American Journal of Transplantation

IntroductionAntibody-mediated rejection (AMR) remains a leading cause of kidney allograft failure, with both HLA and non-HLA antibodies implicated in its pathogenesis. The contribution of non-HLA antibodies (non-HLA Abs) to microvascular inflammation (MVI) and graft outcome, particularly in cases lacking donor-specific anti-HLA antibodies (HLA-DSAs), remains incompletely understood. MethodsWe analyzed 571 post-transplant serum samples from 326 patients with histological features of AMR (AMRh) and 164 stable controls. Non-HLA Abs were detected using the previously developed Non-HLA Antibody Detection Immunoassay (NHADIA), and associations were examined with histological lesions, AMRh persistence, and graft outcomes. Biopsies were scored according to Banff 2022 criteria, and patients were stratified by HLA-DSA and NHADIA status. ResultsNHADIA values were significantly higher in AMRh patients compared to controls (P=0.0001), regardless of HLA-DSA status. NHADIA values correlated with the severity of glomerulitis, peritubular capillaritis and global MVI scores. In AMRh patients with HLA-DSAs, non-HLA Abs remained independently associated with MVI severity. Follow-up biopsies revealed persistent AMR lesions in patients with both HLA-DSAs and non-HLA Abs. Allograft survival was lowest in double-positive patients, and NHADIA positivity independently predicted graft loss (HR=2.25, 95% CI: 1.03-4.92, P=0.042). Incorporating NHADIA into the Banff classification reclassified 61% of AMRh cases as "double-positive AMRh," and identified new subgroups with significant prognostic differences. ConclusionPost-transplant detection of non-HLA antibodies identifies a distinct subset of AMR with more severe histology and worse graft prognosis, particularly when coexisting with HLA-DSAs. Integrating non-HLA Ab testing into current diagnostic frameworks may refine AMR classification and improve risk stratification. TRANSLATIONAL STATEMENTThis study highlights the clinical relevance of non-HLA antibodies, identified using our innovative endothelial cell-based assay (NHADIA), in kidney transplant recipients. Their presence is associated with more severe antibody-mediated rejection (AMR) and poorer graft outcomes, even in the absence of donor-specific HLA antibodies. Incorporating non-HLA antibody detection into routine post-transplant evaluation may allow clinicians to better identify high-risk patients, including those previously classified as DSA-negative AMR. This expanded immunological profiling refines AMR diagnosis, improves risk stratification, and opens new avenues for personalized immunosuppressive strategies, ultimately enhancing long-term graft survival and patient care.

BACKGROUNDChronic antibody-mediated rejection (CAMR) is the main cause of late kidney allograft loss, and no specific effective treatment has been identified so far. Here, we proposed to explore the non-invasive peripheral blood transcriptome signature of CAMR. METHODSFirst, we compared PBMC gene expression from bulk RNA-seq between 35 patients experiencing late CAMR (mean=7.1 years) vs. 43 patients without graft dysfunction at late stages (Stable, mean=4.4 years) to identify the molecular drivers of CAMR. Second, we explored the 1-year gene expression signature in stable patients exhibiting (n=11) or not (n=51) a subsequent CAMR to define possible predictive biomarkers of CAMR. RESULTSWe reported 188 differentially regulated genes during late CAMR (q<0.05). Importantly, CAMR is associated with an upregulation of genes from the degranulation pathway (e.g. MMP9, MMP8 and LCN2) and from the C1q complement complex (e.g. C1QA, C1QB and C1QC), as well as with a downregulation of genes associated with subclinical rejection (e.g. TCL1A). The upregulated degranulation and complement signatures were validated in six independent cohorts gathering a total of 360 stable and 131 chronic rejection patients. Contrary to the injury effect observed during late stages, MMP9 was downregulated at 1-year in PBMCs of patients who later experienced CAMR. CONCLUSIONSThese results suggest a dual role for MMP9 expression with an early protective effect against CAMR and deleterious effects in the later stage. MMP9 peripheral expression appears as a promising biomarker candidate for kidney transplantation follow-up. Translational StatementCAMR is the main cause of late kidney allograft loss, and we aimed to identify molecular targets and biomarkers. By comparing CAMR and stable kidney-transplanted patients prior and during diagnosis, we identified MMP9 as a potential biomarker for both CAMR prognosis and diagnosis. We also highlighted the C1q complement complex and TCL1A in CAMR as potential diagnostic biomarkers. These results provide new insights into CAMR pathophysiology and may guide the development of innovative treatment targeting MMP9 expression in kidney transplanted patients. Ultimately, this work laid the foundation for exploring MMP9 expression kinetics and develop new ways of treating CAMR patients.

The long-term impact of SARS-CoV-2 infection on kidney allograft survival remains incompletely understood, particularly regarding the influence of vaccination, acute kidney injury (AKI), and post-infection immunosuppression. We conducted a retrospective analysis of 129 kidney transplant recipients with confirmed SARS-CoV-2 infection between March 2020 and March 2022 with a median follow-up of 50 months. Among 129 recipients, 106 (82%) received vaccination at any time before or after SARS-CoV-2 infection (82%) while 23 (18%) remained unvaccinated. Unvaccinated patients experienced significantly lower long-term graft survival (52% vs. 85%; p = 0.0004) and patient survival (83% vs. 99%; p = 0.0003) compared with vaccinated recipients. AKI occurred in 15% of recipients and independently predicted graft failure (aHR 2.88; p = 0.0341). Post-SARS-CoV-2 serum creatinine and albuminuria were strong prognostic markers of graft loss. Unvaccinated status independently predicted graft failure in both transplantation-anchored (aHR 2.80; p = 0.0342) and SARS-CoV-2-anchored models (aHR 5.31; p = 0.0004). Continuation of mycophenolate mofetil at post-infection assessment was associated with reduced graft-failure risk (aHR 0.99; p = 0.0193). These findings underscore the importance of sustained vaccination in preserving long-term allograft function.

BackgroundKidney transplant recipients with COVID-19 are at high risk of poor outcome because of comorbidities and immunosuppression. The effects of immunosuppressive therapy reduction are unclear in patients with COVID-19. MethodsWe conducted a retrospective study on 45 consecutive kidney transplant recipients followed at the University Hospital of Modena who tested positive for COVID-19 by RT-PCR analysis. ResultsThe median age of patients was 56.1 (interquartile range, [IQR] 47.3-61.1) years with a predominance of male (64.4%). Kidney transplantation vintage was 10.1 (2.7-16) years, and more than half of patients (55.6%) was on triple immunosuppressive therapy. Early reduction of immunosuppression occurred in 62.8% of patients and included antimetabolite (88.8%) and calcineurin inhibitor withdrawal (22.2%). Of the 45 patients, 88.9% became symptomatic and 40% required hospitalization. Overall mortality was 17.8%. There were no differences in outcomes between full- and reduced-dose immunosuppressive therapy at the end of follow-up. One hospitalized patient experienced irreversible graft failure. There were no differences in serum creatinine level and proteinuria in non-hospitalized patients with COVID-19. Admitted patients had better kidney function after dismission (P=0.019). Risk factors for death were age (odds ratio [OR]: 1.19; 95%CI: 1.01-1.39), and duration of kidney transplant (OR: 1.17; 95%CI: 1.01-1.35). One kidney transplant recipient experienced symptomatic COVID-19 reinfection after primary infection and anti-SARS-CoV-2 mRNA vaccine. ConclusionsDespite the reduction of immunosuppression, COVID-19 affected survival of kidney transplant recipients with COVID-19. Age and duration of kidney transplant were independent predictors of death in COVID-19. Early kidney function was favorable in most survivors after COVID-19.

BackgroundSolid organ transplant recipients have attenuated immune responses to SARS-CoV-2 vaccines. Emerging evidence suggests at least equivalent immunogenicity of heterologous compared with homologous vaccine regimens in the general population. In this study, we report on immune responses to 3rd dose BNT162b2 vaccines in transplant recipients either primed with ChAdOx1 or BNT162b2. Methods700 kidney transplant recipients were prospectively screened for serological responses (median time of 33 (21-52) days) following 3 primary doses of a SARS-CoV2 vaccine. All vaccine doses were received post-transplant, and all 3rd doses were BNT162b2. All participants had serological testing performed post-2nd vaccination at a median time of 34 (IQR 26-46) days following the 2nd inoculation, and at least once prior to their 1st dose of vaccine. Results366/700 (52.3%) participants were primed with BNT162b2, whilst 334/700 (47.7) had received ChAdOx1. Overall, 139/700 (19.9%) participants had evidence of prior infection. Of 561 infection naive participants, 263 (46.9%) had no detectable anti-S following 2-doses of vaccine (V2). 134 (23.9%) participants remained seronegative post 3rd vaccine (V3); 54/291 (18.6%) and 79/270 (29.3%) of participants receiving BNT162b2 and ChAdOx1 respectively, p=0.0029. Median anti-S concentrations were significantly higher post-V3 in patients who had received BNT162b2 compared with ChAdOx1, at 612 (27-234) versus 122 (7.1-1111) BAU/ml respectively, p<0.0001. Cellular responses were investigated in 30 infection naive participants at a median time of 35 (24-46) days post-V3. Eighteen of 30 (60.0%) participants had undetectable T-cell responses. There were neither qualitative or quantitative differences in T-cell responses between those patients who received BNT162b2 or ChAdOx1 as their first 2-doses, with 10/16 (62.5%) and 8/14 (57.1%) respectively having undetectable T-cell responses, p=0.77. ConclusionA significant proportion of transplant recipients remain seronegative following 3 doses of SARS-CoV-2 vaccines, with anti-S concentrations lower in patients receiving heterologous versus homologous vaccinations.

BackgroundSubclinical kidney allograft acute rejection (SCR) corresponds to "the unexpected histological evidence of acute rejection in a stable patient". The diagnosis of SCR relies on surveillance biopsy. Positron emission tomography (PET/CT) after injection of F18-fluorodeoxyglucose ([18F]FDG) has been proposed as a non-invasive screening approach. In the present multicenter prospective study, we assess the diagnostic yield [18F]FDGPET/CT to rule out SCR in stable KTR at 3 months post KTx. MethodsFrom 01/2021 to 03/2025, we prospectively combined surveillance biopsy and [18F]FDGPET/CT at [~]3 months post transplantation in adult kidney transplant recipients from 4 independent imaging centers. The mean standardized uptake value (mSUV) was measured in kidney cortex and referenced as a ratio to psoas muscle mSUV (mSUVR). ResultsOur multicentric 185-patient cohort was categorized upon Banff-2022: normal (n=158); borderline (n=18); SCR (n=9, including 6 T-cell-mediated rejection and 3 microvascular inflammation). No significant correlation was observed between the mSUVR and ti score (R=0.032, p-value=0.67). The mSUVR reached 2.33 [1.97-2.93], 2.71 [2.50-3.33] and 2.42 [2.27-3.14] in normal, borderline and SCR groups, respectively. In multivariate models stratified by center, the risk of non-normal histology (n=27, including borderline and SCR) increased with donor age (OR=1.05 [1.01-1.1], p=0.02) but not with the mSUVR (OR=4.11 [0.91-18.48], p=0.07). The risk of biopsy-proven SCR (n=9) was not significantly associated with mSUVR. ConclusionsThe mSUVR of [18F]FDG PET/CT does not reliably rule out SCR on surveillance biopsy.

We used Digital Spatial Profiling to localize transcripts in glomeruli and tubulointerstitial compartments in a series of 4 biopsies from a patient diagnosed with acute antibody-mediated rejection (AMR). The 4 biopsies included: a baseline protocol biopsy 25 days after transplantation; a 3 month biopsy diagnosed as acute AMR; a biopsy 4 months after treatment with intravenous immunoglobulin (IVIg) showing ongoing AMR with a mild increase in tubulointerstitial fibrosis; and a biopsy 7 months later with resolution of glomerulitis. Glomeruli were captured in regions of interest (ROIs) for whole exome sequencing. Compared to baseline glomeruli, 17 transcripts were increased and 39 decreased in the 3 subsequent biopsies (> 2-fold and p < 0.005). Increased signatures for macrophages correlated with increased numbers of CD68 positive cells imaged in the corresponding glomeruli. The Human Cell Atlas classified the 39 transcripts decreased during the initial rejection as characteristic of podocytes and this gene signature did not recover in the subsequent 2 biopsies. Additional ROIs encompassing areas of tubulointerstitial fibrosis disclosed signatures for memory B cells in the acute AMR sample. Treatment with IVIg did not eliminate the B cell signal in the subsequent biopsy. Collectively these data demonstrate a compartmentalization of injury processes. Innate immune cells including macrophages were located in glomerular and tubulointerstitial compartments, whereas, adaptive immune cells including memory B cells localized to the tubulointerstitial compartment. Furthermore, podocyte transcripts were decreased in glomeruli and did not recover with treatment indicating a vulnerability of these cells to acute AMR.

BackgroundEvidence for involvement of NK cells and monocytes as prime agents in the process of human kidney transplant rejection is expanding quickly. These cells express FCGR3A, used for antibody-dependent cellular cytotoxicity (ADCC) after engagement with donor-specific antibodies on graft cells. A functional genetic polymorphism in the Fc fragment of IgG IIIa receptor (CD16a) gene (FCGR3A) results in a high-affinity (V158 allele) or low-affinity receptor (F158 allele). MethodsWe studied the impact of the FCGR3A F158V genotype on graft histology, function, and outcomes in a large, unselected observational cohort study of 1259 kidney transplants (40% F/F158, 46% F/V158 and 14% V/V158) with 5435 post-transplant transplant biopsies. ResultsPresence of at least one V158 allele was proportionally and significantly associated with a lower rate of C4d without rejection (F/V158, HR=0.51 [0.29-0.92], p=0.026; V/V158, HR=0.28 [0.08-0.90], p=0.033). Additionally, V/V158 was strongly associated with chronic active antibody-mediated rejection (HR=9.13 [1.89-44], p=0.006) and transplant glomerulopathy (HR=1.67 [1.01-2.75], p=0.046). The V/V158 genotype significantly associated with accelerated graft functional decline (-1.68 mL/min/y versus -0.74 mL/min/y (F/F158), p=0.034) and a higher rate of graft failure (HR=1.49 [1.01-2.20], p=0.045). This association was driven by transplants with post-transplant rejection. ConclusionsOur findings suggest that FCGR3A affinity is key in AMR pathobiology. Low affinity of this receptor (F158 allele) might associate with abrogation of effective downstream ADCC, even in the presence of complement activation (C4d deposition), while high affinity of this receptor (V158 allele) might escalate AMR, accelerating evolution to more chronic AMR phenotypes (transplant glomerulopathy). This framework provides an immunobiological link between lower C4d without rejection rates, higher chronic active AMR/ transplant glomerulopathy rates and adverse graft outcomes associated with the FCGR3A V/V158 genotype. Key study findingsO_LIV/V158 associates with higher rates of chronic active AMR/transplant glomerulopathy, and lower rates of C4d without rejection C_LIO_LIV/V158 drives faster graft decline and graft failure, especially in transplants complicated with rejection C_LIO_LIFCGR3A F158V might link complement activation and ADCC in AMR pathobiology, accelerating chronic alloimmune injury C_LI

BACKGROUNDDespite a sharp rise in kidney graft short-term survival rates and better donor-recipient HLA matching, mid- and long-term survival have not sufficiently improved over the past decades. Several studies suggest that non-HLA factors could be involved in kidney allograft injury, but no validated marker has yet been identified. Here, we aimed at finding genetic variations and mismatches associated with graft function and survival. METHODSUsing genome-wide strategies, we tested the recipients and donors common variations (SNPs and CNVs), and the donor-recipient genetic mismatches for association with 1-year kidney graft function and with time-to-death-censored kidney graft failure in a monocentric European cohort of 1,482 complete donor-recipient pairs. We validated our findings through a meta-analysis in two independent European cohorts gathering a total of 1,842 additional complete pairs. RESULTSWe did not identify any significant association with 1-year graft function. However, we discovered four non-HLA mismatches (3 SNPs and 1 CNV) associated with time-to-kidney graft failure. One signal in a regulatory region upstream the TOM1L1 gene (p=6.3x10-9, HR=4.1) was successfully replicated in the validation cohorts (pmeta-analysis=6.7x10-9, HR=2.9) and ranked among the top 50 rejection-specific genes in a pan-organ transcriptomics study. This locus was also associated with time-to-cellular and humoral rejection (p=0.02) in the discovery cohort in patients achieving primary graft function. CONCLUSIONSBy running one of the largest ever performed kidney transplantation genomic analyses, we identified and confirmed a novel donor-recipient genetic mismatch in a biologically relevant non-HLA locus associated with kidney allograft failure. Lay SummaryDespite significant advances in immunosuppression, kidney transplant recipients remain at risk of graft rejection and, in more severe cases, graft failure, which can lead to retransplantation, return to dialysis, or even patient death. Donor-recipient HLA compatibility is integrated into kidney transplant clinical care, as this genetic region is key for immunity and graft tolerance. However, several studies suggest that compatibility outside of the HLA region could also influence graft survival. We aimed at investigating this hypothesis in a cohort of 1,482 donor-recipient pairs and found a novel genetic region involved in kidney graft dysfunction that was validated after meta-analysis of two independent cohorts. These findings contribute to a better understanding of the impact of donor-recipient genetic compatibility on kidney transplant outcomes.

BackgroundThe effects of Banff histological diagnoses on kidney transplant outcome have been well characterized. However, repeated observation of such histological injury across multiple biopsies in kidney transplant recipients remains insufficiently explored. MethodsIn an observational cohort (N=1819 transplantations with 5736 post-transplant biopsies, recurrent event survival models quantified transitions between diagnoses of T-cell mediated rejection (TCMR), antibody-mediated rejection (AMR), DSA-negative C4d-negative microvascular inflammation (MVIDSA-/C4d-), BK polyomavirus nephropathy (BKPyVAN), borderline TCMR (bTCMR), and probable AMR (pAMR), revealing patterns in the disease trajectories. In two observational cohorts (N=1818 transplantations with 5732 biopsies, N=853 transplantations with 975 biopsies), time-dependent cumulative covariates were constructed for TCMR, AMR, MVIDSA-/C4d- and BKPyVAN, enabling estimation of associations of repeated diagnoses with graft failure using multivariable cause-specific Cox models. ResultsThe incidence rate of a diagnosis was most strongly associated with earlier diagnosis of the same type, but associations between different types of diagnoses also occurred. The hazard of kidney graft failure was significantly increased by repeated observation of TCMR in multiple biopsies (HR 7.97, 95% CI 4.94 - 12.86), as well as by repeated AMR (HR 6.19, 95% CI 3.15 - 12.17), repeated MVIDSA-/C4d- (HR 4.53, 95% CI 2.15-9.54) and repeated BKPyVAN (HR 10.90, 95% CI 5.83 - 20.35). The hazard of graft failure was increased more after repeated diagnoses in transplants than after first diagnoses. The effects of repeated TCMR and repeated AMR remained significant even when observed in protocol biopsies in the absence of graft dysfunction. Repeated observation of BKPyVAN was the most detrimental of all diagnoses when observed in indication biopsies, but it was the least harmful when observed in protocol biopsies. ConclusionIncidence of Banff histological diagnoses appears to be affected by earlier diagnoses, especially those of the same type. These repeated observations of a specific diagnosis have an additional effect on the hazard of graft failure, underscoring a critical unmet need for adequate treatment strategies for these recurrent or persistent injury processes. Lay summaryIn two observational cohorts of 1819 and 750 kidney transplant recipients, kidney transplant biopsies were taken at multiple time points after transplantation. Based on the Banff classification for transplant pathology, various post-transplant diseases were diagnosed, often at more than one time point during follow-up. We assessed patterns in the occurrence of diagnoses over time, and related these diagnoses to survival of the kidney grafts using survival models with time-dependent cumulative diagnoses. We found that repeated observation of the same diagnosis was much more common than consecutive observations of different diagnoses. Repeated diagnoses of tissue injury also decreased kidney graft survival more compared to single diagnoses. This indicates that treatment options for patients with repeated or persistent diagnoses are currently inadequate and novel strategies are needed.

Optimizing immunosuppressive therapy remains central to improving long-term outcomes after kidney transplantation. Both induction and maintenance therapies are widely used, yet their comparative effectiveness across heterogeneous populations requires further evaluation. To this end, this national retrospective cohort study analyzed 228,855 deceased-donor kidney transplant recipients using the over two-decade data (2000-2024). We employed multivariable Cox proportional hazards (PH) models for clinical inference and four machine learning (ML) survival models: random survival forest (RSF), support vector machine (SVM), penalized Cox regression (CoxNet), and extreme gradient boosting optimized with the Cox partial likelihood (XGBoost-Cox) to assess predictive performance of death-censored graft failure and all-cause patient mortality. Model performance was evaluated using the concordance index (C-index) and time-dependent area under the curve (tdAUC). Maintenance regimens with calcineurin inhibitors (CNI) and mycophenolate mofetil (MMF) demonstrated protective effects for both graft failure (CNI+MMF: hazard ratio [HR] 0.72, 95% confidence interval [CI] 0.70-0.74; CNI+MMF+steroids: HR 0.84, 95% CI 0.82-0.87) and patient mortality (CNI+MMF: HR 0.78, 95% CI 0.76-0.81; CNI+MMF+steroids: HR 0.90, 95% CI 0.88-0.93). Among induction therapies, antithymocyte globulin (ATG) showed protective associations (HR 0.93 for both outcomes), while interleukin-2 receptor (IL-2R) antagonists and Alemtuzumab demonstrated neutral effects. Combined ATG + IL-2R therapy comparatively increased the hazard of graft failure (HR 1.09). Recipient diabetes, dialysis dependence, older age, and higher Kidney Donor Profile Index (KDPI) were strong adverse predictors. Traditional Cox regression achieved robust discrimination (graft failure concordance index: 0.685; patient mortality concordance index: 0.704) comparable to ML survival models. These findings reinforce CNI and MMF maintenance regimens as foundational to contemporary immunosuppression while demonstrating differential effectiveness across induction strategies. The dual analytical framework, which integrates classical Cox PH modeling with ML survival models, shows that Cox models remain highly competitive for clinical inference while ML approaches offer complementary predictive value to support individualized post-transplant risk stratification.

Neutralizing antibody responses are attenuated in many solid organ transplant recipients (SOTRs) despite SARS-CoV-2 vaccination. Pre-exposure prophylaxis (PrEP) with the monoclonal antibody combination Tixagevimab and Cilgavimab (T+C) might augment immunoprotection, yet activity against Omicron sublineages in vaccinated SOTRs is unknown. Vaccinated SOTRs who received 300+300mg T+C (either single dose or two 150+150mg doses) within a prospective observational cohort submitted pre- and post-injection samples between 1/10/2022-4/4/2022. Binding antibody (anti-receptor binding domain [RBD], Roche) and surrogate neutralization (%ACE2 inhibition; [&ge;]20% connoting neutralizing inhibition, Meso Scale Discovery) were measured against variants including Omicron sublineages BA.1 and BA.2. Data were analyzed using the Wilcoxon matched-pairs signed-rank test and McNemars test. Among 61 participants, median (IQR) anti-RBD increased from 424 (IQR <0.8-2322.5) to 3394.5 (IQR 1403.9-7002.5) U/ml post T+C (p<0.001). The proportion demonstrating vaccine strain neutralizing inhibition increased from 46% to 100% post-T+C (p<0.001). BA.1 neutralization was low and did not increase (8% to 16% of participants post-T+C, p=0.06). In contrast, BA.2 neutralization increased from 7% to 72% of participants post-T+C (p<0.001). T+C increased anti-RBD levels, yet BA.1 neutralizing activity was minimal. Encouragingly, BA.2 neutralization was augmented and in the current variant climate T+C PrEP may serve as a useful complement to vaccination in high-risk SOTRs.

ObjectivesTo evaluate the clinical implications of adjunctive molecular gene expression analysis (MMDx) of biopsy specimens in heart transplant (HT) recipients with suspected rejection. IntroductionHistopathological evaluation remains the standard method for rejection diagnosis in HT. However, the wide interobserver variability combined with a relatively common incidence of "biopsy-negative" rejection has raised concerns about the likelihood of false-negative results. MMDx, which uses gene expression to detect early signs of rejection, is a promising test to further refine the assessment of HT rejection. MethodsSingle-center prospective study of 418 consecutive for-cause endomyocardial biopsies performed between November 2022 and May 2024. Each biopsy was graded based on histology and assessed for rejection patterns using MMDx. MMDx results were deemed positive if borderline or definitive rejection was present. The impact of MMDx results on clinical management was evaluated. Primary outcomes were 1-year survival and graft dysfunction following MMDx-guided clinical management. Secondary outcomes included changes in donor-specific antibodies, MMDx gene transcripts, and donor-derived cell-free DNA (dd-cfDNA) levels. ResultsWe analyzed 418 molecular samples from 237 unique patients. Histology identified rejection in 32 cases (7.7%), while MMDx identified rejection in 95 cases (22.7%). Notably, in 79 of the 95 cases where MMDx identified rejection, histology results were negative, with the majority of these cases being antibody-mediated rejection (62.1%). Samples with rejection on MMDx were more likely to show a combined elevation of dd-cfDNA and peripheral blood gene expression profiling than those with borderline or negative MMDx results (36.7% vs 28.0% vs 10.3%; p<0.001). MMDx results led to the implementation of specific antirejection protocols or changes in immunosuppression in 20.4% of cases, and in 73.4% of cases where histology was negative and MMDx showed rejection. 1-year survival was better in the positive MMDx group where clinical management was guided by MMDx results (87.0% vs 78.6%; log rank p=0.0017). ConclusionsIn our cohort, MMDx results more frequently indicated rejection than histology, often leading to the initiation of antirejection treatment. Intervention guided by positive MMDx results was associated with improved outcomes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/24309444v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19c6035org.highwire.dtl.DTLVardef@1704be4org.highwire.dtl.DTLVardef@1866b99org.highwire.dtl.DTLVardef@24823_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundPlasma cell rich rejection (PCRR) is an uncommon, ill-defined type of renal allograft rejection in the current literature considered a subtype of T cell-mediated rejection (TCMR). PCRR has poorer clinical outcome and is often refractory to classic immunosuppressive therapy. Our study analyzed clinical course, Banff lesion scores and mRNA expression of PCRR compared to (late) rejection. MethodsWe retrospectively scored and reclassified the last known biopsy of 263 renal transplant recipients, morphologically classified as rejection according to the 2019 Banff classification. mRNA expression analysis was performed using the Nanostring B-HOT panel on a subset of cases. PCRR was compared to (late) TCMR, ABMR and mixed rejection for renal function follow-up and graft survival. ResultsmRNA analysis revealed uniquely expressed genes in PCRR including LOX, CPA3, IL4, IL17F, and MMP12. PCRR is enriched for genes related to mast cells, memory B- and T-cells and transcripts involved in NK cells and allograft fibrosis with heterogeneity in gene expression in biopsies with PCRR. PCRR might be a late event compared to late TCMR and ABMR, with a higher degree of total inflammation and fibrosis. Graft survival and renal function was similar to late TCMR and ABMR during a 5-year follow-up period after renal biopsy. ConclusionPCRR represents a distinct late-onset stage of inflammation displaying diverse gene expression patterns, with presence of mainly mast cells, NK cells and transcripts involved in renal allograft fibrosis. Clinical outcomes in patients with PCRR appeared more similar to late TCMR and ABMR.

Background and aimsTissular gene expression profiling has the potential to refine the diagnosis of cardiac allograft rejection. Contrary to whole-transcriptome approaches, targeted molecular profiling applicable to formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies (EMB) can be easily implemented in clinical practice. We aimed to develop and validate the first rejection molecular diagnostic system dedicated to heart transplantation (HTx). MethodsAn international multicenter study was designed, building a deep phenotyped cohort of HTx recipients recruited between 2011 and 2021 at 4 referral centers. Detailed donors, recipients, clinical, immunological, biological, and histological parameters were collected. EMBs were graded according to international working formulations. Tissue gene expression was analyzed on FFPE-EMB using the consensus Banff Human Organ Transplant gene set. Molecular classifiers of antibody-mediated (AMR) and acute cellular rejection (ACR) were built. Discrimination and calibration were assessed in the development and validation sets (NCT06436027). ResultsA total of 591 biopsies were included: 188 AMR (pAMR1(I+): n=51; pAMR1(H+): n=58; pAMR2-3: n=79), 289 ACR (1R n=174; 2-3R n=115) and 114 matched non-rejection cases. Biopsies were split in a derivation (n=476) and a validation set (n=115). AMR top significant transcripts were related to the IFN-gamma inducible pathway, endothelial activation, and monocyte-macrophage recruitment. ACR was characterized by transcripts related to T-cell receptor, CD3 receptor activation, and CD28 signaling. ACR and AMR molecular rejection models were strongly associated with the pathology severity of rejection and accurately identified rejection in the derivation (ROC-AUC: AMR=0.831, ACR:=0.837) and validation sets (ROC-AUC: AMR=0.812; ACR=0.849). Calibration was adequate. The robustness of the molecular classifiers were reinforced by various sensitivity analyzes. An automated report was developed to enhance the reproducibility and clinical applicability of the molecular analysis. ConclusionsIn this study, the first tissue-based rejection molecular diagnostic system applicable to FFPE- EMB and dedicated to heart transplantation rejection was developed and internally validated. This tool has the potential to refine the diagnosis of rejection.

BackgroundIn lung transplantation, de novo immunodominant donor-specific anti-HLA antibodies recognizing HLA-DQ antigens (dn-iDSA-DQ) are predominant and can induce chronic lung allograft dysfunction (CLAD). We previously developed a method to measure the active concentration of dn-iDSA-DQ. We aimed to determine whether this new quantitative biomarker is associated with transplantation outcomes. MethodsThis retrospective multicentre cohort study included 90 lung transplant recipients (LTRs) developing dn-iDSA-DQ, evidenced through single antigen flow beads (SAFB) follow-up. We measured the active concentration of dn-iDSA-DQ at the time of their first detection (T0) for all LTRs, and within the 2 years after DSA detection, whenever possible. SAFB dn-iDSA-DQ characteristics and clinical data were retrieved up to 5 years after DSA detection. ResultsWe tested 184 sera with SPR (n=90 at T0, n=94 within the 2 years after DSA detection), among which 63 (34.4%) had a quantifiable concentration of the dn-iDSA-DQ ([&ge;]0.3 nM). The median SAFB mean fluorescence intensity (MFI) of the dn-iDSA-DQ with a concentration [&ge;]0.3 nM was higher (p<0.0001), yet the correlation between SAFB MFI and active concentration was low (r=0.758, p<0.0001). In multivariate analysis, a concentration of the dn-iDSA-DQ [&ge;]0.3 nM at T0 was independently associated with a lower 2-year CLAD-free survival (HR 2.06, p=0.02). A concentration of the dn-iDSA-DQ [&ge;]0.3 nM within the 2 years from DSA detection was associated with a lower graft survival in univariate analysis. ConclusionsActive concentration of dn-iDSA-DQ appears as a valuable biomarker to identify pathogenic DSA at their first detection because of its association with CLAD.

BackgroundAttenuated immune responses to mRNA SARS-CoV-2 vaccines have been reported in solid organ transplant recipients. Most studies have assessed serological responses alone, and there is limited immunological data on vector-based vaccines in this population. This study compares the immunogenicity of BNT162b2 with ChAdOx1 in a cohort of kidney transplant patients, assessing both serological and cellular responses. Methods920 patients were screened for spike protein antibodies (anti-S) following 2 doses of either BNT162b2 (n=490) or ChAdOx1 (n=430). 106 patients underwent assessment with T-cell ELISpot assays. 65 health care workers were used as a control group. ResultsAnti-S was detected in 569 (61.8%) patients. Seroconversion rates in infection-naive patients who received BNT162b2 were higher compared with ChAdOx1, at 269/410 (65.6%) and 156/358 (43.6%) respectively, p<0.0001. Anti-S concentrations were higher following BNT162b, 58(7.1-722) BAU/ml, compared with ChAdOx1, 7.1(7.1-39) BAU/ml, p<0.0001. Calcineurin inhibitor monotherapy, vaccination occurring >1st year post-transplant and receiving BNT162b2 was associated with seroconversion. Only 28/106 (26.4%) of patients had detectable T-cell responses. There was no difference in detection between infection-naive patients who received BNT162b2, 7/40 (17.5%), versus ChAdOx1, 2/39 (5.1%), p=0.15. There was also no difference in patients with prior infection who received BNT162b2, 8/11 (72.7%), compared with ChAdOx1, 11/16 (68.8%), p=0.83. ConclusionsEnhanced humoral responses were seen with BNT162b2 compared with ChAdOx1 in kidney transplant patients. T-cell responses to both vaccines were markedly attenuated. Clinical efficacy data is still required but immunogenicity data suggests weakened responses to both vaccines in transplant patients, with ChAdOx1 less immunogenic compared with BNT162b2.

The development of de novo donor-specific antibodies (DSAs) against HLA is associated with premature graft failure in kidney transplantation. However, rates and factors influencing de novo DSA formation vary widely across the literature. We aimed to identify pre-transplant factors influencing the development of de novo HLA-specific antibodies following kidney transplantation using machine learning. Data from 460 kidney transplant recipients at a single centre between 2009-2014 were analysed. Pre-transplant variables were collected, and post-transplant sera were screened for HLA antibodies. Positive samples were investigated using Single Antigen Bead (SAB) testing. Machine learning models (Classification and Regression Trees, Random Forest, XGBoost, CatBoost) were trained on a training set of pre-transplant data to predict de novo DSA formation, with and without SMOTE oversampling. Model performance was evaluated on an independent testing set using F1 scores, and feature importance was assessed using SHAP. In the full cohort analysis, XGBoost models performed the best, with F1 scores of 0.54-0.59 without SMOTE and 0.72-0.79 with SMOTE. The strongest predictors were pre-transplant HLA antibodies, number of kidney transplants, cold ischemia time (CIT), recipient age and female gender. SHAP dependence plots showed that pre-existing HLA antibodies and past transplants increased the risk of de novo DSA development. In the unsensitised subgroup analysis, model performance was poor. Machine learning models can be used to identify pre-transplant risk factors for de novo HLA-specific antibody development in kidney transplantation. Monitoring and risk-stratifying patients based on these factors may help guide preventive immunological strategies and recipient selection to improve long-term allograft outcomes. Translational statementThis study identified pre-transplant risk factors for the development of de novo HLA-specific antibody in kidney transplantation. Monitoring and risk-stratifying patients based on these factors may help guide preventive immunological strategies and recipient selection to improve long-term allograft outcomes.

Vascularized composite allotransplantation (VCA) involves immunologically heterogeneous tissues and is associated with high rates of acute rejection (AR), particularly due to the immunogenicity of the skin. This study assessed biomarkers to monitor AR in a recipient of full-face and bilateral hand transplantation. Over 4.6 years postoperatively, we analyzed serial blood and tissue samples, including donor-derived cell-free DNA (dd-cfDNA) in plasma, recipient DNA in allograft biopsies using short tandem repeat (STR) analysis, lymphocyte subsets via flow cytometry, and level of angiotensin II type 1 receptor antibodies (AT1R-Abs). dd-cfDNA and STR showed trends toward elevated recipient signal during acute rejection, though differences were not statistically significant. CD8+ T-cell percentages increased before AR onset, suggesting potential as a prognostic biomarker. AT1R-Ab levels did not differ significantly between AR and non-rejection episodes, possibly due to prophylactic immune cell depletion. While dd-cfDNA and STR levels correlate with rejection episodes and reflect key cellular events within graft tissue, CD8+ T-cell count remains the most robust prognostic biomarker for predicting the onset of cytotoxicity in this patient. These findings highlight the importance of further longitudinal, multi-patient studies to validate emerging biomarkers and improve rejection monitoring strategies in VCA.

The COVID-19 pandemic has reduced access to solid organ transplantation, compounding organ shortages and waitlist mortality. A continued area of uncertainty is the safety of transplanting organs recovered from SARS-CoV-2 infected donors, as autopsies of patients who died with COVID-19 show that the virus can be found in extra-pulmonary organs1. Case reports and series on transplantation of these organs have been published 2, 3, but population-level data is lacking. We queried a national transplant database for recipients of organs recovered from donors recently infected by SARS-CoV-2. For organs with more than 50 cases, these were then propensity-score matched at a ratio of 1:10 to similar recipients of organs recovered from donors who tested negative for SARS-CoV-2 (controls). Data were extracted from the Scientific Registry of Transplant Recipients (SRTR - v2203 - updated March 2022), which collects detailed information on all solid organ transplants in the United States since 1986. Cases were defined as adult ([&ge;] 18 years) recipients of organs recovered from deceased donors who tested positive for SARS-CoV-2 by nasopharyngeal or lower respiratory sample polymerase chain reaction or antigen assay within 7 days of organ transplantation. Multiple organ transplants were excluded. There were 775 kidney, 330 liver, 123 heart, 44 kidney-pancreas, 16 lung, 5 pancreas, and 3 small bowel transplants of organs recovered from 393 deceased donors recently infected by COVID-19. For kidney, liver, and heart transplants, Kaplan-Meier curves of both overall and graft survival at 90 days were similar between cases and controls. Our data shows that transplanting kidneys, livers, and hearts recovered from deceased donors recently infected by SARS-CoV-2 was not associated with increased recipient mortality or worse graft-survival. This should help transplant providers make decisions regarding acceptance of these organs, and counsel transplant candidates on the safety of receiving them. The limited number of kidney-pancreas, lung, pancreas, and intestinal cases precludes significant conclusions for these organs. Our data also strongly supports the notion that donors with recent COVID-19 infection should not be automatically excluded from the donor pool. The limited number of kidney-pancreas, lung, pancreas, and intestinal cases precludes significant conclusions for these organs. Limitations include lack of data on donor infection timeline and estimates of viral load (PCR cycle thresholds), description of donor COVID-19 symptomatology at organ procurement, donor or recipient vaccination or prior COVID-19 infection status, which are not tracked in the database. We did not have information regarding transmission of COVID-19 to transplant recipients. Future analysis of updated versions of the database should help address. Our data strongly support the notion that donors with recent COVID infection should not be automatically excluded from the donor pool. Prospective studies are needed to confirm our findings and provide insights on optimal post-transplant management of these recipients.