Use of Rituximab for persistent EBV DNAemia, and Its effect on donor-specific antibody development in pediatric renal transplant recipients: A case series.

INTRODUCTION: Persistent EBV DNAemia (PEBV) is associated with late-onset PTLD. The efficacy of rituximab in PEBV is not conclusive. We monitored PEBV and DSA in pediatric kidney transplant patients with or without rituximab. METHODS: 13 PEBV patients received standard treatment with immunosuppression reduction and valganciclovir, with or without IVIG; 5/13 were further treated with rituximab. RESULTS: All Rituximab-treated and 6/7 No-Rituximab patients were EBV seronegative at transplant and seroconverted post-transplant. Peak EBV PCR levels were lower in No-Rituximab than Rituximab patients and all No-Rituximab patients cleared PEBV after standard treatment. Additional 1-2 doses of rituximab reduced EBV PCR levels in all 5 Rituximab patients, 3 cleared PEBV. One No-Rituximab patient developed localized PLTD. None of Rituximab patients developed de novo DSA, while 4/8 No-Rituximab patients did: 2/4 had ABMR. 1/5 Rituximab and 5/8 No-Rituximab patients had acute rejection. There was no change in eGFR between pre-EBV DNAemia and follow-up in Rituximab patients, while reduction in No-Rituximab patients was found. There was no difference in graft and patient survival. CONCLUSIONS: While early intervention with rituximab in pediatric patients with PEBV may reduce viral load and PTLD, we observed a slower development of de novo DSA, and rejection and maintenance of eGFR.

Impact and production of Non-HLA-specific antibodies in solid organ transplantation.

Organ transplantation is an effective way to treat end-stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long-term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody-mediated rejection is a major obstacle for long-term graft survival. Donor human leucocyte antigen (HLA)-specific antibodies were initially identified as a major cause for antibody-mediated rejection. Recently, with the development of solid-phase-based assay reagents, the contribution of non-HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non-HLA antibodies, including angiotensin II type 1 receptor (AT1 R), K-α-tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.

Angiotensin II type I receptor antibodies in thoracic transplantation.

Angiotensin II type I receptor (AT1R) is a critical player in regulating vasoconstriction, blood pressure, sodium retention. Even though AT1R has limited polymorphism, AT1R antibodies have been detected in several diseases. The role of AT1R antibodies in transplantation is first reported in kidney transplant, and then identified in heart and lung transplants. Mechanical circulatory support devices (MCS) can also stimulate production of AT1R antibodies. AT1R antibodies may negatively impact graft or patient survival through mechanisms independent of the classical complement activation.

The management of antibodies in heart transplantation: An ISHLT consensus document.

Despite the successes from refined peri-operative management techniques and immunosuppressive therapies, antibodies remain a serious cause of morbidity and mortality for patients both before and after heart transplantation. Patients awaiting transplant who possess antibodies against human leukocyte antigen are disadvantaged by having to wait longer to receive an organ from a suitably matched donor. The number of pre-sensitized patients has been increasing, a trend that is likely due to the increased use of mechanical circulatory support devices. Even patients who are not pre-sensitized can go on to produce donor-specific antibodies after transplant, which are associated with worse outcomes. The difficulty in managing antibodies is uncertainty over which antibodies are of clinical relevance, which patients to treat, and which treatments are most effective and safe. There is a distinct lack of data from prospective trials. An international consensus conference was organized and attended by 103 participants from 75 centers to debate contentious issues, determine the best practices, and formulate ideas for future research on antibodies. Prominent experts presented state-of-the-art talks on antibodies, which were followed by group discussions, and then, finally, a reconvened session to establish consensus where possible. Herein we address the discussion, consensus points, and research ideas.

Revealing a new mode of sensitization induced by mechanical circulatory support devices: Impact of anti-AT1 R antibodies.

BACKGROUND: Increased levels of angiotensin II type 1 receptor (AT1 R) antibody have been shown to be associated with allograft rejection. This study aims to determine the rate of development of antibody to AT1 R after mechanical circulatory support device (MCS) implantation, and if the development of strong binding AT1 R antibodies is associated with survival. METHODS: Eighty-eight patients who had one MCS implantation were accessed based on serum availability. Mechanical circulatory support devices in this cohort included pneumatic bilateral paracorporeal ventricular assist device, continuous flow left ventricular assist device, and total artificial heart. RESULTS: Of 88 patients, seven patients had AT1 R antibodies ≥40 U/mL preimplantation. For 81 patients who had AT1 R antibodies <40 U/mL, the median value was 8 U/mL. Of these 81 patients, AT1 R antibody levels in 55 (68%) patients reached the saturated concentration (≥40 U/mL) postimplantation (P < .0001), with the highest percentage of patients with the saturated level of AT1 R antibody observed in the pneumatic bilateral paracorporeal ventricular assist device group. Compared to patients without the saturated level of AT1 R antibodies, patients with the saturated AT1 R antibody level had lower 18-month survival (P = .040). CONCLUSION: Mechanical circulatory support devices implantation significantly increases AT1 R antibody levels. The saturated level of AT1 R antibodies is associated with lower patient survival postimplantation.

Comprehensive assessment for serum treatment for single antigen test for detection of HLA antibodies.

The single antigen test is widely used in the field of transplantation to determine the specificity of HLA antibodies. It will be beneficial to standardize the procedure of the single antigen test among HLA laboratories. It is not uncommon that single antigen testing on native sera fails to detect antibodies with very high concentrations. It has been shown that cleavage products of activated complement components may mask strongly binding antibodies in single antigen testing. To overcome inhibition by the activated complement products, sera are pretreated with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or heat inactivation before single antigen testing. However, no studies have been published to systemically compare the impact of these treatments on single antigen testing. The aim of this study is to understand the different effects these treatments may have on single antigen test results. We found that mean fluorescence intensity (MFI) obtained from sera treated with EDTA and heat inactivation were nearly identical, while DTT treatment was less potent to remove the inhibition. In addition, sera dilution did not further increase MFI of antibodies after EDTA treatment. Our results provide guidance to choose a pretreatment reagent for single antigen testing, and to compare studies obtained from laboratories using different treatments.

Impact of Non-Human Leukocyte Antigen-Specific Antibodies in Kidney and Heart Transplantation.

The presence of donor human leukocyte antigen (HLA)-specific antibodies has been shown to be associated with graft loss and decreased patient survival, but it is not uncommon that donor-specific HLA antibodies are absent in patients with biopsy-proven antibody-mediated rejection. In this review, we focus on the latest findings on antibodies against non-HLA antigens in kidney and heart transplantation. These non-HLA antigens include myosin, vimentin, Kα1 tubulin, collagen, and angiotensin II type 1 receptor. It is suggested that the detrimental effects of HLA antibodies and non-HLA antibodies synergize together to impact graft outcome. Injury of graft by HLA antibodies can cause the exposure of neo-antigens which in turn stimulate the production of antibodies against non-HLA antigens. On the other hand, the presence of non-HLA antibodies may increase the risk for a patient to develop HLA-specific antibodies. These findings indicate it is imperative to stratify the patient's immunologic risk by assessing both HLA and non-HLA antibodies.

Differences in pathologic features and graft outcomes in antibody-mediated rejection of renal allografts due to persistent/recurrent versus de novo donor-specific antibodies.

Antibody-mediated rejection (ABMR) of renal allografts occurs in two forms. Type 1 ABMR results from persistence and/or a rebound of preexisting donor-specific antibodies in sensitized patients and usually occurs early post-transplantation. Type 2 ABMR is associated with de novo donor-specific antibodies and usually occurs over one year post-transplantation. It is generally accepted that types 1 and 2 also differ with regard to certain pathologic features including the frequencies of C4d positivity and concurrent cell-mediated rejection. However, direct comparison of pathologic, serologic, and clinical features of types 1 and 2 ABMR is lacking. Here we compared these features in 80 cases of ABMR (37 type 1, 43 type 2) diagnosed at our center. Compared with type 1, type 2 ABMR occurred later post-transplantation, was more often associated with donor-specific antibodies against Class II HLA, and was associated with more interstitial fibrosis/tubular atrophy and more frequent cell-mediated rejection, although these did not differ with respect to C4d positivity. By univariate analysis, graft survival was lower with type 2 than type 1 ABMR with borderline significance. Still, among these 80 patients, all but one treated for ABMR following diagnosis, the only two independent predictors of graft failure were at least moderate interstitial fibrosis/tubular atrophy and failure of the donor-specific antibody relative intensity scale score, a measure of the combined strength of all donor-specific antibodies present, to decrease in response to therapy.

Corticosteroid wean after heart transplantation-Is there a risk for antibody formation?

BACKGROUND: Corticosteroid withdrawal after heart transplantation is limited to select immune-privileged patients but it is not known whether this predisposes patients to a higher risk for sensitization. METHODS: A total of 178 heart transplant recipients had panel-reactive antibody (PRA) measurements at transplant and every 6 months and were monitored for rejection with protocol endomyocardial biopsies. Corticosteroid withdrawal was initiated at 6 months post-transplant in select patients. RESULTS: Patients successfully weaned off prednisone (SPW; n=103) had lower PRA compared to those maintained on prednisone (MP; n=51) at pretransplant (34% vs 63%), 6 months (18% vs 49%), 12 months (19% vs 51%), and 18 months (15% vs 47%) after transplant (P<.05). Among 68 nonsensitized patients at transplant in the SPW group, seven (10%) developed de novo PRA at 12 months, compared to four of 19 (21%) of MP patients. Freedom from any treated rejection (97% vs 69% vs 67%), acute cellular rejection (100% vs 86% vs 71%), and antibody-mediated rejection (100% vs 88% vs 88%; all P≤.001) at 2 years was higher in SPW compared to MP and those who failed prednisone wean, respectively. CONCLUSION: Few patients successfully weaned off prednisone after heart transplant develop de novo circulating antibodies but are not at increased risk for developing rejection.

A 3-Center Study Reveals New Insights Into the Impact of Non-HLA Antibodies on Lung Transplantation Outcome.

BACKGROUND: The presence of antibodies to angiotensin type 1 receptor (AT1R) and endothelin type A receptor (ETAR) is associated with allograft rejection in kidney and heart transplantation. The aim of our study was to determine the impact of AT1R and ETAR antibodies on graft outcome in lung transplantation. METHODS: Pretransplant and posttransplant sera from 162 lung recipients transplanted at 3 centers between 2011 and 2013 were tested for antibodies to AT1R and ETAR by the enzyme-linked immunosorbent assay (ELISA) assay. Clinical parameters analyzed were: HLA antibodies at transplant, de novo donor-specific antibodies (DSA), antibody-mediated rejection (AMR), acute cellular rejection, and graft status. RESULTS: Late AMR (median posttransplant day 323) was diagnosed in 5 of 36 recipients with de novo DSA. Freedom from AMR significantly decreased for those recipients with strong/intermediate binding antibodies to AT1R (P = 0.014) and ETAR (P = 0.005). Trends for lower freedom from acute cellular rejection were observed for recipients with pretransplant antibodies to AT1R (P = 0.19) and ETAR (P = 0.32), but did not reach statistical significance. Lower freedom from the development of de novo DSA was observed for recipients with antibodies detected pretransplant to AT1R (P = 0.054), ETAR (P = 0.012), and HLA-specific antibodies (P = 0.063). When the pretransplant antibody status of HLA-specific antibody (hazard ratio [HR], 1.69) was considered together with either strong binding to AT1R or ETAR, an increased negative impact on the freedom from the development of de novo DSA was observed (HR, 2.26 for HLA antibodies and ETAR; HR, 2.38 for HLA antibodies and ETAR). CONCLUSIONS: These results illustrate the increased negative impact when antibodies to both HLA and non-HLA antigens are present pretransplant.

Non-human leukocyte antigen-specific antibodies in thoracic transplantation.

PURPOSE OF REVIEW: Development of donor human leukocyte antigen (HLA)-specific antibodies is associated with graft loss, yet the role of non-HLA antibodies in solid organ transplant needs to be further defined. It is suggested that HLA antibodies and non-HLA antibodies collaborate together to impact graft outcome. This review focuses on the latest findings on antibodies against these non-HLA antigens in thoracic organ transplant. RECENT FINDINGS: These non-HLA antigens include signaling proteins expressed on the cell surface, such as angiotensin II type 1 receptor (AT1R), endothelin type A receptor, and structure proteins, such as myosin, vimentin, and Kα1 tubulin, and extracellular matrix protein collagen. Antibodies against these antigens may impact the allograft in different ways. Although these non-HLA antibodies can damage the allograft through complement-mediated or cell-mediated cytotoxicity, antibodies against AT1R and endothelin type A receptor can also alter the endothelial cell function by activating intracellular signals. The presence of these non-HLA antibodies may predispose the patient to develop HLA-specific antibodies. Recently, it has been shown patients with AT1R antibodies pretransplant have a higher chance to develop de-novo donor-specific HLA antibodies. SUMMARY: The findings suggest it is important to stratify the patient's immunologic risk by assessing both the HLA and non-HLA-specific antibodies.

Antibody-mediated rejection of the lung: A consensus report of the International Society for Heart and Lung Transplantation.

Antibody-mediated rejection (AMR) is a recognized cause of allograft dysfunction in lung transplant recipients. Unlike AMR in other solid-organ transplant recipients, there are no standardized diagnostic criteria or an agreed-upon definition. Hence, a working group was created by the International Society for Heart and Lung Transplantation with the aim of determining criteria for pulmonary AMR and establishing a definition. Diagnostic criteria and a working consensus definition were established. Key diagnostic criteria include the presence of antibodies directed toward donor human leukocyte antigens and characteristic lung histology with or without evidence of complement 4d within the graft. Exclusion of other causes of allograft dysfunction increases confidence in the diagnosis but is not essential. Pulmonary AMR may be clinical (allograft dysfunction which can be asymptomatic) or sub-clinical (normal allograft function). This consensus definition will have clinical, therapeutic and research implications.

CPRA for allocation of kidneys in the US: More candidates ≥98% CPRA, lower positive crossmatch rates and improved transplant rates for sensitized patients.

In 2009 calculated panel reactive antibody (CPRA) replaced PRA as the metric for HLA sensitization in the US kidney allocation system. During the next four years, registrants with at least one unacceptable antigen increased (34-40%) and registrants with ≥98% PRA/CPRA increased from 7% to 9% of the waitlist. These changes were accompanied by a reduction in kidney offers refused for positive crossmatch: 14,137 (1.7%) in 2009 and 3,310 in 2013 (0.4%). Registrants with ≥98% PRA/CPRA had highest rates of refusal but also showed substantial improvement (20% in 2009 vs 8% in 2013). For registrants with ≥98% PRA/CPRA, 45% of accepted offers in 2009 were not transplanted into the intended recipient compared to 11% in 2013. Transplant rates remained low for these patients (∼50/1000 active patient-years), but rates improved for patients with 80-97% PRA/CPRA (223/1000 active patient-years in 2009 vs 354/1000 in 2013). In 2013, 40% regraft candidates had CPRA ≥98% compared to 4% of primary graft candidates. More females than males were ≥98% CPRA (14% vs 7%) and more females had CPRA above 0 (50% vs 28%). In the CPRA era, listing of unacceptable antigens increased, positive crossmatches were diminished and transplant rates for sensitized patients improved.

Optimizing transplantation of sensitized heart candidates using 4 antibody detection assays to prioritize the assignment of unacceptable antigens.

BACKGROUND: The virtual crossmatch relies on the assignment of unacceptable antigens (UAs) to identify compatible donors. The purpose of our study was to identify an algorithm for assignment of UAs such that a negative complement-dependent cytotoxicity (CDC) crossmatch and concomitant negative or weakly positive flow cytometric crossmatch (FXM) are obtained. METHODS: We used 4 antibody methods: (1) Luminex single antigen (LSA), (2) LSA with a 1:8 serum dilution, (3) C1q LSA, and (4) CDC panel. The UAs were prioritized in the following order: (1) all C1q+/CDC+, (2) LSA 1:8 >7,500 median fluorescence intensity, and (3) LSA >10,000 median fluorescence intensity. RESULTS: Of 295 heart transplants that were performed at our center, 69 (23%) recipients had detectable human leukocyte antigen specific antibody at the time of transplant. All donor specific antibodies (DSAs) were avoided for 44 of 69 (64%) (DSA-). There were 25 recipients who had DSA at the time of transplant: 12 (48%) had negative FXM (DSA+/FXM-), and 13 (52%) had positive T-cell and/or B-cell FXM (DSA+/FXM+). Lower freedom from antibody-mediated rejection was observed for the DSA+/FXM+ group compared with the DSA- group (p < 0.0001). DSA remained detectable after transplant in the sera of 14 recipients, and de novo DSA was detected in 32 recipients. Freedom from antibody-mediated rejection was comparable for both groups (p = 0.53) but was lower than the DSA- group (p < 0.0001). Survival was comparable for all groups at 1,200 days post-transplant. CONCLUSIONS: Strategic prioritization of UA assignment has allowed transplantation of highly sensitized patients across the DSA barrier with survival rates comparable to DSA- heart transplant recipients.

A Phase I/II Trial of the Interleukin-6 Receptor-Specific Humanized Monoclonal (Tocilizumab) + Intravenous Immunoglobulin in Difficult to Desensitize Patients.

BACKGROUND: Current desensitization (DES) methods are not always effective. Thus, novel, more effective approaches are desirable. Interleukin (IL)-6 is an attractive target as it promotes B-cell differentiation to plasma cells, is important for immunoglobulin production, and induces Th17 cells. Here, we undertook a phase I/II pilot study of DES using a novel drug (anti-IL-6 receptor (IL-6R),Tocilizumab [TCZ]) + intravenous Ig (IVIg) to assess safety and limited efficacy. METHODS: From July 2012 to November 2013, 10 patients unresponsive to DES with IVIg + Rituximab were treated with IVIg + TCZ. Patients received IVIg on days 0 and 30 at 2 g/kg and TCZ 8 mg/kg on day 15 then monthly for 6 months. If transplanted, patients received IVIg once and TCZ monthly for 6 months. RESULTS: No differences in baseline characteristics were seen in patients not transplanted versus transplanted. Two patients in each group developed serious adverse events: not transplanted- pulmonary congestion with epilepticus (likely not related) versus transplanted infective colitis with colonic perforation and Bell Palsy (both possibly related). Five of 10 patients were transplanted. Mean time to transplant from first DES was 25 +/- 10.5 months but after TCZ was 8.1 +/- 5.4 months. Six-month protocol biopsies showed no antibody-mediated rejection. Donor-specific antibody strength and number were reduced by TCZ treatment. Renal function at 12 months was 60 +/- 25 mL/min. CONCLUSIONS: Tocilizumab and IVIg appear to be safe. From this pilot trial, we are cautiously optimistic that targeting the IL-6/IL-6R pathway could offer a novel alternative for difficult to desensitize patients. Larger controlled studies are essential to prove efficacy

A phase I/II placebo-controlled trial of C1-inhibitor for prevention of antibody-mediated rejection in HLA sensitized patients.

BACKGROUND: Antibody-mediated rejection (AMR) is a severe form of rejection, mediated primarily by antibody-dependent complement (C) activation. C1 inhibitor (C1-INH, Berinert) inhibits the classical and lectin pathways of C activation. We performed a randomized, placebo-controlled study using C1-INH in highly sensitized renal transplant recipients for prevention of AMR. METHODS: Twenty highly sensitized patients desensitized with IVIG+rituximab±plasma exchange were enrolled and randomized 1:1 to receive plasma-derived human C1-INH (20 IU/kg/dose) versus placebo intraoperatively, then twice weekly for 7 doses. Renal function, adverse events (AEs)/serious AEs, C3, C4, and C1-INH levels were monitored and C1q+ HLA antibodies were also blindly assessed. RESULTS: One patient in the C1-INH group versus 2 patients in the placebo group developed serious AEs, but none were related to study drug. Delayed graft function developed in 1 C1-INH subject and 4 in the placebo. The C1-INH trough levels increased with C1-INH treatment. C3 and C4 levels also increased significantly in the C1-INH group compared to placebo. No C1-INH patient developed AMR during the study. Two patients developed AMR after the study. Three placebo patients developed AMR, one during the study. C1q+ donor specific antibodies were reduced in 2 C1-INH treated patients tested, while immunoglobulin G DSA levels showed decreased binding for both groups. CONCLUSIONS: The C1-INH appears safe in the posttransplant period. The C1-INH treatment may reduce ischemia-reperfusion injury. The C1-INH also resulted in significant elevations of C1-INH levels, C3, C4, and reduced C1q+ HLA antibodies. Taken together, the combination of antibody reduction and C1-INH may prove useful in prevention of AMR. Further controlled studies are warranted.

Benefits of rituximab combined with intravenous immunoglobulin for desensitization in kidney transplant recipients.

BACKGROUND: Highly HLA-sensitized (HS) patients have difficulty accessing compatible donors, especially deceased donor (DD) transplants. Desensitization protocols (DES) have evolved, but rigorous evaluation is lacking. Here, we examined the efficacy of rituximab as a DES agent in a placebo-controlled trial. METHODS: Candidates were randomized to IVIG+placebo versus IVIG+rituximab. End points included rates of transplantation, antibody-mediated rejection (ABMR), and renal function. Protocol biopsies were performed at 1 year and analysis of patient and graft survival and donor-specific HLA antibodies (DSA) were performed. RESULTS: Initially, 15 HS DDs were randomized with 13 receiving transplants. However, we discontinued study entry after five serious adverse events were observed. The study was un-blinded and attribution of patients was noted (IVIG+placebo N=7, IVIG+rituximab N=6). No significant differences were seen in DSA levels at transplant. All ABMR episodes occurred in the IVIG+placebo arm and required intense therapy (P=0.06). The two graft losses were in the placebo group. DSA rebound associated with severe ABMR was seen in three patients in the IVIG+placebo group. No rebound was seen in the IVIG+rituximab group. Renal function at 6 and 12 months showed a significant benefit for IVIG+rituximab (P=0.04). CONCLUSIONS: Based on limited assessment with acknowledged limitations, both protocols appear effective in achieving levels of DSA allowable for transplantation. However, IVIG+rituximab appeared more effective in preventing DSA rebound and, more importantly, preventing ABMR and development of transplant glomerulopathy.

Polyomavirus BK viremia in kidney transplant recipients after desensitization with IVIG and rituximab.

BACKGROUND: Desensitization with intravenous immune globulin (IVIG) and rituximab improves transplantation rates. It is unclear if desensitization increases the risk of polyomavirus BK (BKV) viremia. Here, BKV viremia in HLA-sensitized patients after desensitization with IVIG and rituximab was analyzed. METHODS: Baseline characteristics and outcomes were compared in the desensitized group (N=187) and the non-desensitized group (N=284). Surveillance for BKV viremia was done at 1, 2, 3, 6, 9, and 12 months posttransplant. Univariable and multivariable analyses were performed. RESULTS: BKV viremia was observed in 20% of the desensitized and 10% of the non-desensitized (P<0.001) groups by 2 years posttransplant. The desensitized group had more lymphocyte depleting induction and more rejection. They also had a greater degree of viremia with more patients having a peak viral load greater than 10,000 copies per milliliter (P<0.001). However, there was no significant difference in BKV-associated nephropathy or graft loss in the two groups. There was an association of BKV viremia with desensitization and lymphocyte induction. Only desensitization remained a significant predictor in the multivariable model with an adjusted HR of 2.13 (95% CI 1.21-3.77, P=0.009). CONCLUSIONS: Desensitization with IVIG and rituximab is associated with a higher incidence of BKV viremia with high viral copies and was the major predictor of BKV viremia in the multivariable model. More frequent surveillance for BKV viremia and an early, aggressive treatment strategy are essential for preventing high BKV viral loads in this patient population.