Incidence and Role of Recipient-Specific Antibodies in Allogeneic Hematopoietic Cell Transplantation from Mismatched Related Donors.

High titer of donor-specific antibodies (DSAs) increases the risk of graft rejection after mismatched related hematopoietic cell transplantation (HCT). There are no data regarding the incidence of anti-HLA recipient-specific antibodies (RSAs) and their role after transplantation. Here we aimed to identify the incidence of RSAs in a mismatched related hematopoietic cell donor population and their possible impact on immune-mediated complications, such as acute graft-versus-host disease (aGVHD), and complications resulting from endothelial injury, such as transplantation-associated thrombotic microangiopathy (TA-TMA) and veno-occlusive disease (VOD). We prospectively analyzed the incidence of anti-HLA antibodies in 28 mismatched related pairs of recipients and their donors who underwent HCT at our center between 2020 and 2022. In positive samples screened for anti-HLA class I and/or II antibodies, the specificity of the HLA antibodies was analyzed. All recipients had a hematologic malignancy and received a myeloablative conditioning regimen and immunosuppression consisting of post-transplantation cyclophosphamide, tacrolimus, and mycophenolate mofetil. Patients were tested for TA-TMA and aGVHD development during routine post-transplantation visits up to 100 days post-transplantation. We used modified Jodele criteria for TA-TMA diagnosis, and based aGVHD grading on the MAGIC criteria. VOD was assessed using the European Society for Blood and Marrow Transplantation. Anti-HLA antibodies were detected in 12 donors (43%) and in 9 recipients (32%). There were no significant differences between donors and recipients according to age (median, 42 years [range, 17 to 69 years] versus 39 years [range, 8 to 68 years]), sex, or pregnancy history. No transfusion history was noted in the donor group (P < .05). RSA antibodies were present more often than DSAs and were detected in 9 out of 12 (75%) anti-HLA-positive donors and in only 2 out of 9 (22%) recipients, respectively (P < .05). During the follow-up, 11 patients (39%) developed aGVHD, including grade I-II in 9 (32%) and grade III-IV in 2 (7%). Twelve patients (43%) met the criteria for TA-TMA, and only 1 patient (3.5%) was diagnosed with VOD by day 100 post-HCT. RSAs were detected significantly more often in the TA-TMA group; among 12 patients diagnosed with TA-TMA, 7 (58%) had RSAs (P < .05). We did not find a correlation between RSAs and aGVHD. The patient with VOD did not have an RSA-positive donor. There was no difference in membrane attack complex (MAC) concentration in the RSA-positive group on day 30 and day 60 post-HCT; however, there was a trend toward higher MAC concentration in the RSA-positive group on day 100 (median, 912 ng/mL [range, 788 to 1120 ng/mL] versus 616 ng/mL [range, 352 to 1244 ng/mL]; P = .055). Patients with RSA suffered more often from platelet and red blood cell decreases or transfusion refractoriness, and increased lactate dehydrogenase activity was observed in all RSA-positive cases. The donor immune status and the presence of RSA may be associated with higher rates of TA-TMA in mismatched HCT recipients. Antibody-mediated complement activation might be an additional factor influencing TA-TMA occurrence.

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

Hypothesis: The activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment. Methods: The current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs. Results: The number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts. Conclusions: Patients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

In-Depth Analysis of Anti-HLA Antibodies Using C1q Assay.

Alloantibodies are significant biomarkers of posttransplant kidney rejection. With solid-phase assays, the presence of alloantibodies and complement-binding capacities can be tested. It has been noted that complement-binding anti-HLA (C1q) correlates well with high titers of anti-HLA antibodies (single antigen bead, SAB), but we have recently shown that lower SAB titers may be associated with complement in the complement cytotoxicity test. If so, low titers of donor-specific antibodies could be stratified for complement binding and used for better donor-recipient matching. To study this, we tested 268 patients awaiting kidney transplantation for SAB and C1q and stratified them into positive (Allo+) and negative (Allo-) cohorts. Next, all assayed specificities of SAB were matched with the corresponding C1q in order to correlate mean fluorescence intensity levels. We found a strong correlation between SAB and C1q for all HLA loci apart from HLA-DP. Moreover, there was no strict cutoff for C1q prediction on SAB mean fluorescence intensity level. In addition, an unusual laboratory phenotype was found; that is, a positive C1q result without corresponding SAB specificity. We tested this phenomenon and found positive IgM SAB. CONCLUSIONS: Simultaneous C1q and SAB testing may serve as a tool for in-depth analysis of alloantibodies before transplantation.

Anti-HLA immunization of patients qualified for lung transplantation - Single center study.

For lung transplantation, the presence of donor-specific anti-HLA antibodies (DSA) is an important factor of antibody-mediated rejection (AMR) in its hyperacute, acute or chronic form during long-term follow up. The aim of the study was to assess the allosensitization of Polish patients qualified for a lung transplantation in our center. A retrospective study of 161 potential lung allograft recipients, also of 31 patients transplanted in the University Hospital of Gdansk, between June 2018 and December 2020 were performed. 121 potential recipients were thoroughly tested for immunization status before eventual lung transplantation. SAB-testing, PRA-CDC and vPRA assessment, and HLA typing were performed to guide donor-recipient matching and risk stratification. Then 73 patients were separated and qualified for the list of patients awaiting lung transplantation. Then 31 patients were transplanted based on a negative biological crossmatch result. The patients were generally not sensitized, as the median PRA-CDC was 0% (min 0; max 53), and the vPRA, calculated according to HLA ABDR (>2000 cut-off MFI), was 8% (min 0; max 99). If the cut-off was split into 2000 MFI for HLA ABDR, 10,000 MFI for HLAC, and 7000 MFI for HLA-DQ, the vPRA increased to 20% (min 0; max 99). The immunization status was assessed with single antigen-SAB assays. For class I, the number of any detectable alloantibodies was 14 (11.6%) 21 (17.35%) 16 (13.22%) for locus HLA-A/B/C, and 28 (23.14%) 30 (24.8%) 24 (19.8%) for locus HLA-DR/DQ/DP, respectively. The immunization of the transplanted patients was then analyzed in detail. Summarizing, the study is an analysis of the degree of anti-HLA immunization in the population of patients eligible for lung transplantation, which showed that this degree is of low intensity and can be effectively and safely and very precisely diagnosed before transplantation.

KIR Receptors as Key Regulators of NK Cells Activity in Health and Disease.

Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells.

Immune Algorithm Optimization for Organ Transplantation in Poland.

The lack of a uniform method for determining unacceptable HLA mismatches (UAMs) for organ transplantation worldwide has resulted in many different algorithms for donor-recipient matching. Here we present our proposal for changes to the current algorithm for immune evaluation of potential kidney recipients in Poland based on the experience of various transplantation centers. The most important finding of this article is an algorithm that stratifies the pretransplant immunologic risk based on strict laboratory criteria, enabling harmonization between transplant centers in Poland. This is because of a step-by-step algorithm for alloantibody assessment using solid-phase assays (SPA) and clearly defined technical issues, as well as cutoffs for reporting UAMs. Our novel approach focuses on a laboratory testing extension in the scope of HLA typing; detection and characterization of alloantibodies before transplantation; desensitization; and post-transplant monitoring. The proposed changes will allow for the assessment of clinically relevant anti-HLA antibodies with complement binding properties; the determination of UAMs in the potential donor; the calculation of virtual panel reactive antibodies (vPRA); the calculation of the recipient's immunologic rejection risk stratification; the assessment of the donor-recipient virtual cross-match (vXM); and the determination of the final recipient's selection for the biological cross-match testing. Collectively, the optimized algorithm permit for UAM verification is based on laboratory proofed data and will firmly improve organ allocation and transplant outcomes in Poland. We hope that this novel approach also improves the individual patient's risk stratification and future personalized treatment.