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Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in

BackgroundHigh consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown.MethodsWe used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ≥ 18 years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged > 60, Q1 and Q4; 10 men/women per quartile).ResultsWe found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p = 0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94 ng/μl versus 2.22 ng/μl, respectively; p = 0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gcα3-linked glycans, increasing the α3/α6-glycans ratio score.ConclusionsWe found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.

Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah-/- mice with NGpos glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets.

Background:End-stage renal failure occurs in a substantial number of patients having received a nonrenal transplantation (NRT), for whom a kidney transplantation is needed. The medical strategy regarding the use of immunosuppression (IS) for a kidney graft in patients after an NRT is not well established. The prekidney grafts long-term IS advocates for a mild induction, such as using anti-IL-2R antibodies, whereas addition of new incompatibilities and anti-HLA preimmunization may suggest using stronger IS such as induction by polyclonal antithymocyte globulins (ATG). Methods:We performed Cox multivariate and propensity score analysis of our validated transplant database to study the impact of the type of induction therapy on kidney graft survival of recipients of a kidney graft after NRT. Results:We report here that kidney transplantation after NRT treated with an ATG induction has a poorer outcome (kidney and recipient survival) than that with an anti-IL-2R induction. After accounting for potential baseline differences with a multivariate Cox model, or by adjusting on a propensity score, we found that despite patients having received ATG cumulate more risk factors, ATG appears independently involved. As animal-derived biotherapeutics induce antiglycan antibodies and particularly anti-N-glycolylneuraminic acid (Neu5Gc) IgGs which may activate endothelial cells in patients and grafts, we also investigated the magnitude and the nature of the anti-Neu5Gc elicited by the induction and showed that induction was associated with a shift in anti-Neu5Gc IgG repertoire. Possible reasons and mechanisms of a deleterious ATG usage in these patients are discussed. Conclusions:Our study suggests that ATG induction after a kidney transplantation in recipients already under maintenance IS for a NRT should be used cautiously.

Humans have circulating antibodies against diverse glycans containing N-glycolylneuraminic acid (Neu5Gc) due to function-loss mutation of the CMAH gene. This xenogenic non-human carbohydrate is abundant in red meat, xenografts and biotherapeutics. Low levels of diet-derived Neu5Gc is also present on normal human endothelial cells, and together with anti-Neu5Gc antibodies could potentially mediate "xenosialitis" chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a drug containing polyclonal IgG glycoproteins commonly used as an immunosuppressant in human transplantation and autoimmune diseases. In type-1 diabetes patients, infusion of Neu5Gc-glycosylated ATG caused increased global anti-Neu5Gc response. Here, for the first time we explore changes in anti-Neu5Gc IgG repertoire following the immunization elicited by ATG, compared with the basal antibodies repertoire that reflect exposure to dietary-Neu5Gc. We used glycan microarrays with multiple Neu5Gc-glycans and controls to elucidate eventual differences in ATG-elicited repertoire, before/after ATG administration and track their kinetics (0, 1, 18 and 24 months). Response of all basal-pre-existing Neu5Gc-specific antibodies rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18-24 months. Induced-antibodies showed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including endogenous glycolipids, that was further validated by affinity-purified anti-Neu5Gc antibodies from patients' sera. These findings strongly suggest that ATG-induced anti-Neu5Gc IgGs represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies.

BackgroundThe two common sialic acids (Sias) in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients.MethodsWe investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG.ResultsNeu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia linkages revealed Siaα2-3>Siaα2-6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2-3>Siaα2-6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express 4-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3±16.9 pmol Sia/μg protein), with Neu5Ac at 8-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity toward Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts.ConclusionsWe conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation.

Background and aimsThe beneficial effect of one graft on another has been reported in combined transplantation but the associated mechanisms and biological influence of each graft have not yet been established.MethodsIn multiple analyses, we explored the PBMC phenotype and signature of 45 immune-related messenger RNAs and 754 microRNAs from a total of 235 patients, including combined liver-kidney transplant recipients (CLK), patients with a liver (L-STA) or kidney (K-STA) graft only under classical immunosuppression and patients with tolerated liver (L-TOL) or kidney grafts (K-TOL).ResultsCLK show an intermediary phenotype with a higher percentage of peripheral CD19(+) CD24(+) CD38(Low) memory B cells and Helios(+) Treg cells, two features associated with tolerance profiles, compared to L-STA and K-STA (P < 0.05, P < 0.01). Very few miRNA were significantly differentially expressed in CLK vs. K-STA and even fewer when compared to L-STA (35 and 8, P < 0.05). Finally, CLK are predicted to share common miRNA targets with K-TOL and even more with L-TOL (344 and 411, P = 0.005). Altogether CLK display an intermediary phenotype and gene profile, which is closer to that of liver transplant patients, with possible similarities with the profiles of tolerant patients.ConclusionThese data suggest that CLK patients show the immunological influence of both allografts with liver having a greater influence.

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.

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti- TNF, or anti-[[alpha].sub.4][[beta].sub.7] therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled [[alpha].sub.4][[beta].sub.7] integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment., Introduction Inflammatory bowel disease (IBD) consists of 2 major forms, ulcerative colitis (UC) and Crohn's disease (CD), which are chronic relapsing gastrointestinal disorders characterized by chronic intestinal inflammation, dysregulated immune [...]

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4- Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/ sirolimus recipients in the setting of sepsis and a paralyzed INF-[gamma] response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant., Introduction Graft-versus-host disease (GVHD) represents a major cause of mortality after hematopoietic stem cell transplant (HCT) (1-4), and despite substantial effort, the control of GVHD remains one of the key [...]

BACKGROUND. Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS. We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [[SSD.sup.+]] and 803 without SSD [[SSD.sup.-]]) from the Donnees Informatisees et Validees en Transplantation data bank. Two subgroups of [SSD.sup.+] and [SSD.sup.-] patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS. SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, [SSD.sup.+] patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with [SSD.sup.-] recipients. CONCLUSION. In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in [SSD.sup.+] patients. FUNDING. This study was funded by Societe d'Acceleration du Transfert de Technologies Ouest Valorisation, the European FP7 'Translink' research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation., Introduction Polyclonal anti-human T lymphocyte IgGs (antithymocyte globulins [ATGs]) from various animal sources have been used since the very beginning of allotransplantation (1, 2). ATGs are administered either immediately following [...]

T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti-IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4- and anti-CD8-mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation., Introduction T cell depletion by antibodies is one of the most potent immunosuppressive therapies and is increasingly used as an induction therapy in organ transplantation (1). However, T cell homeostasis [...]

Whilst calcineurin inhibitors (CNI) are the cornerstone of immunosuppressive maintenance therapy in kidney transplantation, several studies have investigated the safety of CNI withdrawal in order to avoid their numerous side effects. In this context, we performed several years ago a clinical randomized trial evaluating CNI weaning in stable kidney transplant recipients without anti-HLA immunization. The trial was interrupted prematurely due to a high number of de novo DSA (dnDSA) and biopsy proven acute rejection (BPAR) in patients who underwent tacrolimus weaning, resulting in treatment for rejection and resumption of tacrolimus. We report here the long-term outcomes of patients included in this clinical trial. Ten years after randomization, all patients are alive with a functional allograft. They all receive tacrolimus therapy except one with recurrent cutaneous neoplasia issues. Long-term eGFR was comparable between patients of the two randomized groups (46.4 ml/min vs 42.8 ml/min). All dnDSA that occurred during the study period became non-detectable and all rejections episodes were reversed. The retrospective assessment of HLA DQ single molecule epitope mismatching determined that a majority of patients who developed dnDSA after tacrolimus withdrawal would have been considered at high immunological risk. Minimization of immunosuppression remains a challenging objective, mainly because of the issues to properly select very low immunological risk patients. Valuable improvements have been made the last decade regarding evaluation of the allograft rejection notably through the determination of numerous at-risk biomarkers. However, even if the impact of such tools still need to be clarify in clinical routine, they may permit an improvement in patients' selection for immunosuppression minimization without increasing the risk of allograft rejection. [ABSTRACT FROM AUTHOR]