Aim Antibody-mediated Rejection Research Articles

The efficacy of rituximab treatment for antibody-mediated rejection in liver transplantation: A retrospective Japanese nationwide study.

Antibody-mediated rejection (AMR) has been consistently elucidated in liver transplantation (LT); however, the treatment for AMR, including rituximab, has not been indicated as a strongly recommended therapeutic protocol. This study was conducted as the Japanese multicenter retrospective study to accumulate data on the use of rituximab for AMR among patients undergoing LT between August 2001 and December 2016. Thirteen patients (five children and eight adults) were enrolled. The types of AMR in the pediatric cases were chronic AMR in four cases and indeterminate AMR in one case. Among the pediatric cases, rituximab treatment only showed therapeutic efficacy in two patients with chronic AMR. Among the adult patients, five patients had chronic AMR, and three had acute AMR. Although two patients with chronic AMR died due to graft failure, liver function tests revealed improvement after rituximab treatment in the other patients. Two of the three patients with acute AMR died due to graft failure; rituximab treatment showed no therapeutic efficacy in these cases. Although bacterial infections occurred within 3months after rituximab administration in three patients, rituximab treatment could be safely administered without any direct adverse effects. The indication of rituximab therapy as an additional treatment for mild acute AMR and chronic AMR may be feasible; however, a prospective randomized control study is needed to evaluate the therapeutic efficacy of rituximab treatment for AMR.

OR4 Development of an in vitro model of human antibody mediated rejection

Aim Antibody-mediated rejection (AbMR) remains a leading barrier to long term transplant survival. The immune mechanisms of AbMR are incompletely understood but recent evidence suggests that monocytes and NK cells are potential effectors against HLA ab-coated endothelium. We developed an in vitro model of human AbMR that incorporates the immune components currently known. Methods Human umbilical vein endothelial cells (HUVECs) served as endothelial targets and were pretreated with IFN-g for 48 h to maximize HLA expression. HLA abs were derived from sera pooled from highly sensitized patients on our waitlists. Primary NK cells and monocytes were isolated by immunomagnetic depletion from healthy controls and added to HLA ab-coated HUVECs. RNA was extracted for global gene expression analyses after 8 h. Analyses focused on 3 representative sets of transcripts: IFN-g response, chemokines, and endothelial activation (Table 1). Results NK cell/ HLA ab-mediated attack leads to increased chemokines that promote additional NK cell and monocyte recuitment (Fig. 1), many reflecting endothelial IFN-g effects. Adhesion molecules key to recruitment and leukocyte engagement were also increased, some of these being required for optimal NK cell activation (ICAM1). The presence of monocytes in the system (despite being separated from NK-HUVECs) increased all 3 responses observed with NK cells, particularly endothelial activation. Conclusions Our results suggest that we have a working model of human AbMR that incorporates 2 effector cell types, HLA abs, and HUVEC targets. This model argues for a key effector role by NK cells in AbMR, whose function is greatly enhanced by soluble mediators from monocytes in close proximity.

P060 Antibody-mediated and cellular rejection: How can they seem so similar?

Aim Antibody-mediated rejection (ABMR) involves a cellular component where NK cell CD16 activation plays a pivotal role. In T-cell mediated rejection (TCMR) CD8+ T cells; the most closely related cell to NK cells, are most prevalent. We used global gene expression analyses to determine the similarities in gene expression changes following CD16 and CD3 stimulation of NK cells and CD8+ T cells, respectively and examined their expression in kidney transplant biopsies. Methods Cells were purified from PBMCs by immunomagnetic separation and stimulated in vitro on plates coated with crosslinking antibodies or uncoated plates. NK (N = 3) and CD8+ T cells (N = 3) were cultured for 4 h in the presence of IL2 prior to harvest. RNA was extracted and analyzed on Affymetrix gene expression arrays and supernatants underwent multiplex analysis for soluble mediators. Results We identified 276 transcripts increased (>2X, FDR Conclusion The cognate events that drive the rejection process in ABMR (NK cell CD16 activation) and TCMR (CD8+ T cell TCR/CD3 activation) are surprisingly similar. Our study helps us understand the confusion regarding the accurate diagnosis of rejection due to similarities and invites exploration of T-cell directed therapies for potential treatment of ABMR. P.F. Halloran: Stock Shareholder; Company/Organization; P F Halloran holds shares in Transcriptome Sciences Inc., a company with an interest in molecular diagnostics..

P119 Single-center experience in routine post-transplant antibody testing of kidney transplant recipients without pre-existing donor specific anti-HLA antibodies

Aim Antibody-mediated rejection (AMR) of kidney allografts is thought to follow a stepwise process which includes alloimmune recognition, followed by microscopic changes (subclinical rejection) and then allograft dysfunction (clinical rejection). There is data to suggest that development of de novo donor-specific antibodies (DSA) is associated with subclinical and clinical rejection. Following a recent expert consensus guideline, we implemented routine post-transplant antibody testing for patients to capture de novo DSA to determine how often we detected de novo DSA and whether we could use de novo DSA in the clinical decision making process. Methods The Beth Israel Deaconess Medical Center Transplant program instituted routine post-transplant antibody testing in mid-2014. The frequency of post-transplant antibody testing was determined based on the presence or absence of DSA against kidney allograft. Patients without pre-existing DSA, based on cumulative pre-transplant testing, were considered low-risk for AMR and routinely tested at one year post-transplant for de novo DSA by multiplex bead array solid phase antibody screen. Results From May 2014-June 2015, 84 patients received a kidney allograft, 69 (82%) of whom were considered low-risk (i.e. no DSA ever detected by serial pre-transplant testing). Of the 69 low-risk patients, five patients were not tested for antibody at one-year post-transplant. Four of 64 (6%) low-risk patients were found to develop de novo DSA by one-year post-transplant. In three patients, there was concomitant proteinuria or elevated creatinine found at the time of post-transplant antibody testing. Two of three suffered AMR; one had chronic allograft changes. The fourth patient with de novo DSA had stable kidney function and is being monitored. Conclusions We detected de novo DSA in 6% of low risk renal transplant recipients with routine post-transplant monitoring at one year post-transplant. In three patients, there was concomitant laboratory findings of allograft dysfunction which prompted allograft biopsy. Further studies have to be performed to determine the optimal timing of routine antibody testing in low-risk patients after kidney transplantation.

OR18 C3D-Binding de novo donor-specific HLA antibodies and antibody-mediated rejection of kidney transplants

Aim Antibody-mediated rejection (AMR) is a major cause of kidney graft loss, yet assessment of individual risk at diagnosis is impeded by the lack of a reliable prognosis assay. Here, we tested whether the capacity of HLA antibodies to bind complement component C3d allows accurate risk stratification at the time of AMR diagnosis. Methods Sera from kidney transplant recipients, who underwent a protocol or for-cause kidney biopsy and had detectable de novo DSA (by One Lambda) at the time of biopsy (median 3.8 yrs post-tx), were included in this study. These serum samples were re-tested using the Immucor single antigen beads with and without C3d detection system. Results This study included samples from 123 kidney recipients (70 males; 14 re-Tx; 46 LD) transplanted in our center between 1989 and 2011. Fifty-seven patients (46%) had C3d-binding DSA. Most C3d-binding DSAs were high MFI DSAs (11700 + 5188), and only 4/57 (7%) C3d-binding DSAs had 5000 MFI. Seventy percent of the patients with C3d-binding DSA (40/57) had AMR (18 aAMR and 22 cAMR) and C4d-positive biopsies; twenty-six percent (15/57) had ACR, and four percent (2/57) had negative biopsies. Fifty-two present of the patients (34/66) in the C3d-negative DSA group had DSA with MFI 5000. Some of the weak and moderate DSA detected by One Lambda single antigen bead reagents were not detected with the Immucor SAB. Among the patients with C3d-negative DSA, thirty-five percent (23/66) had AMR (7 aAMR and 16 cAMR); twelve percent (8/66) had ACR and thirty-three percent (22/66) had C4d-positive biopsies. In most cases 15/22 (68%), the C4d-positive biopsies were observed in patients with strong DSA (MFI > 5000). Conclusions Our data indicate a strong correlation between the presence of C3d-binding DSAs and AMR. C3d-binding antibodies seem to be prevalent to stronger antibodies. Further studies are needed to evaluate whether the presence of C3d-binding donor-specific antibodies can predict AMR and identify patients who are at increased risk of allograft failure. Download : Download full-size image

OR37 Antibody ligation of Hla B*44:03 fails to stimulate endothelial cell proliferation due to a failure to recruit integrin beta4

Aim Antibody-mediated rejection (AMR) is an important clinical problem after solid organ transplantation. Recipients producing posttransplant HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Notably, not all patients producing post-transplant Ab develop AMR. We postulated that distinct HLA class I alleles may differ in their capacity to transduce signals. Our previous data showed that HLA-B*44:03 was less efficient than other B locus molecules, including B*44:02, to stimulate signal transduction networks in endothelial cells (EC) despite having a similar binding capacity. We tested the hypothesis that failure of B*44:03 to stimulate signal transduction and EC proliferation was due to its inability to recruit integrin β 4. Method Primary human aortic EC (HAEC) were stimulated with human HLA-B12 allele specific mAb, and phosphorylation of signaling proteins was detected by Western Blot. For protein:protein complex formation, HAEC were stimulated with F(ab’)2 fragment of anti-HLA class I (HLA I) mAb W6/32 or mouse IgG as a control. Cell lysates were immunoprecipitated with a mouse anti-HLA-B12 mAb recognizing both B*44:02 and B*44:03 alleles and complex formation between class I and integrin β 4 was detected by Western Blot. Gene silencing was determined with siRNA transfection. EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Treatment of HAEC with anti-B12 mAb stimulated a increase in phosphorylation of Src, mTOR, ERK and Akt in B*44:02, but not in B*44:03 HAEC. Co-immunoprecipitation experiments showed complex formation between HLA I and integrin β 4 in B*44:02 HAEC, but not in HAEC carrying B*44:03. Furthermore, transfection of EC with integrin β 4 siRNA inhibited HLA-B12 mAb-induced protein phosphorylation and cell proliferation. Conclusions Our results demonstrate that HAEC carrying B*44:03 fail to transduce proliferative signals because they are unable to recruit integrin β 4 into a signaling complex. Disrupting complex formation between integrin β 4 and HLA I may provide a new therapeutic target to prevent AMR.

OR17 Heme oxygenase-1 modulates HLA class I antibody-dependent endothelial cell activation

Aim Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in heart and kidney transplantation. Activation of endothelial cells (ECs) via complement-independent effects of human leukocyte antigen class I (HLA I) antibodies (Abs) plays a major role in the pathogenesis of AMR. As the antioxidant enzyme heme oxygenase (HO)-1 is known to have cell type-specific anti-inflammatory effects in the endothelium, we investigated its role on HLA I Ab-dependent activation of human ECs. Methods Regulation of inducible proinflammatory endothelial adhesion molecules and chemokines (VCAM-1, ICAM-1, IL-8 and MCP-1) by monoclonal pan- and allele-specific HLA I Abs was determined in cell cultures of primary human umbilical venous, aortic macrovascular and microvascular ECs. HO-1 was modulated by pharmacological regulators and siRNA-mediated knockdown. Adherence of THP-1 monocytes to ECs was determined by leukocyte adhesion assay. Results Exposure of human macro- and microvascular EC cultures to HLA I Abs caused endothelial activation, as indicated by up-regulation of VCAM-1, ICAM-1, MCP-1 and IL-8. This up-regulation was mediated via the phosphatidylinositol-3 kinase (PI3K)/Akt and NF- κ B pathways. Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent EC activation. Binding of THP-1 monocytes was enhanced in HLA I Ab-stimulated ECs. This effect was counteracted by HO 1 up-regulation. Conclusion HLA I Ab-dependent EC activation is modulated by specific HO-1 up-regulation. Thus, targeted regulation of endothelial HO-1 may be a novel therapeutic approach for the treatment of AMR in kidney and heart transplantation. S. Immenschuh: Grant/Research Support; Company/Organization; Else Kroner-Fresenius Stiftung EKFS 2012_A309.

OR38: CARFIZOMIB FOR REFRACTORY ANTIBODY MEDIATED REJECTION IN LUNG TRANSPLANTATION THE IMPACT ON DSA

Aim Antibody mediated rejection (AMR) after lung transplantation (LTx) is associated with early and severe chronic rejection and death. Conventional therapies consisting of plasma exchange and IVIG with and without Rituximab are partially effective. Newer therapies with proteasome inhibitors (Carfizomib (CFZ) and Bortezomib (BTZ)) that target both B and plasma cells may be more effective in removing the donor specific antibodies (DSA). CFZ, unlike BTZ, irreversibly inhibits the activity of 20S proteasome and results in less neuropathic side effects. Method We evaluated the impact of CFZ therapy in 7 LTx on DSA level and function and correlated with clinical outcome as measured by pulmonary function test. All 7 patients had DSA IgG specificity and titer measured by single antigen bead Luminex assay and DSA function of complement binding by C1q-screen at baseline and at 1 month and 3 months post-treatment. Results The specificity, strengths and C1q reactivity pre- and post-therapy is summarized in Table below. All 7 patients had class II DSA and 6/7 were DQ specific with C1q reactivity. 5/7 LTx responded to initial treatment with a drop in IgG DSA level, with negative C1q DSA reactivity ( 15,000 MFI the IgG DSA was inhibited (“prozone effect”), however IgG DSA significantly increased in the presence of EDTA (LTx 3, 5, 6, 7). Conclusion In this small cohort of LTx who mostly failed conventional therapies and have persistent DSA, CFZ based approach appears to be useful, initially. However, 5/7 LTx recovered their C1q DSA reactivity upon longer follow-up suggesting that one cycle of CFZ it may not be effective to impact the rebound of circulating DSA.

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Aim Antibody-mediated rejection (AMR) is a clinical problem of solid organ transplantation. Recipients producing anti-HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Anti-HLA Ab contribute to the process of AMR by binding to the HLA molecules on endothelial cells (EC) and triggering signal transduction pathways leading to cell survival and proliferation. We and others have reported that ligation of HLA-I with Ab on EC contributes to the process of transplant vasculopathy. However, the role of HLA-II Ab in this process is unknown. Methods Primary human aortic EC were infected with recombinant adenovirus pAd/PL-DEST encoding CIITA, the regulator of HLA-II expression, or treated with TNFa and IFNg. HLA-II expression was determined by FACS using Ab. CIITA infected or cytokine-treated EC were stimulated with anti-HLA-II Ab, phosphorylation was detected by Western Blot, and proliferation was measured by CSFE labeling and flow cytometry. Results Infection of EC with adenovirus-CIITA induced a 3 to 5-fold increase in HLA-II expression, including DP, DQ, and DR. Similarly, cytokine treatment of EC mediated a 2-fold increase in HLA-II expression on EC. Ligation of HLA-II antigens on CIITA infected and cytokine treated EC stimulated a marked increase in phosphorylation of Src and FAK, activation of mTORC1 pathway including S6K, S6RP, and 4E-BP1, and activation of mTORC2 signaling, Akt and ERK. The highest phosphorylation level was seen using 1.0 μg/ml of Ab. Ligation of HLA with class II mAb on EC also stimulated cell proliferation. Conclusions In this study we found that CIITA adenovirus infection and cytokine treatment can induce HLA-II expression on EC. Ligation of class II with Ab on EC triggered intracellular signal cascades promoting cell survival and proliferation. Our results provide novel methods to study the mechanisms of HLA-II Ab-mediated alterations in endothelial cells. These studies also suggest that HLA-II Ab may contribute to the process of chronic rejection.