Detecting the Humoral Alloimmune Response: We Need More Than Serum Antibody Screening

Abstract: Whereas many techniques exist to detect HLA antibodies in the sera of immunized individuals, assays to detect and quantify HLA-specific B cells are only just emerging. The need for such assays is becoming clear, as in some patients, HLA-specific memory B cells have been shown to be present in the absence of the accompanying serum HLA antibodies. Because HLA-specific B cells in the peripheral blood of immunized individuals are present at only a very low frequency, assays with high sensitivity are required. In this review, we discuss the currently available methods to detect and/or quantify HLA-specific B cells, as well as their promises and limitations. We also discuss scenarios in which quantification of HLA-specific B cells may be of additional value, besides classical serum HLA antibody detection (read more)

Complement-binding Donor-specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem-cell transplantation and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q-binding DSA was done on 22 allosensitized recipients. The presence of C1q+DSA was labeled as C1q positive, and the absence of C1q+DSA was labeled as C1q negative. Twenty-two of 122 patients (18%) had DSA, 19 of which were females (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 MFI versus 2,065 MFI for those who engrafted (p=0.007). Nine patients with DSA were C1q positive in the initial samples with median DSA level 15,279 MFI (range 1,554-28,615), compared with 7 C1q negative patients with median DSA level 2,471 MFI (665-12,254) (p=0.016). Of 9 patients, who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant [all with high DSA levels (median 15,279, range 6,487-22,944)] and experienced engraftment failure, while 4 patients became C1q negative pre-transplant and all engrafted the donor cells (p=0.008). In conclusion, patients with high DSA levels (> 5,000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+ DSA should be assessed in allosensitized patients prior to hematopoietic stem-cell transplantation. Reduction of C1q+DSA levels might prevent engraftment failure in hematopoietic stem cell transplantation. (read more)

Interference of therapeutic antibodies used in desensitization protocols on lymphocytotoxicity crossmatch results

BACKGROUND : Therapeutic antibodies used to desensitize patients awaiting a human leukocyte antigen (HLA) or ABO-mismatched graft are suspected to interfere with the lymphocytotoxicity crossmatch (LCT-XM) test when they are present in the tested sera because of their potential ability to activate or inhibit the complement.
METHODS: The most frequent therapeutic antibodies (Abs) used in desensitization protocols (intravenous immunoglobulins, rituximab, basiliximab, eculizumab, antithymocyte globulin) were added to a negative- or a positive-control serum at various concentrations, and tested in vitro in a LCT-XM test.
RESULTS: Rituximab turned the LCT-XM positive on B cells at 0.2μg/mL and antithymocyte globulin turned the LCT-XM positive with T and B cells at 20μg/mL and 200μg/mL, respectively. Treatment with dithiothreitol sera, supplemented with rituximab (0.2 and 2μg/mL) and antithymocyte globulins (20 and 200μg/mL), partially or totally reduced this positive interference. Intravenous immunoglobulin, eculizumab, and basiliximab did not trigger any interference with the negative control serum. In a positive LCT-XM, eculizumab did not annihilate activation of the rabbit complement. Intravenous immunoglobulins (25g/L) could partially or totally reduced lysis score of positive crossmatch with weak lysis scores.
CONCLUSION: If eculizumab within the serum did not annihilate rabbit complement activation and basiliximab did not interfere with the crossmatch reaction, treatments based on rituximab, antithymocyte globulin and intravenous immunoglobulins need to be taken into account when interpreting a positive or negative crossmatch test (read more)

Nucleic Acid Testing of Organ Donors: Is the Glass Half Empty or Half Full?

In this issue of the American Journal of Transplantation, Suryaprasad and colleagues present 3 cases of hepatitis C virus (HCV) transmission from seronegative, nucleic acid test (NAT)–negative donors to solid organ transplant recipients. This case series represents one of the greatest contributions to the literature on risk mitigation strategies against donor-derived infections and will most definitely add fuel to the ongoing debate about the benefit of NAT in organ donors. Briefly, all three organ donors had evidence of active nonmedical injection drug use up to the time of the event leading to their demise: Donor 1 was hospitalized due to a heroin overdose; donor 2 with evidence of IV track marks on physical exam; and donor 3 with toxicology results positive for opiates. All of the donors had undetectable NAT at the time of initial evaluation: 2 were tested on the second day of hospitalization and one on the fourth day of hospitalization. Subsequent retrospective testing of stored splenocytes or lymphocytes collected during organ procurement detected HCV RNA. Eight of 12 (66.7%) recipients from these three donors acquired a donor-derived HCV infection with testing of the index recipient at day 9, 32, and 66, respectively. With its findings, the case series greatly informs 3 critical issues that have been the center of an ongoing debate within the transplant community: (1) the limitations of donor NAT, (2) the importance of recipient follow-up testing, and (3) how we define donors at increased risk for disease transmission (read more).

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’ (read more)

β2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. β2-glycoprotein I (β2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact β2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize β2GPI/HLA class II complexes in the absence of phospholipids. In situ association between β2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against β2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that β2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis (read more)

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen–antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen–antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength (read more)

Transfer of HLA-Specific Allosensitization From a Highly Sensitized Deceased Organ Donor to the Recipients of Each Kidney

We report for the first time the adoptive transfer of donor HLA-specific allosensitization in two recipients following kidney transplantation from a highly sensitized donor. Kidneys from a donation after circulatory death donor were transplanted into two nontransfused, HLA-specific antibody negative males receiving their first transplant. Antibody screening 7 days after transplant showed high level de novo IgG HLA class I- and class II-specific antibodies in both recipients, with largely overlapping antibody profiles but no antibodies to donor HLA. The unusually rapid appearance of de novo alloantibodies in immunosuppressed nonsensitized recipients and absence of donor HLA-specific antibody prompted testing of stored donor serum that revealed high antibody levels with specificities very similar to those seen in both recipients, but in addition the presence of strong antibodies to each recipient HLA. Alloantibody levels gradually declined but were still detectable at 3 months. These findings suggest that alloreactive passenger B cells/plasma cells within the kidneys of highly sensitized donors may give rise to rapid development of posttransplant de novo HLA-specific alloantibodies. While the clinical significance of this phenomenon is uncertain it provides one explanation for the appearance of de novo HLA-specific antibodies directed against third party but not donor HLA (read more)