anti-HLA Abs Research Articles

OR21: Gamma-delta T CELL EXPANSION ASSOCIATES WITH LESSER RISK OF ALLOANTIBODY DEVELOPMENT IN PEDIATRIC HEART TRANSPLANTATION

Aim To determine whether the expansion of γδ T cells observed in pediatric heart allograft recipients associates with transplant outcomes. Methods We studied a population of 58 pediatric heart transplant recipients under the auspices of the Institutional Review Board. We performed Ab determinations by solid phase assays (SPA-IgG, “LSAB”). Flow cytometry staining was performed to determine the percentage of lymphocytic lineage markers: CD3, CD4, CD8, CD19, CD16& 56, CD45, TCRγδ, TCRαβ. Results Pediatric heart allograft recipients often require total or partial thymectomy at the time of their first heart operation, which leads to lifelong alterations in their T cell repertoire. While the prevailing population of circulating T cells in non-thymectomized children carries the TCRαβ TCR (96 ± 3%), we observed a significant expansion of TCRγδ T cells by Flow Cytometry in 37 of 58 patients studied. Within the population with high circulating fraction of gd T’s (8–20% of lymphocytes, n = 37), 23 patients (23/37, or 62%) developed anti-HLA antibodies detected by SPA-IgG (LSAB), while 14 did not. In patients with normal TCRγδ fractions ( Conclusion Thymectomy in pediatric heart recipients which results in the expansion of γδ T cells is associated with lesser risk of alloantibody development. The finding that patient with low γδ T cells are more likely to produce anti HLA Abs suggests that thymectomy results in a breakdown of immune regulation. Th2 impairment may result in insufficient cytokine production to fully support antibody production in these patients. Although mechanistic studies are needed to understand the causation relationships underpinning this phenomenon, the association alone suggests that monitoring of γδ T cell expansion may constitute a useful tool for Ab development prediction in pediatric heart transplant patients.

P037 : LACK OF C1Q REACTIVITY IN POST-LVAD PATIENT SERA

End stage heart disease can be treated with implantation of a mechanical circulatory support device to assist the left ventricle (LVAD) as a bridge to heart transplantation. During the LVAD operation patients may be exposed to large volumes of blood and blood products resulting in production of anti-HLA antibodies (Abs). This higher degree of HLA antigen exposure/sensitization may result in positive virtual crossmatches, eliminating many potential donor-recipient pairings for transplantation. However, when LVAD patients are transplanted they do not have a different rejection frequency or poorer graft outcome than non-LVAD or non-sensitized heart transplant recipients. We hypothesized that while these LVAD treated patients present with anti-HLA Abs these Abs may not be C1q reactive and therefore may not be functionally deleterious. We evaluated 90 patients (61 male and 29 female with a mean age of 54 years). Seventy-three patient sera were evaluated pre-LVAD and 17 patient sera post-LVAD for the presence of cI and cII HLA Abs and C1q reactivity using One Lambda, Inc. reagents and following manufacturer’s instructions. The cI and cII PRAs pre and post LVAD were divided into three groups. The cI PRAs of 0–5% were seen in 57% of patients pre-LVAD vs. 59% of patients post-LVAD. PRAs of >5–50% were seen in 23% of patients pre-LVAD vs. 29% of patients post-LVAD. Finally, PRAs of >50% were seen in 20% of patients pre-LVAD vs. 12% of patients post-LVAD. Class II PRAs of 0–5% were seen in 83% of patients pre-LVAD vs. 59% of patients post-LVAD. Class II PRAs of >5–50% were seen in 7% of patients pre-LVAD vs. 29% of patients post-LVAD ( p ⩽ 0.05). Finally, PRAs of >50% were seen in 10% of patients pre-LVAD vs. 12% of patients post-LVAD. Pre-LVAD C1q reactivity was seen in 38% of patients with cI HLA Abs and 45% of patients with cII HLA Abs. However, in contradistinction to the pre-LVAD C1q positive reactivity only 8% of both cI and/or cII post-LVAD sera were C1q positive (8% vs. 38%, p < 0.01; 8% vs. 45%, p < 0.01). These data suggest that while HLA antibodies are present in post-LVAD patient sera 92% of these antibodies tested as C1q non-reactive. This may explain why these identifiable HLA antibodies do not adversely impact LVAD treated heart transplant patients.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

TNT003, a Novel Anti-C1s Monoclonal Antibody, Inhibits Anti-HLA Antibody Induced Complement Deposition.

PURPOSE: To determine whether the anti-C1s antibody, TNT003, inhibits complement deposition induced by anti-HLA Class I and II antibodies (Ab). METHODS: HLA reference and transplant patient sera were used as sources of anti-HLA Ab. Complement deposition was evaluated using C1qScreen (OneLambda) and C3d solid phase assays (Immucor/Lifecodes). Briefly, single antigen beads were coated with anti HLA Ab, incubated with normal human serum complement, and C1q or C3d production/deposition were detected by monoclonal PE conjugates and quantified by Luminex analysis. For in vitro studies, either HLA-typed primary human aortic endothelial cells (HAECs) or EBV-immortalized B cells (B cells) were incubated with sera containing cognate anti-HLA Ab and normal human complement serum, and deposition of complement proteins was detected by flow cytometry using monoclonal antibodies. Complement split products produced during deposition reactions were measured by ELISA. TNT003 (True North Therapeutics Inc, CA) was titrated into these experiments in a concentration-dependent manner (0-50 μg/mL). RESULTS: The anti-C1s monoclonal Ab, TNT003, inhibited complement deposition in both solid phase and in vitro assays, and complement split product formation during in vitro assays. TNT003 reduced both anti-HLA Class I and II Ab induced C3d but not C1q deposition on single antigen beads in a concentration-dependent manner. In vitro, increasing concentrations of TNT003 were inversely proportional to levels of complement deposition on ECs that had been treated with anti-HLA Ab; complement split product production was also diminished with TNT003 treatment. Similar results were seen using B cells as a source of HLA, as TNT003 addition to the complement deposition reaction decreased levels of complement proteins on the cell surface, as well as split products in the supernatant of these reactions. CONCLUSIONS: We demonstrate here that TNT003, a novel anti-C1s monoclonal Ab, is capable of inhibiting complement activation, deposition, and split product formation induced by anti-HLA Class I and II Ab in both solid phase and cell based in vitro assays. These data suggest TNT003 may be useful in reducing DSA-induced complement-mediated damage in transplant patients experiencing AMR. DISCLOSURES:Thomas, K.: Grant/Research Support, True North Therapeutics Inc. Valenzuela, N.: Grant/Research Support, True North Therapeutics Inc. Parry, G.: Employee, True North Therapeutics Inc. Panicker, S.: Employee, True North Therapeutics Inc. Reed, E.: Grant/Research Support, Pfizer, True North Therapeutics Inc, Novartis, Immucor.

Suppression of Allo-HLA IgG Secreted By Long-Lived Bmem Cells In Vitro By a HLA-E mAb Versus IVIg.

Introduction: IVIg is used to reduce allo-HLA Abs pre/ post-transplant, but the mechanism is unclear. IVIg reacts with HLA-I alleles, which disappears after HLA-E-reactivity is adsorbed out. Methods: We generated an HLA-E mAb (TFL-007) that mimics the HLA-reactivity of IVIg, and investigated if it suppresses HLA-Abs secretion by activated Bmem cells and immortalized B hybridoma. Results: We found TFL-007 significantly suppressed the secretion of allo-HLA class II IgG produced by the activated long-lived Bmem cells from a woman's blood without Antigen exposure >20 years after postpartum alloimmunization, and this suppression was far superior to that by IVIg. IVIG suppressed the secretion of the primary alloAb against DRB1*0101 only marginally, and did not significantly affect the secondary alloAbs against DRB1*0102.Figure: No Caption available.Importantly, the MFI of other secondary alloAbs actually increased after IVIg treatment. TFL-007 significantly reduced the secretion of both primary and secondary alloAbs. Compared to IVIg, the suppression to the Abs secretion was stronger. This finding was confirmed with HLA-I Ab secreted by B hybridoma from another alloimmunized woman. Neither IVIg preparation evidenced any suppression of the secretion of allo-HLA-B IgG.Figure: No Caption available.In contrast, TFL-007 had a strikingly significant suppressive effect on both HLA-B*0702 and B*8101. It is worth to be noted TFL-007 showed dosimetric suppression of allo-HLA Abs. Conclusions: TFL-007, compared to IVIg, has better effect to suppress the anti-HLA Abs produced by activated long-lived Bmem cells and B hybridoma.

Lack of C1q Reactivity in Post-LVAD Patient Sera.

End stage heart disease can be treated with implantation of a mechanical circulatory support device, often to assist the left ventricle (LVAD) as a bridge to heart transplantation. During the LVAD operation patients may be exposed to large volumes of blood and blood products resulting in production of anti-HLA antibodies (Abs). This higher degree of HLA sensitization results in positive virtual crossmatches, eliminating many potential donors. However, when LVAD patients are transplanted they do not have a different rejection severity or frequency or poorer graft outcome than non-LVAD or non-sensitized heart transplant recipients. We hypothesized that while these LVAD treated patients display anti-HLA Abs these Abs may not be C1q reactive and therefore may not be functionally deleterious. We evaluated 90 patients (61 male, 29 female, mean age of 54 years). Seventy-three patient sera were evaluated pre-LVAD and 17 patient sera post-LVAD for the presence of cI and cII HLA Abs and C1q reactivity using One Lambda, Inc. reagents and following manufacturers instructions. The cI and cII PRAs pre and post LVAD were divided into three groups. For cI PRAs of 0-5% were seen in 57% of patients pre-LVAD vs. 59% of patients post-LVAD. PRAs of > 5-50% were seen in 23% of patients pre-LVAD vs. 29% of patients post-LVAD. Finally, PRAs of > 50% were seen in 20% of patients pre-LVAD vs. 12% of patients post-LVAD. Class II PRAs of 0-5% were seen in 83% of patients pre-LVAD vs. 59% of patients post-LVAD. Class II PRAs of > 5-50% were seen in 7% of patients pre-LVAD vs. 29% of patients post-LVAD (p≤0.05). Finally, PRAs of >50% were seen in 10% of patients pre-LVAD vs. 12% of patients post-LVAD. Pre-LVAD C1q reactivity was seen in 38% of patients with cI HLA Abs and 45% of patients with cII HLA Abs. However, in contradistinction to the pre-LVAD C1q positive reactivity only 8% of both cI and/or cII post-LVAD sera were C1q positive (8% vs. 38%, p<0.01; 8% vs. 45%, p<0.01) These data suggest that while HLA antibodies are present in post-LVAD patient sera 92% of these antibodies tested as C1q non-reactive. This may explain why these identifiable HLA antibodies do not adversely impact LVAD treated heart transplant patients.

A Novel Multiplex Approach to Define Donor Specific and Poly-Reactive B-Cell Receptor HLA Antigen Recognition in Clinical Transplantation.

Study Purpose The role of anti-donor HLA specific B cells which are crucial effectors for generation of donor specific antibodies associated with chronic graft rejection is still unknown. Quantifying the frequency of HLA Ag-specific B cells is of great importance for better understanding of the mechanisms of graft rejection, earlier diagnosis of rejection and to guide specific B cell-targeted therapy. Aim: To quantify and characterize HLA antigen (Ag)-committed B cells identified by single antigen HLA-coated multiplexed beads (SAB) post-transplantation (PT). Methods PBMC from kidney transplant recipients (TR n=6) and healthy volunteers (HV n=6) were analyzed using SAB. Bead-B-lymphocyte Rosette (BBR) frequency was enumerated and their HLA specificities were characterized by flow cytometry. TR serum was tested for circulating anti-HLA antibodies using SAB. Donor specific rosette (DSR) polyreactivity, defined by the percentage of total SAB forming rosettes (Fig 1), were compared with presence or not of DSA of same specificities and their MFI. Results Significantly higher frequency of BBRs were identified in TR with circulating anti-HLA Abs compared to HV ((2.49±0.74 vs 0.27±0.09; p<0.02). Both DSR and Non-DSR were present in higher frequency (1.15 % vs 0.17% of B cells; P<0.05) and correlated with poor graft outcome. Not all DSR were associated with DSA at 12 months PT. Patients with poor graft outcome had wide breadth of B cell polyreactivity compared to recipients with good graft outcome (HLA class I 76.97± 28.3 vs 13.1 ± 3.15; p<0.05. HLA class II 91.9 ± 10.5 vs 13.69 + 10.3; p<0.05). Conclusions This new approach using multiplex beads highlights for the first time a pattern of HLA Ag-specific B cell polyreactivity. The breadth of HLA reactivity may be a determinant of an aggressive clinical course after transplantation. The wide array of available HLA Ags in a single test will help in defining B cell receptor poly & cross-reactivity. In addition it enables phenotyping, functional and gene expression analysis of HLA specific B cells.Figure: No Caption available.

1041-LBP: Establishment of a consistent single antigen bead (SAB) IgG subtype alloantibody assay. Correlation with C1q complement fixing alloantibody

Aim Clinically relevant assays that help assessing the risk of rejection in transplanted patients remain a priority in the transplantation field. In this study we determined the frequency of different complement and non-complement fixing HLA antibody (Ab) subtypes in transplanted patients. We also examined the correlation of these assays with C1q-binding alloantibodies. Methods Sera from patients with high titers of HLA antibodies were tested for standard total IgG SAB and C1q-alloantibody binding according to the manufacturer’s protocol (OneLambda). The frequency of each Ab subtype was determined using the same SAB Luminex technology, but replacing the secondary Ab conjugated to PE for human IgG1, IgG2, IgG3 and IgG4 subclasses from Southern Biotech. A titration analysis was performed using 1:5 and 1:100 dilutions of the standard concentration. Saturating secondary titers were chosen for evaluation. Samples were pre-heat inactivated for all the assays. Results The distribution of IgG1, IgG2, IgG3 and IgG4 in the population analyzed was 59%, 26%, 9% and 6% respectively. Complement-fixing IgG subtypes (IgG1 and IgG3) accounted for 68% of the anti-HLA Ab present in the analyzed patients, while non-fixing complement IgG subtypes (IgG2 and IgG4) accounted for 32%. Correlation between the total IgG SAB and the IgG subtypes assays were 78% for class I and 92% for class II Ab, and 37% for class I and 67% for class II Ab between IgG subtypes and C1q assays. Conclusions This study reveals the potential clinical application of IgG subtype analysis for the risk rejection assessment of transplanted patients.

The Influence Of Anti-HLA Antibodies On Engraftment and Donor's Chimerism Following Allogeneic Hematopoietic Stem Cell Transplantation From HLA-Mismatched Unrelated Donors

Introduction Although anti-HLA Antibodies (Abs) are considered an important factor of graft failure in solid organ transplants, their role in allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still undiscovered. Large polymorphism and immunogenicity of HLA-antigens and heterogeneity of anti-HLA Abs warrant the need of such investigation. The purpose of this study was to define the presence of anti-HLA Abs before allo-HSCT from HLA-mismatched unrelated donors and their impact on engraftment and post-transplant full donor’s chimerism. Material and Methods 70 HLA-mismatched donor/recipient pairs entered the study. Indication for allo-HSCT was: ALL, AML, CML, SAA, PNH, MDS and CLL. Preparative regimen was myeloablative in 68pts (97%) and reduced in 2pts (2.3%). Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (69pts) or Alemtuzumab (1pt). HLA A,B,C,DR,DQ alleles were PCR-typed. Single HLA-antigen was mismatched in 46pts, single HLA-allele in 16pts, double antigens or alleles in 2 pts and another 2 pts had combined antigenic/allelic HLA mismatch. Anti-HLA A,B,C,DR,DQ,DP Abs were identified in sera collected prior to the conditioning treatment with use of automated DynaChip assay utilizing microchips bearing purified class I and class II HLA antigens. Post-transplant chimerism was analyzed using STR-PCR method at 30, 100-days and 1-year after allo-HSCT. Results Anti-HLA Abs pre-formed before allo-HSCT were detected in 32pts: against class I, II or both in 13(18.6%), 7(10%) and 12(17.1%) pts. Anti-HLA Abs were detected after allo-HSCT in 49pts: against class I, II or both in 22(32.4%), 7(10.3%) and 20(29.4%) pts, respectively. Anti-HLA Abs directed against the mismatched HLA antigens were observed in 4 pts before allo-HSCT. Although no Abs specific to mismatched HLA alleles were detected, Abs belonging to the same Cross-Reactive Groups (CREGs) were present in 5pts. No graft failure has been observed (graft failure was defined as absence of neutrophil recovery by day 30 after allo-HSCT or loss of donor’s chimerism). The detection of anti-HLA Abs before allo-HSCT was associated with decrease of post-transplant donor’s chimerism (18/31 vs 11/35, p=0.03). Anti-HLA Abs had no significant impact on engraftment of platelets and neutrophils. The median time to neutrophils engraftment was 16.9 days (range 7-31 days) in pts with and 18.9 days (range 13-30 days) in pts without anti-HLA Abs (p=0.188). The median time to platelets engraftment was 16.9 days (range 9-31 days) in patients with and 18.3 days (range 10-32 days) in pts without anti-HLA Abs (p=0.274). Conclusions Our preliminary results indicate, that anti-HLA Abs are present before transplantation in mismatched allo-HSCT recipients. They influence the post-transplant full donor’s chimerism, but they did not influence engraftment and graft failure. Disclosures: No relevant conflicts of interest to declare.

187-P: ANTI HLA IMMUNIZATION REDUCES ACCESS TO VASCULARIZED COMPOSITE ALLO TRANSPLANTATION FOR EXTENSIVELY BURNED PATIENTS

Aim Vascularized composite allotransplantations (VCA) provide therapeutic options for functional or esthetic damage beyond conventional surgical reconstruction. The objectives of this study were to evaluate the immunological characteristics of patients treated for third degree burns who could benefited from reconstructive transplantation by VCA and to determine their access to transplantation. Methods Serum samples of all the 29 patients treated in a single center from 2007 to 2010 for extensive burns were collected. Fifteen patients received skin allografts, 8 cryopreserved skin allografts (CPSA) and 7 glycerolized preserved skin allografts (GPSA). Anti HLA Ab and C1q binding Ab were detected by LABScreen single-antigen, (One Lambda, Canoga Park, CA), MFI cut offs were respectively 500 and 300 over background. Then cPRA representing the percentage of potential donors expected to present unacceptable mismatches was calculated. Results Among the patients, 97% had anti HLA Ab, 45% had C1q fixing Ab, 62% were considered hyperimmunized with a cPRA above 85%. The average cPRA score of the 1055 patients on the waiting list for kidney transplantation at the same time in the same region was 27.2% ± 37.3 whereas it was 64.7%± 40.8 for burned patients. The risk of being hyper immunized was 3.3 times higher for burned patients than for those on the waiting list for kidney transplantation. Multivariate analysis (stepwise backward before multiple linear regression) identified the use of CPSA (p=0.0043) and the number of previous pregnancies (p=0.0074) to be independent predictive factors for cPRA with an adjusted coefficient of determination (R 2 ) of 0.37 (p = 0.0024). Conclusions Anti-HLA alloimunization is potentially a major obstacle to VCA in extensively burned patients. To improve access to transplantation, glycerolized preserved skin allografts should be preferred to cryopreserved skin allograft for burned patients suffering extensive facial and hand injuries when possible.

180-P: ANTI-HLA-CLASS-I ANTIBODIES AND PLATELET TRANSFUSION REFRACTORINESS

It is known that: 1) anti-class-I-HLA antibodies are responsible for platelet transfusion refractoriness, 2) leucocytes in transfusion products sensitise patients to produce anti HLA antibodies, 3) leucodepleted blood products reduce but do not eliminate HLA sensitisation. It is not clear what the level of platelet refractoriness risk is in HLA-sensitised patients, therefore it is not standard practice to screen for HLA antibodies in platelet transfusion. This is a pilot study to assess that risk and measure the value of HLA-antibody screening in platelet transfusion. This study looks at the relationship between HLA-class-I antibody status and the total number of units of transfused platelets is studied. Since platelets are transfused according to a standard transfusion trigger, the total number of platelets units transfused indirectly measures thrombocytopenia and platelet transfusion refractoriness. Leukaemia patients with anti-HLA-class-I antibodies use on average 36 more units of platelets than patients without antibodies. Sensitised patients: average = 120 units, sd = 120 unit, n = 82. Non-sensitised patients: average = 84 units, sd = 112 units, n = 73. Using a cutoff point of 105 units a 2x2 contingency table shows a likelihood ratio of 2.29 and a chi square of 10.8, statistically significant for a p < 0.005. Conditional probabilities. 72% of patients using more than 105 units of PLT will have anti-HLA Ab 44% of patients using less than 105 units of PLT will have anti-HLA Ab 44% of patients with anti-HLA Ab will use more than 105 units of PLT 19% of patients without anti-HLA Ab will use more than 105 units of PLT. 1) Ab screens reveal the risk of excessive platelet transfusion requirements. 2) Generalised leucodepletion is not by itself sufficient to address the widespread problem of platelet transfusion refractoriness. 3) Optimal platelet transfusion may have to take into account the patient’s Ab status and the HLA and PLT Ag phenotype of both donor and recipient.

61-P: CLINICAL FLOW CYTOMETRY CROSSMATCH: DETECTION OF DONOR-T CELL, -B CELL, AND -ENDOTHELIAL PROGENITOR CELL SPECIFIC (EPC) IgG AND IgM ANTIBODIES IN TRANSPLANT RECIPIENT SERUM

Aim The AbSorber flow cytometry crossmatch (FXM) XM-ONE 3:1 (XM3) test measures binding of IgG and IgM antibodies (ab) in recipient’s serum to donor-T, -B, and -Tie2+ EPC. The XM3 and our laboratory’s Clinical FXM (CFXM) were compared to examine the feasibility of the XM3’s use in clinical settings. Methods Peripheral blood (N=5) and serum (N=5) were collected from human subjects. Tie2+ EPC and lymphocytes were isolated using manufacturer’s instructions. Cells were incubated with either test serum, negative control serum (NCS) or positive control serum (PCS), washed, and stained with a combination of anti-CD3, -CD19, and ab directed at human -IgG, or -IgM. The median channel shift (MCS) from the NCS for T cell, B cell, and EPC FXM for IgG was considered positive if > 40, >100, and >50, respectively. The MCS cut-off for IgM on T cell, B cell, and EPC was considered positive if >80 MCS. The FXM were acquired in a BD FACSCanto II instrument and the data analysis was performed using the DiVa software. Results 1) The XM3 and CFXM for CD3+IgG+ T cells had similar Median Channel Fluorescence (MCF) (271 ± 19 vs. 241 ± 26) and MCS (9 ± 16 vs. 22 ± 19). All crossmatches were negative; 2) The XM3 and CFXM for CD19+IgG+ B cells also had similar MCF (301 ± 40 vs. 273 ± 19) and MCS (114 ± 77 vs. 105 ± 69). Two B cell FXM were positive and the remaining crossmatches were negative by both methods; 3) Three FXM for CD3+IgM+ T cell and CD19+IgM+ B cell by XM3 and CFXM were negative by both methods; and 4) Of five EPC crossmatches performed by XM3, two subjects were positive for IgG and all five subjects were negative for IgM. Conclusions In addition to facilitating the detection of conventional analysis of donor T and B cell specific IgG ab, the XM3 helps identify donor T and B cell specific IgM ab in recipient’s serum. In contrast to anti-HLA ab, donor-EPC specific IgG or IgM ab occur in fewer recipients, but their identification may impact allograft survival. The XM3 identifies EPC specific IgG or IgM ab in human serum.

Early Morning Educational Lecture 1: Differences Between Anti‐HLA Abs and Anti‐carbohydrate Abs

XenotransplantationVolume 20, Issue 5 p. 323-324 Abstracts Early Morning Educational Lecture 1: Differences Between Anti-HLA Abs and Anti-carbohydrate Abs First published: 20 September 2013 https://doi.org/10.1111/xen.12060_6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume20, Issue5Special Issue: IXA 2013 - Joint Congress of IXA and Organ Transplantation in ABO-incompatible and Hyperimmunized Recipients 2013September/October 2013Pages 323-324 RelatedInformation

In Kidney Transplant Patients, Alemtuzumab but Not Basiliximab/Low-Dose Rabbit Anti-Thymocyte Globulin Induces B Cell Depletion and Regeneration, Which Associates with a High Incidence of De Novo Donor-Specific Anti-HLA Antibody Development

In this single-center matched-cohort study, we evaluated the phenotype of repopulating B cells and its correlation with donor-specific anti-HLA Ab development and long-term graft function in 16 renal transplant recipients and 32 age- and gender-matched controls induced with alemtuzumab or basiliximab (Bas)/low-dose rabbit anti-thymocyte globulin (rATG), respectively. Alemtuzumab, but not Bas/rATG, profoundly depleted peripheral B cells in the first 2 mo posttransplantation. Early posttransplant, naive B cells were significantly depleted, whereas Ag-experienced and memory B cells were partially spared. Transitional B cells transiently increased 2 mo posttransplant. At month 6 posttransplant, pregerminal center B cells emerged, a process promoted by increased BAFF serum levels. Thereafter, B cell counts increased progressively, mainly due to expansion of naive B cells. Conversely, Bas/rATG did not modify the B cell phenotype throughout the follow-up period. Alemtuzumab was associated with a higher incidence of de novo DSA compared with Bas/rATG. DSA development was predicted by changes in the B cell compartment and correlated with worse long-term graft function. Thus, alemtuzumab-induced B cell depletion/reconstitution may promote chronic humoral responses against the graft.

Development of De Novo Anti-HLA Antibodies in Pediatric Heart Transplant Recipients

Purpose There is increasing evidence that the de novo development of anti-HLA antibodies (Abs) following solid organ transplantation may be associated with negative outcomes including rejection in the first year and graft loss; especially de novo donor-specific antibodies (DSAs). Little data is available in paediatric heart transplant (HTx) recipients. We sought to prospectively determine the incidence, class and early impact of de novo anti-HLA Abs in a cohort of pediatric HTx recipients. Methods and Materials Following informed consent, serial panel reactive antibody (PRA) testing post-HTx (weeks 1, 2, & 4, and months 6 & 12) was done in 25 patients (14 males) transplanted between Mar 2008 & Mar 2010. Development of DSAs was correlated with rejection, allograft vasculopathy (AV) and survival. Results Median age at HTx was 8 yrs (1.5 mos – 16.3 yrs) with mean follow-up time of 15 mos (1.5 – 31 mos). 4 patients were highly sensitized pre-HTx (cPRA > 10%); 2 to Class II and 2 to both Class I & II. 4 patients had a positive crossmatch (XM), 1 T cell, 3 B cell. 18 (72%) patients developed de novo anti-HLA Abs at a median of 6 wks (IQR: 10 days – 1.7 yrs) post-HTx: anti-Class I in 6 (33%), anti-Class II in 1 (6%), and Both in 11 (61%). De novo DSAs occurred in 12/25 (48%) at a median of 2 mos (IQR: 1 month – 1.6 years) post-HTx: anti-Class I in 5 (42%), anti-Class II in 5 (42%), and Both in 2 (16%). 14 (56%) patients had evidence of acute cellular rejection (ACR) ≥ ISHLT 2 R on biopsy; 6 with multiple episodes. A positive T cell crossmatch (p Conclusions The majority of pediatric HTx recipients develop de novo anti-HLA antibodies within the first 2 years post-HTx with half being donor-specific. Anti-Class I antibodies were associated with cellular rejection but not mortality. Further study is required to determine the long term impact on graft and patient survival.

Lung Transplantation in Patients with Pretransplantation Donor-Specific Antibodies Detected by Luminex Assay

New methods of solid-phase assays, such as Luminex assay, with high sensitivity in detecting anti-human leukocyte antigen (HLA) antibodies (Abs), have increased the proportion of sensitized candidates waiting for lung transplantation (LTx). However, how to apply these results clinically during graft allocation is debated: strict exclusion of candidates with Luminex-positive results can lead to lost opportunities for Tx. We retrospectively analyzed the clinical impact of pre-LTx Luminex-detected Abs on post-LTx outcomes for patients who underwent LTx before the availability of Luminex assay. We analyzed data for 56 successive patients who underwent LTx before 2008 and were considered to not have anti-HLA Abs by then-available methods of detection at the date of their LTx. Pre-LTx sera from these patients were retested by Luminex assay. Using log-rank test, freedom from bronchiolitis obliterans syndrome (BOS) and graft survival were compared between patients with and without pre-LTx Luminex-detected anti-HLA Abs classes I and II and donor-specific Abs (DSA) classes I and II. Freedom from bronchiolitis obliterans syndrome was lower, and mortality was higher for patients with than those without pre-LTx Luminex-detected DSA class II (P=0.004 and P=0.007, respectively) but did not differ for patients with and without DSA class I or anti-HLA Abs class I or II. It suggests to avoid attributing graft with forbidden antigens to sensitized candidates with Luminex-detected DSA class II and to evaluate the role of specific posttransplantation protocols for LTx candidates who require emergency LTx.

A glow of HLA typing in organ transplantation.

The transplant of organs and tissues is one of the greatest curative achievements of this century. In organ transplantation, the adaptive immunity is considered the main response exerted to the transplanted tissue, since the main goal of the immune response is the MHC (major histocompatibility complex) molecules expressed on the surface of donor cells. Cell surface molecules that induce an antigenic stimulus cause the rejection immune response to grafted tissue or organ. A wide variety of transplantation antigens have been described, including the major histocompatibility molecules, minor histocompatibility antigens, ABO blood group antigens and endothelial cell antigens. The sensitization to MHC antigens may be caused by transfusions, pregnancy, or failed previous grafts leading to development of anti-human leukocyte antigen (HLA) antibodies that are important factor responsible for graft rejection in solid organ transplantation and play a role in post-transfusion complication Anti-HLA Abs may be present in healthy individuals. Methods for HLA typing are described, including serological methods, molecular techniques of sequence-specific priming (SSP), sequence-specific oligonucleotide probing (SSOP), Sequence based typing (SBT) and reference strand-based conformation analysis (RSCA) method. Problems with organ transplantation are reservoir of organs and immune suppressive treatments that used to decrease rate of rejection with less side effect and complications.

Anti-HLA Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation From HLA-Mismatched Unrelated Donors

AbstractAbstract 4475 Introduction:Anti-HLA Antibodies (Abs) are considered an important factor in solid organ transplants and transfusion medicine, but role of humoral arm of immunological response to HLA antigens in allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unknown. Large polymorphism and immunogenicity of HLA-antigens and heterogeneity of anti-HLA Abs warrant the need of such investigation. The purpose of this study was to define presence and profiles of anti-HLA Abs detected before or after allo-HSCT from HLA-mismatched unrelated donors and their impact on allo-HSCT results. Material and methods:35 HLA-mismatched donor/recipient pairs entered the study. Indication for allo-HSCT was: ALL (7pts), AML(18pts), CML(5pts), SAA(2pts), CLL(1pt), MDS(1pt) and PNH (1pt). Preparative regimen was myeloablative in 33pts (94.3%) and reduced in 2pts (5.7%). Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (34pts) or Alemtuzumab (1pt). HLA A, B, C, DR, DQ alleles were PCR-typed. 21(60%) pts had mismatch of single HLA-antigen: A-4(11.4%), B-1(2.8%), C-13(37%), DQ-3(8,5%); 10(28.5%) pts had mismatch of single HLA-allele: A-3(8.5%), B(11.4%), DQ-3(8.5%); 4 pts had double antigenic (A+C and A+DQ) or combined antigenic/allelic (A/B and C/A) HLA mismatches. Anti-HLA A, B, C, DR, DQ, DP Abs were identified in sera collected before start of the conditioning treatment and +30 days, +100 days and 1 year after allo-HSCT with use of automated DynaChip assay utilizing microchips bearing purified class I and class II HLA antigens. Results:Anti-HLA Abs pre-formed before allo-HSCT were detected in 17(48.5%) pts: against class I, II or both in 6(35%), 4(24%) and 7(41%) pts. Anti-HLA Abs were detected after allo-HSCT in 25(71.4%) pts, against class I, II or both in 9(36%), 3(12%) and 13(52%) pts, respectively. In 7 pts anti-HLA Abs were not detected neither before nor after allo-HSCT. Anti-HLA Abs directed against the mismatched HLA antigens were observed in 4 pts before and in 10 pts after allo-HSCT, no anti-HLA Abs specific against mismatched alleles were detected. Allo-HSCT results obtained in studied subgroups are presented in the Table below:before allo-HSCTafter allo-HSCTpresentabsentpresentabsentAnti-HLA Abs % (number of pts)48.5% (17)51.5% (18)71.4% (25)28.6% (10)Acute GVHD grade III-IV5.8% (1)5.5% (1)4% (1)10% (1)Chronic GVHD extensive29.4% (5)27.7% (5)33% (8)20% (2)100% donor chimerism88% (15)100% (18)96% (24)100% (10)Chimerism decrease <100%41.1% (7)27.7% (5)44% (11)10% (1)Relapse11.7% (2)16.6% (3)16% (4)10% (1)Death29.4% (5)33.3% (6)28% (7)40% (4)Graft failure5.8% (1)000 Conclusions:Our preliminary results indicate that anti-HLA Abs are present pre- or post-transplant in mismatched allo-HSCT recipients and may be potentially responsible for the occurrence of complications, what needs to be further investigated and analyzed. Disclosures:No relevant conflicts of interest to declare.

Deceased Donor HLA Antibody Incompatible Renal Transplantation without Antibody Removal: High Incidence of Acute Rejection Reduced by T-Cell Depleting Induction Therapy

Introduction: Screening for HLA-specific antibodies (Ab) is routine for patients on kidney transplant waiting lists. In the UK screening is performed at least every 3 months, and HLA antigens to which a patient has detectable Ab are listed as ‘unacceptable mismatches’. Many units use a stringent definition of unacceptable, for example any HLA antigen to which Ab can be detected with an MFI >1000 (using the Luminex® platform). Whilst this approach is effective in preventing a positive crossmatch (XM) following allocation, it also precludes the offer of organs to sensitized patients. Here we describe our experience of deceased donor (DD) renal transplantation knowingly performed in the presence of donor-specific anti-HLA Ab (DSA). Methods: 21 patients received HLA Ab-incompatible (HLAi) DD transplants in one of two circumstances: (1) Patients in whom the threshold for ‘unacceptable’ for any HLA specificity was increased to a Luminex MFI >3000 (n=8), and (2) Patients with a positive flow cytometry B-cell crossmatch (FC-BXM) known to have anti-HLA-DP Ab (n=13). Since donors in the UK are not routinely DP genotyped, the presence of DP DSA was assumed, and confirmed retrospectively by donor DP genotyping. Complement-dependent cytotoxicity crossmatch (CDC-XM) using a recent pre-transplant serum sample was negative in all patients. FC-BXM was positive in 15 (71%). The mean DSA MFI was 5268 (range 1086-16356) and mean follow-up length was 20.8 months (range 1-64). All episodes of antibody-mediated rejection (AMR) and T cell-mediated rejection (TCMR) were analyzed. Results: All patients received tacrolimus, MMF and prednisolone. Induction agents used were Basiliximab (n=8), Alemtuzumab (n=9) and Thymoglobulin (n=4). No planned Ab removal was used. Death-censored 1 year graft survival was 92% (12/13 patients with >12 months follow up). Acute rejection (AR) was significantly more common in those receiving basiliximab at induction compared to a T-cell depleting agent (75% (6/8: 3 with TCMR and 3 with AMR) versus 15% (2/13: both AMR) respectively, p=0.018). Patients developing AMR were more likely to have a positive CDC-XM using historic serum (3/5 with AMR versus 1/16 without AMR, p=0.028). Of the 12 patients with isolated DP DSA, 2 experienced AMR and 2 TCMR. All episodes of AMR were reversed with plasma exchange. Mean creatinine for those patients with graft function at baseline (18/21) was 137μmol/L and at 1 year was 133μmol/L (n=12). There were 3 deaths (post-operative haemorrhage at 2 weeks, disseminated aspergillosis at 3 weeks and a cerebral lymphoma at 4 years) and 1 graft loss (graft rupture with Banff 2B AR at 1 week). Conclusions: HLAi DD renal transplantation is associated with a high incidence of AR, with AMR in 24% of patients despite negative CDC-XM. Our limited experience suggests that: (1) T-cell depleting induction is beneficial, (2) A current negative but historic positive CDC-XM could prompt prospective antibody removal and (3) donor HLA-DP genotyping should be routine practice. Despite high rates of AR, transplantation in these high-risk recipients is still achievable with satisfactory allograft function at up to 5 years post-transplant.

A Novel Concept of Phased Desensitization Using Bortezomib and Rituximab for Highly HLA-Sensitized Kidney Transplantation

Introduction: Donor specific alloantibody producing plasma cells (DSA-PCs) appear resistant to conventional immunosuppressive agents. The recent studies demonstrated the impact of pretransplant desensitization with the proteasome inhibitor bortezomib on DSA-PCs in sensitized renal allograft candidates. However, other studies have showed that one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients. We developed a concept of phased desensitization with rituximab and bortezomib, and applied it in to a highly sensitized kidney transplant candidate. Methods: We assessed the activity of bortezomib against various B cell subpopulations and plasma cells and suppressive effect of that to produce anti-blood group antibodies (Abs) DSA in mice. To elicit Abs against allopeptide (DSA), B6 mice were immunized with thymocytes from Balb/c mice. After the immunization, the mice received intravenous injections of bortezomib (0.75 mg/kg body weight) 3 times a week for 1 week. On the basis of the results, we have established a novel concept of desensitization for a sensitized patient. Results: Bortezomib led to a significant reduction in the subpopulations of CD21-/IgMhigh plasmablasts, and CD138+/CD25-/IgG κ-light chain+ plasma cells in the spleen of Balb/c mice, but not in that of CD21+/IgMhigh marginal-zone B cells and the CD21low/IgMlow follicular B cells. Flow cytometry analyses revealed that bortezomib did not reduce the B cells with receptors for either A-carbohydrates. Thus, bortezomib reduced not only IgG producing plasma cells but also IgM producing cells, although their precursor memory B cells were resistant to this reagent. Taken together with a known fact that rituximab is able to deplete memory B cells, we have proposed a concept of phased desensitization using bortezomib and rituximab, and applied it in to a highly sensitized kidney transplant patient. A 56-year-old man with renal graft failure was referred to our hospital. His first renal graft from his brother remained functioning for 16 years but was lost due to chronic rejection. This allograft loss resulted in sensitization of the recipient against HLA antigens. Unfortunately, his wife who volunteered as a second donor shared the same A2 antigen with the first donor, which might be a target of the preformed anti-HLA Abs. Single antigen flow-based DSA testing revealed 11 anti-HLA class I Abs including anti-donor HLA A2 Ab. He received a desensitization regimen consisting of a single dose of rituximab (375 mg/m2) combined with double-filtration plasmapheresis (DFPP) followed by low doses of intravenous immuno-globulin (DFPP/IVIG treatment). Tacrolimus and mycophenolate mofetil were started 2 weeks before the DFPP/IVIG treatment. Despite 3 DFPP/IVIG sessions, the crossmatch remained positive. Thereafter, he was treated with one cycle of bortezomib (1.3mg/m2 × 4 doses). After 3 DFPP/IVIG sessions, the crossmatch turned negative and renal transplantation was successfully performed. The induction quadruple immunosuppression protocol comprised tacrolimus, mycophenolate mofetil and methylprednisolone. After the transplantation, neither Ab-mediated rejection nor severe acute cellular rejection was encountered in this patient. Conclusions: The current study suggests that phased desensitization with rituximab and bortezomib may be needed for highly HLA-sensitized kidney transplant candidates.