Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

Detection of Acquired Uniparental Disomy (aUPD) with HLA Typing and Microarray Analysis after T Cell-Containing Haploidentical (HI) Hematopoietic Stem Cell Transplantation (HSCT)

Loss of heterozygocity (LOH) in large regions of chromosome 6p encompassing the major histocompatibility complex (MHC) due to de novo acquired aUPD has been demonstrated in leukemic cells of patients who relapse after HI HSCT. This genetic event results in the loss of the unshared haplotype on recipient malignant cells, eliminating donor T cell recognition through MHC, and potentially eliminating donor lymphocyte infusion (DLI) as effective treatment. To confirm aUPD in patients relapsing after treatment with a T cell containing HI HSCT, and to formulate effective treatment plans, we began testing for aUPD primarily in patients with acute myeloid leukemia (AML) who developed post HSCT relapse starting in 2013. All patients underwent HI HSCT on a Jefferson 2 step trial in which every patient after conditioning received 2 x 108/kg T cells (HSCT step 1), followed 2-days later by cyclophosphamide (CY) for bidirectional tolerization. Two days after CY, all patients received a CD 34-selected donor product (HSCT step 2). Upon relapse, HLA typing was to be performed on blood or marrow containing leukemic blasts. An in-house analysis showed that HLA haplotypes were detectable in cells that comprised 10% or more of the analyzed sample. After DNA extraction of the samples, low resolution typing was done followed by high resolution confirmatory typing in cases where the unshared haplotype was not initially detected. MNC (2) and CD 34+ (1) selection was performed on 3 samples. High density single nucleotide polymorphism microarray (MA) analysis for aUPD was performed on the 2 MNC sorted specimens. Eleven patients with AML were eligible for aUPD analysis. One patient with ph+ ALL was also tested for aUPD due to the late timing of relapse. Three of 12 had insufficient samples, bringing the analyzed group to 9 patients. Table 1. Patients Disease at HSCT Post HSCT Relapse Day aUPD Analysis Post Relapse Events No aUPD 1 Secondary AML 174 HLA typing BM with 54% blasts-unshared haplotype present Died after chemo attempt 2 Refractory AML 187 HLA typing blood with 32% blasts-unshared haplotype present Died complications of chemotherapy and DLI 3 AML CR2 465 HLA typing BM with 55% blasts- unshared haplotype present Died-no further therapy 4 Refractory AML 63 HLA typing blood with 95% blasts-unshared haplotype present Died-failed Flt-3 Inhibitor Consistent with aUPD 5 Refractory AML 1902 HLA typing BM with 79% blasts-unshared haplotype not detected Alive 19 months post relapse, chemo then IL-2 x 1 year 6 Ph+ ALL CR1 571 HLA typing blood with 56% lymphoblasts-unshared haplotype not detected Chemo + TKI, NED x > 2 years 7 Refractory AML 274 HLA typing CD 34 selected marrow sample (90% purity) unshared haplotype not detected Died-failed PD-1 8 AML CR2 453 MNC sorted marrow aspirate containing 55% blasts-unshared haplotype not detected on HLA typing. Genomic loss in 6p including MHC antigens on microarray analysis Being reinduced 9 Refractory AML 398 HLA typing not done at time of relapse. Retrospective microarray analysis showed genomic loss in 6p including MHC antigens on microarray analysis Died-failed DLI 5/9 patients, including the patient with ph+ ALL, had findings consistent with aUPD, confirmed by MA analysis in two patients. HLA typing and MA analysis (Figure) performed on the same sample (patient 8) were concordant in findings of aUPD. One patient (#5) without a KIR ligand mismatch with his donor, had aUPD at relapse therefore DLI was not given. The patient achieved CR with chemotherapy, and surprisingly was without evidence of disease for 1 year on low dose IL-2, prior to relapse just after it was tapered. aUPD was associated with late myeloid and lymphoid leukemic relapse after T cell containing HI HSCT. HLA typing is a widely available alternative to MA analysis for the specific purpose of aUPD detection, and can be performed quickly to help guide post relapse therapy in samples with adequate blast counts. Concordance between the 2 studies was demonstrated in 1 patient in our series. Current efforts regard retrospective MA analysis of samples in which the presence or absence of aUPD was determined based on HLA typing alone, to confirm the reliability of HLA typing for identification of aUPD. Intriguingly, low dose IL-2 was associated with maintenance of remission, suggesting a possible avenue of inquiry into the impact of the loss of MHC expression by malignant cells on natural killer cell activity. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

CD8+ T Cells Mediate Antibody-Independent Platelet Clearance Following Transfusion

Background: Alloimmunization to major histocompatibility complex (MHC) antigens is the leading immunological barrier to transplantation and platelet transfusion therapy. In the context of platelet transfusion medicine, alloimmunization to MHC antigens can substantially diminish the therapeutic efficacy of subsequent blood transfusions. Presently, MHC alloantibodies are recognized as the primary mediators of immune-mediated platelet clearance. However, individuals previously exposed to MHC alloantigens can display significant platelet clearance in the absence of detectable anti-platelet antibodies. Given the ability of cellular immunity to mediate rejection of solid organ allografts across MHC differences, we hypothesized that cellular immunity likewise facilitates the clearance of platelets in MHC alloimmunized recipients. Methods: FVB (H-2Kq) MHC-immunized or non-immunized C57BL/6 (H-2Kb) recipients were transfused with filter leukoreduced platelet rich plasma (PRP) isolated from C57BL/6 donors expressing green fluorescent protein (GFP) under a H-2Kb promoter (B6 GFP) or PRP obtained from a FVB X B6 GFP F1 cross, which generates GFP+ H-2Kq+ platelets. Following transfusion, mice were bled at 10 minutes, 1 hour, 2 hours or 24 hours to calculate the percentage of GFP+ transfused platelets remaining using flow cytometric analysis. To test the hypothesis that platelet clearance can occur in an antibody-independent process, FVB H-2Kq MHC-immunized or non-immunized μMT recipients were similarly transfused with B6 GFP or FVB X B6 GFP PRP. CD8+ T cells in immunized μMT recipients were depleted by i.p. injection of anti-CD8 (clone 2.43) antibody for 3 consecutive days prior to transfusion. NK cells were similarly depleted by injection of anti-NK1.1 (clone PK-136) antibody, 1 day prior to transfusion. CD4, CD8 T cell and NK cell depletion was evaluated in the peripheral blood prior to platelet transfusion, and compared to non-treated and isotype control treated recipients. Results: While GFP+ platelets were readily detected following transfusion into non-immunized or MHC matched recipients, transfusion of FVB-B6 platelets into H-2Kq MHC-immunized C57BL/6 recipients resulted in rapid clearance within the first hour following transfusion. In contrast, though little platelet clearance was detected in H-2Kq MHC-immunized μMT recipients within an hour following transfusion, very few transfused platelets were observed after 24 hours. Moreover, despite platelet clearance, anti-MHC antibodies were not detectable in μMT recipients. To determine the cellular component responsible for clearance in alloimmunized μMT recipients, CD8+ T cells or NK cells were depleted prior to transfusion. NK cell depletion failed to impact platelet clearance in alloimmunized μMT recipients; however, depletion of CD8+ T cells prior to platelet transfusion abrogated platelet clearance in immunized μMT recipients. Conclusion: Immunized recipients rapidly clear platelets in an alloantigen specific fashion following platelet transfusion in a murine model. Though platelet clearance occurred at a faster rate in intact immunized C57BL/6 recipients, B cell deficient MHC-immunized μMT recipients possessed the capacity to induce significant clearance at later time points. Evaluation of the cellular populations responsible for antibody-independent clearance in μMT recipients strongly suggest that CD8+ T cells play a key role in antibody-independent immune-mediated platelet clearance. These results suggest that, in addition to antibodies, CD8+ T cells can play a role in the development of platelet refractoriness and may contribute to platelet non-responsiveness in patients with little to no detectable anti-platelet alloantibodies. Disclosures No relevant conflicts of interest to declare.