Chronic lung allograft dysfunction is associated with an increased number of non-HLA antibodies.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the major cause of adverse outcomes in lung transplant recipients. Multiple factors, such as infection, alloimmunity, and autoimmunity, may lead to CLAD. Here, we aim to examine the role of non-human leukocytes antigen (HLA) antibodies in CLAD in a large retrospective cohort. METHODS: We analyzed non-HLA antibodies in the pre- and post-transplant sera of 226 (100 CLAD, 126 stable) lung transplant recipients from 5 centers, and we used a separate cohort to confirm our findings. RESULTS: A panel of 18 non-HLA antibodies was selected for analysis based on their significantly higher positive rates in CLAD vs stable groups. The panel-18 non-HLA antibodies (n > 3) may be positive pre- or post-transplant; the risk for CLAD is higher in the latter. The presence of both non-HLA antibody and HLA donor-specific antibody (DSA) was associated with an augmented risk of CLAD (HR=25.09 [5.52-14.04], p < 0.001), which was higher than that for single-positive patients. In the independent confirmatory cohort of 61 (20 CLAD, 41 stable) lung transplant recipients, the risk for CLAD remained elevated in double-positive patients (HR=10.67 [0.98-115.68], p = 0.052). After adjusting for nonstandard immunosuppression, patients with double-positive DSA/Non-HLA antibodies had an elevated risk for graft loss (HR=2.53 [1.29-4.96], p = 0.007). CONCLUSIONS: Circulating non-HLA antibodies (n > 3) were independently associated with a higher risk for CLAD. Furthermore, when non-HLA antibodies and DSA were detected concomitantly, the risk for CLAD and graft loss was significantly increased. These results show that humoral immunity to HLA and non-HLA antigens may contribute to CLAD development.

Comparison of human leukocyte antigen immunologic risk stratification methods in lung transplantation.

Outcomes after lung transplantation (LTx) remain poor, despite advances in sequencing technology and development of algorithms defining immunologic compatibility. Presently, there is no consensus regarding the best approach to define human leukocyte antigen (HLA) compatibility in LTx. In this study, we compared 5 different HLA compatibility tools in a high-resolution HLA-typed, clinically characterized cohort, to determine which approach predicts outcomes after LTx. In this retrospective single-center study, 277 donor-recipient transplant pairs were HLA-typed using next generation sequencing. HLA compatibility was defined using HLAMatchmaker, HLA epitope mismatch algorithm (HLA-EMMA), predicted indirectly recognizable HLA epitopes (PIRCHE), electrostatic mismatch score (EMS), and amino acid mismatches (AAMMs). Associations with HLA mismatching and survival, chronic lung allograft dysfunction (CLAD), and anti-HLA donor-specific antibody (DSA) were calculated using adjusted Cox proportional modeling. Lower HLA class II mismatching was associated with improved survival as defined by HLAMatchmaker (P < .01), HLA-EMMA (P < .05), PIRCHE (P < .05), EMS (P < .001), and AAMM (P < .01). All approaches demonstrated that HLA-DRB1345 matching was associated with freedom from restrictive allograft syndrome and HLA-DQ matching with reduced DSA development. Reducing the level of HLA mismatching, in T cell or B cell epitopes, electrostatic differences, or amino acid, can improve outcomes after LTx and potentially guide immunosuppression strategies.

Impact of assigning 2-field HLA alleles from real-time PCR on deceased donor assessments and conformance with high resolution alleles.

Waitlisted sensitised transplant recipients with HLA allele level antibodies to their own HLA antigen family are disadvantaged by current deficiencies in HLA typing for deceased donors. This is primarily because at time of organ allocation, HLA typing is provided at antigen level whereas solid phase assays provide allele level antibody definition. The gold standard for HLA allele typing is next generation sequencing (NGS), however time limitations with established NGS systems prevent NGS use for deceased donors. Instead, many labs use a real-time PCR (qPCR) antigen level result for deceased donors, which can disadvantage sensitised patients. Here, we compared assigning qPCR 2-field alleles to qPCR antigen level to determine the impact on virtual crossmatch (VXM) and discuss impact on donor-specific antibody (DSA) assignments. 244 consecutive deceased donors were HLA typed to allelic level by qPCR (LinkSeq SABR) and subsequently by NGS (One Lambda Alltype). The impact of qPCR allele assignments on potential DSA identification was investigated, by retrospectively investigating all 3904 VXMs, where recipient DSA assessments were assessed against donor HLA, was performed within the cohort. There was 96.3% concordance between qPCR and NGS for all allele level loci, with HLA-A; DQB1; and DPB1 having best agreement (99.4%, 98.4% and 99.4% respectively). Of the 3904 VXMs with qPCR allele assignment, there were 13 (<1%) occasions where the potential DSA assignment was impacted, with DQA1 having the most impact. Assigning alleles derived from qPCR to define unacceptable antigens for VXMs, can allow improved access to donor offers for sensitised patients by better defining alleles.

Major technological advances will enhance Australian donor-recipient matching and improve transplant outcomes.

In recent times, numerous and significant technological and supportive changes have taken place in Australian transplantation. These changes are often deployed without the wider clinical community having a full understanding of what has brought about these changes and the impacts they have. Here, we aim to clarify the reasoning behind these changes and shed light on potential future endeavours to improve patient outcomes.

Determining Clinical Thresholds for Donor HLA Eplet Compatibility to Predict Best Outcomes Following Lung Transplantation.

Currently, the assessment of immunological risk in lung transplantation (LTx) does not completely consider HLA compatibility at the molecular level. We have previously demonstrated the association of HLA eplets in predicting chronic lung allograft dysfunction following LTx; however, the associations between HLA eplet mismatch (epMM) loads and overall survival are unknown. Methods: In this retrospective, single-center study, 277 LTx donor-recipient pairs were high resolution HLA typed and analyzed for HLA epMMs using HLAMatchmaker (version 3.1). LTx pairs were also assessed for the presence of the previously described risk epitope mismatches DQ2-DQA1*05 and DQ7-DQA1*05. Results: HLA class I epMMs were not associated with deleterious outcomes; however, lower HLA class II (≤19), DQA1 (≤2), and combined HLA class I and II (≤29) epMM demonstrated an association with increased time to chronic lung allograft dysfunction and improved overall survival. The presence of a risk epitope mismatch was not associated with worse clinical outcomes. Conclusions: HLA epMM can risk-stratify LTx recipients and potentially guide donor-recipient matching and immunosuppression strategies.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation.

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.

Consequences of donor-derived passengers (pathogens, cells, biological molecules and proteins) on clinical outcomes.

It is recognized that donor factors contribute to lung transplant outcomes. Recent observations and studies have started to elucidate potential mechanisms behind explaining these observations. This perspective piece summarizes evolving lung transplant literature on the subject, focusing on donor "passenger" organisms, cells, hormones, and proteins transferred to the recipient. Many extrinsic and intrinsic donor features or properties have important consequences for subsequent allograft function in the recipient. Potentially, a better understanding of these features may provide useful novel therapeutic targets to enhance allograft outcomes.

Transfer of donor anti-HLA antibody expression to multiple transplant recipients: A potential variant of the passenger lymphocyte syndrome?

Antibody-mediated rejection, whereby transplant recipient B cells and/or plasma cells produce alloreactive anti-human leukocyte antigen (HLA) antibodies, negatively influences transplant outcomes and is a major contributor to graft loss. An early humoral immune response is suggested by the production of anti-HLA donor-specific antibodies (DSA) that can be measured using solid phase assays. We report the early posttransplant coexistence of a shared anti-HLA antibody profile in 5 solid organ transplant recipients who received organs from the same donor. Retrospective analysis of the donor's serum confirmed the presence of the same anti-HLA profile, suggesting the transfer of donor-derived anti-HLA antibodies, or the cells that produce them, to multiple solid organ transplant recipients. The time frame and extent of transfer suggest a novel variant of the passenger lymphocyte syndrome. These findings have important implications for the consideration of all posttransplant antibody measurements, particularly the interpretation of non-DSAs in the sera of transplant recipients.

HLA class II Eplet mismatch predicts De Novo DSA formation post lung transplant.

The use of algorithms such as HLAMatchmaker to redefine donor-recipient HLA matching is gaining increasing attention. Our research has previously demonstrated that higher HLA class II eplet mismatches correlated with the development of chronic lung allograft dysfunction (CLAD). In this study of lung transplant recipients we prospectively examined the association between donor-recipient HLA eplet mismatches as defined by HLAMatchmaker (version 2.1) and de-novo anti-HLA donor-specific antibody (DSA) formation, as assessed by single antigen-bead solid phase assay. HLA class II eplet mismatch, when split at the median for the cohort, predicted the development of de-novo HLA class II DSA at 3 months but not at 12 months. Having previously shown that high HLA class II eplet mismatches was associated with CLAD, we now show that the same factors are associated with de-novo HLA class II DSA post-transplant.