Donor-specific Antibodies In Recipients Research Articles

Recipient-Specific Antibodies (RSA) in Mismatched Related Transplants May Induce Transplant-Associated Thrombotic Microangiopathy (TA-TMA)

HLA incompatibility increases the risk of transplant-related complications. Specifically, donor-specific antibodies (DSA) in mismatched related transplants (mm-HCT) increase the risk of graft failure. Less is known about the role of recipient-specific antibodies (RSA) detected in donors, as the immune status of the donor is not routinely examined before transplant with regard to anti-HLA antibodies. These antibodies may be transferred together with transplanted cells or produced by memory donor cells. We hypothesize that donor-derived RSA may induce immune-mediated complement activation in the classical pathway (CH50), leading to overproduction of membrane attack complex (MAC) and exacerbation of endothelial damage. Here we present preliminary epidemiological data regarding the presence of DSA and RSA, and the impact of RSA antibodies on transplant-related complications, especially transplant-associated thrombotic microangiopathy (TA-TMA) and acute graft-versus-host disease (aGvHD). Methods We prospectively analyzed the presence of anti-HLA antibodies in 20 pairs of donors and recipients qualified for mm-HCT. All recipients suffered from hematological malignancies and received a myeloablative conditioning regimen and immunosuppression consisting of post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil. Patients’ and donors’ immunization status were tested with the solid phase method. Tests were carried out using a flow fluorometer across the screening range (Luminex Screen, One Lambda, USA). In the group of positive samples for anti-HLA class I and/or II antibodies, identification of the specificity of the HLA antibodies was performed (Luminex Single Antigen Beads, One Lambda, USA). Reagents enabling the determination of anti-HLA antibodies in the IgG class were used. All results were presented as mean fluorescence intensity (MFI) values. Monitoring of complement activation on days 30, 60, and 100 post-transplant was started after completing epidemiological data in the first 10 pairs. All patients were checked for TA-TMA and GvHD development during routine post-transplant visits. TA-TMA screening included TA-TMA triad: hypertension or kidney injury, LDH increase and decrease in number of platelets or refractoriness to transfusions, and additionally decrease in hemoglobin concentration, proteinuria, and clinical presentation with TA-TMA involved organ damage. Schistocytes as non-objective and nonrepeatable markers were excluded from diagnostic criteria. Results Anti-HLA antibodies were detected in 9/20 (45%) of recipients, and in 9/20 (45%) of the donors despite the lack of any transfusion history in donor group. Neither the presence of DSA nor RSA was affected by gender. RSAs were present more often in donors (7/9; 77%) than DSAs in recipients (2/9; 22%); p=0.052. Data summarizing MFI values and type of antibodies are presented in table 1. Laboratory diagnosis of TA-TMA met 12/20 (60%) patients, with a significant clinical presentation including severe hypertension (3) pericarditis (1), gastrointestinal (5), or neurological (6) impairment in 8/12 (66%). All patients with RSA (7/7; 100%) developed TA-TMA compared to no-RSA group (5/13; 38%) (p<0.01). TA-TMA-mediated organ damage was noticed more often in the RSA population (5/7; 71%) compared to the no-RSA group (3/13, 23%) (p<0.01); (Table 2). A trend for higher activation of CH50 and MAC in RSA compared to a no-RSA group was noted with a mean CH50 93 vs. 82 (p=0,16), and MAC 1428 vs. 1007 (p=0,24) respectively. A trend for higher maximum concentrations of tacrolimus 14 vs 10.7 (p=0.07) was observed in patients with TA-TMA. There was no impact of ABO incompatibility on TA-TMA development. During the time of observation 6 patients (30%) developed aGvHD - grades I, II, and III were diagnosed in 3, 1, and 2 patients respectively. Only 1 patient with RSA developed aGvHD. There was no correlation between RSA and GvHD occurrence (RSA 1/7, no-RSA 5/13, p=0.26) and between aGvHD and TA-TMA. CMV reactivation was documented in 13/20 (65%) patients, with no differences between the RSA and no-RSA groups (p=0.65). Conclusions RSAs occur surprisingly often in the donor population, irrespectively to the donor`s gender and the lack of transfusions history. The exact role of these antibodies is not fully understood; however, our data may suggest their impact on complement activation and TA-TMA induction. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First-in-Human Trial of Bcma-CD19 Compound CAR with Remarkable Donor-Specific Antibody Reduction

Ma: iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Consultancy, Equity Ownership, Research Funding; iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

A Novel Assay to Screen Porcine MHC Class II Antibodies in Potential Xenotransplant Recipients

BackgroundWell‐elucidated in clinical allotransplantation is the role of recipient's donor specific antibodies and graft rejection, specifically the antibodies targeted at Human Leukocyte Antigen (HLA) class II. To determine whether cross‐reactive or preformed antibodies exist to Swine Leukocyte Antigen (SLA) class II, an assay was developed to screen the patients on the Indiana University (IU) transplant waiting list.Methods69 patients on the IU transplant waiting list were selected for study based on their levels of anti‐HLA class II antibodies. Wild‐type pig red blood cells were used to absorb the sera, removing anti‐carbohydrate antibodies and leaving anti‐MHC antibodies. This sera was screened on an immortalized fibroblast cell line transfected with a version of the human class II transactivator (CIITA), producing a constitutively activate SLA class II cell line. A cell that incorporated the antibiotic resistance gene but not the CIITA was chosen as a negative control. Following incubation, the sera was evaluated for presence of IgM and IgG antibody binding by flow cytometry.ResultsCells lacking expression of SLA class II demonstrated decreased IgM and IgG binding compared to cells with 100% SLA class II DR and DQ expression (Figure 1). Of note are several individuals whose sera preferentially binds the class II positive cell (the dots beneath the diagonal line). The difference in binding between a positive and negative cell is statistically different for IgG (p = 0.0048) but not IgM (p = 0.5102). Increased binding is found when evaluating those individuals with anti‐HLA class II antibodies (Figure 2). The statistical significance holds when comparing the change in binding on the SLA class II positive and negative cells for individuals with and without anti‐HLA class II antibodies for IgG (p = 0.0341) and IgM (p = 0.2602).ConclusionsExpression of SLA class II results in increased IgM and IgG human antibody binding, demonstrating the presence of human anti‐pig class II antibodies. The existence of this humoral barrier suggests that antibody‐mediated binding to SLA class II may be an issue faced in clinical xenotransplantation.Support or Funding InformationThis publication was made possible in part by Grant Number UL1 TR001108 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.

Predictors and GVL Effects of UCB Chimerism after Haplo-Cord Transplant

Haplo cord transplant combines an umbilical cord blood unit (CBU) graft with a CD34 selected adult haplo-identical graft for the purpose of establishing long term CBU chimerism, which occurs in most but not all cases. Previously we showed that excessive haplo CD34 doses impair CBU engraftment (Liu et al, Blood 2011). Since then we have utilized a fixed haplo CD34 dose of 5x106 CD34 cells/kg. We also conducted a prospective study of CBU threshold dose de-escalation and established a minimum CBU dose of 1.2 x106 TNC/kg as the threshold for further study (van Besien ASH 2014, 1093).Here- using the patients from our prospective study- we sought to identify additional predictors of CBU engraftment as measured by percentage of CBU chimerism in CD33 and CD3 lineages in patients engrafted and in remission by day 56. We focused on d56 chimerism because durable patterns of chimerism are established by then.83 pts were enrolled, but 23 were excluded from analysis because of early death (6) relapse before day 60 (8), failure of both grafts with residual MDS (4), lost to follow up (1) and no chimerism measurements (3).61 pts were analyzed with median age 62 (18-72 years), 41 AML/MDS, 12 NHL, 5 ALL and 4 others. 24 had ASBMT high risk, 13 intermediate and 23 low risk disease. Conditioning was Flu Mel ATG (n=46), Flu Mel TBI 400 ATG (n=14) and Flu Mel TBI 600 ATG (n=1). All pts received GVHD prophylaxis with tacrolimus until d 180 and mycophenolate until d 30. The haplo dose was fixed at 5 x 106 CD34 cells/kg. Donors targeted by recipient donor specific antibodies (DSA) were avoided if possible. CBU units were selected based on HLA matching in sequential cohorts of decreasing threshold cell dose. Coh 1(n=12), minimum 2.0 x106 TNC/kg, coh 2 min (n=14) 1.5 x106 TNC/kg, coh 3 (n=25) min 1.0 x106 TNC/kg. After min cell dose was established, further enrollment occurred with a min dose of 1.2 x106 TNC/kg (coh 4 n=10). Median CBU TNC across all cohorts was 2.27 x107/kg (1.0-8.3). CBU were matched (HR HLA) at 3/8 (n=1), 4/8 (n=7), 5/8 (n=18), 6/8 (n=14) 7/8 (n=16) and 8/8 (n=5). DSA against CBU were present in 7. Haplo graft was 4/8 (n=42), 5/8 (n=13), 6/8 (n=3) and 7/8 (n=3). DSA against haplo were present in 6. Median follow up for survivors is 16 mo (5 -34).Cord graft characteristics, matching, enrollment cohort, nor presence of DSA were associated with d56 CBU chimerism. There was a strong inverse association between degree of matching of the haplo donor and d56 CBU (Fig 1). 54 pts with ≤ 5/8 haplo HLA match had 68% d56CD33 UCB chimerism vs 14% for the six pts with 6/8 or 7/8 HLA match (p=0.004). There was also a significant association between use of TBI and cord blood engraftment. 15 pts who received TBI had 79% d56CD33 UCB chimerism vs 50% for the 46 pts without TBI (p=0.05).Decreasing d56UCB chimerism was strongly associated with cumulative relapse risk (Fig 2A). It was not associated with TRM. The associations were even stronger if only considering AML/MDS (Fig 2B). Best discrimination was with 40% CD33 and 80% CD3 (Table 1).Table 1Relapse rate at one yearDay 56CD3 <80% (22)CD3>80% (39)p valueCD33<40% (19)CD33>40% (42)p valueAll pts n= 6147%21%0.00652%19%0.0008CD3 <80% (15)CD3>80% (26)p valueCD33<40% (13)CD33>40% (28)p valueAML/MDS n=4160%14%0.000358%16%0.00007Conclusions: The establishment of a durable cord blood graft is heavily influenced by HLA matching of the haplo-graft. 6/8 and 7/8 HLA-matched haplo-graft which occur by chance in about 10% of cases, should probably be avoided. More intensive conditioning with the addition of low dose TBI may further favor establishment of the cord blood graft. Cell dose or HLA matching of the cord blood graft has less effect on its long-term establishment.Increasing cord blood chimerism is associated with a much reduced risk of relapse despite- not shown here- very low rates of chronic GVHD). Low levels of cord blood chimerism are associated with very high rates of relapse. These data provide further support for the GVL effects of CBU grafts, particularly in AML/MDS. [Display omitted] [Display omitted] [Display omitted] Disclosuresvan Besien:Miltenyi Biotec: Research Funding.