Jean Paul Soulillou

Tolerance to Cardiac Allografts Via Local and Systemic Mechanisms After Adenovirus-Mediated CTLA4Ig Expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 ± 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-γ, inducible NO synthase, and TGF-β; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.

Oligoclonal myelin-reactive T-cell infiltrates derived from multiple sclerosis lesions are enriched in Th17 cells.

In this study, acute and chronic brain and spinal cord lesions, and normal appearing white matter (NAWM), were resected post-mortem from a patient with aggressive relapsing-remitting multiple sclerosis (MS). T-cell infiltrates from the central nervous system (CNS) lesions and NAWM were separated and characterized in-vitro. All infiltrates showed a proliferative response against multiple myelin peptides. Studies of the T-cell receptor (TCR)Vbeta and Jbeta usage revealed a very skewed repertoire with shared complementarity-determining region (CDR)3 lengths detected in all CNS lesions and NAWM. In the acute lesion, genomic profiling of the infiltrating T-cells revealed up-regulated expression of TCRalpha and beta chain, retinoic acid-related orphan nuclear hormone receptor C (RORC) transcription factor, and multiple cytokine genes that mediate Th17 cell expansion. The differentially expressed genes involved in regulation of Th17 cells represent promising targets for new therapies of relapsing-remitting MS.

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.

Kidney and Recipient Weight Incompatibility Reduces Long-Term Graft Survival

Long-term function of kidney allografts depends on multiple variables, one of which may be the compatibility in size between the graft and the recipient. Here, we assessed the long-term consequences of the ratio of the weight of the kidney to the weight of the recipient (KwRw ratio) in a multicenter cohort of 1189 patients who received a transplant between 1995 and 2006. The graft filtration rate increased by a mean of 5.74 ml/min between the third and sixth posttransplantation months among patients with a low KwRw ratio (<2.3 g/kg; P<0.0001). In this low KwRw ratio group, the graft filtration rate remained stable between 6 months and 7 years but then decreased at a mean rate of 3.17 ml/min per yr (P<0.0001). In addition, low KwRw ratios conferred greater risk for proteinuria, more antihypertensive drugs, and segmental or global glomerulosclerosis. Moreover, a KwRw ratio<2.3 g/kg associated with a 55% increased risk for transplant failure by 2 years of follow-up. In conclusion, incompatibility between graft and recipient weight is an independent predictor of long-term graft survival, suggesting that avoiding kidney and recipient weight incompatibility may improve late clinical outcome after kidney transplantation.

Impact of graft mass on the clinical outcome of kidney transplants

The effect of nephronic mass reduction of kidney transplants has not been analyzed specifically in a large cohort. Transplant injuries in cadaver kidney graft may have led to an underestimation of the magnitude of this factor. The aim of this study was to analyze the consequences of kidney mass reduction on transplantation outcome. The weights of 1142 kidney grafts were collected prospectively immediately before grafting. Donors and recipients <15 yr of age, simultaneous kidney/pancreas grafts, and technical failures before day 7 were excluded from the analysis. The analysis was performed on Cockroft-calculated creatinine clearance and proteinuria in 964 patients for whom all of the necessary information was available. This study reports that the smallest kidneys transplanted into the largest recipients (donor kidney weight/recipient body weight [DKW/RBW] <2 g/kg, n = 88) increased their clearance by 2.38 ml/min every month for 6 mo (P < 0.0001) and by 0.27 ml/min thereafter (P < 0.0001). Conversely, creatinine clearance did not change for the largest kidneys transplanted into the smallest recipients (DKW/RBW ratios >/=4 g/kg). Next, using a Cox model analysis, it was shown that the risk of having a proteinuria >0.5 g/kg was significantly increased for the low DKW/RBW ratios <2 g/kg with 50% of patients having a proteinuria, compared with DKW/RBW ratios >/=4 g/kg (P < 0.001). In cadaver transplant recipients, graft mass has a rapid impact on graft filtration rate and proteinuria. Avoiding major kidney/recipient inadequacy should have a significant influence on long-term transplant function.

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.

Presence of leukaemia inhibitory factor and interleukin 6 in porcine uterine secretions prior to conceptus attachment

Leukaemia inhibitory factor (LIF) plays an important role in embryo development and implantation. We detected peak LIF activity in porcine uterine luminal fluids (ULF) at day 12 of gestation and during day 7 and 13 of the oestrous cycle. A radio-receptor competition assay showed the presence of a molecule in ULF specifically binding to human LIF receptor (LIF-R). LIF activity was partially neutralized by anti-human LIF antibody. Interleukin-6 (IL-6) activity was detected in ULF throughout the oestrous cycle and pre-implantation period. An anti-murine alpha chain (gp80) of IL-6 receptor (IL-6R) specifically neutralized this activity. LIF and IL-6 mRNA were only detected in day 11 endometrium. The presence of LIF or IL-6 in the uterine cavity has not been previously reported. Our results extend LIF production by endometrium during the oestrous cycle and pre-implantation period to another mammalian species other than mouse.

Production of human interleukin for DA cells (HILDA)/leukemia inhibitory factor (LIF) by activated monocytes

We demonstrate mRNA accumulation and production of HILDA/LIF by human activated Mo, monocyte-derived macrophages and myelomonocytic cell lines. Among the various stimuli tested, the synergistic combination of phorbol diester and VD3 was the most potent inducer of HILDA/LIF gene expression. The kinetics of mRNA accumulation on activated Mo showed a stimulation peak at 24 hr which declined thereafter. HILDA/LIF activity in culture supernatants was detected at 24 hr and reached a plateau at 72 hr of culture. In contrast to Mo, PBL did not accumulate HILDA/LIF mRNA upon culture with PDBu and VD3, whereas PHA and the combination of PDBu and A23187 induced HILDA/LIF mRNA accumulation and secretion in the culture supernatant. To exclude the possibility that HILDA/LIF was produced by contaminating PBL, highly enriched Mo preparations were used, which were devoid of T cells as assessed by the absence of TCR-beta chain mRNA transcripts. HILDA/LIF production by monocytic cells was further documented by the capacity of stimulated U937 cell conditioned medium to compete with 125I-labeled nHILDA/LIF for binding to its receptor on murine M1 cells. Under the synergistic effect of PDBu and VD3 stimulation, Mo-derived macrophages as well as HL-60 and U937 cell lines accumulated HILDA/LIF mRNA and produced this cytokine with identical kinetics as for Mo. Finally, we show that HILDA/LIF mRNA accumulation in U937 cells upon stimulation with PDBu, or the combination of PDBu and VD3, was inhibited in the presence of the protein synthesis inhibitor CHX. These results document for the first time that human Mo, when stimulated appropriately in vitro, can express the HILDA/LIF gene and its product, and that intermediate proteins must be newly synthesized in this process.

Effect of brain-dead donor resuscitation on delayed graft function: results of a monocentric analysis.

Background. We have previously shown that a delayed graft function (DGF) longer than 6 days was a crucial threshold for long-term graft outcome. The aim of this study was to analyze the correlation of DGF ≥6 days with brain-dead donor variables, including those related to resuscitation, in a population of 262 consecutive brain-dead donors from 1990 to 2003. Methods. We used a marginal logistic model in which DGF was considered as a binary variable with a cutoff of 6 days. Results. Monovariate analysis of donor parameters showed that male, age above 35 years, primary history of hypertension, hydroxyethyl starch (HES) fluid greater than 1500 mL or epinephrine infusion during resuscitation were risk factors for prolonged DGF. The multivariate logistic regression model showed that epinephrine use during donor resuscitation (P<0.001, odds ratio [OR]=4.35), cold ischemia time (CIT) ≥16 hr (P=0.01, OR=2.16), and recipient age >55 years (P=0.003, OR=2.75), were associated with a risk of prolonged DGF. A long stay (>40 hr) in intensive care and a large volume of colloids (>1250 mL, except HES) correlated with a lower risk of DGF. Conclusion. Our study shows an impact for only a limited number of brain dead donor resuscitation parameters on DGF duration. We also show that CIT has a much lower threshold (<16 hr) for DGF risk than previously described. Importantly, we show that recipient age is clearly a major independent risk factor for prolonged DGF, whereas donor age seems to act mostly as a dependent risk factor.

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.