Absence Of Donor-specific Antibodies Research Articles

#2811 Factor B inhibition with iptacopan in C3 glomerulopathy recurrence after transplantation: a case presentation

Abstract Background and Aims C3 glomerulopathy (C3G) is a clinical-pathological entity resulting from dysregulation of the alternative pathway of the complement system (APC) in the fluid phase. The recurrence risk of C3G after kidney transplantation is about 70% with high risk of graft loss. Standard treatment relies on nephroprotection and unspecific immunosuppression but there are currently new molecules that target specifically APC under development. A novel factor B inhibitor, iptacopan, has shown promising results both in native kidney and transplant recurrence cases in a phase II trial. Method We present a real-world clinical case of early C3G recurrence after kidney transplantation treated with iptacopan (Novartis MAPs program). Patient was vaccinated for encapsulated bacteria. Iptacopan was initiated 200 mg BID. We also present histological response. Results 38-year-old male with chronic kidney disease secondary to C3G initially presented with nephrotic syndrome and hematuria at the age of 31y following a respiratory infection. Blood test showed low serum C3 levels and the presence of C3 nephritic factor without pathological genetic alterations. Kidney biopsy confirmed membranoproliferative glomerulonephritis (MPGN) with predominant C3 deposition. He progressed to CKD stage G5 requiring hemodialysis instead of nephroprotection and immunosuppression with corticosteroids and mycophenolate; and addition with cyclophosphamide and rituximab after 19 months from onset. Five years later, the patient was admitted as first kidney transplant recipient from deceases donor Maastricht-III. Induction therapy included thymoglobulin, and tacrolimus, mycophenolate, and corticosteroids. He experienced delayed graft function requiring hemodialysis. An early biopsy proved C3G recurrence with endocapillary proliferation without C4d deposit and absence of donor specific antibodies. Despite conventional immunosuppression and addition of rituximab and plasma exchange; hemodialysis requirement still be present. So, because C3G pathophysiological base, iptacopan was begun with successful clinical response as he reached hemodialysis discontinuation; and kidney function stabilized after one year of treatment with an estimated glomerular filtration rate about 30 mL/min/1.73 m2. In the follow-up biopsy 5 months later, deposit of C3 in the glomerulus disappeared compared to the post-transplant recurrence but with persistence of endocapillary and mesangial proliferation (see Fig. 1). However, interestingly, he experienced a clinical recurrence with acute kidney injury and hematuria after unintentional iptacopan discontinuation, which was resolver after reinitiating and recovering its previous graft function. No infectious complications were observed after one year of treatment with factor B inhibitor. Conclusion New molecules under development that specifically target APC (fluid phase) may change the treatment scenario of C3G and its recurrence in kidney transplantation. We expose a real-practice early C3G recurrence after kidney transplantation successfully treated with iptacopan. The patient reached kidney function stabilization and disappearance of C3 deposits on immunofluorescence of the follow-up biopsy, identical results as the phase II clinical trial of iptacopan in C3G. So, we might conclude that iptacopan is an effective and safe option for C3G recurrence after kidney transplantation. But, data from randomized studies are needed in order to confirm the efficacy and safety of new inhibitors of the APC as iptacopan.

Impact of COVID-19 pandemic on transplant laboratories: How to mitigate?

A positive flow cytometry crossmatch (FCXM) due to donor specific antibodies (DSA) constitutes a risk for kidney transplantation; such a finding may indicates an unacceptable donor for this patient. However, positive FCXM in the absence of DSA is considered discordant and need further investigations. During COVID-19 pandemic, we observed 22% discordant results out of 445 FCXM performed during eight months period in our laboratory and another 7% were invalid due to high background negative control (NC). No study has addressed the impact of COVID-19 pandemic on FCXM and the overall pre-kidney transplant workups or described a solution to deal with these non-specific reactivities. Herein, we analyzed all FCXM results in SARS-CoV-2 seropositive patients and addressed how this pandemic affected significantly the pre-kidney transplant workups, highlighting both technical and financial implications. We also shared our modified FCXM procedures using dithiotheritol (DTT) sera treatment or blocking donor cells with negative control human serum (NCS) which we found to be successful to abrogate 98% of all discordant FCXM results and to validate all invalid results due to high background NC.In conclusion, COVID-19 pandemic has affected our HLA laboratory significantly by creating many false positive or invalid crossmatch results. Transplant laboratories must consider this before test interpretations and immune risk assessments. We recommend the use of DTT serum treatment to remove nonspecific bindings in the sera of kidney transplant candidates and the use of NCS-blocked donor cells to correct high background when performing FCXM in transplant candidates or donors with recent history of SARS-CoV-2 immunization respectively.

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

(995) A Contemporary Analysis of Antibody-Mediated Rejection Following Heart Transplantation Based on the Presence or Absence of Donor-Specific Antibodies

(995) A Contemporary Analysis of Antibody-Mediated Rejection Following Heart Transplantation Based on the Presence or Absence of Donor-Specific Antibodies

The impact of induction therapy in low-immunological risk kidney transplant recipients regardless of HLA matching

BackgroundInduction agents have proved to reduce the rate of acute rejection (AR) in kidney transplant recipients (KTRs) without improving long-term graft and patient survival (PS). ObjectiveThis study evaluates the utility of induction therapy in low immunological risk KTRs regardless of donor-to-recipient HLA matching. MethodsWe retrospectively reviewed the records of 218 patients undergoing kidney transplantation (KT). These patients were divided into two groups according to the usage of induction therapy: 82 did not receive any induction therapy (Group I), and 136 patients received either Anti-IL2 receptor antibodies or anti-thymocyte globulin (Group II). All patients had panel reactive antibody (PRA) < 20% and absence of donor-specific antibodies (DSA). The difference in outcomes were assessed at different intervals following KT. ResultsThe rate of bacterial infections at one year (p-value = 0.032) and the frequency of CMV disease (p-value = 0.044) were significantly higher in Group II (with induction therapy). The duration of hospital stay, the rate and severity of acute rejection, the occurrence of delayed graft function, the rate and type of surgical complications at one year, and the graft function and survival at one and three years were similar between the two groups (p-value = NS). In addition, the financial burden is much less in Group I (without induction therapy), reducing the total cost of the transplant procedure. ConclusionWe conclude that induction therapy in low-immunological risk kidney transplant patients is not a must regardless of donor-to-recipient HLA matching. Therefore, induction therapy did not yield significant health results, but had negative financial consequences.

332.4: Microvascular Injury With Macrophage, Neutrophil, And Hla-dr Positive Inflammatory Infiltrate, either in dsa Positive or Negative abmr, Determines the Prognosis in Renal Allografts

Introduction: Antibody-mediated rejection (ABMR) has well-defined histomorphological lesions, like linear C4d staining or microvascular inflammation [(g + ptc) ≥ 2] in peritubular capillaries (PTCs). Although these histologic features of ABMR (ABMRh) are usually compatible with donor-specific antibodies (DSA), it is demonstrated that recipients can develop ABMRh in the absence of DSAs. Indeed, DSA can not be found in 40-60% of patients with marked microvascular inflammation (MVI). We aimed to investigate the relationship between PTC macrophage (M), neutrophil, and HLA-DR positive cell infiltration with variable clinical presentations of ABMR. Methods: A total of 125 patients with acute ABMR were categorized into four groups; Group 1: DSA (+) ABMR, Group 2: DSA (-) ABMR, Group 3: DSA (+) mixed rejection (ABMR+vascular rejection), and Group 4: DSA (-) mixed rejection. The degree of the glomerular and PTC macrophage (CD68+), neutrophil, and HLA-DR positive cells was graded. The loss of HLA-DR expression in PTC endothelium was accepted as PTC destruction. The follow-up biopsies were evaluated for the development of diffuse interstitial fibrosis (IF) and transplant glomerulopathy (TG). Results: The type of ABMR showed a significant correlation with PTC C4d expression, the presence of TMA, PTC destruction, microvascular (PTC and glomerular) macrophage, neutrophil, and DR+ cell infiltration (p<0.01 for all). Group 3 showed the highest microvascular inflammation, PTC destruction, C4d expression, and TMA. Groups 4, 1, and 2 followed Group 3, respectively, and Group 2 showed the best outcome. Also, patients in Group 3 developed the highest and earliest IF and TG development during follow-up (p<0.01). Similarly, Groups 4, 1, and 2 followed Group 3 regarding the development of IF and TG development. PTC C4d staining intensity significantly correlated with PTC destruction and DSA positivity (p<0.001). Overall 5-year graft survival was 86%, 51%, 45%, and 26% for Groups 2, 1, 4, and 3, respectively (p<0.001). Overall 5-year graft survival was 87%, 54%, and 20% for patients with grades 1, 2, and 3 PTC destruction, respectively (p<0.001). Additionally, overall 5-year survival was 100%, 81%, and 21% for grades 1, 2, and 3 C4d depositions in PTCs, respectively (P<0.001). Conclusion: ABMRh comprising characteristic histologic findings without detectable DSA represent a distinct phenotype with superior renal allograft survival than ABMRh with positive DSA. Additionally, PTC C4d deposition and the resulting PTC destruction were shown to significantly indicate graft failure and poor prognosis independent of the DSA positivity. Therefore, we concluded that ABMR should be divided into different subgroups for treatment, considering the presence of DSA and vascular rejection.

Differential Treatment Effects for Renal Transplant Recipients With DSA-Positive or DSA-Negative Antibody-Mediated Rejection.

BackgroundAntibody-mediated rejection (ABMR) is the main cause of renal allograft loss. The most common treatment strategy is based on plasmapheresis plus the subsequent administration of intravenous immunoglobulin (IVIG). Unfortunately, no approved long-term therapy is available for ABMR. The current study was designed to analyze the effect of various ABMR treatment approaches on allograft survival and to compare treatment effects in the presence or absence of donor-specific antibodies (DSAs).MethodsThis single-center study retrospectively analyzed 102 renal allograft recipients who had biopsy-proven ABMR after transplant. DSA was detectable in 61 of the 102 patients. Initial standard treatment of ABMR consisted of plasmapheresis (PS) or immunoadsorption (IA), followed by a single course of IVIG. In case of nonresponse or recurrence, additional immunosuppressive medications, such as rituximab, bortezomib, thymoglobulin, or eculizumab, were administered. In a second step, persistent ABMR was treated with increased maintenance immunosuppression, long-term therapy with IVIG (more than 1 year), or both.ResultsOverall graft survival among transplant patients with ABMR was <50% after 3 years of follow-up. Compared to the use of PS/IA and IVIG alone, the use of additional immunosuppressive medications had no beneficial effect on allograft survival (p = 0.83). Remarkably, allografts survival rates were comparable between patients treated with the combination of PS/IA and IVIG and those treated with a single administration of IVIG (p = 0.18). Renal transplant patients with ABMR but without DSAs benefited more from increased maintenance immunosuppression than did DSA-positive patients with ABMR (p = 0.01). Recipients with DSA-positive ABMR exhibited significantly better allograft survival after long-term application of IVIG for more than 1 year than did recipients with DSA-negative ABMR (p = 0.02).ConclusionsThe results of our single-center cohort study involving kidney transplant recipients with ABMR suggest that long-term application of IVIG is more favorable for DSA-positive recipients, whereas intensification of maintenance immunosuppression is more effective for recipients with DSA-negative ABMR.

Effect of donor-to-recipient HLA matching in low-immunological risk kidney transplant recipients without induction therapy on acute rejection, graft survival, infections, and surgical complications at 3 years: The road towards new recommendations

BackgroundDonor-to-recipient human leukocyte antigen mismatching is considered one of the strongest determinants for graft and patient survival in kidney transplant recipients (KTR). ObjectiveThis retrospective study discusses the impact of HLA matching as low immunological risk KTR without induction therapy. Material and methodsRecords of 80 adult kidney transplant patients were reviewed with three years of the follow-up. All patients had panel reactive antibodies (PRA) < 20%, absence of donor-specific antibodies (DSA) and did not receive the induction therapy. These patients were divided into two groups according to their HLA matching between donor and recipient: 55 patients with ≥ 3 HLA matches (Group I; low immunogenicity) were compared to 25 patients with <3 HLA matches (Group II; high immunogenicity). The primary endpoints included the rate and severity of acute rejection (AR) episodes, graft function (creatinine level), and survival at 1, 3, 6, 12, and 36 months. Secondary endpoints include the rate and type of infections at one-year, surgical complications at one-year, and patient survival at 1, 6, 12, and 36 months after kidney transplantation. Baseline demographic characteristics were comparable between the two groups except for recipient age, donor gender, and pre-transplant dialysis time. ResultsThere was no significant difference observed between two groups at one-year in infection rate, the length of hospital stay, AR severity, the rate of cytomegalovirus infection, and the occurrence of delayed graft function. However, the rate of AR, the graft function upon discharge, and the rate and type of surgical complications at one-year were significantly higher in Group II (high immunogenicity). The patient and graft survival at three years, the death-censored graft survival, and the serum creatinine levels at 1, 3, 6, 12, and 36 months were similar between two groups. Two deaths occurred in each group (NS). ConclusionIn our center, the donor-to-recipient HLA mismatch is not considered an immunological risk factor in low-risk kidney transplant recipients (PRA < 20% and absence of DSA).

Transcriptomic heterogeneity of antibody mediated rejection after heart transplant with or without donor specific antibodies

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnosis previously required the detection of circulating donor specific antibodies (DSA); however, the most recent criteria only require pathological findings. This classification defined a subset of patients with AMR, yet without known antibodies. Here, we sought to evaluate differences in the transcriptome profile associated with different types of AMR. RNA sequencing was used on endomyocardial biopsies to analyze and compare transcriptomic profiles associated with different subtypes of AMR defined by immunopathological and histopathological findings, as well as the presence or absence of DSA. Gene expression profiles were characterized for each diagnostic group. The most divergent gene expression profiles were observed between patients with or without DSA. AMR subtypes associated with DSA showed expression of signature genes involved in monocyte activation and response to interferon. There was also substantial difference between the transcriptomic profiles of AMR defined by histopathological and immunopathological findings, the latter being associated with expression of mucin genes. In contrast, there was no differential RNA expression between patients with pAMR1i without DSA and those without AMR. Likewise, no differential expression was observed between patients with pAMR1h with DSA and pAMR2. Overall, our studies reveal different expression profiles in endomyocardial biopsies in relation to some key criteria used to diagnose AMR. These findings support the view that the diagnosis of AMR encompasses several phenotypes that may rely on distinct mechanisms of injury.

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Immunological Considerations.

Mesenchymal stem cell (MSC)-based therapy for type 1 diabetes mellitus (T1DM) has been the subject matter of many studies over the past few decades. The wide availability, negligible teratogenic risks and differentiation potential of MSCs promise a therapeutic alternative to traditional exogenous insulin injections or pancreatic transplantation. However, conflicting arguments have been reported regarding the immunological profile of MSCs. While some studies support their immune-privileged, immunomodulatory status and successful use in the treatment of several immune-mediated diseases, others maintain that allogeneic MSCs trigger immune responses, especially following differentiation or in vivo transplantation. In this review, the intricate mechanisms by which MSCs exert their immunomodulatory functions and the influencing variables are critically addressed. Furthermore, proposed avenues to enhance these effects, including cytokine pretreatment, coadministration of mTOR inhibitors, the use of Tregs and gene manipulation, are presented. As an alternative, the selection of high-benefit, low-risk donors based on HLA matching, PD-L1 expression and the absence of donor-specific antibodies (DSAs) are also discussed. Finally, the necessity for the transplantation of human MSC (hMSC)-derived insulin-producing cells (IPCs) into humanized mice is highlighted since this strategy may provide further insights into future clinical applications.

Allograft recognition by recipient's natural killer cells: Molecular mechanisms and role in transplant rejection.

The current transplant immunology dogma defends that allograft rejection is initiated by recipient's adaptive immune system. In this prevalent model, innate immune cells in general, and natural killer (NK) cells in particular, are merely considered as downstream effectors which participate in the destruction of the graft only upon recruitment by adaptive effectors: alloreactive T cells or donor-specific antibodies (DSA). Challenging this vision, recent data demonstrated that recipients' NK cells are capable of a form of allorecognition because they can sense the absence of self HLA class I molecules on the surface of graft endothelial cells. Missing-self triggers mTORC1-dependent activation of NK cells, which in turn promote the development of graft microvascular inflammation and detrimentally impact graft survival. The fact that some patients develop chronic vascular rejection in absence of DSA or genetically-predicted missing self suggests that other molecular mechanisms could underly NK cell allorecognition. This review provides an overview of these proven and putative molecular mechanisms and discusses future research directions in this emerging field in organ transplant immunology.

Evaluation of three different laboratory methods to detect preformed human leukocyte antigen antibodies in a South African kidney transplant population

Anti-human leukocyte antigen antibodies (anti-HLA) play a crucial role in graft. Detection of anti-HLA, both pre- and post-transplant is a crucial investigation in clinical organ transplantation. Three methodologies for the detection of lymphocytotoxic antibodies were compared to establish which of these is best suited to optimise pre-transplant donor-recipient matching. Serum samples from 15 renal transplant patients were tested for the presence of anti-HLA by i) cytotoxic-dependent cross-match (CDCXM), ii) flow cytometric cross-match (FCXM) and iii) Luminex-based donor specific antibody cross-match (DSAXM) method, Confirmatory tests for the presence of preformed HLA antibodies were tested using Luminex methodology. Two (13%) of the 15 patients had positive HLA Class I antibodies (Ab) using all 3 methods. An additional 2 HLA Class I Ab were identified with FCXM/CDCXM. DSAXM identified 1 HLA Class I positive, not indicated by CDCXM/FCXM.High HLA Class II positivity (40%), identified by CDCXM, while DSAXM and FCXM identified two and one patients, respectively. CDCXM produced 4 false-positive results confirmed by lymphocyte single antigen (LSA) assay. The DSAXM method appears to add value in pre-transplantation screening to identify pre-sensitised patients that may not reject the donor graft due to the absence of donor-specific antibodies.

Stratifying the humoral risk of candidates to a solid organ transplantation: a proposal of the ENGAGE working group.

Detection of circulating antibodies directed against human leukocyte antigen (HLA) molecules, which corresponds to the current definition of 'sensitized patient', has been shown to have a severe impact on both access to transplantation and, if the anti-HLA antibodies are specific to the selected donor, survival of the graft. However, not all donor-specific antibodies (DSA) are equally harmful to the graft and progress in the understanding of humoral memory has led to the conclusion that absence of DSA at transplantation does not rule out the possibility that the patient has a preformed cellular humoral memory against the graft (thereby defining a category of DSA-negative sensitized recipients). Technological progress has led to the generation of new assays that offer unprecedented precision in exploring the different layers (serological and cellular) of alloimmune humoral memory. Based on this recent knowledge, the EuropeaN Guidelines for the mAnagement of Graft rEcipients (ENGAGE) working group to propose an updated definition of sensitization in candidates for solid organ transplantation - one that moves away from the current binary division towards a definition based on homogenous strata with similar humoral risk.

Immuno-Electrophysiological Mechanisms of Functional Electrical Connections Between Recipient and Donor Heart in Patients With Orthotopic Heart Transplantation Presenting With Atrial Arrhythmias.

[Figure: see text].

Successful transatlantic bilateral hand transplant in a young female highly sensitized to HLA class II antigens

Vascularized composite allografts may be more susceptible to rejection than other types of organ transplants, particularly in sensitized recipients. We describe a successful transatlantic bilateral hand transplant in a 40-year old woman who was highly sensitized to class II HLA antigens including HLA-DPB1 (UNet CPRA = 86%). Prior to transplantation, we selected an upper limb donor based on HLA class II matching and absence of donor specific antibodies, given evidence that class II mismatches are associated with acute cellular rejection in hand transplants. The patient was conditioned using five doses of thymoglobulin, and her immunosuppression included tacrolimus, rapamycin, mycophenolate, and prednisone. Post-transplant, the patient non-DSA anti-HLA antibody levels drastically increased, but only transiently and weak DSAs developed, which became undetectable by two months posttransplant. Following transplantation, periodic biopsies over 6 months indicated no evidence of rejection except for transient Banff grade 1 and one sample with grade 2 acute rejection. There was no evidence of rejection on her recent 1-year follow-up. The patient is currently healthy, has recovered protective sensibility, and is regaining excellent function. This case highlights the importance of pre-transplantation planning, donor selection/compatibility, and ethical considerations in the ultimate success of VCA.

Pretransplant Calculated Panel Reactive Antibody in the Absence of Donor-Specific Antibody and Kidney Allograft Survival.

Panel reactive antibody informs the likelihood of finding an HLA-compatible donor for transplant candidates, but has historically been associated with acute rejection and allograft survival because testing methods could not exclude the presence of concomitant donor-specific antibodies. Despite new methods to exclude donor-specific antibodies, panel reactive antibody continues to be used to determine the choice of induction and maintenance immunosuppression. The study objective was to determine the clinical relevance of panel reactive antibody in the absence of donor-specific antibodies. Retrospective observational study of kidney allograft survival among 4058 zero HLA-A-, B-, DR-, and DQB1-mismatched transplant recipients without antibodies to donor kidney antigens encoded by these HLA gene loci. Among 4058 first and repeat transplant recipients, patients with calculated panel reactive antibody (cPRA) 1%-97% were not at higher risk of transplant failure, compared with patients with cPRA of 0% (death censored graft loss: hazard ratio, 1.07; 95% confidence interval, 0.82 to 1.41). Patients with cPRA ≥98% had a higher risk of graft loss from any cause including death (hazard ratio, 1.39; 95% confidence interval, 1.08 to 1.79) and death censored allograft failure (hazard ratio, 1.78; 95% confidence interval, 1.27 to 2.49). In stratified analyses, the higher risk of graft loss among patients with cPRA ≥98% was only observed among repeat, but not first, transplant recipients. In subgroup analysis, there was no association between cPRA and graft loss among living related transplant recipients. Calculated panel reactive antibody is poorly associated with post-transplant immune reactivity to the allograft in the absence of donor-specific antibody. This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_01_25_CJN13640820_final.mp3.

Kidney re-transplantation in a child across the barrier of persisting angiotensin II type I receptor antibodies

BackgroundApproximately 20% of antibody-mediated rejection (ABMR) episodes in the absence of donor-specific antibodies against human leucocyte antigens (HLA-DSA) in pediatric and adult kidney transplant recipients are associated with, and presumably caused by, antibodies against the angiotensin type 1 receptor (AT1R-Ab). While the role of AT1R-Ab for ABMR and graft failure is increasingly recognized, there is little information available on the management of these patients for re-transplantation over the barrier of persisting AT1R-Ab.CaseWe report on a male patient with kidney failure in infancy due to obstructive uropathy who had lost his first kidney transplant due to AT1R-Ab-mediated chronic ABMR. Because this antibody persisted during 4 years of hemodialysis, for the 2nd kidney transplantation (living-related transplantation from his mother), he underwent a desensitization regimen consisting of 15 plasmapheresis sessions, infusions of intravenous immunoglobulin G and thymoglobulin, as well as pharmacological blockade of the Angiotensin II (AT II) pathway by candesartan. This intense desensitization regimen transiently decreased elevated AT1R-Ab titers, resulting in stable short-term kidney allograft function. The subsequent clinical course, however, was complicated by acute cellular rejection and chronic ABMR due to persistent AT1R-Ab and de novo HLA-DSA, which shortened allograft survival to a period of only 4 years.ConclusionThis case highlights the difficulty of persistently decreasing elevated AT1R-Ab titers by a desensitization regimen for re-transplantation and the detrimental effect of the interplay between AT1R-Ab and HLA-DSA on kidney transplant survival.

Impact of rituximab on the T-cell flow cytometric crossmatch

Rituximab is frequently used in the setting of ABO-incompatible renal transplants, and highly sensitized patients. Its interference with B-cell flow cytometric crossmatch (B-FCXM) is well known. However, its effect on the T-cell flow cytometric crossmatch (T-FCXM) has not been described. We aimed to evaluate the effect of rituximab on the T-FCXM using non-pronase and pronase treated donor lymphocytes and compare results with the single antigen bead (SAB) assay. In this retrospective study, 28 patients on rituximab therapy were evaluated against 30 donors. Using non-pronase treated donor lymphocytes, all 30 FCXMs showed strong B-cell positivity {median (IQR) B-cell ratio: 184.65 (253.17)} which significantly reduced {1.0 (1.18); p < 0.00001} with pronase treatment. ‘T-cell tailing’ phenomenon was observed in 17/30 FCXMs in the non-pronase group as a ‘tail of T-cells’, indicating a rare sub-population. However, it disappeared in the pronase-treated group. SAB assay did not show donor-specific antibodies (DSA) in all 17 patients with ‘T-cell tailing’ phenomenon. Although, rituximab is described to impact only B-FCXM, we have consistently found ‘T-cell tailing’ in 57% of T-FCXMs, which clears with pronase treatment. The ‘T-cell tailing’ led to weak positive T-FCMX ratios due to increased MFI in the FL1 channel. However, the absence of DSA in all recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I DSA. Structural homology of Fc receptors on activated T-cells to CD20 could be a possible explanation of the same and provide insight into a novel mechanism of action of rituximab.

Rapid Engraftment, Immune Recovery, and Resolution of Transfusion Dependence in Treatment-Refractory Severe Aplastic Anemia Following Transplantation with Ex Vivo Expanded Umbilical Cord Blood (Omidubicel)

Introduction Severe Aplastic Anemia (SAA) is a life-threatening bone marrow failure disorder associated with pancytopenia, serious infections, and transfusion dependence. Although long term survival for SAA patients (pts) can be achieved with immunosuppressive therapy (IST), one quarter to one third will fail to respond and about half of responders relapse. Many refractory SAA pts lack an HLA matched donor for salvage allogeneic stem cell transplantation. Although umbilical cord blood transplantation (UCBT) is an alternative approach for SAA pts, the procedure is associated with delayed engraftment and high rejection rates. Further, a substantial portion of UCB units have an insufficient number of TNCs/CD34+ cells for optimal transplant outcomes. To address the need to improve UCBT outcomes for SAA, we instituted a phase II trial exploring the use of nicotinamide (NAM) ex-vivo expanded UCB [omidubicel] to transplant pts with refractory SAA. Methods Eligible pts were 4-55 years and had transfusion dependent SAA. Other criteria included a) failure or intolerance to IST, b) lack of an HLA matched donor, c) having a ≥ 4/8 HLA matched UCB unit with a minimum of 1.8 x 109 and at least 1.8x107/kg TNCs and at least 8 x 106 CD34+ cells, and d) absence of donor specific antibodies to mismatched alleles on the UCB unit. The study was designed to enroll up to 6 pts receiving omidubicel and CD34+ selected haploidentical cells (cohort 1), followed by enrollment of up to 23 pts receiving omidubicel only (cohort 2). The conditioning regimen consists of hATG (40 mg/kg) on D -11 to -8, cyclophosphamide (60 mg/kg) on D -7 and -6, fludarabine (25 mg/m2) on D -5 to -1, and 2 Gy TBI on D -1. Tacrolimus and mycophenolate mofetil were given as GVHD prophylaxis. The primary end point of the study is sustained early engraftment. Secondary endpoints include treatment-related mortality, and standard transplant outcomes. Results A total of 8 pts with SAA refractory to IST and eltrombopag with a median age of 23 years (range 6-45) have been transplanted to date. Pts were heavily transfused (median ferritin 3745 µg/dL), had severe neutropenia with a median pre-transplant ANC of 115 (range 0-680), with 5 (63%) pts having HLA alloantibodies and 1 pt having an invasive fungal infection treated with multiple antifungals and granulocyte transfusions in the peri-transplant period. In cohort 1, the first 3 pts received omidubicel and ~3 x106 CD34+ cells/kg from a haploidentical donor. Since all 3 pts in cohort 1 had sustained engraftment with omidubicel, the study moved to cohort 2, where so far 5 pts have received omidubicel alone. UCB units were matched at a median of 5/8 HLA alleles (range 4-6). Before expansion, units contained a median 1.6 x105 CD34+ cells/kg. At time of transplantation, the expanded units contained a median 75.3 x105 CD34+ cells/kg, representing a median 42-fold expansion. With a median follow-up of 10 months (range 1-35), 7/8 (88%) pts have had early and sustained cord engraftment. One pt in cohort 2 failed to engraft and was salvaged with a haploidentical transplant. Another pt in cohort 2 died on D+ 62 from disseminated adenovirus infection. This pt also developed grade 2 acute GVHD and his IST was stopped early in an effort to boost immunity against adenovirus. The other 7 pts are alive without evidence for acute or chronic GVHD and are transfusion independent. CMV reactivation requiring treatment occurred in 3/6 (50%) of pts at risk. Neutrophil and platelet recovery have been remarkably quick, occurring at a median of 10 days (range 6-14) and 31 days (15-40) respectively. Among the 7 pts with sustained engraftment, six pts had ≥ 95% cord myeloid chimerism by D+ 14 and ≥ 95% T-cell chimerism by D+ 26. Immune recovery has also been remarkably brisk: at D+ 100, the median absolute CD4 count was 186/µL (IQR, 118-340) and mean (±SD) IgG level was 522(±161) mg/dL, respectively (Figure). Conclusions These results provide the first evidence that omidubicel can result in rapid engraftment and sustained hematopoiesis in heavily transfused and allo-immunized SAA pts who are at high risk for graft failure with conventional UCBT. Data showing rapid recovery of IgG levels and rapid reconstitution of CD4, CD8, and NK cell subsets suggests immune recovery may occur quicker with omidubicel compared to conventional UBCT. Taken altogether, our preliminary data suggest omidubicel represents a promising new alternative graft source for SAA pts who lack HLA matched donors. Figure Disclosures No relevant conflicts of interest to declare.

RNA Expression Analysis of Antibody Mediated Rejection after Heart Transplant with or without Donor Specific Antibodies

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnostic criteria previously required serum donor specific antibodies (DSA). However, the most recent criteria only requires pathological findings independent of DSA. This classification defined a subset of patients with AMR without known antibodies. To investigate this complication, we performed RNA sequencing using endomyocardial biopsy samples taken at the time of pathology confirmed AMR in the presence and absence of DSA. We evaluated the transcriptome profile of the paraffin-embedded endomyocardial biopsy specimens of 30 representative heart transplant recipients. Patients were categorized as having no evidence of AMR (controls), immunohistochemical findings of AMR (pAMR1i) in the presence of DSA (1i+D), pAMR1i in the absence of DSA (1i-D), histopathological findings of AMR (pAMR1h) in the presence of DSA (1h+D), pAMR1h in the absence of DSA (1h-D) or presence of both immunohistochemical and histopathological findings of AMR (pAMR2) in the presence of DSA. A total of 5 patients were analyzed per group. All were matched for previous mechanical circulatory support and time from transplant to biopsy. There was no differential RNA expression between patients with no AMR and those in the 1i-D group. There was also no differential expression between patients in the 1h+D and pAMR2 groups. The largest difference was seen between the no AMR and 1i+D groups and the no AMR and pAMR2 groups. In addition, there was RNA expression differences between patients with immunohistochemical evidence of AMR compared to those with histopathological findings, suggesting distinct mechanisms. These studies demonstrate that RNA expression profiling can be performed on endomyocardial biopsy tissue preserved in paraffin from the time of a rejection episode and that different expression patterns are observed dependent on the criteria used to diagnose AMR. In particular, AMR in the presence of DSA showed a different RNA expression profile than AMR without DSAs. The RNA expression associated with histological markers of AMR also appeared different than that associated with immunological markers. Overall, our findings suggest the existence of different subtypes of AMR that may rely on distinct mechanisms of injury.