Donor-specific Antibody Titers Research Articles

TBI-Containing with T-Cell Replete Full Haplotype Mismatched Hematopoietic Stem Cell Transplantation in Adult Patients with De Novo Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the 2nd most frequent developing hematologic malignancy with deep heterogeneity, but unfortunately the majority of patients relapse even after allogeneic hematopoietic stem cell transplantation (HSCT) in complete remission (CR). We have developed allogeneic HSCT from familial mismatched/haploidentical donors (FMT) in adult patients with a single disease entity of AML lacking of both HLA-identical siblings and well-matched unrelated donors (WMUD) from the mid 1990s. Previously, several pilot studies of FMT performed with various total body irradiation (TBI)-based conditioning regimens (2~12 gray) with or without post-transplant cyclophosphamide (PT-Cy) as a graft-versus-host disease (GvHD) prophylaxis were reported. Here, we demonstrate about the encouraging outcomes of FMT using unmanipulated peripheral blood donor stem cells (PBSCs) and de-escalated dose of TBI (4~8 gray) containing conditioning regimen without T-cell depletion manner. Although PT-Cy as a specific GvHD prophylaxis regimen is increasingly used for both adult and pediatric FMT, our group investigated the clinical outcomes in adult patients with de novo AML who received full haplotype mismatched HSCT after TBI containing preparation regimen consisted of fractionated TBI (total 4 gray for elderly and 8 gray for <60 years old), fludarabine (150 mg/m 2/day), busulfex (6.4mg/kg/day), and anti-thymocyte globulin (ATG). For GvHD prophylaxis, rabbit ATG (thymoglobulin, Genzyme, 1.25mg/kg/day for 4 days at D-4 to D-1) and a combination of tacrolimus and short-term methotrexate was used. G-CSF mobilized unmanipulated PBSCs were used with a median CD34+ dose of 6.5 x 10 6/kg (1~25.6). From 2008 to the end of 2022, total 429 adult patients were enrolled, and data demonstrated that our trial of intermediate-dose of ATG and reduced toxicity of TBI-containing conditioning regimen to reduce graft-failure even for high levels of donor-specific antibody titer, early transplant-related mortality, critical cytomegalovirus (CMV) disease, and emerging post-transplant lymphoproliferative disease with rapid immune recovery has shown very successful outcomes. Briefly, the median absolute neutrophil count recovery was 12 days (10~26) and the platelet of 14 days (5~38). There was no either primary or secondary graft-failures at all. With a median follow-up of 36.5 months (2~242) for survivors, overall survival rate (OS) and relapse-free survival (RFS) for patients in any CR (n=338) and relapse/refractory status (n=91) were 62.9% vs. 34.3% and 59.5% vs. 32.2%, respectively. Although there was no significant difference in the subgroup analysis, de novo and young aged patients in any CR showed the best OS 73% (Figure 1). Cumulative incidences of acute GvHD and chronic GvHD were 66% (59-73), 70% (63-76), respectively. The incidences of moderate to severe acute GvHD and chronic GvHD were 16.1%, 30.5%, respectively. By univariate and multivariate analyses, patient's age <54 (p=0.043), pre-HSCT CR1 (p <0.001), absence of mod to severe chronic GvHD (p <0.001), and use of post-transplant iron-chelating agent (p=0.001) were the statistically significant in the study. The GvHD-free and relapse-free survival rate in CR1 was 62.4% for younger patients (age <54) and 47.9% for older patients (p=ns). The non-relapse mortality rate was 27.2% for all enrolled patients and 16% for CR patients. The rapidity of immune recovery was also compared with our old control cohort when we performed T-cell depletion using the CliniMACS system, and it showed a significant reconstitution of CD4+ cells (>300/ul) at 3 months post-FMT compared to the much delayed T-cell depleted FMT. Of note, CMV DNAemia was relatively higher (76.9%) than reported in other studies, likely due to the use of ATG and the endemic region of our country. Therefore, we suggest that the strategic reduction of fractionated TBI 4~8 gray-based conditioning with unmanipulated PBSCs graft without PT-Cy seems feasible with favorable outcomes for adult patients with de novo AML undergoing FMT in CR, especially even in a certain subgroup of elderly patients with AML.

Additional Benefits of Rituximab and Plasma Exchange on Top of Standard Induction Therapy in Kidney Transplant Recipients With a Negative CDC Crossmatch but High Preformed Donor Specific Antibody Titer.

Optimal induction strategy in highly sensitized kidney transplant recipients (KTRs) is still a matter of debate. The place of therapies, such as plasma exchange and rituximab, with potential side effects and high cost, is not clearly established. We compared two induction strategies with (intensive) or without (standard) rituximab and plasma exchange in KTRs with high levels of preformed DSA transplanted between 2012 and 2019. Sixty KTRs with a mean age of 52.2 ± 12.2 years were included, 36 receiving standard and 24 intensive induction. Mean fluorescence intensity of immunodominant DSA in the cohort was 8,903 ± 5,469 pre-transplantation and similar in both groups. DSA level decrease was similar at 3 and 12months after transplantation in the two groups. An intensive induction strategy was not associated with better graft or patient survival, nor more infectious complications. The proportion of patients with rejection during the first year was similar (33% in each group), but rejection occurred later in the intensive group (211 ± 188 days, vs. 79 ± 158 days in the standard group, p < 0.01). Our study suggests that an intensive induction therapy including rituximab and plasma exchanges in highly sensitized kidney recipients is not associated with better graft survival but may delay biopsy-proven rejection.

Low Incidence of Acute Antibody-Mediated Rejection after HLA Desensitization in Living Donor Kidney Transplant Recipients.

Desensitization allows the performance of human leukocyte antigen (HLA)-incompatible transplants. However, the incidence of acute rejection (AR) is high. This study aims to analyze the incidence of AR after transplantation with HLA-incompatible living donors in patients who underwent desensitization. Patients were immunosuppressed with tacrolimus, mycophenolic acid derivatives, and steroids after being desensitized with rituximab, plasma exchange, and/or immunoadsorption with specific cytomegalovirus immunoglobulins. A negative complement-dependent cytotoxicity or flow cytometry crossmatch and a donor-specific antibody titer < 1000 mean fluorescence intensity (MFI) were used to determine desensitization efficacy. A total of 36 patients underwent desensitization, and 27 (75%) were transplanted. After a follow-up of 58 ± 58 months (Min−Max: 0.13−169.5), five episodes of AR occurred: two antibody-mediated and three T-cell-mediated. No differences were found in baseline calculated panel-reactive antibodies (cPRA), class I or II MFI, number of antibodies, or Relative Intensity Scale (RIS) between AR and non-AR patients. Patients with antibody-mediated AR had higher cPRA (NS), MFI class I (p = 0.07) and class II (p = 0.006), and RIS (p = 0.01). The two patients with antibody-mediated AR and one patient with T-cell-mediated AR lost their grafts. In conclusion, the incidence of acute antibody-mediated rejection after desensitization was 7.4%, which occurred early post-transplantation in patients with high MFI and was associated with early graft loss.

In-Depth Analysis of Anti-HLA Antibodies Using C1q Assay

Alloantibodies are significant biomarkers of posttransplant kidney rejection. With solid-phase assays, the presence of alloantibodies and complement-binding capacities can be tested. It has been noted that complement-binding anti-HLA (C1q) correlates well with high titers of anti-HLA antibodies (single antigen bead, SAB), but we have recently shown that lower SAB titers may be associated with complement in the complement cytotoxicity test. If so, low titers of donor-specific antibodies could be stratified for complement binding and used for better donor-recipient matching. To study this, we tested 268 patients awaiting kidney transplantation for SAB and C1q and stratified them into positive (Allo+) and negative (Allo-) cohorts. Next, all assayed specificities of SAB were matched with the corresponding C1q in order to correlate mean fluorescence intensity levels. We found a strong correlation between SAB and C1q for all HLA loci apart from HLA-DP. Moreover, there was no strict cutoff for C1q prediction on SAB mean fluorescence intensity level. In addition, an unusual laboratory phenotype was found; that is, a positive C1q result without corresponding SAB specificity. We tested this phenomenon and found positive IgM SAB. CONCLUSIONS: Simultaneous C1q and SAB testing may serve as a tool for in-depth analysis of alloantibodies before transplantation.

Molecular Signature of Antibody-Mediated Chronic Vasculopathy in Heart Allografts in a Novel Mouse Model

Cardiac allograft vasculopathy (CAV) limits the long-term success of heart transplants. Generation of donor-specific antibodies (DSAs) is associated with increased incidence of CAV clinically, but mechanisms underlying development of this pathology remain poorly understood. Major histocompatibility complex-mismatched A/J cardiac allografts in B6.CCR5-/- recipients have been reported to undergo acute rejection with little T-cell infiltration, but intense deposition of C4d in large vessels and capillaries of the graft accompanied by high titers of DSA. This model was modified to investigate mechanisms of antibody-mediated CAV by transplanting A/J hearts to B6.CCR5-/- CD8-/- mice that were treated with low doses of anti-CD4 monoclonal antibody to decrease T-cell-mediated graft injury and promote antibody-mediated injury. Although the mild inhibition of CD4 T cells extended allograft survival, the grafts developed CAV with intense C4d deposition and macrophage infiltration by 14 days after transplantation. Development of CAV correlated with recipient DSA titers. Transcriptomic analysis of microdissected allograft arteries identified the Notch ligand Dll4 as the most elevated transcript in CAV, associated with high versus low titers of DSA. More importantly, these analyses revealed a differential expression of transcripts in the CAV lesions compared with the matched apical tissue that lacks large arteries. In conclusion, these findings report a novel model of antibody-mediated CAV with the potential to facilitate further understanding of the molecular mechanisms promoting development of CAV.

Heat shock protein 90 is a new potential target of anti-rejection therapy in allotransplantation.

The critical roles of heat shock protein 90 (HSP90) in immune reactions associated with viral infection and autoimmune disease are well known. To date, however, its roles in the alloimmune response and the immunosuppressive effect of HSP90 inhibitors in allotransplantation have remained unknown. The purpose of this study was to examine the therapeutic efficacy of the HSP90 inhibitor 17-DMAG in allotransplantation models. C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donors for and recipients of skin and heart transplantation, respectively. Treatment with 17-DMAG (daily i.p.) or a vehicle was initiated 3days before transplantation. Immunological outcomes were assessed by histopathological examinations, flow cytometric analysis, quantitative RT-PCR, ELISA, ELISPOT assay, and MLR. 17-DMAG treatment significantly prolonged the survival of both skin and heart allografts. In 17-DMAG-treated mice, donor-reactive splenocytes producing IFN-γ were significantly reduced along with the intragraft mRNA expression level and serum concentration of IFN-γ. Intragraft mRNA expression of cytokines and chemokines associated with both innate and adaptive immunity was suppressed in 17-DMAG-treated group. MLR showed suppression of the donor-specific proliferation of CD4 + T and CD19 + B cells in the spleens of 17-DMAG-treated mice. 17-DMAG treatment also reduced the number of activated NK cells. Furthermore, the treatment lowered the titers of donor-specific antibodies in the serum and prolonged a second skin allograft in mice sensitized by previous skin transplantation. HSP90 inhibition by 17-DMAG can affect various immune responses, including innate immunity, adaptive immunity, and humoral immunity, suggesting its therapeutic potential against acute rejection in allotransplantation.

Desensitization Regimen Consisting of High-Dose Intravenous Immunoglobulin, Plasmapheresis, and Rituximab (an Anti-CD20 Antibody), Without Eculizumab and/or Bortezomib, in 41 Highly Sensitized Kidney Transplant Recipients.

Antibody-mediated rejection in patients with positive crossmatches can be severe and result in sudden onset of oliguria, leading to graft loss. In an attempt to prevent posttransplant oliguria, we adopted a preoperative desensitization protocol involving the use of high-dose intravenous immunoglobulin/plasmapheresis and the anti-CD20 antibody, rituximab, in 41 transplant recipients with positive crossmatch test results. We retrospectively examined the clinical courses of the 41 kidney transplant recipients, paying special attention to renal graft function, urine volume, and changes in the titers of donor-specific antibodies. Four grafts were lost during an average of 4.5-year follow-up. Average graft function was excellent, with a serum creatinine level of 1.3 ± 0.4 mg/dL. Sufficient urine output, with no oliguria or anuria, was achieved postoperatively in 40 of the 41 patients. However, among the 34 patients who underwent graft biopsies, the biopsies revealed acute antibody-mediated rejection in 21 patients (62%), and chronic antibodymediated rejection in 10 patients (30%). The high-dose intravenous immunoglobulin treatment included in our desensitization protocol was shown to be safe and effective for achieving successful transplant outcomes and allowed the avoidance of more aggressive B-cell-targeted treatments, such as C5 inhibitors and/or proteosome inhibitors, for preventing posttransplant oliguria and anuria.

Measuring human leukocyte antigen alloantibodies: beyond a binary decision

Accurate measurement of human leukocyte antigen antibodies is critical for making clinical decisions treating patients awaiting transplantation or monitoring them post transplantation. Single antigen bead assay results are given as Mean Fluorescence Intensity, falling short of providing the required quantitative measure. Titration studies were shown to circumvent the limitation of target-saturation that affect interpretation of single antigen bead assays especially in highly sensitized patients with strong antibodies. In fact, titration information can serve to measure efficacy of antibody removal during pretransplant desensitization using plasmapheresis/intravenous immunoglobulin (PP/IVIg) approaches. Moreover, recent studies indicate that knowing the donor-specific antibody titer has prognostic value that can guide PP/IVIg desensitization treatments. Newer data demonstrates an additional layer of information obtained by titration studies allowing to stratify patients with very high cPRA (>99%) based on the strength of the antibodies present, rather than the breadth. This data can thereby identify patients that are more likely to benefit from desensitization approaches on the transplant wait-list. Titration studies have a prognostic value with regards to quantifying antibody strength. Obtaining this information does not require performing the complete set of dilutions. In fact, performing two to three specific dilutions can provide relevant information while maintaining practical cost.

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.

Kidney Allograft Recipient Absence of Myeloperoxidase Decreases Donor-Specific Antibody Titers and Attenuates Antibody-Mediated Allograft Rejection

Abstract The incidence of antibody-mediated rejection (AMR) in clinical kidney transplants is increasingly observed. We have reported that strong donor-specific antibody (DSA) responses are induced in B6.CCR5−/−mice transplanted with complete MHC mismatched A/J kidney allografts and that NK cells play a critical role in acute injury and graft failure. Since this acute AMR is accompanied by intense macrophage infiltration into the allograft, we tested the role of recipient-derived myeloperoxidase (MPO) production on kidney allograft survival. B6.CCR5−/−and B6.CCR5−/−MPO−/−mice were transplanted with complete MHC mismatched A/J kidney grafts. Allografts were rejected between days 18 and 25 post-transplant in B6.CCR5−/−recipients but not until days 46–54 in B6.CCR5−/−MPO−/− recipients. DSA titers in B6.CCR5−/−MPO−/−recipients were 4–5 fold lower than those induced in the B6.CCR5−/− recipients. There was also a 60% decrease in the number of donor-reactive T cells producing IFN-g in the spleens of the B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients on day 7 post-transplant. Despite the extended survival, qPCR analyses indicated slight increases in mRNA encoding TNFa, IL-6 and FasL in kidney allografts on day 14 post-transplant in B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients as well as the pro-fibrogenic factors P-selectin and connective tissue growth factor on day 50 post-transplant. These results suggest MPO regulates the magnitude of the DSA and T cell response in kidney transplant recipients and that the decreased DSA titers attenuate acute AMR but induce the indolent development of interstitial fibrosis and glomerular injury that will eventually lead to graft dysfunction and failure at later times.

Donor-specific antibody mediates chronic, but not acute, kidney allograft rejection in the absence of natural killer cells

Abstract Dysregulated donor-specific antibody (DSA) responses are induced in B6.CCR5−/− mice transplanted with complete MHC mismatched kidney allografts and NK cells play a critical role in acute graft injury and failure. The current study investigated the consequence of high DSA titers on graft outcomes in the presence vs. absence of NK cell activation within the kidney graft. B6.CCR5−/− recipients of complete MHC mismatched A/J allografts or semi allogeneic (A/J x B6)F1 kidney grafts responded with equivalent serum DSA titers that reached peak by day 14 post-transplant. B6.CCR5−/− recipients rejected A/J allografts between days 15–30, whereas B6 isografts and (A/J x B6)F1 semi allogeneic grafts survived past day 65. On day 7 post-transplant NK cell infiltration into A/J allografts was composed of distinct Ly49b+ cell populations expressing low and high levels of NK1.1 and both NK1.1high and NK1.1low cells increased in A/J allografts with time to rejection. In contrast, NK cell infiltration into (A/J x B6)F1 grafts on day 7 post-transplant was composed entirely of NK1.1high cells that decreased to background/isograft numbers thereafter. (A/J x B6)F1 grafts at day 65 post-transplant had severe chronic injury with prominent interstitial fibrosis, glomerulopathy and arteriopathy that was accompanied by gene expression of profibrogenic factors. These results indicate that NK cells synergize with DSA to cause acute kidney allograft failure whereas high titers of DSA in the absence of NK cell activation cannot cause acute antibody-mediated rejection but induce the indolent development of interstitial fibrosis and glomerular injury that leads to graft dysfunction and failure at later times after transplantation.

Delayed Donor Bone Marrow Infusion Induces Liver Transplant Tolerance

Nonmyeloablative conditioning followed by donor bone marrow infusion (BMI) to induce tolerance has not been robustly tested in liver transplantation (LT) and may be unsafe at the time of LT. We hypothesized T cell-depleted BMI is effective in inducing tolerance when delayed after LT, resulting in potentially safer future clinical applications. Nonimmunosuppressed syngeneic (Lewis to Lewis) and allogeneic (ACI to Lewis) rat LT transplants were initially performed as controls. Three experimental allogeneic LT groups were treated with tacrolimus (TAC) for 3 to 4 weeks and then underwent: (1) TAC withdrawal alone; (2) nonmyeloablative conditioning (anti-αβTCR mAb + total body irradiation [300 cGy]) followed by TAC withdrawal; (3) Nonmyeloablative conditioning + donor BMI (100 × 10 T cell-depleted bone marrow cells) followed by TAC withdrawal. All group 1 recipients developed chronic rejection. Group 2 had long-term survival but impaired liver function and high donor-specific antibody (DSA) levels. In contrast, group 3 (conditioning + BMI) had long-term TAC-free survival with preserved liver function and histology, high mixed chimerism and blood/liver/spleen CD4 + CD25 + Foxp3+ regulatory T cells, and low DSA titers, similar to syngeneic grafts. While donor-specific tolerance was observed post-BMI, graft-versus-host disease was not. These results support that donor-specific tolerance can be achieved with BMI even when delayed after LT and this tolerance correlates with increased mixed chimerism, regulatory T cell generation, and diminished DSA.

P157 Accelerated graft loss due to non-complement fixing donor-specific antibody against a single DP epitope

Aim The clinical significance of donor-specific antibody (DSA) solely to DP antigens remains controversial. Here we report a case of accelerated graft loss attributed to DSA against a single DPB epitope which was exacerbated by the “peanut butter” phenomenon observed with the solid-phase single antigen bead (SAB) assays. Methods A 48-yo white female with history of ESRD due to hypertension received her 3rd renal transplant (TX) from a deceased donor with 5/6 ABDR mismatch and a 100% cPRA. Her other sensitizations included a pregnancy and multiple transfusions. Results Despite the presence of DSA to DPB1 ∗ 02:01 at 7100 MFI, TX was proceeded in light of clearly negative flow crossmatches (FCXM). Post-TX urine output initially improved but trended down on post-operative day (POD) 2 with elevated serum creatinine ⩾ 6 mg/dL. While SAB on POD3 indicated a decline of DP2 DSA to 4200 MFI, biopsy revealed a Banff IA rejection, with peritubular capillaritis and endothelial cell and vascular injury, suggestive of humoral components. Subsequent SAB on POD13 showed the addition of de novo DSA to B37 at 4300 MFI. Despite immediate intervention with ATG/IVIG/Rituxan/plasmapheresis, the patient developed a profound septic syndrome and a nephrectomy was performed on POD24. Allograft biopsy showed strong evidence of AMR with diffuse cortical necrosis and hemorrhage, arterial thromboses, intimal arteritis, with C4d+ in the peritubular capillaries. Epitope analysis suggested the preexisting DP DSA was in fact directed to a single epitope, 56EE, shared amongst 9 beads comprising 6 DPB1 alleles (02:01/04:02/10:01/18:01/28:01), thereby inadvertently diluting the actual DSA titer as shown on individual beads (“the peanut butter effect”). While it was not being detected by FCXM, the cumulative pre-TX MFI of the DP DSA was indeed > 65,000. Retrospective testing indicated that this DP DSA was never complement fixing, suggesting an involvement of antibody dependent cell-mediated cytoxicity. An intermediate level of anti-AT1R antibody was detected post-TX, which may have further accelerated the graft rejection process. Conclusions This case illustrates the deleterious effect of DSA solely to DP antigens. Epitope analysis may facilitate a more accurate assessment of the actual DSA titer, especially for the highly sensitized patients.

Acquired Downregulation of Donor-Specific Antibody Production After ABO-Incompatible Kidney Transplantation.

The mechanism of long-term B cell immunity against donor blood group antigens in recipients who undergo ABO-incompatible (ABOi) living-donor kidney transplantation (LKTx) is unknown. To address this question, we evaluated serial anti-A and anti-B antibody titers in 50 adult recipients. Donor-specific antibody titers remained low (≤1:4) in 42 recipients (84%). However, antibodies against nondonor blood group antigens were continuously produced in recipients with blood type O. We stimulated recipients' peripheral blood mononuclear cells invitro to investigate whether B cells produced antibodies against donor blood group antigens in the absence of graft adsorption invivo. Antibodies in cell culture supernatant were measured using specific enzyme-linked immunosorbent assays (ELISAs). Thirty-five healthy volunteers and 57 recipients who underwent ABO-compatible LKTx served as controls. Antibody production invitro against donor blood group antigens by cells from ABOi LKTx patients was lower than in the control groups. Immunoglobulin deposits were undetectable in biopsies of grafts of eight recipients with low antibody titers (≤1:4) after ABOi LKTx. One patient with blood type A1 who received a second ABOi LKTx from a type B donor did not produce B-specific antibodies. These findings suggest diminished donor-specific antibody production function in the setting of adult ABOi LKTx.

Anti-huCD20 Antibody Therapy for Antibody-Mediated Rejection of Renal Allografts in a Mouse Model

We have reported that B6.CCR5(-/-) mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody-mediated rejection (AMR). B6.huCD20/CCR5(-/-) mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long-term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

Renal Allograft Rejection

Renal Allograft Rejection

Efficacy of CTLA4-Ig in Preventing Acute Rejection of Fully Allogeneic Heart Allografts in Sensitized Recipients.

Belatacept, a high-affinity variant of CTLA4-Ig, is a recently approved immunosuppressive agent for the prevention of acute rejection. It functions as a potent antagonist of the CD28-B7 interactions, thereby inhibiting T cell co-stimulation and activation. Experimental data involving the transient use of CTLA4-Ig to induce allograft tolerance have shaped the current paradigm that CTLA-4Ig is ineffective at inhibiting memory T cell responses, and by extension T-dependent alloantibody responses. This resistance is explained by the acquisition of additional activation-induced costimulatory molecules by memory T cells. We noted that the efficacy of CTLA4-Ig as a continuously administered drug for controlling rejection in sensitized recipients has not been investigated. Here we report on the efficacy of CTLA4-Ig in preventing acute rejection in sensitized C57BL/6 recipients that had received BALB/c spleen cell transfusion 3-6 months previously. We set up four experimental groups. Group 1 (N=5): sensitized mice with HTX and CTLA4-Ig treated (Day -2, Day 0, Day2, then weekly (1 mg/dose) Group 2 (N=3): sensitized mice with HTX and no CTLA4-Ig, Group 3: (N=10) naive mice with HTX and CTLA4-Ig treated, Group 4 (N=10): naive mice with HTX and no CTLA4-Ig. In the untreated Groups 2 & 4, allografts were rejected within 7 days post-transplantation, whereas in Group 3 of naive mice receiving CTLA4-Ig, all the grafts survived long-term (>90 days). In Group 1 of sensitized recipients receiving CTLA4-Ig, the allografts survived for 42, 42, 49, >90, >90 days. The pre-transplant donor-specific antibody (DSA) titers in the recipients that went on to reject their graft was significantly higher compared to those that accepted their grafts. Furthermore, DSA titers increased post-transplantation in the recipients that went on to reject their allografts, whereas DSA titers decreased in those that accepted their grafts. In conclusion, these observations illustrate the efficacy of CTLA-4Ig treatment at controlling rejection in sensitized recipients, and underscore the barrier pre-transplantation DSA poses to long-term graft acceptance. This experimental model also provides a unique opportunity for testing if DSA promotes antibody-mediated rejection or alloreactive memory T cell priming and T cell-mediated rejection in CTLA4-Ig treated recipients, and for identifying therapies that promote long-term graft survival in sensitized recipients.

Eosinophils Are Not Required for the Induction and Maintenance of an Alloantibody Response

Eosinophil-produced cytokines have been shown to participate in the maintenance of antigen-specific plasma cells (PC) in bone marrow (BM), suggesting that eosinophils are required in the development and/or maintenance of alloantibody responses posttransplant. To test this hypothesis, we sensitized eosinophil-deficient ΔdblGATA1 mice and wild-type (WT) control mice with allogeneic splenocytes or with allogeneic heart grafts and compared the kinetics and titers of serum donor-specific antibodies (DSA), as well as BM and spleen CD130 + B220 low PC populations between groups. Spleen cells from naïve ΔdblGATA1 BALB/c mice contained higher percentages of PC than WT without detectable differences in BM PCs. After sensitization with allogeneic splenocytes, BALB/c ΔdblGATA1 mice contained fewer BM PCs but more splenic PCs compared to controls. These differences were associated with modestly lower titers of serum DSA 4 and 12 weeks after sensitization but secondary immunizations induced similar increases in both groups. Moreover, the kinetics and strength of DSA did not differ in WT and ΔdblGATA1 BALB/c mice transplanted with B6 cardiac allografts, nor did they differ in transplanted ΔdblGATA1 and WT mice on a B6 background. Therefore, eosinophils are not required for alloantibody formation or maintenance in mice and are thus unlikely to be effective targets for antibody desensitization.

Transient Lymphopenia Breaks Costimulatory Blockade-Based Peripheral Tolerance and Initiates Cardiac Allograft Rejection

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long-term graft acceptance. After vascularized MHC-mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti-CD4 plus anti-CD8 mAb. Long-term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44high effector/memory T cells in lymphatic organs and strong recovery of donor-reactive T cell responses, indicating lymphopenia-induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4+ Foxp3+) cell and B cell numbers as well as donor-specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor-reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.

Treatment of Antibody-Mediated Rejection in Kidney Transplant Recipients: A Single-Center Experience With a Bortezomib-Based Regimen

Antibody-mediated rejection after kidney transplant is less responsive to conventional antirejection therapies. The proteasome inhibitor bortezomib has activity against mature plasma cells that produce damaging donor-specific antibodies. We present our experience of using a bortezomib-based regimen in patients with severe antibody-mediated rejection. A retrospective chart review was performed on patients with biopsy-proven antibody-mediated rejection after kidney transplant at our institution over 12 months. Diagnosis of antibody-mediated rejection was made on the basis of positive peritubular capillary C4d staining along with either histologic evidence of acute rejection or positive donor-specific antibody titers. Treatment for antibody-mediated rejection included plasmapheresis, intravenous immunoglobulin, steroids, single-dose rituximab (375 mg/m²) along with bortezomib (1.3 mg/m²) on days 1, 4, 8, and 11. Antibody-mediated rejection was diagnosed in 6 patients. Patients received induction with either alemtuzumab (n=4) or rabbit-antithymocyte globulin (n=2) and were maintained on a tacrolimus/mycophenolate mofetil/early steroid withdrawal protocol. Four of 6 patients responded to treatment. Patients had stable kidney function during followup (median 14 months) after bortezomib therapy. In this series, we demonstrated the effectiveness of a bortezomib-based treatment regimen in achieving reduction of donor-specific antibody titers and stable renal function in patients experiencing severe antibody-mediated rejection.