Donor-specific Antibody Production Research Articles

Does donor age matter? Biological and immunogenic differences between infant and aged mesenchymal stromal cells

Over the last decades, mesenchymal stromal cells MSC) have emerged as a promising therapeutic tool for cell-based therapies. However, their biological characteristics and beneficial potential may decline with patients’ age. Because most patients amenable for regenerative medicine have increased age, this could dramatically limit the autologous use of MSCs. We therefore sought to characterize the biological, immunological, and angiogenetic capacities of infant (iMSCs) versus aged MSCs (aMSCs). Both iMSCs and aMSCs showed typical spindle-shaped morphology, plastic adherence, and the same potential to differentiate into osteogenic, chondrogenic and adipogenic lineages. iMSCs and aMSCs expressed MSC-typical surface markers such as CD90 and CD105 and were negative for CD45, CD34, CD31 and CD117. iMSCs showed faster in vitro proliferation (p<0.001), migration (p<0.001), and stronger endothelial adhesion (p=0.018) than aMSC. There were no differences in HLA class I, β2 microglobulin, HLA class II, and co-stimulatory molecule expression between iMSCs and aMSCs. Both immunogenic HLA class II and CD54 and tolerogenic HLA-E expression were upregulated by IFNγ. iMSCs showed significantly higher CD29 signals and stronger endothelial adhesion than aMSCs. Interestingly, iMSCs outperformed aMSCs in different angiogenesis parameters. If not used in an autologous setting, iMSCs and aMSCs similarly provoked TH1 and TH2 responses in unidirectional ELISPOT assays and donor-specific IgM antibody production. Our results thus suggest that donor age does not affect immunogenicity and immunomodulatory properties, but markedly reduces biological properties of proliferation, migration, adhesion, and limits their angiogenic effects.

(909) - HLA Donor Specific Antibody Production in ABO-Compatible Versus Incompatible Heart Transplant Recipients

A prior study showed lack of production of donor specific antibodies to HLA antigens (DSAs) in non-sensitized ABO-incompatible (ABO-i) and similar age ABO-compatible (ABO-c) recipients. We examined whether ABO compatibility has an effect on de novo DSA production in an American cohort regardless of pre-transplant sensitization status. A retrospective cohort study of recipients < 1 year of age at transplant with > 6 months of follow-up, transplanted between 1/2004-10/2013 at a single U.S. pediatric heart transplant center. Outcomes included development of DSAs and history of rejection. DSA positivity was defined as either the lowest level detectable by assay, mean fluorescence intensity (MFI) >500, or suggestive of clinical relevance, MFI>5000. Of 47 patients, 33 received ABO-c transplants and 14 received ABO-i transplants (median age 5.5 mo vs. 4.2 mo, p=0.577). Median duration of follow-up was 45 mo for ABO-c and 47 mo for ABO-i (p=0.745). There were no significant differences between the two groups in gender, cardiac diagnosis, prior open heart surgery, prior homograft, crossmatch, pre-transplant sensitization to anti-HLA antibodies, number of HLA mismatches, and immunosuppression regimen. Compared to ABO-c recipients, the ABO-i group consistently showed a trend of less frequent de novo DSA positivity and rejection (see Table). For the entire cohort, there was a significant association between antibody-mediated rejection and DSA positivity (36.8% in DSA-positive vs. 7.1% in DSA-negative using MFI>5000 as the cutoff, p=0.021; and 31% vs. 0% using MFI>500, p=0.008). A similar though not statistically significant trend was seen for cellular rejection. Our data suggest that patients receiving ABO-i heart transplants within the first year of life develop fewer DSAs compared to recipients of ABO-c transplants, which may lead to fewer rejections. Larger sample sizes with adjustment for covariates will be needed to confirm results from this pilot study.

Anti-Thymocyte Gamma-Globulin May Prevent Antibody Production After Heart Transplantation

BackgroundRabbit anti-thymocyte globulin (RATG) has been used as induction therapy in heart transplantation. RATG is polyclonal and has been postulated to have anti-humoral properties by preventing the production of circulating antibodies after heart transplant. Thus, we reviewed our patients who received RATG induction therapy and compared them with those who did not receive therapy for post-transplant de novo antibody production. MethodsBetween January 1, 2006, and January 1, 2013, we assessed 196 non-sensitized heart transplant recipients and divided them into those who received 3 to 5 days of RATG induction therapy mostly due to renal insufficiency (n = 35) versus patients who did not receive therapy (n = 161). All patients were given tacrolimus, mycophenolate mofetil, and corticosteroids. Post-transplant circulating antibodies were routinely monitored at 1, 3, 6, and 12 months after heart transplantation; 1-year and 3-year end points were assessed. ResultsThe RATG-treated group had a significantly higher 12-month freedom from de novo antibody production compared with the patients who did not receive RATG induction (89% vs 71%, log-rank P = .043); however there was no significant difference for 12-month freedom from de novo donor-specific antibody production (91% vs 88%, log-rank P = .541). Treated rejection rates in the first-year were comparable in both groups; 3-year actuarial survival, freedom from cardiac allograft vasculopathy, and freedom from non-fatal major adverse cardiac events were also similar between both groups. ConclusionsRATG induction therapy appears to reduce the production of de novo circulating antibodies in non-sensitized patients during the first year after heart transplantation. Although there were no short-term clinical differences between groups, there were imbalances in group characteristics and relatively short follow-up, which are limitations to this study. A randomized, clinical trial with longer follow-up in a larger cohort of patients is warranted.

Donor-specific antibodies in allograft recipients: etiology, impact and therapeutic approaches.

Kidney transplantation remains the treatment of choice for patients with end-stage renal failure. However, despite significant advancements in detection of donor-specific human leukocyte antigen antibodies, improved immunosuppression and patient management, the durability of this life-saving therapy has not improved. This results in increased morbidity and mortality as well as increased cost to the healthcare system. The identification of immune-pathogenic pathways responsible for allograft failure coupled with targeted interventions will represent one of the most important future objectives of transplant immunologist and physicians. The development of sensitive donor-specific antibody (DSA) detection techniques and advancements in renal allograft pathology assessments have revealed the importance of humoral immunity in mediating allograft failure. This is especially true for complement activating DSAs (C1q+). Our current understanding suggests that reduction of immunosuppressive medications or medication nonadherence is now the major causes of DSA development and attendant pathology. Other important factors in initiation of de-novo DSA production include viral infections, human leukocyte antigen-DR/DQ mismatches and autoimmune diseases. Therapies aimed at antibody reduction, B-cell depletion and modification of the complement system will likely usher in new therapeutic approaches for prevention and treatment of DSA-mediated allograft dysfunction.

Efficacy and Safety of Everolimus Based Immunosuppression On De Novo Kidney Transplantation With 5 Years Follow-Up Especially in Protocol Biopsy Findings and Donor Specific Antibody Production.

Purpose: The impact of everolimus (EVR) based immunosuppression in De Novo Kidney Transplantation was evaluated in clinical outcomes, protocol biopsies findings and donor specific antibody (DSA) production.Methods: During 2008 and August 2009, we enrolled twenty-four living donor kidney transplant patients in a 2-year, multicenter, randomized phase 3 study (RAD001A1202 study) to compare the safety and efficacy between EVR based and mycophenolate mofetile (MMF) based immunosuppression. We extended further three years and evaluated clinical outcome in a single center.EVR group received reduced-exposure cyclosporine (target C0 25-50ng/ml after 6 months) + steroid, and EVR-C0 were adjusted 3-8ng/ml. MMF group received standard-exposure cyclosporine (target C0 100-250ng/ml after 6 months) + steroid. Both group received basiliximab induction.Results: All patients continued in each regimen. With a mean observation period of 60.1 (43.8-69.4) months, current patient and graft survival is 100% in both groups (EVR; n=13, MMF; n=11). Renal function expressed as eGFR (ml/min/1.73m2) was similar 40.7±14.6 in EVR group and 37.4±8.6 in MMF group. Significant proteinuria, more than 300mg/day, were observed more in EVR group (76.9%) than in MMF group (54.5%), however the proteinuria in EVR group was successfully treated with angiotensin-II receptor blockade. None of EVR and MMF group was treated for clinical T cell mediated rejection, similar incidence of Banff borderline change on 6 or 12 months protocol biopsies were observed in 7.7% of EVR group and 18.2% of MMF group, but none of both groups revealed peritubular capillaritis. Luminex solid phase assay revealed the similar incidence of class II DSA production in both groups, 7.7% of EVR group at 2 years after transplant and 15.4% of MMF group at 3 years. Protocol oral glucose tolerance test at 6 and 12 months revealed more, but no significantly, worsen profile in 46.1% of EVR group to 18.2% in MMF group.Conclusions: EVR based immunosuppression provides equivalent clinical outcomes as well as the incidence of De Novo DSA production with MMF based immunosuppression with 5 years follow-up.

Defining and Refining Tfh Cells in Antibody Responses and The Role of OX40 Costimulation.

Follicular helper T cells (Tfh) specifically provide help to the maturation and development of B cells in secondary lymphoid tissue follicle. They assist the antigen experienced B cells to form antibody producing cells, and they also provide help to the class switch of antibodies. As the master transcriptional factor, Bcl6 expression is instrumental to Tfh development. However, memory Tfh are defined as Bcl6-CXCR5+ CD4+ T cells, suggesting complexity of Bcl6 in Tfh subsets. In the follicle there is another CXCR5+ CD4 T cell subset which also expresses Foxp3. These follicular regulatory T cells (Tfr) cells inhibit the germinal center formation and antibody production mediated by Tfh. Besides antigen specific TCR interaction, the priming of Tfh also requires assistant from co-stimulation signal from antigen presenting cells. OX40, a member of the TNFR-superfamily, is a co-stimulation receptor presenting on the activated CD4 T cells surface. It is known to be related to the development of several CD4 subsets. Here we demonstrated decreased Tfh development and antibody production in OX40-/- mice after immunization. When given antigen and excess OX40 ligand (OX40L) as stimulation signal, there are more Tfh and Tfr cells both developed in OTII mice. In OX40L transgenic mice, there are more memory Tfh cells developed and enhanced secondary response with antigen re-boost, but the excess OX40 signal can not increase Tfh development in P52-/- mouse as in wild type mice. So it indicates the OX40 signaling mediated Tfh development is activated through non-canonical NFkB pathway. In the allograft transplantation model, donor graft provides antigen and triggers strong immune responses. In both the skin and heart transplantation model, we observed increased Tfh development and donor specific antibody production. In the skin transplantation model, the secondary transplantation will recall both Tfh and antibody production to a higher level. The allograft tissue was rejected faster than the first transplantation. All of these indicate the Tfh might be related to the antibody mediated allograft rejection. We uncovered the relationship of OX40 signaling with Tfh development, further studies are ongoing to investigate its function in regulating allograft rejection in transplantation model. These studies will be important in developing better therapies to prevent antibody-mediated graft damage.

Angiotensin II Type I Receptor Antibodies and Kidney Graft Outcome.

Purpose. In kidney transplantation, antibodies specific for non-HLA antigens has been associated with augmented risk for antibody mediated rejection (AMR) because of their ability to determine vascular lesions and consequently allograft injury. Some studies associated antibodies specific for Angiotensin II Type-1 Receptor (AT1R) with antibody-mediated kidney allograft rejection. To analyzed the role of HLA and non-HLA antibodies on graft survival, we studied de novo development of AT1R-antibodies in kidney transplanted patients showing or not donor-specific anti-HLA antibody (DSA) production and correlated the results of antibody analysis with kidney graft outcome. Methods. Sera from 52 kidney transplanted patients were analyzed for AT1R-antibodies using a new ELISA assay. Thirty-two of the 52 patients had had developed post-transplant DSA; in 27 of these we also tested the ability of DSA to fix C1q. As for graft outcome, 31 patients suffered graft failure/dysfunction for AMR defined by clinical data and/or histopathologic findings according to the Banff classification. Data on C4d staining of biopsies were available in 22 patients only. Results. Sixteen (31%) of the 52 kidney transplanted patients resulted DSA negative/AT1R-antibody negative and did not show AMR: 3 of these suffered graft dysfunction for chronic allograft nephropathy, and 6 had hypertension. Twenty (38%) patients were DSA positive/AT1R-antibody negative: 16 suffered graft failure/dysfunction for AMR, and 4 had hypertension. Thirteen of the 15 DSA positive/AT1R-antibody negative patients were tested for C1q antibodies and showed positivity for C1q fixing DSA. Twelve (23%) patients resulted DSA positive/AT1R-antibody positive: all showed graft failure/dysfunction for AMR, and 11 were C1q-DSA positive also. The remaining 4 (8%) patients showed DSA negative/AT1R-antibody positive results: 3 suffered graft dysfunction for AMR. The production of DSA only or DSA plus AT1R-antibodies showed significant associations with kidney graft failure/dysfunction (P=0.0006 and P<0.0001 respectively). Conclusion. Our study confirms the crucial role of HLA-DSA production in kidney transplantation and underlines the synergic effects of AT1R-antibody development. Monitoring for anti-AT1R should help in patients risk assessment and represents a non-invasive tool for identifying patients who need specific therapies to prolong graft survival.

Either Abatacept or Belatacept in Combination With Donor Bone Marrow Infusion Induces Immune Tolerance Across a Full MHC Barrier in a Translational Large Animal Vascularized Composite Allotransplantation Model.

BACKGROUND: The establishment of tolerance protocols would avoid the debilitating effects of chronic immunosuppression and render Vascularized Composite Allotransplantation (VCA) a routine procedure. We investigated graft survival and tolerance induction using co-stimulation blockade with either abatacept or the higher-affinity equivalent drug belatacept. METHODS: Fully MHC- and gender mismatched MGH miniature swine underwent heterotopic hind-limb transplantation. Recipient animals received a short course (30 days) of tacrolimus monotherapy, +/- donor BM infusion, and CTLA4Ig (either abatacept or belatacept). Tacrolimus only and untreated animals served as controls. Recipients were assessed for: chimerism (SRY-gene PCR analysis), alloreactivity against donor antigens (in vitro CFSE-MLR assays), and donor-specific antibodies (DSA) production (serum flow cytometry analysis). Robust immune tolerance in vivo was assessed by skin grafting. Tolerance maintenance upon allograft removal was tested by CFSE-MLR. RESULTS: Both regimens using abatacept or belatacept plus donor BM infusion resulted in indefinite graft survival (>200 days). While only transient peripheral chimerism was observed, stable micro-chimerism in various graft and recipient tissues was achieved. An increase in intragraft Tregs was seen in tolerant animals as compared to controls. Tolerant animals displayed unresponsiveness to donor but not to third party allogeneic controls in CFSE-MLRs. Donor-matched skin grafts were accepted while third party grafts where quickly rejected indicating donor-specific tolerance. No signs of chronic rejection or donor-specific antibodies were seen in tolerant recipients in either group (abatacept vs belatacept). Graft removal, however, was associated with loss of donor-unresponsiveness 5 weeks post-graftectomy. CONCLUSIONS: Combined costimulation blockade and donor BM cell infusion induced robust donor-specific immune tolerance in a translational fully MHC-mismatched hind limb transplant model.

Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.

(442) - Local Production of Donor Specific Antibodies (DSA) By Intrapulmonary De Novo Lymphoid Tissue Associated With Allograft Airway Rejection

De novo lymphoid tissue formation has been recognized in allograft lungs affected by obliterative bronchiolitis (OB). The aim of the study is to investigate local production of DSA by intrapulmonary de novo lymphoid tissue. A rat intrapulmonary tracheal transplant model of OB was used (allotransplant; Brown-Norway (BN) to Lewis, isotransplant; Lewis to Lewis). Recipient rats were sacrificed at postoperative day 2 (d2), day 14 (d14), day 28 (d28), day 84 (d84) (n=4-5/group) to recover the left lung, spleen and serum. Sliced lung and spleen (200 mg in weight) were cultured for 4 days without further stimulus. DSA levels in the supernatant or serum were measured by flowcytometry after incubation with splenocytes (Lewis or BN-derived) and then with labeled anti-rat IgG. DSA levels at d2 were used as a baseline. De novo lymphoid tissue formation was minimal in isotransplant with no graft obliteration over time, while allograft recipient lungs exhibited dense lymphoid tissue with complete graft obliteration by d28. DSA produced in the spleen showed about a 3.5-fold increase at d14, and then decreased over time with significant difference at d14 and d28 (p=0.006 and 0.024, respectively; Fig.1). Recipient serum also showed significantly high levels of DSA at d14 and d28 (p=0.022 and 0.003, respectively) and then decreased to an insignificant level. Interestingly, however, DSA produced in the lung showed continuous increase with significance after day 28 (d28, p=0.003; d84, p<0.001), reaching a 5-fold increase at d84. We first demonstrated local DSA production in the lung in an animal model of OB. In contrast to only temporal increase in DSA in the spleen and serum, DSA produced in the lung persisted over time. These results suggest the possibility that DSA is continuously produced by de novo lymphoid tissue in the lung and contributes to graft damage even after the serum level of DSA becomes undetectable.

Antibody-Mediated Rejection of Arterialised Venous Allografts Is Inhibited by Immunosuppression in Rats

Objectives and DesignWe determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts.Materials and MethodsFlow cytometry was used for the analysis of day 0, 14 and 30 sera obtained from Lewis recipients of isogeneic iliolumbar vein grafts (group A) or Brown-Norway grafts (group B, C) for the presence of donor specific anti-MHC class I and II antibodies. Tacrolimus 0.2 mg/kg daily was administered from day 1 to day 30 (group C). Histology was performed on day 30.ResultsSera obtained preoperatively and on day 30 were compared in all groups. The statistically significant decrease of anti MHC class I and II antibody binding was observed only in allogenic non-immunosuppressed group B (splenocytes: MHC class I - day 0 (93%±7% ) vs day 30 (66%±7%), p = 0.02, MHC class II - day 0 (105%±3% ) vs day 30 (83%±5%), p = 0.003; B-cells: MHC class I - day 0 (83%±5%) vs day 30 (55%±6%), p = 0.003, MHC class II - day 0 (101%±1%) vs day 30 (79%±6%), p = 0.006; T-cells: MHC class I - day 0 (71%±7%) vs day 30 (49%±5%), p = 0.04). No free clusters of immunoglobulin G deposition were detected in any experimental group.ConclusionArterialized venous allografts induce strong donor-specific anti-MHC class I and anti-MHC class II antibody production with subsequent immune-mediated destruction of these allografts with no evidence of immunoglobulin G deposition. Low-dose tacrolimus suppress the donor-specific antibody production.

Costimulation Blockade Alters Germinal Center Responses and Prevents Antibody-Mediated Rejection

De novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.

The Effect of Anti-Human Leukocyte Antigen, Anti-Major Histocompatibility Complex Class 1 Chain-Related Antigen A, and Anti-Glutathione Transferase-T1 Antibodies on the Long-Term Survival of Renal Allograft

BackgroundOne of the most important mechanisms of allograft rejection is the production of donor-specific antibodies (DSA). Anti-major histocompatibility complex class-I chain-related antigen A (MICA) and anti-glutathione S transferase-T1 (GSTT1) antibodies cause graft dysfunction and reduce graft survival. The aim of this study was to examine the effects of anti-human leukocyte antigen class I–II, anti-MICA, and anti-GSTT1 antibodies in development of antibody-mediated rejection. MethodsAmong the 32 renal transplant patients included in this study 65% experienced antibody-mediated rejection (AMR; chronic active AMR [CAMR], n = 17; acute AMR [AAMR], n = 4) and 35%, ACR. The anti-HLA class I–II and anti-MICA antibodies were determined by using LUMINEX, anti-GSTT1 antibodies by enzyme-linked immunosorbent assay. GSTT1 genotyping of patients and donors was performed by polymerase chain reaction. ResultsAntibody was detected in 19 of 21 patients undergoing antibody-mediated rejection (90%). We detected anti-GSTT1 in 4, anti-MICA in 8, anti-HLA class I in 5, and anti-HLA class II in 9 patients with CAMR (P = .007). If the patients were divided into 2 groups according to being C4d+ and C4d− both anti-HLA class I and class II antibodies were found significantly more frequently among the C4d+ group (P = .019, P = .024). No difference was determined between AMR and ACR groups in terms of anti-GSTT1 and anti-MICA antibodies. ConclusionsIn this study, we observed the role of anti-HLA class II antibodies in the development of CAMR and in long-term allograft survival. It is observed that anti-MICA and anti-GSTT1 antibodies showed no effect on rejection mechanisms.

Immunobiology of SCNT Derived Embryonic Stem Cells: Pivotal Role of mtDNA in Rejection of Transplanted Stem Cells

Background: For regenerative therapies, somatic cell nucleus transfer (SCNT) can be used to transfer the nucleus of a somatic cell into the cytoplasm of an enucleated egg. The resulting SCNT-embryo has the capability to develop to the blastocyst stage. Subsequently, embryonic stem cells (ESCs) can be derived from the inner cell mass of these blastocysts. These ESCs are genetically identical with the donor nucleus, with the exception of mitochondrial DNA (mtDNA) from the receiving egg. This study aimed to investigate the role of mtDNA in ESC transplantation and its immunological recognition. Methods: SCNT-ESCs were generated by transferring donor cells from Balb/C background into enucleated oocytes from BDF1 background. 1×106 of these SCNT-ESCs or SCNT-ESC-derived cardiomyocytes (CMs) were injected into Balb/C (mitochondria-incompatible) or BDF1 (nucleus-incompatible) mice (n=10). After 5 days, cellular and humoral responses were investigated by ELISPOT, donorspecific antibodies, histopathology, and confocal immunofluorescence microscopy. Immunodeficient SCIDbeige mice were used as controls (n=6). In vivo bioluminescence imaging was used to visualize cell survival longitudinal. The amount of mitochondria in undifferentiated ESCs was compared to ESC-derived CMs using the TMRM assay (Figure 1).[Figure 1]Results: Using ELISPOT assays for Th1, Th2, and Th17-cells, we found that foreign mitochondria caused a similar immune response compared to ESCs with foreign nuclear DNA, both being significant higher than in SCIDbeige (p< 0.001). Similar results were observed for the humoral response. Re-injection of ESCs resulted in an adaptive immune response, highlighting the role of T-cell mediated rejection as shown by ELISPOT assays and in vivo bioluminescence imaging. Antigenicity of ESCs increased with their differentiation into CMs, correlating with increased MHCI-expression and amount of mitochondrial antigens (Figure 1). Conclusion: mtDNA is coding for 13 proteins, which surprisingly resulted in an adaptive immune response and donorspecific antibody production after transplantation. Upon ESC differentiation into CMs we observed an increase of mitochondrial antigens resulting in a greater immunological recognition of transplanted cells. In summary, SCNT-derivates are not sufficient immune privileged and undergo immune rejection after transplantation.

Clinical Relevance of C1q-Fixing HLA Antibodies in Kidney Transplantation

Introduction: De novo production of donor-specific HLA antibodies (DSA) represents the major risk factor of graft failure in kidney transplantation. However, some patients show persistent presence of circulating DSA without occurrence of graft dysfunction/loss. The complement-dependent citotoxicity (CDC) assay has demonstrated low sensitivity in detecting DSA. Newer solid phase assays, such as Luminex Single Antigen (LSA) beads (microbeads coated with single HLA class I or class II molecules), are highly sensitive but less predictive of transplant outcome because of detection of both complement-fixing and less clinically relevant no complement-fixing HLA antibodies. Methods: Using a modified LSA assay that identifies antibodies able to fix C1q, we investigate the clinical relevance of de novo HLA-DSA in relation to their capability to fix complement. In serum samples from 40 kidney transplanted patients who had developed IgG-LSA DSA after transplantation, we measured C1q-fixing ability of detected DSA by Class I and Class II LSA beads (One Lambda, CA). As for transplant outcome, 22 (55%) patients suffered graft failure (within 10±1 months from DSA appearance) and 18 (45%) had good graft function (GF) during all the follow up (mean follow up from DSA appearance: 54±34 months). Results: Twenty-three of the 40 patients showed production of C1q-positive DSA; the remaining 17 patients produced C1q-negative DSA. Correlating graft outcome and capability of DSA to fix C1q, we evidenced that graft failure due to antibody-mediated rejection occurred in 20 of the 23 patients showing C1q-positive DSA; only 2 of the 17 patients showing C1q-negative DSA suffered graft failure (87% vs. 12% respectively, P< 0.0001; RR = 7.39; Sensitivity=0.91; Specificity=0.83; PPV=0.87; NPV=0.88). It is to underlay that both C1q-positive and C1q-negative DSA were mainly specific for DQ mismatched molecules of the donor (74% and 53% respectively). Analyzing the target molecules of DQ-specific DSA in relation to the ability to fix complement, we evidenced that 53% (9/17) of anti-DQ C1q-positive DSA were specific for DQ1 molecules while 75% (6/8) of anti-DQ C1q-negative DSA were specific for DQ2 molecules. Conclusion: Our findings demonstrate that C1q-LSA assay shows the capability to identify the subset of IgG-LSA DSA strongly associated to antibody-mediated rejection and graft loss in kidney transplantation. Moreover the ability of C1q assay in distinguishing less harmful no complement-fixing DSA from clinically relevant C1q-fixing DSA represent a non-invasive tool for identifying patients that need specific immunosuppressive-therapy strategy to prolong graft survival.

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In 2008, an international collaborative study of post-transplant monitoring was launched with 44 transplant centers from 17 countries. One aim of the project was to study the time course of antibody development, from time of transplant to months and years post-transplantation, to examine the incidence of antibodies, time to antibody appearance, and time to clinical events. We also investigated possible factors affecting antibody development across multiple centers. As of May 2012, antibody data for 5,057 serial sera from 470 kidney and 97 heart recipients were submitted. For each patient, 3 to 12 sera were collected at various intervals from 1 mo to 14 yrs post-transplantation. Antibody testing was done by luminex methodology. Patient data including age, ethnicity, HLA mismatches, immunosuppression, serum creatinine, and clinical events were collected. An Antibody Tracking Program was developed to analyze antibody profile. At the time of this report, analysis was done for 224 patients. Figure shows an example of antibody plot generated by the tracking program. Incidence of donor-specific antibodies (DSA) at first year was approx. 10%. Median time to antibody appearance was 34 mo. Factors increasing DSA development included deceased donors and level of HLA mismatches. We are continuing to receive data from centers, and further investigation of factors affecting DSA production is underway. In studying natural history of antibody development across multiple centers, we are now able to identify some of the common factors that are predictive of DSA production, and gain an insight as to the time course of antibody development and clinical outcome [Fig. 1]. Ozawa: One Lambda Inc.: Employee. Everly: One Lambda Inc.: Employee. Terasaki: One Lambda Inc.: Stockholder.

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Aim De novo production of donor-specific HLA antibodies (DSA) represents the major risk factor of graft failure in kidney transplantation. However, some patients show persistent presence of circulating DSA without occurrence of graft dysfunction/loss. Newer solid phase assay Luminex Single Antigen (LSA) beads, is highly sensitive respect to complement-dependent citotoxicity assay but less predictive of transplant outcome because of detection of both complement-fixing and less clinically relevant no complement-fixing HLA antibodies. Methods Using Class I and II C1q-LSA assay (One Lambda,CA), that identifies antibodies able to fix C1q, we investigate the clinical relevance of de novo HLA-DSA in 40 kidney transplanted patients. As for transplant outcome, 22 patients suffered graft failure (within 10 ± 1 months from DSA appearance) and 18 had good graft function during all the follow up (mean 54 ± 34 months from DSA appearance). Results Twenty-three patients showed production of C1q-positive DSA while 17 produced C1q-negative DSA. Correlating graft outcome and capability of DSA to fix C1q, graft failure occurred in 20/23 C1q-positive DSA patients; only 2/17 C1q-negative DSA patients suffered graft failure (87% vs.12%, P Conclusions C1q-LSA assay showed the capability to identify the subset of IgG-LSA DSA strongly associated to antibody-mediated rejection and graft loss in kidney transplantation; moreover, its ability in distinguishing less harmful no complement-fixing DSA from clinically relevant C1q-fixing DSA, allows to identify patients that need specific immunosuppressive strategy to prolong graft survival.

Using Donor-Specific Antibodies to Monitor the Need for Immunosuppression

Experience with tolerance protocols has shown that none is perfect and that each escape from tolerance must be identified early to prevent graft failure. In addition, some test is needed for patients who are weaned off immunosuppression (IS) to forewarn of weaning failure. The usual measures of function--such as serum creatinine levels--are not sensitive enough to detect rejection in a timely manner. A study was carried out on 72 patients who received living-donor kidney transplants with clonal deletion protocol (total lymphoid irradiation or bortezomib), and followed with reduced doses of maintenance IS. Every month or every 2 months, a test was performed for donor-specific antibodies (DSA) using Luminex mixed and/or single antigen beads. After transplantation, DSA developed in 17% of the patients at 6 months, 41% at 1 year, and 57% at 2 years, with 95% confidence limits of 10%, 28%; 30%, 55%; and 44%, 71%, respectively. Fifty-three percent of patients weaned IS to less than 10 mg prednisone daily experienced DSA within 3 months. Furthermore, prednisone dose (per 2.5 mg) and years after transplantation were inversely associated with DSA production (risk ratio 0.92 [95% confidence limits: 0.85, 0.99], and 0.70 [0.49, 1.00]). DSA monitoring is highly effective for detecting escape from tolerance and reemergence of the immune response in weaned patients. DSA appearance was inversely proportional to the level of maintenance drugs in the weaning process. Measurement of DSA on a monthly basis is adequate for detection of the change in immune reactivity.

Luminex and antibody detection in kidney transplantation

Preformed anti-human leukocyte antigen (HLA) antibodies have a negative effect on kidney transplantation outcome with an increased rejection rate and reduction in survival. Posttransplantation production of donor-specific anti-HLA antibodies is indicative of an active immune response and risk of transplantation rejection. For many years the primary technique for anti-HLA antibody detection was complement-dependent cytotoxicity (CDC), which has been integrated by solid-phase assays as HLA antigen-coated bead methods (Luminex). This new technological approach has allowed identification of anti-HLA antibodies, not detectable using conventional CDC method, in patients awaiting kidney transplantation. Moreover, use of Luminex technology has enabled better definition of acceptable or unacceptable antigens favoring transplantation in highly immunized patients. However, there are still many unresolved issues, including the clinical relevance of antibodies detected with this system.

Antibody-mediated rejection of the heart in the setting of autoimmune demyelinating polyneuropathy: a case report and review of the literature.

Background. Antibody-mediated rejection (AMR) is caused by the production of donor-specific antibodies (DSA) which lead to allograft injury in part via complement activation. The inflammatory demyelinating polyneuropathies (IDP) are inflammatory disorders of the nervous system, involving both cellular and humoral immune mechanisms directed against myelin. Case Report. A 58-year-old man five years after heart transplant presented with progressive dyspnea, imbalance, dysphagia, and weakness. Nerve conduction studies and electromyogram were consistent with IDP. Plasmapheresis and high-dose steroids resulted in improvement in neurologic symptoms. Within two weeks, he was readmitted with anasarca and acute renal failure, requiring intravenous furosemide and inotropic support. Echocardiogram and right heart catheterization revealed reduced cardiac function and elevated filling pressures. DSA was positive against HLA DR53, and endomyocardial biopsy revealed grade 1R chronic inflammation, with strong capillary endothelial immunostaining for C4d. Plasmapheresis and intravenous immunoglobulin (IVIG) were initiated. His anasarca and renal failure subsequently resolved, echocardiogram showed improved function off inotropes, and anti-DR53 MFI was reduced by 57%. Conclusions. This is an example of a single immune-mediated process causing concurrent IDP and AMR. The improvement in cardiac function and neurologic symptoms with plasmapheresis, IVIG, and high-dose steroids argues for a unifying antibody-mediated mechanism.