Organ transplantation is the only effective approach to treat the patients with end-stage organ failure. However, large number of patients die worldwide because there are not enough available donated organs for transplant.1 For example, in the United States, although 20 000 patients received renal transplantation in 2017, there are still around 120 000 patients in the waiting lists, and the waiting list length kept increasing in the past 10 y. Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem several decades ago.2 Although nonhuman primate organs were used in several xenotransplantation clinic trails between the 1960s and 1990s, nowadays, pig has already been considered as the most suitable species to offer xenogeneic organ because of its easy breeding characteristics, physiologic similarity, and close organ size/function.3,4
Robust xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation,5,6 in which hyperacute rejection (HAR) which is mainly induced by pre-existing antibodies in Old World Monkey hosts, that targets xenoglycan α-Gal antigens on the surface of most porcine cells, causing very rapid pig graft rejection in nonhuman primates.7 Elimination of the α-1,3-galactose transferase gene glycoprotein galactosyltransferase α 1, 3 (GGTA1) in pigs successfully overcame the HAR problem and greatly prolonged the survival of transplanted pig organs in nonhuman primate recipients.8 Recently, the survival times of pig kidney and heart (life supporting) in nonhuman primates have been expanded to 499 and 195 d, respectively, making the pig organ transplantation closer to clinical translation.9,10 However, other than HAR, T-cell–mediated xenograft rejection can still induce a series of complications.11
Major histocompatibility complex (MHC) molecules are the main antigens that cause T-cell–mediated rejection in allo-/xenotransplantation, in which MHC class I and II molecules induced CD8+ and CD4+ T-cell–mediated rejection, respectively.11,12 Previous studies showed that elimination of swine leukocyte antigen class II molecules by transgenic expression of the MHC class II transactivator (CIITA)-dominant negative protein was capable to alleviate human CD4+ T-cell responses to pig aortic endothelial cells.13,14 Moreover, long-term survival was achieved in the genetic engineered–induced pluripotent stem cells, whose MHC-I and MHC-II molecules were depleted, in allogeneic recipients.15 These results suggest that the knockout of porcine SLA-I and SLA-II would be an effective strategy to compromise xenogeneic T-cell responses, which, to our knowledge, has not been tested yet.
Herein, we simultaneously eliminated α-Gal, SLA-I, and SLA-II molecules by CRISPR-Cas9 techniques that target GGTA1, β2-microglobulin (β2M), and CIITA genes and generated GGTA1−/−β2M−/−CIITA−/− Triple Gene Knockout Pigs. We revealed that the depletion of SLA-I and SLA-II molecules not only alleviated human T-cell responses to pig cells in vitro but also prolonged the pig skin graft survivals in unconditioned immune competent mice.
MATERIALS AND METHODS
Animals and Human Samples
NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NOD/SCID IL2rg−/− or NCG) mice, housed in a specific pathogen-free microisolator environment, were purchased from Nanjing Biomedical Research Institute of Nanjing University and used at 10–12 wk of age. C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co and used at 10–12 wk of age. Bama miniature pigs were raised and cloned at the Beijing Farm Animal Research Center. Human blood was obtained from healthy volunteers. Protocols related to the use of animals and human tissues were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University and the Institute of Zoology of Chinese Academy of Sciences. All the experiments were performed in accordance with the protocols.
PUC19-pCAG-SpCas9-2A-GFP and pUC19-U6-sgRNA vectors were obtained from Qi Zhou laboratory. All vectors were constructed as previously reported.16,17 In brief, the oligonucleotides of each single guide RNA were annealed and ligated with U6-sgRNA backbone, which was digested by endonuclease a kind of endonuclease (New England Biolabs [NEB]) and then gel purified. All the oligonucleotide sequences are shown in Table S1 (SDC, http://links.lww.com/TP/B891).
Generation of GGTA1−/−β2M−/−CIITA−/− Pig Embryonic Fibroblast Cell Lines
Primary pig embryonic fibroblasts (PEFs) were isolated according to a standard experimental procedure. Cas9-gRNA plasmids for GGTA1, β2M, and CIITA genes were cotransfected into cultured PEF cells by nucleofection. Cells were procured 48 h after transfection for fluorescence-activated cell sorting. Cas9 positive cells were plated in 96-well plates using the MoFlo XDP cell sorter and cultured for 1 wk in Dulbecco’s Modified Eagle Medium (Gibco, Grand Island, NY) containing 1 mmol/L glutamine (Gibco) and 1% nonessential amino acids (Gibco) and supplemented with 2.5 ng/mL of basic fibroblast growth factor (R&D Minneapolis, MN ). The medium was replaced every 3 d. Monoclonal cells were amplified, and 2 cell lines were identified as containing GGTA1, β2M, and CIITA knockout by polymerase chain reaction (PCR) and sequencing.
Nuclear Transfer and Embryo Transfer
This experiment was performed according to previously described methods.18 All cloned piglets were delivered by natural birth.
Genotyping of the triple gene knockout PEF cell lines and pigs was performed by PCR and Sanger sequencing. The DNA samples were extracted from PEF cells and tissues of the pigs using a Mouse Direct PCR Kit (B40015; Bimake, Houston, TX). The reaction consisted of 5 steps: 95°C, 5 min; 95°C, 30 s; 60°C, 30 s; 72°C, 45 s, 35 cycles; and 72°C, 10 min. All primers are shown in Table S2 (SDC, http://links.lww.com/TP/B891).
Reverse Transcription PCR
RNA was isolated by trizol, and cDNA was synthesized by HiScript II Reverse Transcriptase (R223-01; Vazyme, Nanjing, Jiangsu, China). The reverse transcription PCR primers used were SLA-DR-α-chain 5′-TATCTCCCCTTCATGCCCTCA-3′ (forward) and 5′-GTCCATTCCCTGCAAGCACCT-3′ (reverse). The PCR steps were as follows: 95°C, 5 min; 95°C, 30 s; 58°C, 30 s; 72°C, 30 s, 35 cycles; and 72°C, 10 min. Each sample was measured 3×.
Mixed Lymphocyte Reaction
Mixed lymphocyte reaction (MLR) assay was used to quantify human xenoimmune responses to pig cells. In brief, carboxyfluorescein succinimidyl ester (CFSE)-labeled human PBMCs (hPBMCs) were cocultured with 30 Gy irradiated wild-type (WT) or GBC-3KO pig splenocytes (2:1 ratio) in complete Roswell Park Memorial Institute 1640 medium (Gibco) at 37°C for 6 d. The proliferation and activation of human T cells were determined by flow cytometry by measuring CFSE dilution and CD25 expression.19
Bromodeoxyuridine Incorporation Assay
hPBMCs and pig splenocytes were cocultured (2:1 ratio) in complete RPMI 1640 medium in a humidified incubator at 37°C with 5% CO2 for approximately 120 h. Then, bromodeoxyuridine (BrdU) was added for another 18-h incubation period. The cells were fixed, and the anti-BrdU-POD was added according to the manufacturer’s instructions (Cell Proliferation ELISA, BrdU; Roche, Rotkreuz, Switzerland). After cell washing and substrate addition, the reaction was quantified by the OD values measured at 450 nm (reference wavelength, 690 nm) using a spectrophotometric multiwall ELISA plate reader.
Pig skin transplantation was performed as previously described.20,21 In brief, porcine skin (split thickness of 2.3 mm) was rinsed with phosphate buffered saline 3×, immersed in RPMI 1640 medium with 10 U/mL penicillin and 10 μg/mL streptomycin for 12–18 h, and then transplanted on the dorsum of C57BL/6 mice. Skin graft photographs were taken daily, and rejection was defined when <10% of the graft remained viable.
Expression of GGTA1, SLA-I, and SLA-II molecules on pig splenocytes was determined by flow cytometry using lectin from Bandeiraea simplicifolia (BS-IB4-lectin, isolectin-B4-FITC; Sigma, St. Louis, MO), antihuman β2-microglobulin (BioLegend, San Diego, CA), and anti-SLA-DR (BD Pharmingen, San Diego, CA). After the MLR assays, human T-cell phenotypes were measured by flow cytometry using fluorescent-conjugated antihuman CD3, CD4, CD8, and CD25 antibodies (BD Pharmingen). FCM analysis was performed in LSR Fortessa (BD Biosciences, San Diego, CA). Dead cells were excluded from the analysis by gating lower forward scatter levels and high propidium iodide-retaining cells signals.
Statistical analysis was performed using Prism8 (Graphpad Software Inc.). Differences between specific points were determined by Student t test or, when appropriated, the nonparametric Mann–Whitney test. One-way ANOVA test was used to determine differences between groups. Multiple comparisons between levels were determined with Tukey post hoc tests. To analyze grafts survival and determine median survival times (MSTs), the Kaplan–Meier/log-rank test was used; P values <0.05 were considered significant.
Generation of GGTA1−/−β2M−/−CIITA−/− Triple Gene Knockout Pigs
The schematic profile of the generation GGTA1−/−β2M−/−CIITA−/− triple gene knockout pigs is shown in Figure 1A. To obtain triple gene knockout PEFs cells, 4 target sites were designed in each of the following exons: exon 8 of GGTA1, exon 2 of β2M, and exon 9 of CIITA (Figure 1B). Cas9 and all 12 sgRNA vectors were cotransfected into the PEFs, and Cas9 positive cells were seeded in 96-well plates by fluorescence-activated cell sorting. We obtained 54 cell lines for identification by PCR genotyping and sequencing. The results confirmed that 2 cell lines (NO.3 and NO.21) were GGTA1−/−β2M−/−CIITA−/− triple gene knockout. After nuclear transfer, a total of 1346 cloned embryos were transferred into the oviducts of 12 recipient surrogates. Five pregnancies were established and went to term, of which 2 were executed for analysis at day 65 and day 95 of pregnancy, 1 resulted in miscarriage on day 70, and 2 gave birth by natural delivery to 5 male piglets (Table S3, SDC, http://links.lww.com/TP/B891). Photographs and the physiologic index of the GBC-3KO pigs are shown in Figure 1C and Table S4 (SDC, http://links.lww.com/TP/B891), respectively. GBC-3KO pigs were genotyped by PCR and T7EI (Figures 1D; and Figure S1, SDC, http://links.lww.com/TP/B891) and Sanger sequencing (Figure 1E).
Loss of α-Gal, SLA-I, and SLA-II Expression in GBC-3KO Pig Cells
The cell surface expression of α-Gal, SLA-I, and SLA-II on WT and GBC-3KO porcine splenocytes was measured by flow cytometry using fluorescent-conjugated BS-IB4-Lectin and antibodies against pig β2-microglobulin and SLA-DR. The results showed that WT fetal pig splenocytes were positive for α-Gal, SLA-I, and SLA-II (circa 96.33%, 62.07%, and 25.73%, respectively). In contrast, GBC-3KO fetal pig splenocytes were negative for α-Gal and SLA-I antigens, and only a small portion of these cells (5.48% in average) showed a markedly reduced SLA-II expression (Figure 2A and B). reverse transcription PCR revealed that SLA-II expression remained detectable in GBC-3KO pig heart and kidney, but the levels were remarkably reduced compared with WT pigs (Figure S2A, SDC, http://links.lww.com/TP/B891). Similarly, significantly reduced expression of α-Gal, SLA-I, and SLA-II was observed in neonatal GBC-3KO piglets (Figure S2B, SDC, http://links.lww.com/TP/B891).
CD8 T-cell Deficiency in GBC-3KO Pigs
Considering the important roles of MHCs in T-cell development, we measured the combinations of CD3+ T cells, CD4+, and CD8+ T subsets in neonatal and fetal GBC-3KO pigs. In neonatal PBMCs, the ratios of CD3+ T cells in CD45+ immune cells in GBC-3KO pigs (58.3%) are close to the ones in WT pig (53.8%), whereas, compared with WT pig (35.9%), the ratio of CD8+ T subsets within CD3+ T cells in GBC-3KO pig greatly reduced (2.39%), demonstrating an impairment for CD8 T-cell development in GBC-3KO pig (Figure S2C, SDC, http://links.lww.com/TP/B891). Similarly, CD8 T-cell deficiency was also found in GBC-3KO pigs at fetal stages, evidenced by the reduction of CD8 T subsets ratios in total CD3 T cells (Figure S2C, SDC, http://links.lww.com/TP/B891) and a significant decline of CD8 versus CD4 ratios in GBC-3KO pig spleen than WT pig spleen (Figure 2C).
GBC-3KO Pig Cells Exhibit a Reduced Capacity to Stimulate Xenoimmune Responses
To determine the efficacy of GBC-3KO to reduce the ability to stimulate xenogeneic immune responses, we first compared MLR responses of human T cells to GBC-3KO versus WT pig cells. In brief, CFSE-labeled hPBMCs were cocultured for 6 d with GBC-3KO or WT fetal pig splenocytes (D65) that were irradiated with 30 Gy, and then analyzed for human T-cell proliferation and activation by measuring CFSE dilution and CD25 expression in CD3+ T cells, and CD4+ and CD8+ T-cell subsets (Figure S3A, SDC, http://links.lww.com/TP/B891). Significant less CFSElow/− proliferated subsets as well as CD25+ activated subsets were detected in human CD3+ T cells that cocultured with GBC-3KO pig cells than the ones cocultured with WT pigs (Figure S3B–E, SDC, http://links.lww.com/TP/B891). Similarly, proliferation of the human CD4+ T cells and CD8+ T cells cocultured with GBC-3KO pig cells (8.883% and 1.898%, respectively) was significantly less than the counterparts cocultured with WT pig cells (21.25% and 9.317%, respectively) (Figure 3A and B), whereas the upregulation of CD25 was less extensive in human CD4+ T cells and CD8+ T cells cocultured with GBC-3KO pig cells (24.68% and 5.98%, respectively) than the ones cocultured with WT pig cells (12.76% and 1.10%, respectively) (Figure 3C and D). To further confirm the influences of gene editing for human immune responses, we compared the BrdU incorporation value in hPBMCs in the presence of GBC-3KO or WT pig cells. Consistent with our previous results, significant less extent of proliferation was found for the hPBMCs in GBC-3KO groups than the WT groups (Figure 3E and F). These data demonstrate that the deletion of β2M and CIITA genes in pigs significantly compromises their immunogenicity to human T-cell responses.
To evaluate the impact of SLA-I and SLA-II engineering for pig grafts survival in xenogeneic recipients, we transplant GBC-3KO and WT pig skins in unconditioned C57BL/6 mice. Compared with WT pig skin grafts (MST, 13.5 d), GBC-3KO pig skin grafts showed a significantly prolonged survival (MST, 16 d) in immunocompetent mice (Figure 3G and H). However, either WT or GBC-3KO pig skin xenografts were rejected in immunocompromised (NCG) mice that lacking T, B, and NK cells (Figure 3G and H). These results indicate that elimination of β2M and CIITA genes can alleviate xenogeneic immune responses.
Robust immunologic response is a big challenge for xenotransplantation.2 Although HAR has been largely conquered by eliminating glycan antigen and prescreening of nonhuman primates for preexisting antibody as hosts, T-cell–mediated responses may still raise many complications, such as graft rejection, the requirement of intensive immune suppressive drugs, and so on.11 Currently, there are no reports on animal model and xenogeneic immune reactions of eliminating HAR and T-cell immunity simultaneously. Herein, we generated α-Gal, SLA-I, and SLA-II triple-deficient GBC-3KO pigs through simultaneous knockout of the GGTA1, β2M, and CIITA genes by CRISPR-Cas9 technology. Flow cytometric results using fluorescent-conjugated BS-IB4-Lectin, β2-microglobulin, and, SLA-DR antibodies indicate the loss of α-Gal, SLA-I and, significantly, reduction of SLA-II expression in the splenocytes of GBC-3KO pigs compared with the WT ones, consistently, few SLA-DR transcripts were also found in GBC-3KO pig heart and kidney, which confirmed the triple knockout phenotype as designed. The immunogenicity of GBC-3KO pigs to human T cells was evaluated by MLR assays. Significantly less proliferation and activation were found in human CD4+ and CD8+ responder T cells that cocultured with GBC-3KO pig cells than WT pig cells. Moreover, GBC-3KO pig skin showed a significantly prolonged survival in unconditioned C57BL/6 mice. These results demonstrate that the immunogenicity of GBC-3KO pig cell/tissues was lower than the ones from WT pigs.
MHC molecules play crucial roles in immune cell differentiation, education, and function.22 In this study, we found that pig CD8/CD4 was markedly downregulated, and few pig CD8+ T cells can be detected in the PBMCs and spleens of GBC-3KO pigs, which may be due to missing pig SLA-I mediated T-cell–positive selection in thymus. It has been reported that the depletion of CIITA cannot eliminate SLA-II molecule expression on mouse thymic epithelial cells,23 which may explain why large number of pig CD4+ T cells can still be detected in GBC-3KO pigs. Other than T cells, MHC-I is also important for the differentiation and development of natural killer cells,24 thus missing SLA-I in GBC-3KO bone marrow stromal cells may result in generation of incompetent NK cells. Accordingly, GBC-3KO pigs are weaker and more susceptible to infections than WT pigs; to have healthy organ/cell GBC-3KO donors, decontaminated facilities would be required to execute certain standard procedures, such as cesarean delivery and breeding.
Skin grafting, as a rigorous test, is often used to detect the xenogeneic rejection.25,26 In agreement with the results of the β2M-deficient pigs, we found that skin grafts from GBC-3KO pigs exhibited remarkably prolonged survival to mice.26 Rejection of GBC-3KO pig skin in C57BL/6 mice but not NOD/SCID IL2rg−/− mice indicates that eliminating β2M and CIITA cannot completely prevent xenogeneic immune rejection. Because although depletion of SLA-I and SLA-II may downregulate T-cell activation induced by donor dendritic cells–mediated “direct antigen presentation” process, host dendritic cells can still activate host T cells through “indirect antigen presentation” way.27 In addition, incompatibility of immune regulatory molecules between species could also induce innate immune responses, then arise T-cell immunity.11 For example, human macrophages phagocytose pig cells because pig CD47 molecules cannot crossreact with human signal regulatory protein α.28,29 Transgenic expression of human immune regulatory protein, such as CD47 and human leukocyte antigen E, may need to be included to further alleviate xenogeneic immune responses.30
Therefore, in the next steps, we will try to knock-in multiple human immune and physiology regulatory genes, such as human CD55, thrombomodulin, CD47, and so on, on GBC-3KO pig background to improve their immunologic and coagulation compatibility to primates. In addition, solid organs from these pigs, such as kidney and heart, will be transplanted into nonhuman primates to evaluate the xenogeneic immune responses in vivo.
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