Design, cohort profile and comparison of the KTD-Innov study: a prospective multidimensional biomarker validation study in kidney allograft rejection.

There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018-2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9-13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable.

Subclinical rejection-free diagnostic after kidney transplantation using blood gene expression.

We previously established a six-gene-based blood score associated with operational tolerance in kidney transplantation which was decreased in patients developing anti-HLA donor-specific antibodies (DSA). Herein, we aimed to confirm that this score is associated with immunological events and risk of rejection. We measured this using quantitative PCR (qPCR) and NanoString methods from an independent multicenter cohort of 588 kidney transplant recipients with paired blood samples and biopsies at one year after transplantation validating its association with pre-existing and de novo DSA. From 441 patients with protocol biopsy, there was a significant decrease of the score of tolerance in 45 patients with biopsy-proven subclinical rejection (SCR), a major threat associated with pejorative allograft outcomes that prompted an SCR score refinement. This refinement used only two genes, AKR1C3 and TCL1A, and four clinical parameters (previous experience of rejection, previous transplantation, sex of recipient and tacrolimus uptake). This refined SCR score was able to identify patients unlikely to develop SCR with a C-statistic of 0.864 and a negative predictive value of 98.3%. The SCR score was validated in an external laboratory, with two methods (qPCR and NanoString), and on 447 patients from an independent and multicenter cohort. Moreover, this score allowed reclassifying patients with discrepancies between the DSA presence and the histological diagnosis of antibody mediated rejection unlike kidney function. Thus, our refined SCR score could improve detection of SCR for closer and noninvasive monitoring, allowing early treatment of SCR lesions notably for patients DSA-positive and during lowering of immunosuppressive treatment.

IL-7 is expressed in malignant mesothelioma and has a prognostic value.

Malignant pleural mesothelioma (MPM) is an aggressive cancer mainly related to asbestos exposure. Despite recent therapeutic advances, notably immunotherapies, the benefit remains limited and restricted to a small percentage of patients. Thus, a better understanding of the disease is needed to identify new therapeutic strategies. Recently, interleukin 7 receptor (IL-7R) has been described as being expressed by MPM cells and associated with poorer patient survival. Thus, the aim of this work was to study the IL-7R/IL-7 pathway in MPM using patient samples. We found that, although more than 40% of MPM cells expressed IL-7R, IL-7 had no effect on their intracellular signaling. Accordingly, the addition of IL-7 to the culture medium did not affect MPM cell growth. Using The Cancer Genome Atlas (TCGA) database, we showed that high IL7 gene expression in MPM tumors was associated with a higher overall patient survival and an induction of genes involved in the immune response. In pleural effusions (PEs), we found that IL-7 concentration was not a good diagnostic biomarker. However, we observed that high IL-7 levels in PEs were associated with shorter survival of MPM patients, but not of lung cancer patients. The prognostic value of IL-7 was also conserved when only patients with epithelioid mesothelioma, the most common histological type of MPM, were analyzed. Taken together, our study suggests that, although the IL-7R/IL-7 signaling pathway is not functional in MPM cells, IL-7 expression in PEs may have prognostic value in MPM patients.

Efficient Expansion of Human Granzyme B-Expressing B Cells with Potent Regulatory Properties.

Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25- effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.

Targeting the interleukin-7 receptor alpha by an anti-CD127 monoclonal antibody improves allergic airway inflammation in mice.

BACKGROUND: Interleukin-7 (IL-7) is the most important cytokine for T-cell homeostasis. IL-7 signals through the IL-7 receptor (IL-7R) which is composed of an alpha chain (IL-7Rα), also called CD127 and a common gamma chain. T lymphocytes, especially T helper type 2, play a crucial role in the pathobiology of allergic asthma. OBJECTIVE: To study the effects of an anti-CD127 monoclonal antibody (mAb) in a murine model of allergic airway inflammation induced by house dust mite (HDM). METHODS: Allergic airway inflammation was induced in mice using a protocol comprising 4 weekly percutaneous sensitizations followed by 2 weekly intranasal challenges with total HDM extracts and treated by intraperitoneal injections of an anti-CD127 mAb. Because CD127 is shared by both IL-7R and the receptor for thymic stromal lymphopoietin (TSLP), a group of mice was also treated with an anti-IL-7 mAb to block only the IL-7 signalling pathway. RESULTS: Anti-CD127 mAb-treated mice showed significantly lower airway resistance in response to methacholine and improvement in lung histology compared with isotype mAb-treated animals. Anti-CD127 mAb treatment significantly decreased the mRNA expression of Th2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CCL5/RANTES) in lung tissue, decreased the secretion of Th2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CXCL1 and CCL11/eotaxin) in bronchoalveolar lavage fluid (BALF), decreased serum HDM-specific IgE, and reduced the number of total leucocytes and leucocyte subpopulations such as eosinophils, macrophages, lymphocytes, T lymphocytes, and ILC2 in BALF and lung tissue. Mice treated with anti-IL-7 mAb also showed less allergic airway inflammation as evidenced by significantly lower airway resistance and fewer leucocytes in BALF and lung tissue compared to mice treated with the corresponding isotype control mAb. CONCLUSION AND CLINICAL RELEVANCE: Targeting the IL-7Rα by an anti-CD127 mAb improves allergic airway inflammation in mice and presents as a potential therapeutic approach for allergic asthma.

Interleukin-7 receptor blockade by an anti-CD127 monoclonal antibody in nonhuman primate kidney transplantation.

IL-7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL-7 signaling pathway has been shown to induce long-term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti-human cluster of differentiation 127 (CD127) mAb administered in combination with low-dose tacrolimus or thymoglobulin in a life-sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti-CD127 mAb to the treatment protocols did not prolong graft survival compared to low-dose tacrolimus alone or thymoglobulin alone. Anti-CD127 mAb administration led to full CD127 receptor occupancy during the follow-up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti-CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL-7 is not a limiting factor for T cell homeostasis in primates.

Elicited and pre-existing anti-Neu5Gc antibodies differentially affect human endothelial cells transcriptome.

Humans cannot synthesize N-glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet-induced anti-Neu5Gc antibodies (Abs) develop early in human life. While the interaction of Neu5Gc and diet-induced anti-Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti-Neu5Gc Abs following a challenge with animal-derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity-purified anti-Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc-glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti-Neu5Gc Abs, compared with pre-existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti-Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre-existing or anti-human T-cell globulin (ATG)-elicited anti-Neu5Gc Abs. Compared with pre-existing anti-Neu5Gc Abs, which are normal component of ECs environment, elicited anti-Neu5Gc Abs down-regulated 66 genes, including master genes of EC function. Furthermore, elicited anti-Neu5Gc Abs combined with complement-containing serum down-regulated most transcripts mobilized by serum alone. Both types of anti-Neu5Gc Abs-induced a dose- and complement-dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre-existing anti-Neu5Gc Abs, ATG-elicited anti-Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro-inflammatory profile in vitro.

Poor Patient and Graft Outcome After Induction Treatment by Antithymocyte Globulin in Recipients of a Kidney Graft After Nonrenal Organ Transplantation.

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.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.