Comparative Inhibitory Effects of Tacrolimus, Cyclosporine, and Rapamycin on Human Anti-Pig Xenogeneic Mixed Lymphocyte Reactions.

BACKGROUND: Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . METHODS: A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/ß4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. RESULTS: PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200 ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. CONCLUSION: Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

Immunotherapeutic Agents for Intratumoral Immunotherapy.

Immunotherapy using systemic immune checkpoint inhibitors (ICI) and chimeric antigen receptor (CAR) T cells has revolutionized cancer treatment, but it only benefits a subset of patients. Systemic immunotherapies cause severe autoimmune toxicities and cytokine storms. Immune-related adverse events (irAEs) plus the immunosuppressive tumor microenvironment (TME) have been linked to the inefficacy of systemic immunotherapy. Intratumoral immunotherapy that increases immunotherapeutic agent bioavailability inside tumors could enhance the efficacy of immunotherapies and reduce systemic toxicities. In preclinical and clinical studies, intratumoral administration of immunostimulatory agents from small molecules to xenogeneic cells has demonstrated antitumor effects not only on the injected tumors but also against noninjected lesions. Herein, we review and discuss the results of these approaches in preclinical models and clinical trials to build the landscape of intratumoral immunotherapeutic agents and we describe how they stimulate the body's immune system to trigger antitumor immunity as well as the challenges in clinical practice. Systemic and intratumoral combination immunotherapy would make the best use of the body's immune system to treat cancers. Combining precision medicine and immunotherapy in cancer treatment would treat both the mutated targets in tumors and the weakened body's immune system simultaneously, exerting maximum effects of the medical intervention.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Effects of hepatocyte growth factor on porcine mammary cell growth and senescence.

Background: The porcine mammary glands share morphological and physiological similarities with human ones, making primary porcine mammary cells (PMC) suitable for biomedical research and a potential cellular therapeutic for breast cancer xenogeneic cell immunotherapy. Primary cells isolated from tissues remain the physiological functions of origin tissues but their self-renewal ability is restricted and cells acquire senescence during in vitro expansion. To overcome these drawbacks, here we sought to establish an approach to efficiently increase PMC's in vitro growth. We studied the effects of the hepatocyte growth factor (HGF) to maintain the expansion capacity of porcine mammary cells and identify the possible mechanisms. Purpose: HGF could allow for the increase in vitro proliferation capacity of primary epithelial cells isolated from tissue samples. To effectively produce cells for biomedical research and xenogeneic cell therapy, we planned to study the effects of HGF and its potential mechanisms of action to stimulate cell growth for PMC expansion. Methods: After HGF treatment, the growth, cell cycle, senescence and the cell marker gene expression of PMCs were analyzed in standard 10% FBS and low serum 1% FBS containing medium. Results: HGF significantly enhanced the cell proliferation by shifting the cell cycle population from G1 phase into S phase to increase cell division, reduced the senescent cells and reprogrammed gene expression profiles. Conclusion: We demonstrated that HGF could maintain the expansion capacity of PMCs by increasing cell growth and anti-senescence capability, suggesting its potential application in optimizing the long-term culture of primary cells. Adding a specific growth factor such as HGF in culture allows enhanced expansion of heterogeneous cell populations from normal porcine mammary glandular tissues in vitro. We believe that this cell culture approach will efficiently provide cells for studying mammary cell function and supply cells for therapeutic uses.

Kidney Xenotransplantation: Are We Ready for Prime Time?

PURPOSE OF REVIEW: With the exponential increase in interest and great strides toward clinical application, many experts believe we are ready for kidney xenotransplant human trials. In this review, we will examine the obstacles overcome and those yet to be conquered, discussing the human trials performed and the questions they raised. Additionally, we will revisit overlooked aspects that may be crucial for improvements and suggest future approaches for xenotransplant research. RECENT FINDINGS: Improving survival in pig-to-non-human-primate models with the identification of an ideal immunosuppression regimen led to 3 cases of kidney xenotransplant in brain-dead humans with limited follow-up and a single clinical case of pig-to-human heart xenotransplant with 2-month survival. With limited human results and unlimited potential, xenotransplantation shines a beacon of hope for a brighter future. However, we must navigate through the complexities of balancing scientific progress and patient welfare, avoiding being blinded by xenotransplantation's unquestionable potential.

First clinical-grade porcine kidney xenotransplant using a human decedent model.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

Utilizing Xenogeneic Cells As a Therapeutic Agent for Treating Diseases.

The utilization of biologically produced cells to treat diseases is a revolutionary invention in modern medicine after chemically synthesized small molecule drugs and biochemically made protein drugs. Cells are basic units of life with diverse functions in mature and developing organs, which biological properties could be utilized as a promising therapeutic approach for currently intractable and incurable diseases. Xenogeneic cell therapy utilizing animal cells other than human for medicinal purpose has been studied as a new way of treating diseases. Xenogeneic cell therapy is considered as a potential regenerative approach to fulfill current unmet medical needs because xenogeneic cells could be isolated from different animal organs and expanded ex vivo as well as maintain the characteristics of original organs, providing a versatile and plenty cell source for cell-based therapeutics beside autologous and allogeneic sources. The swine species is considered the most suitable source because of the similarity with humans in size and physiology of many organs in addition to the economic and ethical reasons plus the possibility of genetic modification. This review discusses the old proposed uses of xenogeneic cells such as xenogeneic pancreatic islet cells, hepatocytes and neuronal cells as a living drug for the treatment of degenerative and organ failure diseases. Novel applications of xenogeneic mesenchymal stroma cells and urothelial cells are also discussed. There are formidable immunological barriers toward successful cellular xenotransplantation in clinic despite major progress in the development of novel immunosuppression regimens and genetically multimodified donor pigs. However, immunological barriers could be turn into immune boosters by using xenogeneic cells of specific tissue types as a novel immunotherapeutic agent to elicit bystander antitumor immunity due to rejection immune responses. Xenogeneic cells have the potential to become a safe and efficacious option for intractable diseases and hard-to-treat cancers, adding a new class of cellular medicine in our drug armamentarium.

Dot blots of solubilized extracellular matrix allow quantification of human antibodies bound to epitopes present in decellularized porcine pulmonary heart valves.

BACKGROUND: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild-type (WT) and α-Gal-KO (GGTA1-KO) pigs in human sera. METHODS: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti-α-Gal and anti-Neu5Gc antibodies detected by a sensitive ELISA. RESULTS: The ultimate protocol had an inter-assay variance of 9.5% and an intra-assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1-KO tissue. CONCLUSIONS: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long-term survival similar to current allotransplantation settings.

Combination of novel intravesical xenogeneic urothelial cell immunotherapy and chemotherapy enhances anti-tumor efficacy in preclinical murine bladder tumor models.

BACKGROUND: Immune checkpoint inhibitors induce robust and durable responses in advanced bladder cancer (BC), but only for a subset of patients. Xenovaccination has been proposed as an effective immunotherapeutic approach to induce anti-tumor immunity. Thus, we proposed a novel intravesical xenogeneic urothelial cell immunotherapy strategy to treat advanced BC based on the hypothesis that implanted xenogeneic urothelial cells not only provoke xeno-rejection immune responses but also elicit bystander anti-tumor immunity. METHODS: Mouse advanced bladder cancer models were treated with vehicle control, intravesical xenogeneic urothelial cells, cisplatin + gemcitabine, or the combination and assessed for tumor responses to treatments. Tumors and spleens samples were collected for immunohistological staining, cellular and molecular analysis assessed by antibody staining, ELISA, cytotoxicity, and flow cytometry, respectively. RESULTS: The combination treatment of xenogeneic urothelial cell immunotherapy with chemotherapy was more efficacious than either single therapy to extend survival time in MBT-2 graft bladder tumor model and to suppress tumor progression in murine carcinogen BBN-induced bladder tumor model. The single-cell immunotherapy and combined therapy increased more tumor-infiltrating immune cells in MBT-2 graft tumors compared to vehicle control and chemotherapy treatment groups. The activated T-cell proliferation, cytokine production, and cytotoxicity capacities were also higher in mice with xenogeneic urothelial cell immunotherapy and combination treatments. CONCLUSIONS: Our results suggest the potential for a novel xenogeneic urothelial cell-based immunotherapy alone and synergy with chemotherapy in the combination therapy. Therefore, our study supports developing xenogeneic urothelial cells as an immunotherapeutic agent in combination with chemotherapy for BC treatment.

Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation.

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (Sus scrofa domestica) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1, CMAH, ß4GalNT2, vWF, ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.

The final obstacle to successful pre-clinical xenotransplantation?

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

Applications of CRISPR technologies in transplantation.

In transplantation, the ever-increasing number of an organ's demand and long-term graft dysfunction constitute some of the major problems. Therefore, alternative solutions to increase the quantity and quality of the organ supply for transplantation are desired. On this subject, revolutionary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology holds enormous potential for the scientific community with its expanding toolbox. In this minireview, we summarize the history and mechanism of CRISPR/Cas9 systems and explore its potential applications in cellular- and organ-level transplantation. The last part of this review includes future opportunities as well as the challenges in the transplantation field.

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype-Effects on immune status and susceptibility to human immune responses.

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of ß2 -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti-xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft-infiltrating Th17 cells and activated B cells.

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig-to-humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45+ cells comprising activated human B cells, CD4+ CD44+ IL-17+ Th17 cells, and CD68+ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long-term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI-SP), and peri-transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI-SP treatment in controlling human immune cells in promoting long-term islet xenograft survival.

Anti-tumour effects of a xenogeneic fibroblast activation protein-based whole cell tumour vaccine in murine tumour models.

The clinical benefit of cancer immunotherapy, including tumour vaccines, is influenced by immunosuppressive factors in the tumour microenvironment. Among these factors, cancer-associated fibroblasts (CAFs) and their products, such as fibroblast activation protein-α (FAPα), greatly affect tumourigenesis, development, metastasis and treatment tolerance, which make them promising immunotherapy targets for cancer patients. Our previous study reported that a whole cell tumour vaccine (WCTV) expressing FAPα inhibited tumour growth by simultaneously attacking cancer cells and CAFs. This study aimed to improve WCTVs with xenoantigens to end immune tolerance and to further activate the adaptive immune system. In the present study, we designed a WCTV by transducing a vector encoding human FAPα (hFAPα) into murine tumour cells and evaluated its efficacy in multiple solid tumour models. Immunotherapy with this WCTV effectively delayed tumour growth and prevented recurrence. The anti-tumour responses were clearly linked to antigen-specific cytotoxic T cells, whereas CD4(+) T lymphocytes also played a role. Humoural immune responses were activated because the adoptive transfer of immunoglobulins induced abscopal anti-tumour effects, and autoantibodies against FAPα were specifically detected in the sera of immunized mice. Moreover, an increased number of apoptotic tumour cells along with a reduced number of CAFs within the tumours suggest that xenogeneic FAPα-based WCTV has the potential to drive T cell and antibody responses against cancer cells and CAFs. This finding could offer an advanced strategy to treat multiple solid tumours with individualized cancer immunotherapy techniques.