Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells

Ezzelarab C, Ayares D, Cooper DKC, Ezzelarab MB. Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells. Xenotransplantation 2012; 19: 311–316. © 2012 John Wiley & Sons A/S. Abstract: Background: Thrombin formation is a key feature in the activation of coagulation in pig xenograft recipients. As thrombin is known to activate endothelial and immune cells, we explored whether thrombin activation of pig endothelial cells (EC) was associated with an increased human T‐cell response. Methods: α1,3‐galactosyltransferase gene‐knockout (GTKO) pig aortic EC (pAEC) were activated by porcine interferon‐gamma (pIFNγ), human (h)IFN‐γ, or thrombin. Swine leukocyte antigen (SLA) class I and class II expression were measured. Human peripheral blood mononuclear cells (PBMC) and CD4⁺ T‐cell proliferation in response to activated pAEC, the effect of thrombin on pig CD80/CD86 mRNA, and the effect of thrombin inhibition by hirudin were evaluated. Results: After pAEC activation, SLA I expression did not change, and only pIFNγ upregulated SLA II expression. PBMC proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. CD4⁺ T‐cell proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. Thrombin inhibition by hirudin reduced thrombin‐induced upregulation of pAEC CD86 mRNA, and significantly reduced human PBMC proliferation to pAEC in comparison with thrombin alone (P < 0.05). Conclusions: Thrombin upregulates CD86 mRNA on pAEC, which is associated with increased human T‐cell proliferation against pAEC. Hirudin reduces CD86 mRNA in thrombin‐activated pAEC and is associated with downregulation of the human T‐cell proliferative response. The transplantation of organs from GTKO pigs transgenic for human thrombomodulin, and/or endothelial protein C receptor, in addition to therapeutic regulation of thrombin activation may reduce the cellular response to a pig xenograft and thus reduce the need for intensive immunosuppressive therapy.     

Association of high IFN‐γ plasma levels with low B‐cell counts in renal transplant recipients with stable long‐term graft function

Daniel V, Naujokat C, Sadeghi M, Renner FC, Weimer R, Opelz G. Association of high IFN‐γ plasma levels with low B‐cell counts in renal transplant recipients with stable long‐term graft function.
Clin Transplant 2009 DOI: 10.1111/j.1399‐0012.2009.01067.x
© 2009 John Wiley & Sons A/S. Abstract: Recently, we reported that patients with long‐term stable good graft function had higher interferon‐gamma (IFN‐γ) and lower IL‐4 plasma levels late as compared with early post‐transplant. These patients had more often detectable CD3⁺CD4⁺CD25⁺IFN‐γ⁺Foxp3⁺ peripheral blood lymphocytes (PBL) late post‐transplant than patients with impaired graft function. We therefore speculated that high plasma IFN‐γ late post‐transplant might contribute to the maintenance of graft acceptance. Using ELISA and four‐color flow cytometry, plasma cytokines and PBL subpopulations were measured in 65 renal transplant recipients with stable graft function late post‐transplant. High IFN‐γ plasma levels were associated with low CD19⁺ B PBL (r = ?0.329; p = 0.009) and low activated CD3⁺CD8⁺DR⁺ T PBL (r = ?0.266; p = 0.035). Plasma IFN‐γ increased with time post‐transplant (r = 0.288; p = 0.022) and was not associated with the dose of immunosuppressive drugs (p = n.s.). High plasma IFN‐γ was not associated with serum creatinine (r = 0.038; p = 0.765). Five patients showed evidence of chronic allograft nephropathy in previous biopsies and none of them exhibited increased plasma IFN‐γ. In patients with good long‐term graft function, high IFN‐γ plasma levels were associated with low numbers of B PBL and activated CD8⁺ T PBL. High IFN‐γ plasma levels might prevent the development of an immunological alloresponse and thereby contribute to the maintenance of graft acceptance.     

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights,innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets. Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade. Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups. We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to >250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.     

Cross‐Validation of IFN‐γ Elispot Assay for Measuring Alloreactive Memory/Effector T Cell Responses in Renal Transplant Recipients

Assessment of donor‐specific alloreactive memory/effector T cell responses using an IFN‐γ Elispot assay has been suggested to be a novel immune‐monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross‐validation data of the IFN‐γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN‐γ plates assessing intra‐ and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count‐settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN‐γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen‐specific primed/memory T cell alloresponses. Interestingly, significant high donor‐specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF‐γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

Interferon‐γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1^?/?). Compared with their wild‐type littermates (IFNγR1^+/+), IFNγR1^?/? mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1^?/? mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Interferon Gamma and Contact‐dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell–Mediated Antibody‐dependent Chronic Rejection

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor‐specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody‐mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN‐γ) versus the contact‐dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune‐deficient C57BL/6 rag^?/?γc^?/? recipients, who also received monoclonal anti–major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild‐type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN‐γ–deficient NK cells or host IFN‐γ neutralization led to amelioration of these lesions. Use of either perforin‐deficient NK cells or CD95 (Fas)–deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell–derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN‐γ production and contact‐dependent cytotoxic activity are rate‐limiting effector pathways that contribute to this form of antibody‐induced chronic allograft vasculopathy.     

Induction of porcine‐specific regulatory T cells with high specificity and expression of IL‐10 and TGF‐β1 using baboon‐derived tolerogenic dendritic cells

Background

Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system.

Method

Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4⁺ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity.

Results

TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4⁺ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3⁺ CD4⁺ CD25⁺ CD127^low/? CD45RA^low Foxp3⁺ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff.

Conclusion

In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.     

Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response

Sun L, Yi S, O’Connell PJ. Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response. Xenotransplantation 2010; 17: 121–130. © 2010 John Wiley & Sons A/S. Abstract: Backgrounds: Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Foxp3 expressing human naturally occurring CD4+CD25+ regulatory T cells (nTregs) have been shown to suppress pathological and physiological immune responses, including the CD4+ T‐cell‐mediated anti‐pig xenogeneic response in vitro. Although Foxp3 is required for nTreg development and their function, the precise role of Foxp3 in regulating Treg suppressive function in xenoimmune response remains to be identified. Methods: In vitro expanded human nTregs were transfected with fluorescein isothiocyanate ‐conjugated Foxp3 small interfering RNA (siRNA) by Lipofectamine 2000. Transfected nTregs were sorted by fluorescence‐activated cell sorting, and then analyzed for Foxp3 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25? T cells were stimulated with xenogeneic pig peripheral blood mononuclear cell in the presence or absence of nTregs in a coculture or transwell system for evaluation of nTreg suppressive activity. The production of effector cytokines by xenoreactive CD4+CD25? T cells as well as suppressive cytokine by nTregs in their cocultures was examined by ELISA. Results: The siRNA‐mediated Foxp3 knockdown resulted in impaired nTreg anergic state, downregulated expression of nTreg function associated molecules, and reduced production of suppressive cytokines by nTregs, which together leading to impaired nTreg‐mediated suppression of CD4+CD25? T‐cell proliferation and their effector cytokine production in response to xenogeneic stimulation. Conclusions: This study demonstrates that Foxp3 expression is required for human nTregs to maintain their suppressive function in the xenoimmune response.     

Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts

We have previously demonstrated that indoleamine 2, 3‐dioxygenase (IDO) expressed by dermal fibroblasts generated a tryptophan deficient environment in which immune cells, but not skin cells, undergo apoptosis. However, the mechanism by which primary skin cells such as fibroblasts and keratinocytes are resistant to this culture environment is not elucidated. Here, we asked the question of whether the activity of the general control nonderepressing‐2 (GCN2) kinase pathway in primary immune and skin cells is differently regulated in response to IDO‐induced tryptophan deficient environment. Before addressing this question, the expression of IDO in IDO‐adenoviral infected fibroblasts, as a source of IDO expression, was validated. We then demonstrated a significant immunosuppressive effect of IDO expression in primary human T cells co‐cultured with IDO expressing fibroblasts in the presence of allogeneic pieces of either epidermis or full thickness skin. Evaluating the mechanism by which skin cells, but not T cells, are resistant to IDO induced low tryptophan environment, we then co‐cultured IDO‐expressing fibroblasts with bystander human T cells, the fibroblasts, or keratinocytes for 3 days. The results showed a significant activation of apoptotic pathway as analyzed by caspase‐3 induction as well as the expression of CHOP, a downstream effector of GCN2 kinase pathway in T cells, but not in skin cells.     

Liver Transplantation in a Patient With CD40 Ligand Deficiency and Hyper‐IgM Syndrome: Clinical and Immunological Assessments

Monoclonal antibodies that disrupt CD40–CD40 ligand (CD40L) interactions are likely to have use in human transplantation. However, the extent of the immunosuppressive effects of CD40–CD40L blockade in humans is unknown. Hyper‐IgM syndrome (HIGM) is a rare primary immunodeficiency syndrome characterized by defects in the CD40–CD40L pathway, severe immune deficiency (IgG), and high or normal IgM levels. However, the effects of CD40L deficiency on T‐ and natural killer (NK)‐cell function is not established. Here, we present a patient with HIGM syndrome who underwent liver transplantation for hepatitis C virus infection. Posttransplantation, NK‐cell antibody‐dependent cytokine release (γ‐interferon) to alloantigens and T cell responses to viral antigens and mitogens were assessed and showed normal CD4⁺, CD8⁺, and NK‐cell responses. We also examined antibody‐dependent cellular cytotoxicity against a CD40⁺ and HLA‐expressing cell line. These experiments confirmed that the patient's NK cells were equivalent to those of normal subjects in mediating antibody‐dependent cellular cytotoxicity despite the absence of CD40–CD40L interactions. Mitogenic stimulation of the patient's peripheral blood mononuclear cells showed no expression of CD40L on T and NK cells compared with increased expression in normal subjects. Taken together, these data suggest that absence of CD40L expression is responsible for aberrant B cell immunity but had little impact on NK‐ and T cell immune responses in vitro.     

Cytokine Interferon‐γ suppresses the function of capsule myofibroblasts and induces cell apoptosis

Myofibroblasts (MFs), a contractile subset of fibroblasts, play a pivotal role in physiological wound healing and in the development of many fibroconnective disorders. The complex cytokine network regulating the function of MFs in joint stiffness is still poorly understood. In this in vitro study, we investigated the effect of the cytokine Interferon‐gamma (IFN‐γ) on MFs isolated from human joint capsules. MFs were cultivated either in the presence of increasing concentrations of IFN‐γ alone or in combination with IFN‐γ neutralizing antibodies. Cell viability, cytotoxicity, apoptosis, and mRNA gene expression of the MF markers alpha‐smooth muscle actin (α‐SMA) and collagen type I were analyzed in MF cultures. Contraction potential was analyzed in an established collagen gel contraction assay simulating the extracellular matrix. Using immunofluorescence staining, we could verify that MFs express IFN‐γ‐receptor (R)‐1 on their membrane. IFN‐γ decreased MF viability and significantly elevated the apoptosis rate in a dose‐dependent manner. IFN‐γ down‐regulated α‐SMA and collagen type I mRNA expression which was associated with a diminished MF mediated contraction of the gel matrices. These effects were suppressed by simultaneous treatment of cells with a neutralizing IFN‐γ antibody. Our experiments confirm the hypothesis that the cytokine IFN‐γ is a crucial component of the regulatory network of capsule MFs. IFN‐γ notably influences the ability of MFs to contract collagen matrices by suppressing α‐SMA gene expression. IFN‐γ is toxic for MFs in high concentrations and may negatively regulate the number of pro‐fibrotic MFs during the healing process via induction of cell apoptosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2524–2533, 2017.

     

Monocytes and γδ T cells control the acute‐phase response to intravenous zoledronate: Insights from a phase IV safety trial

Aminobisphosphonates (NBPs) are used widely against excessive bone resorption in osteoporosis and Paget's disease as well as in metastatic bone disease and multiple myeloma. Intravenous NBP administration often causes mild to severe acute‐phase responses (APRs) that may require intervention with analgesics and antipyretics and lead to treatment noncompliance and nonadherence. We here undertook a phase IV safety trial in patients with osteoporosis to investigate the APR of otherwise healthy individuals to first‐time intravenous treatment with the NBP zoledronate. This study provides unique insight into sterile acute inflammatory responses in vivo, in the absence of confounding factors such as infection or cancer. Our data show that both peripheral γδ T cells and monocytes become rapidly activated after treatment with zoledronate, which ultimately determines the clinical severity of the APR. Our study highlights a key role for IFN‐γ in the zoledronate‐induced APR and identifies pretreatment levels of monocytes and central/memory Vγ9/Vδ2 T cells as well as their responsiveness to zoledronate in vitro as predictive risk factors for the occurrence of subclinical and clinical symptoms. These findings have diagnostic and prognostic implications for patients with and without malignancy and are relevant for Vγ9/Vδ2 T‐cell–based immunotherapy approaches. © 2012 American Society for Bone and Mineral Research. © 2013 American Society for Bone and Mineral Research.     

Local expression of indoleamine 2,3‐dioxygenase suppresses T‐cell‐mediated rejection of an engineered bilayer skin substitute

Engineered skin substitutes (ESSs) comprising both keratinocytes and fibroblasts can afford many advantages over the use of autologous keratinocyte grafts for the treatment of full‐thickness and partial‐thickness burns. In this study, we investigated the efficacy of a novel ESS containing both genetically altered fibroblasts that express the immunosuppressive factor indoleamine 2,3‐dioxygenase (IDO) and primary keratinocytes from a nonautologous source to confer immune protection of xenogeneic cells cultured in a bilayer ESS. The results show that engraftment of IDO expressing skin substitutes on the back of rats significantly improves healing progression over 7 days compared with both nontreated and non‐IDO‐expressing skin substitutes (p<0.001). Immuno‐staining of CD3 and CD31 suggests that IDO‐expressing skin substitutes significantly suppress T cell infiltration (p<0.001) and improve neovascularization by four‐fold (12.6±1.2 vs. 3.0±1.0 vessel‐like structure/high power field), respectively. In conclusion, we found that IDO expression can improve the efficacy of nonautologous ESS for the purpose of wound healing by mitigating T‐cell infiltration as well as promoting vascularization of the graft.     

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.     

Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase expression of interferon-γ–treated skin cells in a co-culture system

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme possessing various immunosuppressive characteristics. In this study, we report the possible use of this enzyme in an allogenic skin substitute to suppress the proliferation of immune cells. Human fetal skin fibroblasts and keratinocytes were treated with the cytokine interferon-gamma to induce expression of IDO mRNA and protein. IDO enzyme activity was evaluated by measurement of kynurenine levels in the interferon-gamma-treated and -untreated cells. Results of Northern analysis showed a dose-dependent response in expression of IDO mRNA to the various concentrations of interferon-gamma used. Northern blot analysis also showed a time-dependent expression of IDO in response to different durations of interferon-gamma treatment. The level of kynurenine measured, as the bioactivity of IDO enzyme, was significantly higher in the interferon-gamma-treated fibroblasts and keratinocytes compared to those of controls (p < 0.001). To illustrate the immunosuppressive effects of IDO on immune cell proliferation, IDO-expressing fibroblasts were cocultured with human peripheral blood mononuclear cells for a period of 5 days. Results of 3H-thymidine incorporation assays showed a significant reduction in proliferation of the mononuclear cells cocultured with IDO-expressing skin cells compared to monocytes cocultured with control (non-IDO-expressing) skin cells (p < 0.001). Furthermore, addition of the IDO-inhibitor (1-methyl-D-tryptophan) significantly reversed the immunosuppressive effects of IDO on monocyte proliferation (p < 0.001). In conclusion, suppression of peripheral blood mononuclear cell proliferation due to interferon-gamma-induced IDO-expression in allogenic human skin cells might shed new light on developing a nonrejectable allogenic skin substitute.     

Galectin‐1 Prolongs Survival of Mouse Liver Allografts From Flt3L‐Pretreated Donors

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin‐1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin‐1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin‐1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin‐1 pathway via neutralizing antigalectin‐1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal‐1 significantly prolonged survival of liver allografts from Flt3L‐pretreated donors and ameliorated Flt3L‐triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1‐associated cytokines (IL‐12, IL‐2 and IFN‐γ), Th17‐associated cytokines (IL‐23 and IL‐17) and granzyme B, in parallel with selectively increased IL‐10 expression in liver allografts. In vitro, galectin‐1 inhibited Flt3L‐differentiated DC‐mediated proliferation of allo‐CD4⁺ T cells and production of IFN‐γ and IL‐17. These data provide new evidence of the potential regulatory effects of galectin‐1 in alloimmune responses in a murine model of liver transplantation.     

Clinical validation of a novel enzyme‐linked immunosorbent spot assay‐based in vitro diagnostic assay to monitor cytomegalovirus‐specific cell‐mediated immunity in kidney transplant recipients: a multicenter,longitudinal, prospective,observational study

Impaired cytomegalovirus (CMV)‐specific cell‐mediated immunity (CMV‐CMI) is a major cause of CMV reactivation and associated complications in solid‐organ transplantation. Reliably assessing CMV‐CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T‐Track^® CMV, a novel IFN‐γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE‐I CMV proteins, to monitor CMV‐CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate‐risk renal transplant recipients. CMV‐CMI, CMV viral load, and clinical complications were monitored over 6 months post‐transplantation. Ninety‐five percent and 88–92% ELISpot assays were positive pre‐ and post‐transplantation, respectively. CMV‐specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65‐specific response was ninefold higher in patients with self‐clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T‐Track^® CMV is a highly sensitive IFN‐γ ELISpot assay, suitable for the immunomonitoring of CMV‐seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV‐related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).