Regulatory B Cells Expressing Granzyme B from Tolerant Renal Transplant Patients: Highly Differentiated B Cells with a Unique Pathway with a Specific Regulatory Profile and Strong Interactions with Immune System Cells.

The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB+ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB+ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB+ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB+ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB+ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB+ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB+/- natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB+ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB+ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.

Evaluation of non-invasive biomarkers of kidney allograft rejection in a prospective multicenter unselected cohort study (EU-TRAIN).

Non-invasive biomarkers are promising tools for improving kidney allograft rejection monitoring, but their clinical adoption requires more evidence in specifically designed studies. To address this unmet need, we designed the EU-TRAIN study, a large prospective multicentric unselected cohort funded by the European Commission. Here, we included consecutive adult patients who received a kidney allograft in nine European transplant centers between November 2018 and June 2020. We prospectively assessed gene expression levels of 19 blood messenger RNAs, four antibodies targeting non-human leukocyte antigen (HLA) endothelial antigens, together with circulating anti-HLA donor-specific antibodies (DSA). The primary outcome was allograft rejection (antibody-mediated, T cell-mediated, or mixed) in the first year post-transplantation. Overall, 412 patients were included, with 812 biopsies paired with a blood sample. CD4 gene expression was significantly associated with rejection, while circulating anti-HLA DSA had a significant association with allograft rejection and a strong association with antibody-mediated rejection. All other tested biomarkers, including AKR1C3, CD3E, CD40, CD8A, CD9, CTLA4, ENTPD1, FOXP3, GZMB, ID3, IL7R, MS4A1, MZB1, POU2AF1, POU2F1, TCL1A, TLR4, and TRIB1, as well as antibodies against angiotensin II type 1 receptor, endothelin 1 type A receptor, C3a and C5a receptors, did not show significant associations with allograft rejection. The blood messenger RNAs and non-HLA antibodies did not show an additional value beyond standard of care monitoring parameters and circulating anti-HLA DSA to predict allograft rejection in the first year post-transplantation. Thus, our results open avenues for specifically designed studies to demonstrate the clinical relevance and implementation of other candidate non-invasive biomarkers in kidney transplantation practice.

Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies.

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.

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte.

INTRODUCTION: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. METHODS: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. RESULTS: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. CONCLUSION: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

Blood Gene Signature as a Biomarker for Subclinical Kidney Allograft Rejection: Where Are We?

The observation decades ago that inflammatory injuries because of an alloimmune response might be present even in the absence of concomitant clinical impairment in allograft function conduced to the later definition of subclinical rejection. Many studies have investigated the different subclinical rejections defined according to the Banff classification (subclinical T cell-mediated rejection and antibody-mediated rejection), overall concluding that these episodes worsened long-term allograft function and survival. These observations led several transplant teams to perform systematic protocolar biopsies to anticipate treatment of rejection episodes and possibly prevent allograft loss. Paradoxically, the invasive characteristics and associated logistics of such procedures paved the way to investigate noninvasive biomarkers (urine and blood) of subclinical rejection. Among them, several research teams proposed a blood gene signature developed from cohort studies, most of which achieved excellent predictive values for the occurrence of subclinical rejection, mainly antibody-mediated rejection. Interestingly, although all identified genes relate to immune subsets and pathways involved in rejection pathophysiology, very few transcripts are shared among these sets of genes, highlighting the heterogenicity of such episodes and the difficult but mandatory need for external validation of such tools. Beyond this, their application and value in clinical practice remain to be definitively demonstrated in both biopsy avoidance and prevention of clinical rejection episodes. Their combination with other biomarkers, either epidemiological or biological, could contribute to a more accurate picture of a patient's risk of rejection and guide clinicians in the follow-up of kidney transplant recipients.

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.

Fine-tuning tumor- and allo-immunity: advances in the use of immune checkpoint inhibitors in kidney transplant recipients.

Cancer is a common complication after kidney transplantation. Kidney transplant recipients (KTR) have a 2- to 4-fold higher risk of developing cancer compared to the general population and post-transplant malignancy is the third most common cause of death in KTR. Moreover, it is well known that certain cancer types are overrepresented after transplantation, especially non-melanoma skin cancer. Immune checkpoint inhibitors (ICI) have revolutionized the treatment of cancer, with remarkable survival benefit in a subgroup of patients. ICI are monoclonal antibodies that block the binding of specific co-inhibitory signaling molecules. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), and its ligand programmed cell death ligand 1 (PD-L1) are the main targets of ICI. Solid organ transplant recipients (SOTR) have been excluded from clinical trials owing to concerns about tumor response, allo-immunity, and risk of transplant rejection. Indeed, graft rejection has been estimated as high as 48% and represents an emerging problem. The underlying mechanisms of organ rejection in the context of treatment with ICI are poorly understood. The search for restricted antitumoral responses without graft rejection is of paramount importance. This review summarizes the current knowledge of the use of ICI in KTR, the potential mechanisms involved in kidney graft rejection during ICI treatment, potential biomarkers of rejection, and how to deal with rejection in clinical practice.

Blood MMP-9 measured at 2 years after lung transplantation as a prognostic biomarker of chronic lung allograft dysfunction.

BACKGROUND: Long-term outcomes of lung transplantation (LTx) remain hampered by chronic lung allograft dysfunction (CLAD). Matrix metalloproteinase 9 (MMP-9) is a secretory endopeptidase identified as a key mediator in fibrosis processes associated with CLAD. The objective of this study was to investigate whether plasma MMP9 levels may be prognostic of CLAD development. METHODS: Participants were selected from the Cohort in Lung Transplantation (COLT) for which a biocollection was associated. We considered two time points, year 1 (Y1) and year 2 (Y2) post-transplantation, for plasma MMP-9 measurements. We analysed stable recipients at those time points, comparing those who would develop a CLAD within the 2 years following the measurement to those who would remain stable 2 years after. RESULTS: MMP-9 levels at Y1 were not significantly different between the CLAD and stable groups (230 ng/ml vs. 160 ng/ml, p = 0.4). For the Y2 analysis, 129 recipients were included, of whom 50 developed CLAD within 2 years and 79 remained stable within 2 years. MMP-9 plasma median concentrations were higher in recipients who then developed CLAD than in the stable group (230 ng/ml vs. 118 ng/ml, p = 0.003). In the multivariate analysis, the Y2 MMP-9 level was independently associated with CLAD, with an average increase of 150 ng/ml (95% CI [0-253], p = 0.05) compared to that in the stable group. The Y2 ROC curve revealed a discriminating capacity of blood MMP-9 with an area under the curve of 66%. CONCLUSION: Plasmatic MMP-9 levels measured 2 years after lung transplantation have prognostic value for CLAD.

Polyclonal antibodies selectively inhibit tumor growth and invasion and synergize with immune checkpoint inhibitors.

Heterologous polyclonal antibodies (pAb) were shown to possess oncolytic properties a century ago with reported clinical responses. More recent preclinical models confirmed pAb efficacy, though their ability to tackle complex target antigens reduces susceptibility to tumor escape. Owing to the recent availability of glyco-humanized pAb (GH-pAb) with acceptable clinical toxicology profile, we revisited use of pAb in oncology and highlighted their therapeutic potential against multiple cancer types. Murine antitumor pAb were generated after repeated immunization of rabbits with murine tumor cell lines from hepatocarcinoma, melanoma, and colorectal cancers. Antitumor pAb recognized and showed cytotoxicity against their targets without cross-reactivity with healthy tissues. In vivo, pAb are effective alone; moreover, these pAb synergize with immune checkpoint inhibitors like anti-PD-L1 in several cancer models. They elicited an antitumor host immune response and prevented metastases. The anticancer activity of pAb was also confirmed in xenografted NMRI nude mice using GH-pAb produced by repeated immunization of pigs with human tumor cell lines. In conclusion, the availability of bioengineered GH-pAb allows for revisiting of passive immunotherapy with oncolytic pAb to fight against solid tumor and cancer metastasis.

Effect of Swine Glyco-humanized Polyclonal Neutralizing Antibody on Survival and Respiratory Failure in Patients Hospitalized With Severe COVID-19: A Randomized, Placebo-Controlled Trial.

Background: We evaluated the safety and efficacy of XAV-19, an antispike glyco-humanized swine polyclonal neutralizing antibody in patients hospitalized with severe coronavirus disease 2019 (COVID-19). Methods: This phase 2b clinical trial enrolled adult patients from 34 hospitals in France. Eligible patients had a confirmed diagnosis of severe acute respiratory syndrome coronavirus 2 within 14 days of onset of symptoms that required hospitalization for low-flow oxygen therapy (<6 L/min of oxygen). Patients were randomly assigned to receive a single intravenous infusion of 2 mg/kg of XAV-19 or placebo. The primary end point was the occurrence of death or severe respiratory failure between baseline and day 15. Results: Between January 12, 2021, and April 16, 2021, 398 patients were enrolled in the study and randomly assigned to XAV-19 or placebo. The modified intention-to-treat population comprised 388 participants who received full perfusion of XAV-19 (199 patients) or placebo (189 patients). The mean (SD) age was 59.8 (12.4) years, 249 (64.2%) individuals were men, and the median time (interquartile range) from symptom onset to enrollment was 9 (7-10) days. There was no statistically significant decrease in the cumulative incidence of death or severe respiratory failure through day 15 in the XAV-19 group vs the placebo group (53/199 [26.6%] vs 48/189 [25.4%]; adjusted risk difference, 0.6%; 95% CI, -6% to 7%; hazard ratio, 1.03; 95% CI, 0.64-1.66; P = .90). In the safety population, adverse events were reported in 75.4% of 199 patients in the XAV-19 group and in 76.3% of 190 patients in the placebo group through D29. Conclusions: Among patients hospitalized with COVID-19 requiring low-flow oxygen therapy, treatment with a single intravenous dose of XAV-19, compared with placebo, did not show a significant difference in terms of disease progression at day 15.

Human granzyme B regulatory B cells prevent effector CD4+CD25- T cell proliferation through a mechanism dependent from lymphotoxin alpha.

Introduction: Human 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. Methods: We 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. Results: We 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. Discussion: We 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.

Pre-transplant immune profile defined by principal component analysis predicts acute rejection after kidney transplantation.

Background: Acute rejection persists as a frequent complication after kidney transplantation. Defining an at-risk immune profile would allow better preventive approaches. Methods: We performed unsupervised hierarchical clustering analysis on pre-transplant immunological phenotype in 1113 renal transplant recipients from the ORLY-EST cohort. Results: We identified three immune profiles correlated with clinical phenotypes. A memory immune cluster was defined by memory CD4+T cell expansion and decreased naïve CD4+T cell. An activated immune cluster was characterized by an increase in CD8+T cells and a decreased CD4/CD8 ratio. A naïve immune cluster was mainly defined by increased naïve CD4+T cells. Patients from the memory immune profile tend to be older and to have diabetes whereas those from the activated immune profile were younger and more likely to have pre-transplant exposure to CMV. Patients from the activated immune profile were more prone to experience acute rejection than those from other clusters [(HR=1.69, 95%IC[1.05-2.70], p=0.030) and (HR=1.85; 95%IC[1.16-3.00], p=0.011). In the activated immune profile, those without previous exposure to CMV (24%) were at very high risk of acute rejection (27 vs 16%, HR=1.85; 95%IC[1.04-3.33], p=0.039). Conclusion: Immune profile determination based on principal component analysis defines clinically different sub-groups and discriminate a population at high-risk of acute rejection.

Kidney allograft rejection is associated with an imbalance of B cells, regulatory T cells and differentiated CD28-CD8+ T cells: analysis of a cohort of 1095 graft biopsies.

Introduction: The 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. Methods: Based 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 discussion: We 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.

Immunosuppressant drugs and quality-of-life outcomes in kidney transplant recipients: An international cohort study (EU-TRAIN).

Introduction: Patient-Reported Outcomes (PRO) integrate a wide range of holistic dimensions that arenot captured within clinical outcomes. Particularly, from induction treatment to maintenance therapy, patient quality-of-life (QoL) of kidney transplant recipients have been sparsely investigated in international settings. Methods: In a prospective, multi-centric cohort study, including nine transplant centers in four countries, we explored the QoL during the year following transplantation using validated elicitation instruments (EQ-5D-3L index with VAS) in a population of kidney transplant patients receiving immunosuppressive therapies. Calcineurin inhibitors (tacrolimus and ciclosporin), IMPD inhibitor (mycophenolate mofetil), and mTOR inhibitors (everolimus and sirolimus) were the standard-of-care (SOC) medications, together with tapering glucocorticoid therapy. We used EQ-5D and VAS data as QoL measures alongside descriptive statistics at inclusion, per country and hospital center. We computed the proportions of patients with different immunosuppressive therapy patterns, and using bivariate and multivariate analyses, assessed the variations of EQ-5D and VAS between baseline (i.e., inclusion Month 0) and follow up visits (Month 12). Results: Among 542 kidney transplant patients included and followed from November 2018 to June 2021, 491 filled at least one QoL questionnaire at least at baseline (Month 0). The majority of patients in all countries received tacrolimus and mycophenolate mofetil, ranging from 90.0% in Switzerland and Spain to 95.8% in Germany. At M12, a significant proportion of patients switched immunosuppressive drugs, with proportion varying from 20% in Germany to 40% in Spain and Switzerland. At visit M12, patients who kept SOC therapy had higher EQ-5D (by 8 percentage points, p < 0.05) and VAS (by 4 percentage points, p < 0.1) scores than switchers. VAS scores were generally lower than EQ-5D (mean 0.68 [0.5-0.8] vs. 0.85 [0.8-1]). Discussion: Although overall a positive trend in QoL was observed, the formal analyses did not show any significant improvements in EQ-5D scores or VAS. Only when the effect of a therapy use was separated from the effect of switching, the VAS score was significantly worse for switchers during the follow up period, irrespective of the therapy type. If adjusted for patient characteristics and medical history (e.g., gender, BMI, eGRF, history of diabetes), VAS and EQ-5D delivered sound PRO measures for QoL assessments during the year following renal transplantation.

An automated histological classification system for precision diagnostics of kidney allografts.

For three decades, the international Banff classification has been the gold standard for kidney allograft rejection diagnosis, but this system has become complex over time with the integration of multimodal data and rules, leading to misclassifications that can have deleterious therapeutic consequences for patients. To improve diagnosis, we developed a decision-support system, based on an algorithm covering all classification rules and diagnostic scenarios, that automatically assigns kidney allograft diagnoses. We then tested its ability to reclassify rejection diagnoses for adult and pediatric kidney transplant recipients in three international multicentric cohorts and two large prospective clinical trials, including 4,409 biopsies from 3,054 patients (62.05% male and 37.95% female) followed in 20 transplant referral centers in Europe and North America. In the adult kidney transplant population, the Banff Automation System reclassified 83 out of 279 (29.75%) antibody-mediated rejection cases and 57 out of 105 (54.29%) T cell-mediated rejection cases, whereas 237 out of 3,239 (7.32%) biopsies diagnosed as non-rejection by pathologists were reclassified as rejection. In the pediatric population, the reclassification rates were 8 out of 26 (30.77%) for antibody-mediated rejection and 12 out of 39 (30.77%) for T cell-mediated rejection. Finally, we found that reclassification of the initial diagnoses by the Banff Automation System was associated with an improved risk stratification of long-term allograft outcomes. This study demonstrates the potential of an automated histological classification to improve transplant patient care by correcting diagnostic errors and standardizing allograft rejection diagnoses.ClinicalTrials.gov registration: NCT05306795 .

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation.

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (<100 lymphocytes/L) but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of GH-ALG might present advantages in induction treatment during organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

Aglycosylated extracellular loop of inwardly rectifying potassium channel 4.1 (KCNJ10) provides a target for autoimmune neuroinflammation.

Multiple sclerosis is an autoimmune disease of the central nervous system. Yet, the autoimmune targets are still undefined. The extracellular e1 sequence of KCNJ10, the inwardly rectifying potassium channel 4.1, has been subject to fierce debate for its role as a candidate autoantigen in multiple sclerosis. Inwardly rectifying potassium channel 4.1 is expressed in the central nervous system but also in peripheral tissues, raising concerns about the central nervous system-specificity of such autoreactivity. Immunization of C57Bl6/J female mice with the e1 peptide (amino acids 83-120 of Kir4.1) induced anti-e1 immunoglobulin G- and T-cell responses and promoted demyelinating encephalomyelitis with B cell central nervous system enrichment in leptomeninges and T cells/macrophages in central nervous system parenchyma from forebrain to spinal cord, mostly in the white matter. Within our cohort of multiple sclerosis patients (n = 252), 6% exhibited high anti-e1 immunoglobulin G levels in serum as compared to 0.7% in the control cohort (n = 127; P = 0.015). Immunolabelling of inwardly rectifying potassium channel 4.1-expressing white matter glia with the anti-e1 serum from immunized mice increased during murine autoimmune neuroinflammation and in multiple sclerosis white matter as compared with controls. Strikingly, the mouse and human anti-e1 sera labelled astrocytoma cells when N-glycosylation was blocked with tunicamycin. Western blot confirmed that neuroinflammation induces Kir4.1 expression, including its shorter aglycosylated form in murine experimental autoencephalomyelitis and multiple sclerosis. In addition, recognition of inwardly rectifying potassium channel 4.1 using mouse anti-e1 serum in Western blot experiments under unreduced conditions or in cells transfected with the N-glycosylation defective N104Q mutant as compared to the wild type further suggests that autoantibodies target an e1 conformational epitope in its aglycosylated form. These data highlight the e1 sequence of inwardly rectifying potassium channel 4.1 as a valid central nervous system autoantigen with a disease/tissue-specific post-translational antigen modification as potential contributor to autoimmunity in some multiple sclerosis patients.

Systems prediction of chronic lung allograft dysfunction: Results and perspectives from the Cohort of Lung Transplantation and Systems prediction of Chronic Lung Allograft Dysfunction cohorts.

Background: Chronic 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. Methods: To 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. Results: Clinicome: 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. Conclusion: Systems 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.

Regulation of CD8 T cell by B-cells: A narrative review.

Activation of CD4 T cells by B cells has been extensively studied, but B cell-regulated priming, proliferation, and survival of CD8 T cells remains controversial. B cells express high levels of MHC class I molecules and can potentially act as antigen-presenting cells (APCs) for CD8 T cells. Several in vivo studies in mice and humans demonstrate the role of B cells as modulators of CD8 T cell function in the context of viral infections, autoimmune diseases, cancer and allograft rejection. In addition, B-cell depletion therapies can lead to impaired CD8 T-cell responses. In this review, we attempt to answer 2 important questions: 1. the role of B cell antigen presentation and cytokine production in the regulation of CD8 T cell survival and cell fate determination, and 2. The role of B cells in the formation and maintenance of CD8 T cell memory.

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.