A New Look at Blockade of T-cell Costimulation: A Therapeutic Strategy for Long-term Maintenance Immunosuppression

Activated T cells orchestrate the immune response that results in graft rejection; therefore, a common goal among current immunosuppressive therapies is to block T-cell activation, proliferation and function. Current immunosuppressive regimens that inhibit T cells and immune cells have greatly reduced the incidence of acute rejection following solid-organ transplant. However, the expected improvements in long-term outcomes have not been realized. This may be related to the non-immune side effects of current maintenance immunosuppressants, which target ubiquitously expressed molecules. The focus in transplantation research is shifting in search of maintenance immunosuppressive regimens that might offer improved long-term outcomes by providing efficacy in prevention of acute rejection combined with reduced toxicities. An emerging therapeutic strategy involves an immunoselective maintenance immunosuppressant that inhibits full T-cell activation by blocking the interaction between costimulatory receptor–ligand pairs. This review describes costimulatory pathways and the development of molecules, which inhibit them in the context of transplantation research. Recent clinical data using the selective costimulation blocker, belatacept (LEA29Y), as a part of a CNI-free maintenance immunosuppressive regimen in renal transplantation is highlighted.     

Bystander activation of iNKT cells occurs during conventional T-cell alloresponses

It is well established that iNKT cells can be activated by both exogenous and a limited number of endogenous glycolipids. However, although iNKT cells have been implicated in the immune response to transplanted organs, the mechanisms by which iNKT cells are activated in this context remain unknown. Here we demonstrate that iNKT cells are not activated by allogeneic cells per se, but expand, both in vitro and in vivo, in the presence of a concomitant conventional T-cell response to alloantigen. This form of iNKT activation was found to occur independently of TCR-glycolipid/CD1d interactions but rather was a result of sequestration of IL-2 produced by conventional alloreactive T cells. These results show for the first time that IL-2, produced by activated conventional T cells, can activate iNKT cells independently of glycolipid/CD1d recognition. Therefore, we propose that the well-documented involvement of iNKT cells in autoimmunity, the control of cancer as well as following transplantation need not involve recognition of endogenous or exogenous glycolipids but alternatively may be a consequence of specific adaptive immune responses.     

Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation

Abstract: Background: Experience with non‐antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non‐Gal endothelial cell (EC) antigens. Methods:  To gauge the diversity of the non‐Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T‐cell immunosuppression, Group A (n = 4) and Gal knockout (GT‐KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non‐Gal antibody was measured by flow cytometry and by western blots using GT‐KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results:  Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non‐Gal antibody response induced by GT+ and GT‐KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions:  These experiments indicate that the non‐Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non‐Gal antibody responses.     

Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells

Tsuyuki S, Kono M, Bloom ET. Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells. Xenotransplantation 2002; 9:410–421. © Blackwell Munksgaard, 2002
Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas⁺ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas⁺ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L -transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.     

猪到猕猴异种心脏移植超急性排斥反应的免疫学及病理学变化

目的观察猪到猕猴异种心脏移植超急性排斥反应时的免疫学及病理学变化。方法采用猪到猕猴腹腔内异位心脏移植模型,检测发生超急性排斥反应者的血液中补体、天然抗体及T淋巴细胞亚群的变化,并对移植心脏进行免疫组化(测定C3、C4、C5b9、IgG及IgM的沉积)及病理学分析。结果发生超急性排斥反应时,血清补体C3、C4的含量、总补体活性及抗猪内皮细胞天然抗体均有一定程度的下降;CD4 /CD8 T淋巴细胞的比率也有所下降;移植心脏中均有补体C3、C4、C5b9的沉积,IgG及IgM也均有沉积,但IgG和IgM沉积强度的差异无统计学意义;病理学改变主要为心肌间质弥漫性出血、水肿,毛细血管内普遍淤血。结论补体通过经典途径激活参与猪到猕猴异种心脏移植超急性排斥反应;超急性排斥反应时受者血中天然抗体水平明显下降;CD4 T淋巴细胞可能参与异种移植超急性排斥反应过程并有所消耗;发生超急性排斥反应的移植物突出病理表现为间质出血。     

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals.     

Fates of CD4+ T cells in a tolerant environment depend on timing and place of antigen exposure

In experimental organ transplantation, tolerance is induced by administration of anti-CD40L mAb in conjunction with donor-specific splenocyte transfusion. Multiple, sometimes conflicting mechanisms of action resulting from this treatment have been reported. To resolve these issues, this study assessed the fates of graft reactive cells at different times and locations in the tolerant environment. Alloantigen-specific CD4(+) T cells transferred at time of tolerance induction (7 days before transplantation) became activated, expressed CD69 and CD44, and proliferated. Importantly, a large subset of this population became Foxp3(+) , more so in the lymph nodes than spleen, indicative of differentiation to a regulatory phenotype. In contrast, graft reactive CD4(+) T cells transferred to tolerogen-treated recipients at the time of transplantation failed either to proliferate or to differentiate, and instead were deleted via apoptosis. In untreated rejecting recipients graft reactive CD4(+) T cells became activated, proliferated and differentiated mainly in the spleen, and many of these cells were eventually deleted. These data resolve many apparent contradictions in the literature by showing that the timing of antigen exposure, the immunologic status of the recipients and secondary lymphoid organ location act together as key factors to determine the fate of graft reactive CD4(+) T cells.     

Porcine PD-L1: cloning, characterization, and implications during xenotransplantation

BACKGROUND: Effective intervention achieved by manipulating cell-mediated xenogeneic immune responses would critically increase the clinical feasibility of xenotransplantation as immediate hyperacute rejections become controllable through genetic modulations of donor organs. Endogenous negative regulatory signals like the programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) system are candidate targets for the control of cell-mediated xenogeneic immune response. METHODS: A porcine PD-L1 molecule was cloned using RACE (rapid amplification of cDNA ends) technology based on the human PD-L1 sequence. The functional effects of cloned porcine PD-L1 were tested on human CD4(+) T cell activation using porcine PD-L1-transfected bystander cells. Cellular proliferation was monitored by [3H] thymidine incorporation, and human T cell apoptosis was measured by flow cytometry. RESULTS: Porcine PD-L1 (GenBank accession number AY837780) was found to have 73.8% sequence homology with human PD-L1 and to contain two immunoglobulin domains in its extracellular region. Moreover, porcine PD-L1 expressed on Chinese hamster ovary (CHO) cells inhibited human CD4(+) T cell proliferation stimulated with anti-CD3 only or anti-CD3 plus anti-CD28. Percentages of apoptotic activated human T cells increased by over 30% in the presence of porcine PD-L1/CHO cells, and the addition of recombinant human PD-1-Fc fusion proteins during human T cell activation reversed the inhibitory effects of porcine PD-L1. CONCLUSIONS: Cloned porcine PD-L1 showed high sequence homology with human PD-L1 and a similar molecular structure. Moreover, porcine PD-L1 inhibited human CD4(+) T cell activation in human PD-1-dependent manner, and this involved activated T cell apoptosis. The authors suggest that PD-1-PD-L1 might play an important endogenous immune regulatory role during xenogeneic transplantation, and that the effective application of this system would improve transplanted xenogeneic organ survival.     

The pig-to-primate immune response: relevance for xenotransplantation

Abstract:  Background:  The allotransplantation of some solid organs can be associated with a graft‐vs.‐host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig‐to‐primate organ xenotransplantation. Methods:  The responses of 16 pigs (six farm‐housed wild‐type and five wild‐type housed under high herd health conditions [all designated WT], and 5 α1,3‐galactosyltransferase gene‐knockout [GT‐KO] housed under high herd health conditions) to human (n = 6) and baboon (n = 6) peripheral blood mononuclear cells (PBMC) were determined. Assays included flow cytometry, complement‐dependent cytotoxicity, and mixed lymphocyte reaction. Results:  Anti‐primate cytotoxic IgM antibodies were detected in the sera of all pigs, but anti‐primate IgG antibodies were minimal. All pigs demonstrated a cellular proliferative response to primate PBMC that was equivalent to, or greater than, the allo response. The strength of the pig‐to‐primate GVH responses was proportional to the health status of the pigs, those from a high health status herd, particularly from a specific pathogen‐free herd maintained under clean husbandry conditions, where colonization of the gastrointestinal tract may be reduced, having lower responses. Conclusions:  After pig organ transplantation in a primate, if the organ is from an early‐weaned, early‐segregated GT‐KO pig, the strength of a GVH response is likely to be relatively weak. Although not investigated here, any GVH response is likely to be suppressed by the immunosuppressive therapy administered to the recipient to suppress the anti‐donor immune response.     

T regulatory cells in xenotransplantation

Abstract: The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long‐term graft survival. In this regard, the induction of donor‐specific tolerance through isolation and expansion of xenoantigen‐specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4⁺CD25⁺Foxp3⁺ T cells, CD8⁺CD28^? Treg, double negative CD4^?CD8^? T cells, and natural killer Treg. Although only studied in vitro so far, human CD4⁺CD25⁺Foxp3⁺ Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8⁺CD28^? Treg and double negative CD4^?CD8^? Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4⁺Vα14⁺ Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4⁺CD25⁺Foxp3⁺ Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.     

Changes in cardiac gene expression after pig-to-primate orthotopic xenotransplantation

Byrne GW, Du Z, Sun Z, Asmann YW, McGregor CGA. Changes in cardiac gene expression after pig‐to‐primate orthotopic xenotransplantation. Xenotransplantation 2011; 18: 14–27. © 2011 John Wiley & Sons A/S. Abstract: Background: Gene profiling methods have been widely useful for delineating changes in gene expression as an approach for gaining insight into the mechanism of rejection or disease pathology. Herein, we use gene profiling to compare changes in gene expression associated with different orthotopic cardiac xenotransplantation (OCXTx) outcomes and to identify potential effects of OCXTx on cardiac physiology. Methods: We used the Affymetrix GeneChip Porcine Genomic Array to characterize three types of orthotopic cardiac xenograft outcomes: 1) rejected hearts that underwent delayed xenograft rejection (DXR); 2) survivor hearts in which the xenograft was not rejected and recipient death was due to model complications; and 3) hearts which failed to provide sufficient circulatory support within the first 48 h of transplant, termed “perioperative cardiac xenograft dysfunction” (PCXD). Gene expression in each group was compared to control, not transplanted pig hearts, and changes in gene expression > 3 standard deviations (±3SD) from the control samples were analyzed. A bioinformatics analysis was used to identify enrichments in genes involved in Kyoto Encyclopedia of Genes and Genomes pathways and gene ontogeny molecular functions. Changes in gene expression were confirmed by quantitative RT‐PCR. Results: The ±3SD data set contained 260 probes, which minimally exhibited a 3.5‐fold change in gene expression compared to control pig hearts. Hierarchical cluster analysis segregated rejected, survivor and PCXD samples, indicating a unique change in gene expression for each group. All transplant outcomes shared a set of 21 probes with similarly altered expression, which were indicative of ongoing myocardial inflammation and injury. Some outcome‐specific changes in gene expression were identified. Bioinformatics analysis detected an enrichment of genes involved in protein, carbohydrate and branched amino acid metabolism, extracellular matrix–receptor interactions, focal adhesion, and cell communication. Conclusions: This is the first genome wide assessment of changes in cardiac gene expression after OCXTx. Hierarchical cluster analysis indicates a unique gene profile for each transplant outcome but additional samples will be required to define the unique classifier probe sets. Quantitative RT‐PCR confirmed that all transplants exhibited strong evidence of ongoing inflammation and myocardial injury consistent with the effects of cytokines and vascular antibody‐mediated inflammation. This was also consistent with bioinformatic analysis suggesting ongoing tissue repair in survivor and PCXD samples. Bioinformatics analysis suggests for the first time that xenotransplantation may affect cardiac metabolism in survivor and rejected samples. This study highlights the potential utility of molecular analysis to monitor xenograft function, to identify new molecular markers and to understand processes, which may contribute to DXR.     

ABO-incompatible allotransplantation as a basis for clinical xenotransplantation

The 8th Congress of the International Xenotransplantation Association (IXA), held in Goteborg in September, 2005, immediately followed the 2nd International Symposium on ABO-Incompatibility in Transplantation, both congresses organized by Michael Breimer and Lennart Rydberg. The Proceedings of the Symposium on ABO-Incompatibility in Transplantation have been published (Xenotransplantation 2006; 13 (2)). The present paper provides an overview of a workshop held at the 8th Congress of the IXA, and highlights the immunological concepts emerging from ABO-incompatible allotransplants and discusses them in relation to xenotransplantation. Using specified immunomodulatory protocols, ABO-incompatible solid organ allotransplantation has become a clinical reality for a small number of patients over the last two decades. ABO-incompatible adult kidney and infant heart transplants have similar patient and graft survivals as their ABO-compatible counterparts. In contrast, ABO-incompatibility is present in up to 30% of all patients receiving hematopoietic stem cell transplants in the absence of specific immunomodulation, without affecting overall survival. Consequently, ABO-incompatible solid organ transplants and hematopoietic stem cell transplants may serve as in vivo models to elucidate the immunological mechanisms of accommodation and/or tolerance in the clinical setting. Because of similarities in the immunological hurdles that need to be overcome, knowledge obtained from ABO-incompatible allotransplantation might further promote advances in the field of xenotransplantation. The similarities and differences between ABO-incompatible allotransplantation and xenotransplantation are discussed.     

共刺激信号通路在异种移植中的作用研究进展

器官短缺是限制器官移植发展的重要因素,异种移植有望解决器官短缺问题,因此成为新的研究热点。T细胞调节相关的共刺激信号通路研究是当前异种移植免疫方面的热点话题。自从共刺激分子CD28发现以来,已经发现了多种共刺激分子,包括共刺激和共抑制受体及其相关配体。针对移植供体的特异性T细胞活化是引起急性免疫排斥反应的关键因素,不同免疫阶段共刺激分子在T细胞上表达和诱导有所不同,这些共刺激分子在维持T细胞耐受性以及T细胞免疫反应的平衡中发挥着关键作用。目前共刺激信号通路在器官移植领域的作用越来越受到重视,本文就异种移植免疫相关的共刺激信号通路的最新研究进行综述,以期为异种移植免疫抑制方案优化提供参考。     

T-cell-mediated immunological barriers to xenotransplantation

Xenotransplantion remains the most viable option for significant expansion of the donor organ pool in clinical transplantation. With the advent of nuclear transfer technologies, the production of transgenic swine has become a possibility. These animals have allowed transplant investigators to overcome humoral mechanisms of hyperacute xenograft rejection in experimental pig-to-non-human primate models. However, other immunologic barriers preclude long-term acceptance of xenografts. This review article focuses on a major feature of xenogeneic rejection: xenogeneic T cell responses. Evidence obtained from both small and large animal models, particularly those using either islet cells or kidneys, have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. Additionally, both the direct and indirect pathway of antigen presentation appear to be involved in these anti donor processes. Enhanced understanding of (i) CD47 and its role in transduced xeno-bone marrow (ii) CD39 and its role in coagulation dysregulation and (iii) thymic transplantation have provided us with encouraging results. Presently, experiments evaluating the possibility of xenogeneic tolerance are underway.     

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models

Dufrane D, D'hoore W, Goebbels R‐M, Saliez A, Guiot Y, Gianello P. Parameters favouring successful adult pig islet isolations for xenotransplantation in pig‐to‐primate models.
Xenotransplantation 2006; 13: 204–214. © Blackwell Munksgaard, 2006 Abstract: Background: In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin‐producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre‐clinical results and data in the pig‐to‐primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. Methods: Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K⁺/Na⁺ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15 000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. Results: By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P<0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short‐term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm² (r = 0.708, P<0.01) or with the percentage of large islets (r = 0.680, P<0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm² and more than 42% of large islets (>100 μm) thus enabled more than 120 000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C‐peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post‐transplantation, however, loss of islet function was associated with graft destruction and immune reaction. Conclusions: Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.     

Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses

Ezzelarab M, Ezzelarab C, Wilhite T, Kumar G, Hara H, Ayares D, Cooper DKC. Genetically‐modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T‐cell xenoresponses. Xenotransplantation 2011; 18: 183–195. © 2011 John Wiley & Sons A/S. Abstract: Background: Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non‐human primate T‐cell responses has not been studied. Methods: Mesenchymal stromal cells from wild‐type (WT), α1,3‐galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement‐regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T‐cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T‐cell proliferation and the production of pro‐inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. Results: α1,3‐galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T‐cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, <1% of GTKO pMSC expressed SLA II, compared with 2.5% of GTKO pAEC. After pig interferon‐gamma (pIFN‐γ) activation, 99% of GTKO pAEC upregulated SLA II expression, compared with 49% of GTKO pMSC. Only 3% of GTKO pMSC expressed CD80 compared with 80% of GTKO pAEC without activation. After pIFN‐γ activation, GTKO pAEC upregulated CD86 mRNA level stronger than GTKO pMSC. The human CD4⁺ T‐cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN‐γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4⁺T‐cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T‐cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL‐2, IFN‐γ, TNF‐α, and sCD40L correlated with the downregulation of T‐cell proliferation by all types of MSC. Conclusion: Genetically modified pMSC is significantly less immunogenic than WT pMSC. GTKO/CD46 pMSC downregulates the human T‐cell responses to pig antigens as efficiently as human MSC, which can be advantageous for therapeutic cell xenotransplantation.