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Circulating immune cells are critical mediators of the response to inflammation upon recruitment to the tissue but how gene expression state influences recruitment is not well known. Here we report the longitudinal single-cell transcriptome profiling of blood mononuclear cells in patients undergoing kidney transplantation rejection. We identify a gene expression module which is associated to transcriptional regulation, homing and early activation in multiple cell types. The circulating cells expressing this module are reduced in patients undergoing graft rejection. This reduction was confirmed in a pig model of acute kidney transplantation rejection. In connection with this, the module expression drastically increased in the kidney grafts undergoing rejection indicating a preferential recruitment of cells highly expressing this module. We identify the receptor CXCR4 within the module and its ligand CXCL12 expressed in the graft as a likely recruitment mechanism between circulating cells and the tissue. We then explore publicly available transcriptomics data in circulating cells and show that this module is generally expressed in healthy individuals and more importantly is associated with the response to infection, including SARS Covid-19. Moreover, we find that module expression is predictive of immune mediated diseases. In summary, we find a gene expression module in circulating immune cells which enables preferential recruitment to inflamed tissues to mediate effector function.

Leukocyte immunoglobulin-like receptors (LILRs) are a family of inhibitory or stimulatory receptors expressed by immune cell types belonging to both myeloid and lymphoid lineage. Several members of the LILR family recognize major histocompatibility complex class I and thus play important roles in a range of clinical situations including pregnancy. Moreover, paired immunoglobulin-like receptors (PIRs), the murine orthologs of LILRs, are implicated in experimental transplant allorecognition by monocytes and contribute to the induction of donor-specific monocyte-memory. After non-self recognition, activating PIRs are transiently overexpressed at the surface of monocytes and participate in donor-specific monocyte recruitment, leading to graft rejection in vivo. In the present study, we mapped LILR expression and also their respective reported ligands at single cell level in the renal allograft and circulating cells in the context of kidney transplant rejection. Recipient-derived monocytes were shown to infiltrate the donor tissue and to differentiate into macrophages. We thus also investigate LILR expression during in vitro monocyte-to-macrophage differentiation in order to characterize the myeloid population that directly contribute to allorecognition. Altogether our results emphasize non-classical monocytes and CD68+ M1 macrophages as key players in LILRs-ligand interaction in kidney transplantation.

Operationally tolerant kidney transplant recipients harbor an immunological signature, associated with low rejection risk, and focused on B lymphocytes. Here, we investigated whether patients with long-term transplantation and still on immunosuppressive therapy would present such a signature of low immunological rejection risk, compared to more recently transplanted patients. Of 114 kidney transplant recipients enrolled, 38 with more than 25 years of graft survival and stable graft function under calcineurin inhibitors, were matched with two different groups of transplanted patients (10-15 and 5-7 years after transplantation). Three phenotypes associated with low immunological rejection risk (Tfh, B and regulatory T cells), initially found in operationally tolerant kidney transplant recipients, and the composite score of tolerance (combination of six transcriptomic markers, age at transplantation and age at sampling) were analyzed. We found that very long-term patients were characterized by a significantly lower percentage of total B cells, a significantly higher proportion of CD24HiCD38Lo memory B cells, significantly fewer CD24LoCD38Lo naive B cells, and a significantly lower proportion of PD1HiCCR7Lo Tfh lymphocytes than more recently transplanted patients. This phenotype is associated with a positive composite score of tolerance in patients transplanted for more than 25 years. Thus, our study suggests a dual phenotype in very long-term kidney transplanted patients with an immunological profile associated with low rejection risk.

Kidney transplant injury processes are associated with molecular changes in kidney tissue, primarily related to immune cell activation and infiltration. How these processes are reflected in the circulating immune cells, whose activation is targeted by strong immunosuppressants, is poorly understood. To study this, we analyzed the molecular alterations in 384 peripheral blood samples from four European transplant centers, taken at the time of a kidney allograft biopsy, selected for their phenotype, using RNA-sequencing. In peripheral blood, differentially expressed genes in 136 rejection and 248 no rejection samples demonstrated upregulation of glucocorticoid receptor and nucleotide oligomerization domain-like receptor signaling pathways. Pathways enriched in antibody-mediated rejection (ABMR) were strongly immune-specific, whereas pathways enriched in T cell-mediated rejection were less immune related. In polyomavirus infection, upregulation of mitochondrial dysfunction and interferon signaling pathways was seen. Next, we integrated the blood results with transcriptomics of 224 kidney allograft biopsies which showed consistently upregulated genes per phenotype in both blood and biopsy. In single-cell RNASeq (scRNASeq) analysis of seven kidney allograft biopsies, the consistently overexpressed genes in ABMR were mostly expressed by infiltrating leukocytes in the allograft. Similarly, in peripheral blood scRNASeq analysis, these genes were overexpressed in ABMR in immune cell subtypes. Furthermore, overexpression of these genes in ABMR was confirmed in independent cohorts in blood and biopsy. Thus, our results highlight the immune activation pathways in peripheral blood leukocytes at the time of kidney allograft pathology, despite the use of current strong immunosuppressants, and provide a framework for future therapeutic interventions. ispartof: KIDNEY INTERNATIONAL vol:102 issue:1 pages:183-195 ispartof: location:United States status: published

The liver displays a strong capacity to induce tolerance toward hepatic antigens. However, hepatic tolerance can be overcome with the development of local autoimmune diseases such as autoimmune hepatitis (AIH). This chronic inflammatory disorder leads to a progressive destruction of liver parenchyma if non-treated. Although the CD4+ T cell response seems a key player of this immune disorder, the dynamics and biology of emerging liver antigen-specific CD4+ T cells are poorly described. Here, we developed a new murine model which mimics hepatic autoreactivity allowing the study and monitoring of antigen-specific CD4+ T cells from their emergence to local immune response. We show that the induction of the expression of an antigen in the liver in non-inflammatory condition leads to antigen tolerance. In inflammatory condition, using viral vector transduction, we observe the development of a complete adaptive immune response concomitant with the antigen expression in the liver. The presence of antigen-specific CD4+ T cells in the liver is associated to transient hepatic damages. Interestingly, the neo-antigen expression by hepatocytes after peripheral immunisation induces the recruitment of antigen-specific CD4+ T cells and hepatic damages. These data demonstrate that the recruitment of antigen-specific CD4+ T cells in the liver is conditioned by an immune coordination between surface antigen expression by hepatocytes and peripheral immune response and mimics the first step of a local autoreactive process. In the long-term, we observe that the hepatic environment has the capacity to control the local, but not the systemic, antigen-specific CD4+ T cells. Additional immune events might be involved in the long-term chronic immune reactivity in the liver, following the first steps described in this study.Key pointsAntigen expression in the liver in non-inflammatory condition leads to antigen toleranceAntigen expression in the liver in immunization condition (concomitant or pre-existing) is sufficient to induce liver recruitment of antigen-specific CD4+ T cells and hepatic damagesThis model mimics the first step of an autoreactive process against a liver antigenIn the long-term, the hepatic environment induces a local tolerance toward the antigen expression and the clearance of liver-infiltrating, but not peripheral, antigen-specific CD4+ T cellsThis new murine model can be of interest for the analysis of the immunomodulatory pathways implicated in liver tolerization of autoreactive CD4+ T cells and identification of potential extrinsic factors implicated in an acute-to-chronic transition as observed during autoimmune hepatitis

International audience; T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

Background Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. Methods We investigated the frequency and function of CD8+ T cell subsets-including effector memory (EM) and terminally differentiated EM (TEMRA) CD8+ T cells-in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8+ T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. Results Increased frequency of circulating TEMRA CD8+ T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8+ T cells associated with reduced risk of graft failure. A distinct TEMRA CD8+ T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8+ T cells, CD16 engagement resulted in selective activation of TEMRA CD8+ T cells, which mediated antibody-dependent cytotoxicity. Conclusions At 1 year post-transplant, the composition of memory CD8+ T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8+ T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8+ T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8+ T cell monitoring for predicting risk of kidney transplant failure.

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

Research advances in Tribbles homolog (TRIB) genes have established the consensus that this protein family plays roles in diverse biological conditions and regulates intracellular signaling networks and several human diseases. In this review, we focus on one member of the family, TRIB1, and its role at the crossroads of immune signaling. TRIB1 directly interacts with transcription factors such as FOXP3 and C/EBPα, with several signaling molecules such as MEK1 and MALT1 and directly acts on key cell signaling pathways such as the MAPK and NF-κB pathways. Altogether, these interactions emphasize that TRIB1 is at the center of major cell signaling pathways while TRIB1 has cell-specific roles, potentially depending on the expressing cells and binding partners. In this review, we describe its roles in immune cells and highlight the interacting partners explaining these functions which suggests TRIB1 as a precise mediator of cellular homeostasis as well as in different cancers and immune-related disorders.

We assessed the pharmacokinetics and safety of XAV-19, a swine glyco-humanized polyclonal antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in coronavirus disease 2019 (COVID-19)-related moderate pneumonia. The objective was to evaluate the optimal dose and safety of XAV-19 during this first administration to patients with COVID-19-related moderate pneumonia. In this phase IIa trial, adults with COVID-19-related moderate pneumonia with a duration of ≤10 days were randomized to receive an infusion of XAV-19 at 0.5 mg/kg of body weight at day 1 and day 5 (group 1), 2 mg/kg at day 1 and day 5 (group 2), or 2 mg/kg at day 1 (group 3) or placebo. Eighteen patients (n = 7 for group 1, n = 1 for group 2, n = 5 for group 3, and n = 5 for placebo) were enrolled. Baseline characteristics were similar across groups; median XAV-19 serum concentrations (ranges) at the time of the maximum serum concentration of the drug (Cmax) and at day 8 were 9.1 (5.2 to 18.1) and 6.4 (2.8 to 11.9) μg/ml, 71.5 and 47.2 μg/ml, and 50.4 (29.1 to 55.0) and 20.3 (12.0 to 22.7) μg/ml for groups 1, 2, and 3, respectively (P = 0.012). The median terminal half-life (range) was estimated at 11.4 (5.5 to 13.9) days for 2 mg/kg of XAV-19 at day 1. Serum XAV-19 concentrations were above the target concentration of 10 μg/ml (2-fold the in vitro 100% inhibitory concentration [IC100]) from the end of perfusion to more than 8 days for XAV-19 at 2 mg/kg at day 1. No hypersensitivity or infusion-related reactions were reported during treatment, and there were no discontinuations for adverse events and no serious adverse events related to the study drug. A single intravenous dose of 2 mg/kg of XAV-19 demonstrated high serum concentrations, predictive of potent durable neutralizing activity with good tolerability. (This study has been registered at ClinicalTrials.gov under identifier NCT04453384.)

BackgroundWe assessed the pharmacokinetics and safety of XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2, in COVID-19-related moderate pneumonia. In vitro, 100% neutralization activity is seen with XAV-19 concentrations above 5 μg/mL.MethodsIn this phase 2a trial, adults with COVID-19-related moderate pneumonia of ≤10 days duration were randomized to infusion of XAV-19 0.5 mg/kg at day 1 and day 5 (group 1), 2 mg/kg at day 1 and day 5 (group 2), 2 mg/kg at day 1 (group 3) or placebo.ResultsEighteen patients (n=7 for group 1, n=1 for group 2, n=5 for group 3, and n=5 for placebo) were enrolled. Baseline characteristics were similar across groups, XAV-19 serum concentrations (μg/mL, median, range) at Cmaxand at day 8 were 9.1 (5.2-18.1) and 6.4 (2.8-11.9), 71.5 and 47.2, and 50.4 (29.1-55.0) and 20.3 (12.0-22.7) for groups 1, 2 and 3, respectively (p=0.012). Terminal half-life (median, range) was estimated at 11.4 (5.5-13.9) days for 2 mg/kg of XAV-19 at day 1. Serum XAV-19 concentrations were above the target concentration of 10 μg/mL (tow fold the in vitro 100% inhibitory concentration [IC100]) from the end of perfusion to more than 8 days for XAV-19 2 mg/kg at day 1. No hypersensitivity or infusion-related reactions were reported during treatment, there was no discontinuation for adverse events and no serious adverse events related to study drug.ConclusionsSingle intravenous dose of 2 mg/kg of XAV-19 demonstrated high serum concentrations, predictive of potent durable neutralizing activity with good tolerability.Trial registrationClinicalTrials.gov Identifier:NCT04453384Main pointIn this first-in-human trial including patients with COVID-19-related pneumonia, a single 2mg/kg dose of a swine glyco-humanized polyclonal anti-SARS-CoV-2 antibody, achieved serum concentrations above the target of neutralization threshold for 8 days in all patients, with good tolerability and safety.

BANK1 transcript is upregulated in whole blood after kidney transplantation in tolerant patients. In comparison to patients with rejection, tolerant patients display higher level of regulatory B cells (Bregs) expressing granzyme B (GZMB+) that have the capability to prevent effector T cells proliferation. However, BANK1 was found to be decreased in these GZMB+ Bregs. In this article, we investigated seven different transcriptomic studies and mined the literature in order to make link between BANK1, tolerance and Bregs. As for GZMB+ Bregs, we found that BANK1 was decreased in other subtypes of Bregs, including IL10+ and CD24hiCD38hi transitional regulatory B cells, along with BANK1 was down-regulated in activated/differentiated B cells, as in CD40-activated B cells, in leukemia and plasma cells. Following a reductionist approach, biological concepts were extracted from BANK1 literature and allowed us to infer association between BANK1 and immune signaling pathways, as STAT1, FcγRIIB, TNFAIP3, TRAF6, and TLR7. Based on B cell signaling literature and expression data, we proposed a role of BANK1 in B cells of tolerant patients that involved BCR, IP3R, and PLCG2, and a link with the apoptosis pathways. We confronted these data with our experiments on apoptosis in total B cells and Bregs, and this suggests different involvement for BANK1 in these two cells. Finally, we put in perspective our own data with other published data to hypothesize two different roles for BANK1 in B cells and in Bregs.

Background: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. Methods: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. Results: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. Conclusions: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Lymphocytes constitute an essential and potent effector compartment of the immune system. Therefore, their development and functions must be strictly regulated to avoid inappropriate immune responses, such as autoimmune reactions. Several lines of evidence from genetics (e.g. association with multiple sclerosis and primary biliary cirrhosis), human expression studies (e.g. increased expression in target tissues and draining lymph nodes of patients with autoimmune diseases), animal models (e.g. loss of functional protein protects animals from the development of collagen-induced arthritis, experimental autoimmune encephalomyelitis, type 1 diabetes, bleomycin-induced fibrosis) strongly support a causal link between the aberrant expression of the lymphocyte-restricted transcriptional regulator BOB.1 and the development of autoimmune diseases. In this review, we summarize the current knowledge of unusual structural and functional plasticity of BOB.1, stringent regulation of its expression, and the pivotal role that BOB.1 plays in shaping B- and T-cell responses. We discuss recent developments highlighting the significant contribution of BOB.1 to the pathogenesis of autoimmune diseases and how to leverage our knowledge to target this regulator to treat autoimmune tissue inflammation.

International audience; Humans cannot synthesizeN-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 activa-tion 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 com-plement‐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.

International audience; Resolution of inflammation is elicited by proresolving lipids, which activate GPCRs to induce neutrophil apoptosis, reduce neutrophil tissue recruitment, and promote macrophage efferocytosis. Transcriptional analyses in up to 300 patients with Inflammatory Bowel Disease (IBD) identified potential therapeutic targets mediating chronic inflammation. We found that ChemR23, a GPCR targeted by resolvin E1, is overexpressed in inflamed colon tissues of severe IBD patients unresponsive to anti-TNF alpha or anti-alpha 4 beta 7 therapies and associated with significant mucosal neutrophil accumulation. We also identified an anti-ChemR23 agonist antibody that induces receptor signaling, promotes macrophage efferocytosis, and reduces neutrophil apoptosis at the site of inflammation. This ChemR23 mAb accelerated acute inflammation resolution and triggered resolution in ongoing chronic colitis models, with a significant decrease in tissue lesions, fibrosis and inflammation-driven tumors. Our findings suggest that failure of current IBD therapies may be associated with neutrophil infiltration and that ChemR23 is a promising therapeutic target for chronic inflammation.

BackgroundWeb-based data analysis and visualization tools are mostly designed for specific purposes, such as data from whole transcriptome RNA sequencing or single-cell RNA sequencing. However, limited efforts have been made to develop tools designed for data of common laboratory data for non-computational scientists. The importance of such web-based tool is stressed by the current increased samples capacity of conventional laboratory tools such as quantitative PCR, flow cytometry or ELISA.ResultsWe provide a web-based application FaDA, developed with the R Shiny package providing users to perform statistical group comparisons, including parametric and non-parametric tests, with multiple testing corrections suitable for most of the standard wet-lab analyses. FaDA provides data visualization such as heatmap, principal component analysis (PCA) and receiver operating curve (ROC). Calculations are performed through the R language.ConclusionsFaDA application provides a free and intuitive interface allowing biologists without bioinformatic skills to easily and quickly perform common lab data analyses. The application is freely accessible at https://shiny-bird.univ-nantes.fr/app/FadaAbbreviationsAUC: Area Under the Curve; FaDA: Fast Data Analysis; GEO: Gene Expression Omnibus; ELISA: enzyme-linked immunosorbent assay; PCA: Principal Component Analysis; qPCR: quantitative PCR; ROC: Receiver Operating Curve.

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.

Regulatory B cells (Bregs) have the ability to regulate inflammation in various pathological situations, making them key players in immune regulation. Several mechanisms have been described and we recently identified a GZMB expressing Breg population in kidney transplanted patients who tolerate a kidney graft. To further investigate their biology and mechanisms, we conducted a transcriptomic analysis by RNAseq of these cells and we performed the first weighted meta-analysis of publicly available transcriptomic data from published Breg studies both in humans and mice. We identified two distinct and unique transcriptional signatures of 126 and 93 genes, respectively, associated with these Bregs. While we highlighted genes coding for proteins with potent involvement in regulatory functions, proliferation, and coding for transcription factors, the comparison between humans and mice did not allow identifying a common pattern. Thus, our results suggest distinct species-restricted Breg transcriptional signatures in humans and mice.

Renal operationally tolerant patients (TOL) display a defect in B cell differentiation, with a deficiency in plasma cells. Recently described, T follicular helper cells (Tfh) play a critical role in B cell differentiation. We analyzed blood Tfh subsets in TOL and transplanted patients with stable graft function under immunosuppression (STA). We observed a reduced proportion of blood activated and highly functional Tfh subsets in TOL, without affecting Tfh absolute numbers. Functionally, Tfh from TOL displayed a modified gene expression profile, failed to produce IL-21 and were unable to induce IgG production by naive B cells. This Tfh defect is linked to a low incidence of post graft de novo DSA (dnDSA) immunization, suggesting that the lack of Tfh in TOL may induce a pro-tolerogenic environment with reduced risk of developing dnDSA. Finally, we showed that elevated Tfh in STA precedes the occurrence of dnDSA during an alloresponse. These data provide new insights into the mechanisms of antibody response in operational tolerance. Disrupted homeostasis and impaired Tfh function in TOL could lead to a reduced risk of developing dnDSA and suggest a predictive role of blood Tfh on the occurrence of dnDSA in transplant recipients. This article is protected by copyright. All rights reserved.

It is now widely accepted that in order to improve long-term graft function and survival, a more personalized immunosuppressive treatment of transplant patients according to the individual anti-donor immune response status is needed. This applies to the identification of potentially "high-risk" patients likely to develop acute rejection episodes or display an accelerated decline of graft function, patients who might need immunosuppression intensification, and operationally tolerant patients suitable for immunosuppression minimization or weaning off. Such a patient stratification would benefit from biomarkers, which enable categorization into low and high risk or, ideally, identification of operational tolerant patients. Here, we report on recent developments regarding identification and performance analysis of noninvasive biomarkers such as mRNA and miRNA expression profiles, chemokines, or changes in immune cell subsets in either blood or urine of renal transplant patients. We will also discuss which future steps are needed to accelerate their clinical implementation.

BACKGROUND: Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. METHODS: We investigated the frequency and function of CD8(+) T cell subsets—including effector memory (EM) and terminally differentiated EM (TEMRA) CD8(+) T cells—in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8(+) T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. RESULTS: Increased frequency of circulating TEMRA CD8(+) T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8(+) T cells associated with reduced risk of graft failure. A distinct TEMRA CD8(+) T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8(+) T cells, CD16 engagement resulted in selective activation of TEMRA CD8(+) T cells, which mediated antibody-dependent cytotoxicity. CONCLUSIONS: At 1 year post-transplant, the composition of memory CD8(+) T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8(+) T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8(+) T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8(+) T cell monitoring for predicting risk of kidney transplant failure.

International audience; B cells secreting granzyme B (GZMB+) with suppressive properties have been discovered in a growing number of immunological contexts, autoimmunity, chronic infection, and neoplasias, as well as in healthy volunteers under physiological conditions.To date, no phenotype has been proposed for these GZMB+ B cells, and their biology also remains uncharacterized.These data provide new insights into GZMB+ B cell biology and function that emerge as one component of a complex regulatory network. Successfully expanding these cells while maintaining their potent immunosuppressive properties provides new clues for novel future cell therapies.

Application of Microarrays Technology to organ transplantation in human with the aid of statistical and non-statistical analysis, including interaction-based methods, is reviewed here.