Gilles Blancho

Long-term IgG response to porcine Neu5Gc-antigens without transmission of PERV in burn patients treated with porcine skin xenografts

Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti–non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti–non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons

Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade to prevent/cure this rejection. Here, we used a baboon model of preimmunization to explore the prevention of acute antibody-mediated rejection by an early inhibition of the classical complement pathway using human recombinant C1-inhibitor. Baboons were immunized against peripheral blood mononuclear cells from allogeneic donors and, once a specific and stable immunization had been established, they received a kidney from the same donor. Rejection occurred at day 2 posttransplant in untreated presensitized recipients, with characteristic histological lesions and complement deposition. As recombinant human C1-inhibitor blocks in vitro cytotoxicity induced by donor-specific antibodies, other alloimmunized baboons received the drug thrice daily intravenously during the first 5 days after transplant. Rejection was prevented during this treatment but occurred after discontinuation of treatment. We show here that early blockade of complement activation by recombinant human C1-inhibitor can prevent acute antibody-mediated rejection in presensitized recipients. This treatment could also be useful in other forms of acute antibody-mediated rejection caused by induced antibodies.

Rapid failure of pig islet transplantation in non human primates

Abstract: We have previously demonstrated that adult pig islets of Langerhans are not destroyed in vitro by primate sera. Whether these islets can function when placed into the liver of non‐human primates is not known. We now report on the outcome of pig islet xenotransplantation into five non diabetic primates (four baboons and one macacus fascicularis) receiving intraportally purified adult pig islets. The average number of islet‐equivalent per graft was 110000 (60–180000). All animals received associations of ATG, cyclosporine or LF 195 (a deoxyspergualin analog), mycophenolate mofetil and corticosteroids. A specific porcine C‐peptide (C‐pep) RIA test was used to monitor insulin secretion. Two hours after grafting, porcine C‐peptide was positive (from 0.37 to 4.25 ng/ml) in all monkeys except one. Primate C‐pep was normal in all cases. Only two monkeys had detectable levels of porcine C‐pep on day 1 or 2 with undetectable levels thereafter, even after glucagon challenge between days 6 and 10. Several normal islets with moderate inflammatory infiltration were observed in one animal liver on day 2 (the time of necropsy) as well as islets with IgM and complement deposition. Among animals sacrificed on days 14, 16 and 38, some residual islet cells could be identified only in livers collected on day 14. Partial glycaemic control was achieved in some rats receiving islets from the same preparations. In conclusion, adult pig islets are not able to maintain insulin secretion for more than 24 h when injected intraportally into non diabetic immunosuppressed monkeys, suggesting immediate islet xenograft destruction.

Preclinical efficacy and immunological safety of FR104, an antagonist anti-CD28 monovalent Fab' antibody.

Antagonist anti‐CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA‐4 and PDL‐1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti‐CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti‐CD28 Fab′ antibody fragment presenting a long elimination half‐life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti‐CD3 antibodies or when cross‐linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft‐versus‐host disease, which was prevented by administration of FR104 in a CTLA4‐dependent manner. Interestingly, administration of high doses of CTLA4‐Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA‐4, as compared to CD80/CD86 antagonists.

Control of Transplant Tolerance and Intragraft Regulatory T Cell Localization by Myeloid-Derived Suppressor Cells and CCL5

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that are believed to inhibit immune responses in the contexts of cancer and organ transplantation, in association with regulatory T cells (Treg). However, the way in which MDSC cooperate with Treg remains elusive. In this study, we used DNA microarrays to analyze gene expression in blood-derived MDSC from rat recipients of kidney allografts. We found CCL5 (Rantes), a chemotactic C-C motif 5 chemokine, to be strongly downregulated after treatment with a tolerizing regimen. The amount of CCL5 protein was also lower in the plasma of tolerant recipients, whereas intragraft CCL5 was unchanged. Because CCL5 is chemotactic for Treg, we hypothesized that a gradient of CCL5 between the graft and peripheral blood might contribute to the intragraft localization of Treg in tolerant animals. To test this hypothesis, we treated tolerant rat recipients of kidney allografts with recombinant rat CCL5 to restore normal plasma concentrations. This led to a strong reduction in intragraft Treg monitored by immunohistofluorescence and by quantitative real-time PCR measurement of Foxp3 mRNA. Ultimately, this treatment led to an increase in serum creatinine concentrations and to kidney graft rejection after about a month. The kidney function of syngeneic grafts was not affected by a similar administration of CCL5. These data highlight the contribution of MDSC to the establishment of a graft-to-periphery CCL5 gradient in tolerant kidney allograft recipients, which controls recruitment of Treg to the graft where they likely contribute to maintaining tolerance.

Lack of Immunotoxicity After Regional Intravenous (RI) Delivery of rAAV to Nonhuman Primate Skeletal Muscle

In the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle.

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.

Myeloid-derived suppressor cells: mechanisms of action and recent advances in their role in transplant tolerance.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses in infection, chronic inflammation, cancer, and autoimmunity. In this paper, we review recent findings detailing their mode of action and discuss recent reports that suggest that MDSC are also expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection as well as graft-versus-host disease.

Xenotransplantation of Galactosyl-Transferase Knockout, CD55, CD59, CD39, and Fucosyl-Transferase Transgenic Pig Kidneys Into Baboons

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

T cell response in xenorecognition and xenografts: a review

Xenotransplantation has recently become a subject of interest for the transplantation community due to the current organ shortage, which could be partially or even totally solved by the development of this strategy. The humoral response, which arises as a result of species disparities, is the major obstacle to the success of xenotransplantation. However, if the use of different strategies such as plasmapheresis, immunoadsorption, the utilization of organs from transgenic pigs for complement regulatory molecules and new immunosuppressive drugs, may allow to overcome or reduce the early antibody mediated rejections (hyperacute or acute vascular rejection), delayed responses based on cellular activations will still occur. In this review, despite the fact that different cell populations have been shown to be implicated in these phenomena (NK, granulocytes, macrophages), we will focus on recent published information concerning T cell response only, in xenorecognition.