BACKGROUND: Donor human leukocyte antigen (HLA)-specific antibodies (DSA) and non-HLA antibodies can cause allograft injury, possibly leading to chronic lung allograft dysfunction (CLAD) after lung transplantation. It remains unclear whether these antibodies are produced locally in the graft or derived solely from circulation. We hypothesized that DSA and non-HLA antibodies are produced in CLAD lungs.
METHODS: Lung tissue was prospectively collected from 15 CLAD patients undergoing retransplantation or autopsy. 0.3 g of fresh lung tissue was cultured for 4 days without or with lipopolysaccharide or CD40L: lung culture supernatant (LCS) was sampled. Protein eluate was obtained from 0.3 g of frozen lung tissue. The mean fluorescence intensity (MFI) of DSA and non-HLA antibodies was measured by Luminex and antigen microarray, respectively.
RESULTS: LCS from all 4 patients who had serum DSA at lung isolation were positive for DSA, with higher levels measured after CD40L stimulation (CD40L+LCS). Of these, only 2 had detectable DSA in lung eluate. MFI of non-HLA antibodies from CD40L+LCS correlated with those from lung eluate but not with those from sera. Flow cytometry showed higher frequencies of activated lung B cells in patients whose CD40L+LCS was positive for DSA (n = 4) or high non-HLA antibodies (n = 6) compared to those with low local antibodies (n = 5). Immunofluorescence staining showed CLAD lung lymphoid aggregates with local antibodies contained larger numbers of IgG+ plasma cells and greater IL-21 expression.
CONCLUSIONS: We show that DSA and non-HLA antibodies can be produced within activated B cell-rich lung allografts.

BACKGROUND: Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, subclinical, or EMB-negative rejection events. While non-human leukocyte antigen (non-HLA) antibodies have emerged as important risk factors for antibody-mediated rejection after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has the potential to guide efforts to overcome deficiencies of EMB in rejection monitoring.
METHODS: Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included the number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and donor-specific anti-human leukocyte antigen antibody (HLA-DSA) responses.
RESULTS: A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support versus against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-major histocompatibility complex class I chain-related gene A (MICA) and anti-angiotensin II type I receptor antibodies (AT1R) in HT rejection, evidence for their involvement was equivocal. Conversely, the strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies.
CONCLUSIONS: This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

The presence of anti-Glutathione S-transferase T1 (GSTT1) antibodies (abs) has been hypothesized as a pathogenic contributor in antibody-mediated rejection (AMR).
We aimed to evaluate the relationship between genetic variants of GSTT1, anti-GSTT1 abs and AMR in a cohort of 87 kidney transplant (KTx) patients using Immucor\'s non-HLA Luminex assay. Patients were classified according to biopsy-proven AMR and HLA-DSA status: AMR with positive anti-HLA-DSAs (AMR/DSA+, n = 29), AMR but no detectable anti-HLA-DSAs (AMR/DSA-, n = 28) and control patients with stable allograft function and no evidence of rejection (n = 30).
At an MFI cut-off of 3000, the overall prevalence of anti-GSTT1 abs was 18.3%. The proportion of patients with anti-GSTT1 abs was higher in the AMR/DSA- group (25%), compared to the control (13.3%) and AMR/DSA+ group (3.4%) (p = 0.06). Among patients with anti-GSTT1 abs, the MFI was higher in AMR/DSA- and GSTT1-Null patients. Of 81 patients who underwent GSTT1 genotyping, 19.8% were homozygotes for the null allele (GSTT1-Null). GSTT1-Null status in the transplant recipients was associated with the development of anti-GSTT1 abs (OR, 4.49; 95%CI, 1.2-16.7). In addition, GSTT1-Null genotype (OR 26.01; 95%CI, 1.63-404) and anti-GSTT1 ab positivity (OR 14.8; 95%CI, 1.1-190) were associated with AMR. Within AMR/DSA- patients, the presence of anti-GSTT1 abs didn\'t confer a higher risk of failure within the study observation period.
The presence of anti-GSTT1 abs and GSTT1-Null genotype is associated with AMR, but do not appear to lead to accelerated graft injury in this cohort of early allograft injury changes, with a limited period of follow-up.

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system.

BACKGROUND: Immune response to several kidney self-antigens (KSAg) such as Collagen IV (Col-IV), Perlecan (PL), and Fibronectin (FN) have been associated with antibody-mediated damage and poor allograft survival. Thus, the aim of this study was to determine if humoral immune responses to KSAg correlates with progression of chronic immune injury (CII) changes at 1 year or 2 years.
METHODS: Kidney transplant recipients who underwent 1- or 2-year biopsies, with chronic interstitial inflammation (ci > 1) and/or glomerular membrane double contouring (cg > 0) were analyzed with matched controls. Sera were analyzed retrospectively for antibodies against KSAg using ELISA. The presence of antibodies to KSAg were compared at 0, 4, 12, and 24 months using logistic regression.
RESULTS: We identified a cohort of 214 kidney transplant recipients. Of these, we identified 33 cases and matched 66 controls. Logistical regression showed an odds ratio of 1 with the confidence interval crossing 1 for the presence of response to KSAg at all the time points.
CONCLUSIONS: Humoral immune responses to either KSAg alone or in combination with donor-specific anti-HLA antibodies are not associated with progression to CII at 1 and 2 years after kidney transplantation.

Allo- and autoimmune mechanisms are involved in kidney allograft rejection and loss. This study investigates the impact of anti-angiotensin II type-1 receptor antibodies (anti-AT1RAbs) detected alone or in association with HLA donor-specific antibodies (HLA-DSAs) on the outcome of kidney transplantation (KTx). Anti-AT1RAbs and HLA-DSAs were detected in 71 kidney transplant (KT) recipients who developed biopsy-proven acute or chronic active T-cell rejection (TCMR) (n = 51) or antibody-mediated rejection (ABMR) (n = 20), forming the rejection group (RG). The control group (CG) included 71 KTx recipients with comparable characteristics without rejection. All patients had been transplanted with negative T/B flow crossmatch (T/BFCXM). The median follow-up period was 3.7 years. Antibodies were determined pre- and periodically post-KTx by Luminex method for HLA-DSAs and enzyme-linked immunosorbent assay for anti-AT1RAbs. Before KTx, twenty-three (32.4%) patients in the RG, sixteen with TCMR and seven with ABMR, were found anti-AT1Rabs-positive (≥10 U/mL) versus eleven (15.5%) patients in the CG (p = 0.031). Simultaneous detection of preformed anti-AT1RAbs and HLA-DSAs was found in five patients of the RG versus two of the CG (p = 0.355). At the time of transplant biopsy, fifteen (21.1%) patients, four with ABMR and eleven with TCMR, were positive for anti-AT1RAbs. Anti-AT1RAbs and HLA-DSAs were detected simultaneously in 7/15 (46.7%) cases, three with ABMR and four with TCMR. During the follow-up, thirteen (18.3%) patients in the RG, eight with ABMR and five with TCMR, lost their graft compared to one patient (1.4%) in the CG (p = 0.001). Six out of thirteen (46.2%) RG patients who lost the graft were found positive for anti-AT1RAbs pretransplant. Patient survival with functioning graft did not differ significantly between anti-AT1Rabs-positive and negative KT recipients (log-rank p = 0.88). Simultaneous detection of anti-ATR1Abs and HLA-DSAs did not have a significant influence on patient survival with functioning graft (log-rank p = 0.96). Graft function at the end of the follow-up was better, but not significantly, in anti-AT1Rabs-negative patients, with serum creatinine 1.48 [1.20-1.98] mg/dL and eGFR (CKD-EPI) 48.5 [33.5-59.0] mL/min/1.73 m2, compared to anti-AT1Rabs-positive ones who had serum creatinine 1.65 [1.24-2.02] mg/dL (p = 0.394) and eGFR (CKD-EPI) 47.0 [34.8-60.3] mL/min/1.73 m2 (p = 0.966). Anti-AT1RAbs detection pretransplant characterizes KT recipients at increased risk of cellular or antibody-mediated rejection. Furthermore, anti-AT1RAbs, detected alone or simultaneously with HLA-DSAs, appear to be associated with impaired graft function, but their role in graft survival has not been documented in this study. Screening for these antibodies appears to complement pretransplant immunological risk assessment.

Immunological injury to the allograft, specifically by antibodies to de novo donor specific human leukocyte antigen (dnDSA) and antibody mediated injury and rejection are the major limitations to graft survival after heart transplantation (HT). As such, our approach to allosensitization remains limited by the inability of contemporaneous immunoassays to unravel pathogenic potential of dnDSA. Additionally, the role of dnDSA is continuously evaluated with emerging methods to detect rejection. Moreover, the timing and frequency of dnDSA monitoring for early detection and risk mitigation as well as management of dnDSA remain challenging. A strategic approach to dnDSA employs diagnostic assays to determine relevant antibodies in conjunction with clinical presentation and injury/rejection of allograft to tailor therapeutics. In this review, we aim to outline contemporary knowledge involving detection, monitoring and management of dnDSA after HT. Subsequently, we propose a diagnostic and therapeutic approach that may mitigate morbidity and mortality while balancing adverse reactions from pharmacotherapy.

There is growing evidence on the important role of non-human leukocyte antigen (HLA) antibodies in lung and heart transplant rejection. Since data on the prevalence and clinical significance of non-HLA antibodies in the Asian population are scarce, we analyzed non-HLA antibodies in heart and lung transplant patients.
We used the Luminex method to measure non-HLA antibodies in patients who underwent heart transplantation (N = 28) or lung transplantation (N = 36) between 2016 and 2019. We evaluated the association between pre-existing non-HLA antibodies and acute rejection-free days in these recipients.
Of 64 patients, 27 (42.2%) patients underwent rejection, with 26 (40.6%) acute cellular rejection and one (1.6%) acute antibody-mediated rejection. Among 33 identified different non-HLA antibodies, only the anti-glutathione S-transferase theta-1 (GSTT1) antibody positive rate was significantly higher in patients with acute rejection compared to those without rejection (14.8% vs. 0%, p = 0.016). The angiotensin II type I receptor positive rate was not significantly different between the two groups (40% vs. 18.5%, p = 0.129). In the multivariate Cox regression analysis, anti-GSTT1 antibody-positive patients had a higher risk of acute allograft rejection (hazard ratio, 4.19; 95% confidence interval [CI], 1.41-12.49; p = 0.010). The Kaplan-Meier curve showed that anti-GSTT1 antibody-positive patients had fewer acute rejection-free days (χ2 = 7.892; p = 0.005). Additionally, patients who underwent platelet transfusion (odds ratio, 1.49; 95% CI, 1.16-1.91; p = 0.002) before transplantation were more likely to be positive for anti-GSTT1 antibody.
Patients with antibodies against GSTT1 before heart or lung transplantation have an increased risk of acute rejection.

Kidney transplantation is the treatment of choice for patients with end-stage renal disease. Although an improvement in graft survival has been observed in the last decades with the use of different immunosuppressive drugs, this is still limited in time with antibody-mediated rejection being a main cause of graft-loss. Immune monitoring and risk assessment of antibody-mediated rejection before and after kidney transplantation with useful biomarkers is key to tailoring treatments to achieve the best outcomes. Here, we provide a review of the rationale and several accessible tools for immune monitoring, from the most classic to the modern ones. Finally, we end up discussing a practical proposal for alloimmune risk assessment in kidney transplantation, including histocompatibility leukocyte antigen (HLA) and non-HLA antibodies, HLA molecular mismatch analysis and characterization of peripheral blood immune cells.

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.