not as dense as the tumors of IL2RG KO pigs (B, right). (B′) CD45 staining. CD45+ cells were evident and abundant within the tumor. Scale bars: black, 10 mm; red, 100 μm. (C) Lymphocyte infiltration. The number of CD45+ lymphocytes was counted and compared between the IL2RG KO and WT groups as the rate of CD45+ lymphocytes in the tumor area. In WT pigs, CD45+ cells comprised 46.7% of the tumor area, which was significantly higher than that in the IL2RG KO pigs (∗p < 0.05). Data are presented as the mean ± SD of six independent experiments.

Fig. 3

Histological analysis of the transplanted cells in WT pigs 1 week after transplantation. (A–G) Tumors derived from transplanted cells within the subcutis of the ear. The right panels show an enlarged image of each stain. (A) HE staining. Moderately encapsulated and infiltrative tumors were visible. (B–F) Lymphocyte markers were verified by immunostaining. (B) T lymphocyte marker, CD3. (C) B lymphocyte marker, CD20. (D) NK cell marker, CD56. (E) Monocyte/macrophage lineage and dendritic cell marker, Iba1. (F) Cytotoxic T cell and NK cell marker, granzyme B. (G) Major histocompatibility complex class II molecule marker, HLA-DR. Numerous lymphoid cells infiltrated the tumor and lymphocyte activation was confirmed; therefore, rejection of transplanted cells by pigs with an intact immune system occurred at the transplantation site. These phenomena were confirmed in all WT pigs. Scale bar: 100 μm.

Fig. 4

Histological analysis of the transplanted cells in IL2RG KO pigs 3 weeks after transplantation. (A–G) Tumors derived from transplanted cells within the subcutis of the ears. The right panels show the enlargement of each staining. (A) HE staining. There is a focal, moderately demarcated area composed of nests and packets of transplanted cells within the subcutis of the ears. (B–F) Lymphocyte markers were verified by immunostaining. (B) T lymphocyte marker, CD3. (C) B lymphocyte marker, CD20. (D) NK cell marker, CD56. (E) Monocyte/macrophage lineage and dendritic cell marker, Iba1. (F) Cytotoxic T cell and NK cell marker, granzyme B. (G) Major histocompatibility complex class II molecule marker, HLA-DR. In IL2RG KO pigs, scarce lymphoid-related markers were confirmed at all transplanted sites. The transplanted cells were not rejected and formed tumors at the transplanted sites. Scale bar: 100 μm.

Fig. 5

Transplantation of HeLa S3 cells into the pancreas of DPF IL2RG KO pigs. Two 7-day-old IL2RG KO pigs underwent laparotomy in an isolator unit under sterile conditions. tdTomato-expressing HeLa S3 cells were transplanted into the tail of the pancreas in the pigs. (A and B) Broad bright-field photographs of tumors at 3 (Y446-4) and 4 weeks (Y446-2) after transplantation. (A and B, left panels) Macroscopic appearance of the pancreas. The yellow lines show the outline of each pancreas. (A and B, middle panels) Macroscopic appearance of a dissected pancreas. A large tumor was observed in the pancreatic tail. (A and B, right panels) Dissected tumors. (A′ and B′) Fluorescence-stereomicroscopic image of the tumor in IL2RG KO pigs. The tumor was inspected with a fluorescent lamp and filter to verify whether it emitted tdTomato. The transplanted cells in the pancreas appeared bright red. (A′ and B′, left and middle panels). The dissected tumor exhibited a strong red signal (A′ and B′, right panels). (C, C′) Histological appearance of a tumor (Y446-4). The tumor was HE stained and labeled with CD45. Each right lower panel is an enlargement of the images in C and C′. C and C′ are lower magnifications of the dissected tumor. (B) HE staining. The tumors exhibited central necrosis. (C′) A small number of CD45+ cells were verified at the boundary of the tumor, but they did not infiltrate. The time after birth is indicated weeks. Scale bars: black = 10 mm; yellow = 100 μm.

Fig. 6

Comparison between IL2RG KO pigs and other immunodeficient models. Three different immunodeficient pigs were produced by the following operations: K302 underwent thymectomy, splenectomy, and gastrostomy. K303 underwent thymectomy and gastrostomy. K304 underwent gastrostomy. (A) Body weights at specified times. The body weight gain was unstable in two pigs after transplantation (K302 and K304). Yellow line: K302; red line: K303; purple line: K304. The time after birth is indicated as days and weeks. ∗ Production of different immunodeficient pigs, ∗∗ Treated with immunosuppressive drugs, ∗∗∗ Transplantation. (B–D) The transplanted cells were verified by the red tdTomato color at the transplanted sites using a fluorescent lamp and filter. In two pigs, K302 and K304, red signals were observed 2 weeks after transplantation. No tumor was identified in the ears of K303 at 1 week post-transplantation. Scale bar: 10 mm.

Supplementary Fig. S1

Schematic representation of the strategy used to stably obtain the IL2RG KO pigs

Supplementary Fig. S2

Schematic illustration of the isolator units for the uterectomy-isolated rearing (U-iR) method DPF pigs were produced in novel biocontainment facilities. Each excised uterus was transferred to an aseptic isolator (recovery unit) via a disinfecting tub filled with 0.05% peracetic acid, and fetuses were recovered from the uterus within 5 min. The recovered piglets were then transferred to the second isolator unit (rearing unit, 1–5 piglets/unit) for artificial nursing with a γ-irradiated milk substitute and kept in the novel biocontainment rearing unit. Upper panel: isolator unit for recovery of newborn piglets from excised uterus. A: inflow of sterile air, B: disinfection tub through which the excised uterus is transferred into the isolator, C: acrylic glovebox unit for recovering piglets from the uterus, D: heating and filtering unit for air inflow, E: filtering unit for air outflow, F: connecting port. Lower panel: Rearing unit for artificial nursing of piglets A: rearing chamber; B: plastic glovebox unit for handling piglets; C: blow and filtering unit for air inflow; D: filtering unit for air outflow; E: connecting port for transferring piglets from the isolator unit; F, G: dotted lines indicate a drainboard floor (F) and wire mesh ceiling (G), respectively

Supplementary Fig. S3

Blood concentration levels of immunosuppressive drugs. Three immunodeficient pigs were produced through three different operations including OIDP 16 days before transplantation. Tacrolimus hydrate (0.5 mg/kg/day), MMF (60 mg/kg/day), and prednisolone (20 mg/body/day) were administered through a gastric tube 6 days before transplantation to induce immunodeficiency in pigs. All pigs were reared in conventional housing. The cells were transplanted into both ears. The day of transplantation was defined as day 0. (A) Blood tacrolimus hydrate concentration (B) Blood concentrations of MMF. Purple zones show the therapeutic ranges. K302 and K303 did not reach the therapeutic effective blood concentration of MMF from day 0–2 weeks after transplantation. Blue line: K302; orange line: K303; green line: K304. The time after birth is indicated as days