Immunogenicity of third dose COVID-19 vaccine strategies in patients who are immunocompromised with suboptimal immunity following two doses (OCTAVE-DUO): an open-label, multicentre, randomised, controlled, phase 3 trial.

BACKGROUND: The humoral and T-cell responses to booster COVID-19 vaccine types in multidisease immunocompromised individuals who do not generate adequate antibody responses to two COVID-19 vaccine doses, is not fully understood. The OCTAVE DUO trial aimed to determine the value of third vaccinations in a wide range of patients with primary and secondary immunodeficiencies. METHODS: OCTAVE-DUO was a prospective, open-label, multicentre, randomised, controlled, phase 3 trial investigating humoral and T-cell responses in patients who are immunocompromised following a third vaccine dose with BNT162b2 or mRNA-1273, and of NVX-CoV2373 for those with lymphoid malignancies. We recruited patients who were immunocompromised from 11 UK hospitals, aged at least 18 years, with previous sub-optimal responses to two doses of SARS-CoV-2 vaccine. Participants were randomly assigned 1:1 (1:1:1 for those with lymphoid malignancies), stratified by disease, previous vaccination type, and anti-spike antibody response following two doses. Individuals with lived experience of immune susceptibility were involved in the study design and implementation. The primary outcome was vaccine-specific immunity defined by anti-SARS-CoV-2 spike antibodies (Roche Diagnostics UK and Ireland, Burgess Hill, UK) and T-cell responses (Oxford Immunotec, Abingdon, UK) before and 21 days after the third vaccine dose analysed by a modified intention-to-treat analysis. The trial is registered with the ISRCTN registry, ISRCTN 15354495, and the EU Clinical Trials Register, EudraCT 2021-003632-87, and is complete. FINDINGS: Between Aug 4, 2021 and Mar 31, 2022, 804 participants across nine disease cohorts were randomly assigned to receive BNT162b2 (n=377), mRNA-1273 (n=374), or NVX-CoV2373 (n=53). 356 (45%) of 789 participants were women, 433 (55%) were men, and 659 (85%) of 775 were White. Anti-SARS-CoV-2 spike antibodies measured 21 days after the third vaccine dose were significantly higher than baseline pre-third dose titres in the modified intention-to-treat analysis (median 1384 arbitrary units [AU]/mL [IQR 4·3-7990·0] compared with median 11·5 AU/mL [0·4-63·1]; p<0·001). Of participants who were baseline low responders, 380 (90%) of 423 increased their antibody concentrations to more than 400 AU/mL. Conversely, 166 (54%) of 308 baseline non-responders had no response after the third dose. Detectable T-cell responses following the third vaccine dose were seen in 494 (80%) of 616 participants. There were 24 serious adverse events (BNT612b2 eight [33%] of 24, mRNA-1273 12 [50%], NVX-CoV2373 four [17%]), two (8%) of which were categorised as vaccine-related. There were seven deaths (1%) during the trial, none of which were vaccine-related. INTERPRETATION: A third vaccine dose improved the serological and T-cell response in the majority of patients who are immunocompromised. Individuals with chronic renal disease, lymphoid malignancy, on B-cell targeted therapies, or with no serological response after two vaccine doses are at higher risk of poor response to a third vaccine dose. FUNDING: Medical Research Council, Blood Cancer UK.

Application of HLA molecular level mismatching in ethnically diverse kidney transplant recipients receiving a steroid-sparing immunosuppression protocol.

Defining HLA mismatch at the molecular compared with the antigen level has been shown to be superior in predicting alloimmune responses, although data from across different patient populations are lacking. Using HLA-Matchmaker, HLA-EMMA and PIRCHE-II, this study reports on the association between molecular mismatch (MolMM) and de novo donor-specific antibody (dnDSA) in an ethnically diverse kidney transplant population receiving a steroid-sparing immunosuppression protocol. Of the 419 patients, 51 (12.2%) patients had dnDSA. De novo DSA were seen more frequently with males, primary transplants, patients receiving tacrolimus monotherapy, and unfavorably HLA-matched transplants. There was a strong correlation between MolMM load and antigen mismatch, although significant variation of MolMM load existed at each antigen mismatch. MolMM loads differed significantly by recipient ethnicity, although ethnicity alone was not associated with dnDSA. On multivariate analysis, increasing MolMM loads associated with dnDSA, whereas antigen mismatch did not. De novo DSA against 8 specific epitopes occurred at high frequency; of the 51 patients, 47 (92.1%) patients with dnDSA underwent a pretreatment biopsy, with 21 (44.7%) having evidence of alloimmune injury. MolMM has higher specificity than antigen mismatching at identifying recipients who are at low risk of dnDSA while receiving minimalist immunosuppression. Immunogenicity consideration is important, with more work needed on identification, especially across different ethnic groups.

SARS-CoV-2-specific immune responses and clinical outcomes after COVID-19 vaccination in patients with immune-suppressive disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and infection outcomes were evaluated in 2,686 patients with varying immune-suppressive disease states after administration of two Coronavirus Disease 2019 (COVID-19) vaccines. Overall, 255 of 2,204 (12%) patients failed to develop anti-spike antibodies, with an additional 600 of 2,204 (27%) patients generating low levels (<380 AU ml-1). Vaccine failure rates were highest in ANCA-associated vasculitis on rituximab (21/29, 72%), hemodialysis on immunosuppressive therapy (6/30, 20%) and solid organ transplant recipients (20/81, 25% and 141/458, 31%). SARS-CoV-2-specific T cell responses were detected in 513 of 580 (88%) patients, with lower T cell magnitude or proportion in hemodialysis, allogeneic hematopoietic stem cell transplantation and liver transplant recipients (versus healthy controls). Humoral responses against Omicron (BA.1) were reduced, although cross-reactive T cell responses were sustained in all participants for whom these data were available. BNT162b2 was associated with higher antibody but lower cellular responses compared to ChAdOx1 nCoV-19 vaccination. We report 474 SARS-CoV-2 infection episodes, including 48 individuals with hospitalization or death from COVID-19. Decreased magnitude of both the serological and the T cell response was associated with severe COVID-19. Overall, we identified clinical phenotypes that may benefit from targeted COVID-19 therapeutic strategies.

Application of the Banff Human Organ Transplant Panel to kidney transplant biopsies with features suspicious for antibody-mediated rejection.

The Banff Classification for Allograft Pathology includes the use of gene expression in the diagnosis of antibody-mediated rejection (AMR) of kidney transplants, but a predictive set of genes for classifying biopsies with 'incomplete' phenotypes has not yet been studied. Here, we developed and assessed a gene score that, when applied to biopsies with features of AMR, would identify cases with a higher risk of allograft loss. To do this, RNA was extracted from a continuous retrospective cohort of 349 biopsies randomized 2:1 to include 220 biopsies in a discovery cohort and 129 biopsies in a validation cohort. The biopsies were divided into three groups: 31 that fulfilled the 2019 Banff Criteria for active AMR, 50 with histological features of AMR but not meeting the full criteria (Suspicious-AMR), and 269 with no features of active AMR (No-AMR). Gene expression analysis using the 770 gene Banff Human Organ Transplant NanoString panel was carried out with LASSO Regression performed to identify a parsimonious set of genes predictive of AMR. We identified a nine gene score that was highly predictive of active AMR (accuracy 0.92 in the validation cohort) and was strongly correlated with histological features of AMR. In biopsies suspicious for AMR, our gene score was strongly associated with risk of allograft loss and independently associated with allograft loss in multivariable analysis. Thus, we show that a gene expression signature in kidney allograft biopsy samples can help classify biopsies with incomplete AMR phenotypes into groups that correlate strongly with histological features and outcomes.

Antibody prevalence after three or more COVID-19 vaccine doses in individuals who are immunosuppressed in the UK: a cross-sectional study from MELODY.

BACKGROUND: In the UK, additional COVID-19 vaccine booster doses and treatments are offered to people who are immunosuppressed to protect against severe COVID-19, but how best to choose the individuals that receive these vaccine booster doses and treatments is unclear. We investigated the association between seropositivity to SARS-CoV-2 spike protein with demographic, disease, and treatment-related characteristics after at least three COVID-19 vaccines in three cohorts of people who are immunosuppressed. METHODS: In a cross-sectional study using UK national disease registries, we identified, contacted, and recruited recipients of solid organ transplants, participants with rare autoimmune rheumatic diseases, and participants with lymphoid malignancies who were 18 years or older, resident in the UK, and who had received at least three doses of a COVID-19 vaccine. The study was open to recruitment from Dec 7, 2021, to June 26, 2022. Participants received a lateral flow immunoassay test for SARS-CoV-2 spike antibodies to complete at home, and an online questionnaire. Multivariable logistic regression was used to estimate the mutually adjusted odds of seropositivity against each characteristic. FINDINGS: Between Feb 14 and June 26, 2022, we screened 101 972 people (98 725 invited, 3247 self-enrolled) and recruited 28 411 (27·9%) to the study. 23 036 (81·1%) recruited individuals provided serological data. Of these, 9927 (43·1%) were recipients of solid organ transplants, 6516 (28·3%) had rare autoimmune rheumatic diseases, and 6593 (28·6%) had lymphoid malignancies. 10 485 (45·5%) participants were men and 12 535 (54·4%) were women (gender was not reported for 16 [<0·1%] participants), and 21661 (94·0%) participants were of White ethnicity. The median age of participants with solid organ transplants was 60 years (SD 50-67), with rare autoimmune rheumatic diseases was 65 years (54-73), and with lymphoid malignancy was 69 years (61-75). Of the 23 036 participants with serological data, 6583 (28·6%) had received three vaccine doses, 14 234 (61·8%) had received four vaccine doses, and 2219 (9·6%) had received five or more vaccine doses. IgG anti-spike antibodies were undetectable in 2310 (23·3%) of 9927 patients with solid organ transplants, 922 (14·1%) of 6516 patients with rare autoimmune rheumatic diseases, and 1366 (20·7%) of 6593 patients with lymphoid malignancies. In all groups, seropositivity was associated with younger age, higher number of vaccine doses (ie, five vs three), and previous COVID-19. Immunosuppressive medication reduced the likelihood of seropositivity: the lowest odds of seropositivity were found in recipients of solid organ transplants receiving a combination of an anti-proliferative agent, a calcineurin inhibitor, and steroids, and those with rare autoimmune rheumatic diseases or lymphoid malignancies treated with anti-CD20 therapies. INTERPRETATION: Approximately one in five recipients of solid organ transplants, individuals with rare autoimmune rheumatic diseases, and individuals with lymphoid malignancies have no detectable IgG anti-spike antibodies despite three or more vaccine doses, but this proportion decreases with sequential booster doses. Choice of immunosuppressant and disease type is strongly associated with serological response. Antibody testing using lateral flow immunoassay tests could enable rapid identification of individuals who are most likely to benefit from additional COVID-19 interventions. FUNDING: UK Research and Innovation, Kidney Research UK, Blood Cancer UK, Vasculitis UK and the Cystic Fibrosis Trust.

Diagnostic application of transcripts associated with antibody-mediated rejection in kidney transplant biopsies.

BACKGROUND: The diagnosis of antibody-mediated rejection (AMR) is reached using the Banff Classification for Allograft Pathology, which now includes gene expression analysis. In this study, we investigate the application of 'increased expression of thoroughly validated gene transcripts/classifiers strongly associated with AMR' as diagnostic criteria. METHOD: We used quantitative real-time polymerase chain reaction for 10 genes associated with AMR in a retrospective cohort of 297 transplant biopsies, including biopsies that met the full diagnostic criteria for AMR, even without molecular data (AMR, n = 27), biopsies that showed features of AMR, but that would only meet criteria for AMR with increased transcripts [suspicious for AMR (AMRsusp), n = 49] and biopsies that would never meet criteria for AMR (No-AMR, n = 221). RESULTS: A 10-gene AMR score trained by a receiver-operating characteristic to identify AMR found 16 cases with a high score among the AMRsusp cases (AMRsusp-high) that had significantly worse graft survival than those with a low score (AMRsusp-low; n = 33). In both univariate and multivariate Cox regression analysis, the AMR 10-gene score was significantly associated with an increased hazard ratio (HR) for graft loss (GL) in the AMRsusp group (HR = 1.109, P = 0.004 and HR = 1.138, P = 0.012, respectively), but not in the whole cohort. Net reclassification index and integrated discrimination improvement analyses demonstrated improved risk classification and superior discrimination, respectively, for GL when considering the gene score in addition to histological and serological data, but only in the AMRsusp group, not the whole cohort. CONCLUSIONS: This study provides evidence that a gene score strongly associated with AMR helps identify cases at higher risk of GL in biopsies that are suspicious for AMR but do not meet full criteria.

Multi-arm Trial of Inflammatory Signal Inhibitors (MATIS) for hospitalised patients with mild or moderate COVID-19 pneumonia: a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The primary objective of MATIS is to determine the efficacy of ruxolitinib (RUX) or fostamatinib (FOS) compared to standard of care (SOC) with respect to reducing the proportion of hospitalised patients progressing from mild or moderate to severe COVID-19 pneumonia. Secondary objectives, at 14 and 28 days, are to: Determine the efficacy of RUX or FOS to reduce mortality Determine the efficacy of RUX or FOS to reduce the need for invasive ventilation or ECMO Determine the efficacy of RUX or FOS to reduce the need for non-invasive ventilation Determine the efficacy of RUX or FOS to reduce the proportion of participants suffering significant oxygen desaturation Determine the efficacy of RUX or FOS to reduce the need for renal replacement therapy Determine the efficacy of RUX and FOS to reduce the incidence of venous thromboembolism Determine the efficacy of RUX and FOS to reduce the severity of COVID-19 pneumonia [graded by a 9-point modified WHO Ordinal Scale* Determine the efficacy of RUX or FOS to reduce systemic inflammation Determine the efficacy of RUX or FOS to the incidence of renal impairment Determine the efficacy of RUX or FOS to reduce duration of hospital stay Evaluate the safety of RUX and FOS for treatment of COVID-19 pneumonia. TRIAL DESIGN: A multi-arm, multi-stage (3-arm parallel-group, 2-stage) randomised controlled trial that allocates participants 1:1:1 and tests for superiority in experimental arms versus standard of care. PARTICIPANTS: Patients will be recruited while inpatients during hospitalisation for COVID-19 in multiple centres throughout the UK including Imperial College Healthcare NHS Trust. INCLUSION: Patients age ≥ 18 years at screening Patients with mild or moderate COVID-19 pneumonia, defined as Grade 3 or 4 severity by the WHO COVID-19 Ordinal Scale Patients meeting criteria: Hospitalization AND SARS-CoV2 infection (clinically suspected or laboratory confirmed) AND Radiological change consistent with COVID-19 disease CRP ≥ 30mg/L at any time point Informed consent from patient or personal or professional representative Agreement to abstain from sexual intercourse or use contraception that is >99% effective for all participants of childbearing potential for 42 days after the last dose of study drug. For male participants, agreement to abstain from sperm donation for 42 days after the last dose of study drug. EXCLUSION: Requiring either invasive or non-invasive ventilation including CPAP or high flow nasal oxygen at any point after hospital admission but before baseline, not related to a pre-existing condition (e.g., obstructive sleep apnoea) Grade ≥ 5 severity on the modified WHO COVID-19 Ordinal Scale, i.e. SpO2 < 90% on ≥ 60% inspired oxygen by facemask at baseline; non-invasive ventilation; or invasive mechanical ventilation In the opinion of the investigator, progression to death is inevitable within the next 24 hours, irrespective of the provision of therapy Known severe allergic reactions to the investigational agents Child-Pugh B or C grade hepatic dysfunction Use of drugs within the preceding 14 days that are known to interact with any study treatment (FOS or RUX), as listed in the Summary of Product Characteristics Pregnant or breastfeeding Any medical condition or concomitant medication that in the opinion of the investigator would compromise subjects' safety or compliance with study procedures. Any medical condition which in the opinion of the principal investigator would compromise the scientific integrity of the study Non-English speakers will be able to join the study. If participants are unable to understand verbal or written information in English, then hospital translation services will be requested at the participating site for the participant where possible. INTERVENTION AND COMPARATOR: RUXOLITINIB (RUX) (14 days): An oral selective and potent inhibitor of Janus Associated Kinases (JAK1 and JAK2) and cell proliferation (Verstovek, 2010). It is approved for the treatment of disease-related splenomegaly or constitutional symptoms in myelofibrosis, polycythaemia vera and graft-versus-host-disease. RUX will be administered orally 10mg bd Day 1-7 and 5mg bd Day 8-14. FOSTAMATINIB (FOS) (14 days): An oral spleen tyrosine kinase inhibitor approved for the treatment of thrombocytopenia in adult participants with chronic immune thrombocytopenia. FOS will be administered orally 150mg bd Day 1-7 and 100mg bd Day 8-14. Please see protocol for recommended dose modifications where required. COMPARATOR (Standard of Care, SOC): experimental arms will be compared to participants receiving standard of care. It is accepted that SOC may change during a rapidly evolving pandemic. Co-enrolment to other trials and rescue therapy, either pre- or post-randomisation, is permitted and will be accounted for in the statistical analysis. MAIN OUTCOMES: Pairwise comparison (RUX vs SOC and FOS vs SOC) of the proportion of participants diagnosed with severe COVID-19 pneumonia within 14 days. Severe COVID-19 pneumonia is defined by a score ≥ 5 on a modified WHO COVID-19 Ordinal Scale, comprising the following indicators of disease severity: Death OR Requirement for invasive ventilation OR Requirement for non-invasive ventilation including CPAP or high flow oxygen OR O2 saturation < 90% on ≥60% inspired oxygen RANDOMISATION: Participants will be allocated to interventions using a central web-based randomisation service that generates random sequences using random permuted blocks (1:1:1), with stratification by age (<65 and ≥65 years) and site. BLINDING (MASKING): No participants or caregivers are blinded to group assignment. Clinical outcomes will be compared blind to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): For an early informal dose examination by the Data Monitoring Committee a minimum of 30 participants will be recruited. For Stage 1 of this multi-arm multi-stage study, 171 participants will be randomised, with 57 participants in each arm. If at least one experimental intervention shows promise, then Stage 2 will recruit a further 95 participants per arm. Sample size calculations are given in the protocol. TRIAL STATUS: Recruitment is ongoing and started 2nd October 2020. We anticipate completion of Stage 1 by July 2021 and Stage 2 by April 2022. The current protocol version 2.0 of 11th February 2021 is appended. TRIAL REGISTRATION: EudraCT: 2020-001750-22 , 9th July 2020 ClinicalTrials.gov: NCT04581954 , 9th October 2020 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest of expediting dissemination of this material, familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Molecular assessment of antibody-mediated rejection in human pancreas allograft biopsies.

Pancreas transplant longevity is limited by immune rejection, which is diagnosed by graft biopsy using the Banff Classification. The histological criteria for antibody-mediated rejection (AMR) are poorly reproducible and inconsistently associated with outcome. We hypothesized that a 34-gene set associated with antibody-mediated rejection in other solid organ transplants could improve diagnosis in pancreas grafts. The AMR 34-gene set, comprising endothelial, natural killer cell and inflammatory genes, was quantified using the NanoString platform in 52 formalin-fixed, paraffin-embedded pancreas transplant biopsies from 41 patients: 15 with pure AMR or mixed rejection, 22 with T cell-mediated rejection/borderline and 15 without rejection. The AMR 34-gene set was significantly increased in pure AMR and mixed rejection (P = .001) vs no rejection. The gene set predicted histological AMR with an area under the receiver operating characteristic curve (ROC AUC) of 0.714 (P = .004). The AMR 34-gene set was the only biopsy feature significantly predictive of allograft failure in univariate analysis (P = .048). Adding gene expression to DSA and histology increased ROC AUC for the prediction of failure from 0.736 to 0.770, but this difference did not meet statistical significance. In conclusion, assessment of transcripts has the potential to improve diagnosis and outcome prediction in pancreas graft biopsies.

Donor-specific antibodies detected by single antigen beads alone can help risk stratify patients undergoing retransplantation across a repeat HLA mismatch.

Whether reexposure to mismatched HLA antigens (RMM) in the setting of a negative crossmatch is associated with increased immunological risk remains an area of uncertainty. This is due to evidence derived predominantly from registry data, which lacks comprehensive information on alloantibody and rejection. In this study, we analyze the impact of low-level preformed donor-specific antibodies (DSA) against an RMM on transplant outcomes. From 1988 consecutive renal transplant recipients, we analyzed 179 patients undergoing retransplantation, of whom 55 had a RMM. All patients were crossmatch negative and preformed DSA were detected by single antigen beads alone. Multivariate analysis revealed that patients with preformed DSA against an RMM were independently at risk of antibody-mediated rejection (HR 8.70 [3.42-22.10], P < .0001) and death-censored allograft loss (HR 3.08 [1.17-8.14], P = .023). In addition, prior transplant nephrectomy (HR 2.04 [1.00-4.17], P = .0495) was also associated with allograft failure, whereas receiving a retransplant that was matched at HLA class II was associated with a favorable outcome (HR 0.37 [0.14-0.99], P = .047). In the absence of preformed DSA, an RMM was not associated with de novo DSA development, rejection, or allograft loss. In conclusion, an RMM portends increased immunological risk only in the presence of a preformed DSA in patients undergoing retransplantation.

Predicting long-term renal and patient survival by clinicopathological features in elderly patients undergoing a renal biopsy in a UK cohort.

BACKGROUND: Several publications have demonstrated the use of renal biopsy in elderly patients in establishing a diagnosis and enabling directed therapy. However, evidence on the long-term outcomes following biopsies is lacking. The aim of this study is to describe the renal and patient outcomes in elderly patients according to indication for biopsy, clinical parameters and the histological diagnosis. METHODS: We performed a retrospective cohort study of 463 patients >70 years old who underwent a renal biopsy at our centre between 2006 and 2015. RESULTS: The median age of the patients was 74.8 (range 70.0-89.6) years. The most frequent primary diagnoses were pauci-immune crescentic glomerulonephritis (GN; 12%), acute interstitial nephritis (10.8%) and membranous GN (7.1%). Death-censored renal survival at 1 and 5 years following the index biopsy was 85.2 and 75.9%, respectively, and patient survival at 1 and 5 years was 92.2 and 71.6%, respectively. Patients who progressed to end-stage renal disease (ESRD) were at higher risk of dying compared with patients who did not require dialysis [hazard ratio 2.41 (95% confidence interval 1.58-3.68; P < 0.001]. On multivariate analysis, factors associated with the risk of progression to ESRD were creatinine (P < 0.001), heavy proteinuria (P = 0.002) and a non-chronic kidney disease (CKD) biopsy indication (P = 0.006). A histological diagnosis of primary GN (P = 0.001) or tubulointerstitial nephritis (P = 0.008) was associated with a favourable renal outcome, while patients with vasculitis and paraprotein-related renal disease (PPRD) had the highest risk of requiring dialysis (P = 0.0002 and P = 0.003, respectively). PPRD was also an independent risk factor for death. CONCLUSIONS: This study demonstrates that renal biopsies in the elderly not only enable directed therapy, but also provide prognostic information on renal and patient survival.

Clinical-pathological correlations in post-transplant thrombotic microangiopathy.

AIMS: Post-transplant thrombotic microangiopathy (TMA) is a rare and clinically challenging finding in renal transplant biopsies. In addition to recurrent atypical haemolytic uraemic syndrome, TMA in renal transplants is associated with various conditions, such as calcineurin inhibitor (CNI) treatment, antibody-mediated rejection (ABMR), viral infections, sepsis, pregnancy, malignancies, and surgery. The therapeutic implications of this diagnosis are considerable. In order to better understand post-transplant TMA and to identify histological or clinical differences between associated causes, we conducted a multicentre retrospective study. METHODS AND RESULTS: Clinical parameters and transplant renal biopsy findings from 81 patients with TMA were analysed. Biopsies from 38 patients were also analysed with electron microscopy. On the basis of clinical-pathological correlation, TMA was attributed to a main aetiology, whenever possible. TMA occurred at a median of 30 days post-transplantation. Systemic features of TMA were present in only 18% of cases. Twenty-two per cent of cases were attributed to CNI and 11% to ABMR. Although other potentially contributing factors were found in 56% of patients, in most cases (63%) no clearly attributable cause of TMA was identified. Histological differences between groups were minimal. The detection of ultrastructural features that are usually associated with ABMR may help to establish ABMR as the cause of TMA. CONCLUSIONS: Although CNI and ABMR appear to be the main contributors to post-transplant TMA, the aetiology of most cases is probably multifactorial, and TMA cannot be unequivocally attributed to a single underlying aetiology. Morphological features of TMA are not discriminating, but electron microscopy may help to identify ABMR-associated TMA.

Molecular Assessment of C4d-Positive Renal Transplant Biopsies Without Evidence of Rejection.

INTRODUCTION: Immunohistochemical staining for C4d in peritubular capillaries has been part of antibody-mediated rejection (AbMR) definition in the Banff Classification for Allograft Pathology since 2003. However, it has limited sensitivity and specificity, therefore the clinical significance of C4d-positive biopsies without evidence of rejection (C4d+ WER) is unknown. We investigated the transcript levels of genes associated with AbMR in C4d+ WER biopsies from both ABO-compatible and incompatible renal transplant patients. METHODS: RNA was extracted from formalin-fixed paraffin-embedded renal transplant biopsies (n = 125) and gene expression analysis of 35 AbMR-associated transcripts carried out using the NanoString nCounter system. RESULTS: AbMR-associated transcripts were significantly increased in samples with AbMR or suspicious AbMR. A subgroup of 17 of 35 transcripts that best distinguished AbMR from C4d-negative biopsies without evidence of rejection was used to study C4d+ WER samples. There was no differential expression between C4d-negative and C4d+ WER from both ABO-incompatible and -compatible transplants. The geometric mean of 17 differentially expressed genes was used to assign the C4d+ WER biopsies a high- or low-AbMR transcript score. Follow-up biopsies showed AbMR within 1 year of initial biopsy in 5 of 7 high-AbMR transcript patients but only 2 of 46 low-AbMR transcript patients. In multivariate logistic regression analysis, elevated transcript levels in a C4d+ WER biopsy were associated with increased odds for biopsy-proven AbMR on follow-up (P = 0.032, odds ratio 16.318), whereas factors including donor-specific antibody (DSA) status and time since transplantation were not. CONCLUSION: Gene expression analysis in C4d+ WER samples has the potential to identify patients at higher risk of developing AbMR.

Alemtuzumab dose adjusted for body weight is associated with earlier lymphocyte repletion and less infective episodes in the first year post renal transplantation - a retrospective study.

The optimal dose of alemtuzumab for renal transplant induction is not known, and the doses reported in the literature vary. This study compares two separate dosing regimens of alemtuzumab in renal transplantation. The first is a standard fixed dose of 30 mg (SD), and the second is a dose adjusted for body weight at 0.4 mg/kg (AD). In this first year post-transplant, there was no difference in patient [HR 0.64 (0.22-1.86), P = 0.39] or allograft survival [HR 1.18 (0.48-2.90), P = 0.72] between the two groups. There was also no difference in overall rejection-free survival [HR 1.12 (0.79-1.58), P = 0.53]. However, absolute lymphocyte count was significantly higher at all measured time points in the first year in the AD group. There were also less episodes of urosepsis [HR 1.38 (1.03-1.85), P = 0.037] and fungal infection [HR 5.15 (2.00-13.28), P = 0.015] in the AD group compared with the SD group. This study shows that AD alemtuzumab is associated with earlier lymphocyte repletion and less infective episodes in the first year postrenal transplant, without increasing the risk of rejection. This work highlights the need for studies into the optimal dosing of monoclonal antibodies used in transplantation.

Tubuloreticular Inclusions in Renal Allografts Associate with Viral Infections and Donor-Specific Antibodies.

The presence of tubuloreticular inclusions (TRIs) in native glomerular endothelial cells associates with viral infections and lupus nephritis. However, the associations of TRIs in renal transplant biopsy specimens are not known. We analyzed data from 316 patients who had a transplant biopsy with electron microscopy examination; 41 of 316 (13.0%) patients had TRIs. Patients with TRIs had significantly lower allograft survival rates (50.9%) than patients without TRIs (74.3%; P=0.03). Transplant glomerulopathy-free survival was also inferior in the TRI-positive group (57.5%) compared with the TRI-negative group (87.3%; P=0.002). Serologically, hepatitis C associated with the presence of TRIs (P=0.04) along with donor-specific antibodies (P=0.01). Furthermore, patients who were TRI positive were more likely than patients who were TRI negative to have had a previous rejection episode (P=0.02). On multivariate analysis, TRIs associated with prior rejection, viral infections, and class 1 HLA donor-specific antibodies. These results show that the presence of TRIs in renal allograft biopsy specimens associates with poor allograft outcomes and serologic evidence of viral infections and alloimmunity. The association with alloimmunity is a novel finding that warrants additional investigation.

Peritubular Capillary Basement Membrane Multilayering in Renal Allograft Biopsies of Patients With De Novo Donor-Specific Antibodies.

BACKGROUND: Severe peritubular capillary basement membrane multilayering (PTCBML) is part of the Banff definition of chronic antibody-mediated rejection. We retrospectively investigated whether assessment of the mean number of layers of basement membrane (BM) around peritubular capillaries (PTC) can be used in a cohort of patients with de novo donor-specific antibodies (dnDSA) as an early marker to predict long-term antibody-mediated injury. METHODS: This is a retrospective cohort study with 151 electron microscopy samples from 54 patients with dnDSA, assessed at around 1 year after transplantation, for a mean number of BM layers around PTC and in serial biopsies. Graft survival and time to transplant glomerulopathy (TG) development were estimated in survival analyses. RESULTS: We found that a mean PTCBML count greater than 2.5 layers assessed in a sample of 25 PTCs around 1 year after transplantation is indicative of the development of TG in patients with dnDSA (P = 0.001). In addition, in patients with serial biopsies available for electron microscopy analysis, we could distinguish 2 groups: patients with a mean PTCBML count of 2.5 or less on all biopsies, and patients who developed greater than 2.5 layers at any time after transplantation. The latter group reflected dnDSA patients at risk for TG development (P < 0.001). In patients with dnDSA, PTCBML score added significantly to the sensitivity and specificity of prediction of TG compared with microcirculation injury score alone. CONCLUSIONS: Our results highlight the potential value of assessing the mean number of BM in PTC for early prediction of progression to chronic antibody-mediated injury.

Use of Quantitative Real Time Polymerase Chain Reaction to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants.

INTRODUCTION: Microarray studies have shown elevated transcript levels of endothelial and natural killer (NK) cell-associated genes during antibody-mediated rejection (AMR) of the renal allograft. This study aimed to assess the use of quantitative real-time polymerase chain reaction as an alternative to microarray analysis on a subset of these elevated genes. METHODS: Thirty-nine renal transplant biopsies from patients with de novo donor-specific antibodies and eighteen 1-year surveillance biopsies with no histological evidence of rejection were analyzed for expression of 11 genes previously identified as elevated in AMR. RESULTS: Expression levels of natural killer markers were correlated to microcirculation inflammation and graft outcomes to a greater extent than endothelial markers. Creating a predictive model reduced the number of gene transcripts to be assessed to 2, SH2D1b and MYBL1, resulting in 66.7% sensitivity and 89.7% specificity for graft loss. DISCUSSION: This work demonstrates that elevated gene expression levels, proposed to be associated with AMR, can be detected by established quantitative real-time polymerase chain reaction technology, making transition to the clinical setting feasible. Transcript analysis provides additional diagnostic information to the classification schema for AMR diagnosis but it remains to be determined whether significant numbers of centres will validate transcript analysis in their laboratories and put such analysis into clinical use.

Acute cellular rejection: impact of donor-specific antibodies and C4d.

BACKGROUND: Mixed rejection in kidney transplantation consists of histologic and/or serological evidence of both cellular and humoral components. As it is not confined to a distinct category in the Banff classification, how to best manage these patients is not clear. The aim of this study was to determine the incidence and outcome of morphological T-cell-mediated rejection (TCMR) with a humoral component, defined as the presence of either DSA or C4d, compared with the outcome of pure TCMR. METHODS: We retrospectively studied 922 consecutive renal transplant recipients and analyzed patients with TCMR according to the evidence of a humoral component. RESULTS: A total of 147 cases of morphological TCMR were analyzed. Of these, 92 (62.6%) had "pure" TCMR and 55 (37.4%) had "mixed" TCMR on the index biopsy. On univariant analysis, diffuse C4d (odds ratio [OR]=10.9, 95% confidence interval [CI]=1.8-66.9, P=0.01) and DSA positivity at the time of index biopsy (OR=2.8, 95% CI=1.2-6.6, P=0.02) were associated with allograft loss, whereas arteritis (OR=0.5, 95% CI=0.2-1.2, P=0.11) and glomerulitis (OR=0.9, 95% CI=0.4-2.1, P=0.8) were not. Arteritis was associated with subsequent antibody-mediated rejection (OR=4.9, 95% CI=1.1-20.8, P=0.03), and glomerulitis was associated with the development of transplant glomerulopathy (OR=10.7, 95% CI=3.1-37.1, P<0.01). On the multivariate analysis, only patients with C4d and DSA were at risk of graft failure (OR=4.9, 95% CI=2.0-12.0, P<0.01) in the medium term. CONCLUSION: TCMR with a humoral component has a worse prognosis when compared with pure TCMR. As such, it is important to test for alloantibody in cases of morphological TCMR to optimize patient management. Such cases might benefit from more aggressive immunotherapy.

A case of chronic antibody-mediated rejection in the making.

A kidney transplant recipient developed chronic antibody-mediated rejection (ABMR) with clinically significant transplant glomerulopathy while under careful clinical monitoring. The patient developed a de novo donor-specific antibody (DSA) posttransplantation, and a protocol renal biopsy showed C4d deposition with no histological evidence of rejection. Subsequently he developed peritubular capillary basement membrane multilayering, with negative C4d and DSA. Finally, he developed proteinuria and transplant glomerulopathy, with reappearance of DSA and C4d. Despite having a de novo antibody and progressive antibody-mediated damage, this patient under close histological and serological surveillance did not fulfill Banff criteria for acute or chronic ABMR until his disease was advanced. This case illustrates the limitations of current Banff criteria in this setting, due to the fluctuating nature of DSA and C4d staining.

Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.

BACKGROUND.: Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. METHODS.: We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. RESULTS.: C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). CONCLUSIONS.: The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.