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Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

IntroductionHuman Granzyme B (GZMB) regulatory B cells (Bregs) have suppressive properties on CD4+ effector T cells by a mechanism partially dependent on GZMB. Moreover, these cells may be easily induced in vitro making them interesting for cell therapy.MethodsWe characterized this population of in vitro induced GZMB+Bregs using single cell transcriptomics. To investigate their regulatory properties, Bregs or total B cells were also co-cultured with T cells and scRNAseq was used to identify receptor ligand interactions and to reveal gene expression changes in the T cells.ResultsWe find that Bregs exhibit a unique set of 149 genes differentially expressed and which are implicated in proliferation, apoptosis, metabolism, and altered antigen presentation capacity consistent with their differentiated B cells profile. Notably, Bregs induced a strong inhibition of T cell genes associated to proliferation, activation, inflammation and apoptosis compared to total B cells. We identified and validated 5 receptor/ligand interactions between Bregs and T cells. Functional analysis using specific inhibitors was used to test their suppressive properties and we identified Lymphotoxin alpha (LTA) as a new and potent Breg ligand implicated in Breg suppressive properties.DiscussionWe report for the first time for a role of LTA in GZMB+Bregs as an enhancer of GZMB expression, and involved in the suppressive properties of GZMB+Bregs in human. The exact mechanism of LTA/GZMB function in this specific subset of Bregs remains to be determined.

BackgroundRenal operational tolerance is a rare and beneficial state of prolonged renal allograft function in the absence of immunosuppression. The underlying mechanisms are unknown. We hypothesized that tolerance might be driven by inherited protein coding genetic variants with large effect, at least in some patients.MethodsWe set up a European survey of over 218,000 renal transplant recipients and collected DNAs from 40 transplant recipients who maintained good allograft function without immunosuppression for at least 1 year. We performed an exome-wide association study comparing the distribution of moderate to high impact variants in 36 tolerant patients, selected for genetic homogeneity using principal component analysis, and 192 controls, using an optimal sequence-kernel association test adjusted for small samples.ResultsWe identified rare variants of HOMER2 (3/36, FDR 0.0387), IQCH (5/36, FDR 0.0362), and LCN2 (3/36, FDR 0.102) in 10 tolerant patients vs. 0 controls. One patient carried a variant in both HOMER2 and LCN2. Furthermore, the three genes showed an identical variant in two patients each. The three genes are expressed at the primary cilium, a key structure in immune responses.ConclusionRare protein coding variants are associated with operational tolerance in a sizable portion of patients. Our findings have important implications for a better understanding of immune tolerance in transplantation and other fields of medicine.ClinicalTrials.gov, identifier: NCT05124444.

IntroductionThe human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells.MethodsBased on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models.Results and discussionWe found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

BackgroundChronic lung allograft dysfunction (CLAD) is the leading cause of poor long-term survival after lung transplantation (LT). Systems prediction of Chronic Lung Allograft Dysfunction (SysCLAD) aimed to predict CLAD.MethodsTo predict CLAD, we investigated the clinicome of patients with LT; the exposome through assessment of airway microbiota in bronchoalveolar lavage cells and air pollution studies; the immunome with works on activation of dendritic cells, the role of T cells to promote the secretion of matrix metalloproteinase-9, and subpopulations of T and B cells; genome polymorphisms; blood transcriptome; plasma proteome studies and assessment of MSK1 expression.ResultsClinicome: the best multivariate logistic regression analysis model for early-onset CLAD in 422 LT eligible patients generated a ROC curve with an area under the curve of 0.77. Exposome: chronic exposure to air pollutants appears deleterious on lung function levels in LT recipients (LTRs), might be modified by macrolides, and increases mortality. Our findings established a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant. Immunome: a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and associated with a higher level of interleukin 17A; Immune cells support airway remodeling through the production of plasma MMP-9 levels, a potential predictive biomarker of CLAD. Blood CD9-expressing B cells appear to favor the maintenance of long-term stable graft function and are a potential new predictive biomarker of BOS-free survival. An early increase of blood CD4 + CD57 + ILT2+ T cells after LT may be associated with CLAD onset. Genome: Donor Club cell secretory protein G38A polymorphism is associated with a decreased risk of severe primary graft dysfunction after LT. Transcriptome: blood POU class 2 associating factor 1, T-cell leukemia/lymphoma domain, and B cell lymphocytes, were validated as predictive biomarkers of CLAD phenotypes more than 6 months before diagnosis. Proteome: blood A2MG is an independent predictor of CLAD, and MSK1 kinase overexpression is either a marker or a potential therapeutic target in CLAD.ConclusionSystems prediction of Chronic Lung Allograft Dysfunction generated multiple fingerprints that enabled the development of predictors of CLAD. These results open the way to the integration of these fingerprints into a predictive handprint.

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.

Donor-specific anti-HLA antibodies (DSAs) are a major risk factor associated with renal allograft outcomes. As a trigger of B cell antibody production, T follicular helper cells (Tfhs) promote DSA appearance. Herein, we evaluated whether circulating Tfhs (cTfhs) are associated with the genesis of antibody-mediated rejection. We measured cTfh levels on the day of transplantation and 1 year after transplantation in blood from a prospective cohort of 237 renal transplantation patients without DSA during the first year post-transplantation. Total cTfhs were characterized as CD4+CD45RA−CXCR5+, and the three following subsets of activated cTfh were analyzed: CXCR5+PD1+, CXCR5+PD1+ICOS+, an CXCR5+PD1+CXCR3−. Immunizing events (previous blood transfusion and/or pregnancy) and the presence of class II anti-HLA antibodies were associated with increased frequencies of activated CXCR5+PD1+, CXCR5+PD1+ICOS+, and CXCR5+PD1+CXCR3− cTfh subsets. In addition, ATG-depleting induction and calcineurin inhibitor treatments were associated with a relative increase of activated cTfh subsets frequencies at 1 year post-transplantation. In multivariate survival analysis, we reported that a decrease in activated CXCR5+PD1+ICOS+ at 1 year after transplantation in the blood of DSA-free patients was significantly associated with the risk of developing de novo DSA after the first year (p = 0.018, HR = 0.39), independently of HLA mismatches (p = 0.003, HR = 3.79). These results highlight the importance of monitoring activated Tfhs in patients early after transplantation and show that current treatments cannot provide early, efficient prevention of Tfh activation and migration. These findings indicate the need to develop innovative treatments to specifically target Tfhs to prevent DSA appearance in renal transplantation.

A growing number of patients with end-stage lung disease have benefited from lung transplantation (LT). Improvements in organ procurement, surgical techniques and intensive care management have greatly increased short-term graft survival. However, long-term outcomes remain limited, mainly due to the onset of chronic lung allograft dysfunction (CLAD), whose diagnosis is based on permanent loss of lung function after the development of irreversible lung lesions. CLAD is associated with high mortality and morbidity, and its exact physiopathology is still only partially understood. Many researchers and clinicians have searched for CLAD biomarkers to improve diagnosis, to refine the phenotypes associated with differential prognosis and to identify early biological processes that lead to CLAD to enable an early intervention that could modify the inevitable degradation of respiratory function. Donor-specific antibodies are currently the only biomarkers used in routine clinical practice, and their significance for accurately predicting CLAD is still debated. We describe here significant studies that have highlighted potential candidates for reliable and non-invasive biomarkers of CLAD in the fields of imaging and functional monitoring, humoral immunity, cell-mediated immunity, allograft injury, airway remodeling and gene expression. Such biomarkers would improve CLAD prediction and allow differential LT management regarding CLAD risk.

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

The involvement of TEMRA CD8 is evident in a large array of immunological conditions ranging from auto- to allo-immunity. Nevertheless, the factors leading to their accumulation and activation remain ill-defined and, efficient therapeutics to control their inflammatory response is lacking. Here, we show that IL-15-stimulated TEMRA from kidney-transplant (KT) recipients promote inflammation by inducing the expression of CX3CL1 by endothelial cells in an IFN-γ- and TNF-α-dependent manner. The responsiveness of TEMRA to IL-15 is not restricted to chronic stimulation, as TEMRA from healthy volunteers respond earlier and faster when compared to effector memory (EM). IL-15 induces antiapoptotic signals and promotes proliferation dependent of PI3K/Akt, MAPK, and ERK pathways. Without ex vivo stimulation, TEMRA cells are metabolically more active than naive and EM, as shown by their high ATP reservoir and a high expression of genes involved in glycolysis, glutaminolysis, and the Pentose Phosphate Pathway. Upon stimulation, TEMRA adapt their metabolism by sustaining an increased mitochondrial respiration and glycolysis. Finally, we show that the inhibition of glycolysis is highly effective in preventing endothelial inflammation induced by TEMRA from KT recipients. Together, our findings highlight the metabolic fitness that tightly regulates the immune function of TEMRA in physiological and pathogenic situations.