Augusto Tagliamacco

Posttransplant De Novo Donor-Specific HLA Antibodies Identify Pediatric Kidney Recipients at Risk for Late Antibody-Mediated Rejection

The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti‐HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor‐specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical–pathologic data. At 4.3‐year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non‐DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA‐DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody‐mediated rejection (AMR), and four C4d‐negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1‐year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab‐negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.

Acquisition of C3d‐Binding Activity by De Novo Donor‐Specific HLA Antibodies Correlates With Graft Loss in Nonsensitized Pediatric Kidney Recipients

Alloantibody‐mediated graft injury is a major cause of kidney dysfunction and loss. The complement‐binding ability of de novo donor‐specific antibodies (dnDSAs) has been suggested as a prognostic tool to stratify patients for clinical risk. In this study, we analyzed posttransplant kinetics of complement‐fixing dnDSAs and their role in antibody‐mediated rejection development and graft loss. A total of 114 pediatric nonsensitized recipients of first kidney allograft were periodically monitored for dnDSAs using flow bead assays, followed by C3d and C1q assay in case of positivity. Overall, 39 patients developed dnDSAs, which were C1q+ and C3d+ in 25 and nine patients, respectively. At follow‐up, progressive acquisition over time of dnDSA C1q and C3d binding ability, within the same antigenic specificity, was observed, paralleled by an increase in mean fluorescence intensity that correlated with clinical outcome. C3d‐fixing dnDSAs were better fit to stratify graft loss risk when the different dnDSA categories were evaluated in combined models because the 10‐year graft survival probability was lower in patients with C3d‐binding dnDSA than in those without dnDSAs or with C1q+/C3d− or non‐complement‐binding dnDSAs (40% vs. 94%, 100%, and 100%, respectively). Based on the kinetics profile, we favor dnDSA removal or modulation at first confirmed positivity, with treatment intensification guided by dnDSA biological characteristics.

DQ molecules are the principal stimulators of de novo donor‐specific antibodies in nonsensitized pediatric recipients receiving a first kidney transplant

Data on the different HLA‐antibody (Ab) categories in pediatric kidney recipients developing de novo donor‐specific Abs (DSA) after transplantation are scarce. We retrospectively evaluated 82 consecutive nonsensitized pediatric recipients of a first kidney graft for de novo HLA Ab occurrence and antigen specificity. At a median follow‐up of 6 years, 29% of patients developed de novo DSA, while 45% had de novo non‐DSA. DSA appeared at 25‐month median time post‐transplant and were mostly directed toward HLA‐DQ antigens. Considering each HLA antigen, the estimated rate of DQ DSA (7.55 per 100 person‐years) was much higher than the rates observed for non‐DQ DSA. The HLA‐DQ Ab recognized determinants of the DQβ chain in 70% of cases, α chain in 25% of cases, and both chains in one patient. Non‐DSA peaked earlier than DSA, and were largely directed against HLA class I specificities that belonged to HLA‐A‐ and HLA‐B‐related cross‐reacting epitope groups (CREG) in 56% of cases. Our results indicate a need for evaluating HLA‐DQ compatibilities in kidney allocation, in order to minimize post‐transplant development of de novo DSA, known to be responsible for antibody‐mediated rejection and graft loss.

The Transcription Factor RFX Protects MHC Class II Genes against Epigenetic Silencing by DNA Methylation

Classical and nonclassical MHC class II (MHCII) genes are coregulated by the transcription factor RFX (regulatory factor X) and the transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein “enhanceosome” complex on MHCII promoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome in recruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome was revealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA was found to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was associated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNA methylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of the absence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQB promoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-type fibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA-independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retaining MHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors of APC and nonprofessional APC before induction with IFN-γ.

Cytokine mRNA expression in chronically rejected human renal allografts

Abstract:  Although both immunologic and non‐immunologic components may cause kidney allograft chronic rejection (KGCR), also referred to as chronic allograft nephropathy (CAN), its pathogenesis is largely not yet understood. To explore relevant immunologic mechanisms occurring in KGCR, we have analyzed in surgically removed KG the transcription of the following cytokine and apoptotic molecule genes: interleukin (IL)‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, tumor necrosis factor (TNF)‐α, IFN‐γ, FAS, and FAS‐L. Semiquantitative RT‐PCR was used and KG explants were obtained from two groups of transplanted patients. Group 1 was represented by CR/CAN KG, removed for: (a) superimposed symptoms of acute lesions (SAL) due to tapering or suspension of immunosuppression (subgroup 1a, eight cases); (b) causes other than SAL (two cases, subgroup 1b). Group 2 comprised explanted kidneys with no CR/CAN (three cases – vascular thrombosis, intrarenal hemorrhage and vascular thrombosis). The results showed that in group 1 IL‐ 6 was detectable in seven of 10, IL‐10 in six of 10, IFN‐γ in five of 10, and IL‐3 in four of 10 cases with a variable pattern of reciprocal association. IL‐2 and TNF‐α were represented in one of 10 cases only. Particularly, in the subgroup 1b IL‐10 was never detected. Among the most represented cytokines of group 1, IL‐10 as well as IL‐3 were never found in group 2. The peculiar expression of IL‐10 and IL‐3 and partially IL‐6 seems to support the hypothesis that a Th2 pattern predominantly characterizes KGCR, thus indicating that Th2 cytokines, likely produced by different intragraft cell types including T cells, macrophages and natural killer (NK) cells, may represent an important component in the pathogenesis of this process. Moreover, IL‐10 seems to exquisitely characterize a group of CR/CAN kidney grafts more prone to immunologic assaults.

Kidney Intragraft Homing of De Novo Donor-Specific HLA Antibodies Is an Essential Step of Antibody-Mediated Damage but Not Per Se Predictive of Graft Loss

Donor‐specific HLA antibody (DSA)‐mediated graft injury is the major cause of kidney loss. Among DSA characteristics, graft homing has been suggested as an indicator of severe tissue damage. We analyzed the role of de novo DSA (dnDSA) graft homing on kidney transplantation outcome. Graft biopsy specimens and parallel sera from 48 nonsensitized pediatric kidney recipients were analyzed. Serum samples and eluates from graft biopsy specimens were tested for the presence of dnDSAs with flow bead technology. Intragraft dnDSAs (gDSAs) were never detected in the absence of serum dnDSAs (sDSAs), whereas in the presence of sDSAs, gDSAs were demonstrated in 72% of biopsy specimens. A significantly higher homing capability was expressed by class II sDSAs endowed with high mean fluorescence intensity and C3d‐ and/or C1q‐fixing properties. In patients with available sequential biopsy specimens, we detected gDSAs before the appearance of antibody‐mediated rejection. In sDSA‐positive patients, gDSA positivity did not allow stratification for antibody‐mediated graft lesions and graft loss. However, a consistent detection of skewed unique DSA specificities was observed over time within the graft, likely responsible for the damage. Our results indicate that gDSAs could represent an instrumental tool to identify, among sDSAs, clinically relevant antibody specificities requiring monitoring and possibly guiding patient management.

De Novo Donor-Specific HLA Antibodies Developing Early or Late after Transplant Are Associated with the Same Risk of Graft Damage and Loss in Nonsensitized Kidney Recipients

De novo posttransplant donor-specific HLA-antibody (dnDSA) detection is now recognized as a tool to identify patients at risk for antibody-mediated rejection (AMR) and graft loss. It is still unclear whether the time interval from transplant to DSA occurrence influences graft damage. Utilizing sera collected longitudinally, we evaluated 114 consecutive primary pediatric kidney recipients grafted between 2002 and 2013 for dnDSA occurrence by Luminex platform. dnDSAs occurred in 39 patients at a median time of 24.6 months. In 15 patients, dnDSAs developed within 1 year (early-onset group), while the other 24 seroconverted after the first posttransplant year (late-onset group). The two groups were comparable when considering patient- and transplant-related factors, as well as DSA biological properties, including C1q and C3d complement-binding ability. Only recipient age at transplant significantly differed in the two cohorts, with younger patients showing earlier dnDSA development. Late AMR was diagnosed in 47% of the early group and in 58% of the late group. Graft loss occurred in 3/15 (20%) and 4/24 (17%) patients in early- and late-onset groups, respectively (p = ns). In our pediatric kidney recipients, dnDSAs predict AMR and graft loss irrespective of the time elapsed between transplantation and antibody occurrence.

Serum complement inactivation unveiled prepregnancy donor-specific HLA antibodies leading to postpartum kidney graft loss.

Dear Sirs, Pregnancy in solid organ-transplanted patients can stimulate acute cellular rejection and formation of de novo donor-specific antibodies (DSA) that may cause antibodymediated rejection (AMR) and graft loss [1,2]. Allostimulation in transplanted pregnant patients has been in some cases ascribed to HLA antigens shared between the organ donor and fetus and not expressed by the mother [3,4]. However, de novo DSA may also precede pregnancy and possibly lead to graft damage. Here, we report the case of a postpregnancy graft loss due to AMR in an unsensitized young recipient of a first kidney graft, in whom prepregnancy de novo DSA, previously undetected and retrospectively revealed by serum complement inactivation, were the principal risk factor for the induction of graft damage after pregnancy. An 18-year-old-female dialysis patient underwent kidney transplantation to treat renal hypodysplasia. The patient, who was unsensitized and never transfused before the transplant, was grafted with a kidney from a deceased donor. HLA mismatches with the donor are shown in Table 1a. Immunosuppression consisted of induction with anti-CD25 Mab, followed by maintenance therapy with cyclosporin A, mycophenolate mofetil and prednisone. The post-transplant period was characterized by prompt recovery of renal function and by absence of adverse events. At 3 years post-transplant, the patient, still on triple therapy (cyclosporin A 225 mg/day, mycophenolate mofetil 1000 mg/day, prednisone 5 mg each other day), asked to plan a pregnancy. Consequently, patient sera were tested for the presence of de novo HLA antibodies as part of the prepregnancy counseling. HLA antibodies were analyzed by the LABScreen Mixed kit and Class I and Class II Single Antigen kit (One Lambda Inc., Canoga Park, CA, USA) [5]. Two prepregnancy serum samples resulted negative for HLA antibodies (Table 1b). The husband’s HLA typing revealed the presence of three inheritable mismatch antigens shared between donor and husband, two of which were related to DQB1*06 and DQA1*01 broad specificities, respectively: donor DQB1*0603 versus husband DQB1*0602; donor DQA1*0103 versus husband DQA1*0102. The patient confirmed her willingness to plan a pregnancy and mycophenolate mofetil was substituted with azathioprine. Forty-three months post-transplant, the patient became pregnant and underwent, due to rupture of membranes, a spontaneous premature delivery of a healthy male at 24 weeks. During this period, her renal function remained stable and good. The analysis of a serum sample, collected during pregnancy, showed the presence of a DSA against DQB1*0201 specificity, expressed by the donor only (Table 1b). The neonate’s HLA typing demonstrated that all possible repeated shared mismatches were inherited (Table 1a). Seven months after the delivery, concurrently with an initial deterioration of graft function, DSA analysis detected five further DSA specific for antigens expressed by the donor only or directed against shared antigens (Table 1b). Medication nonadherence was never suspected due to regular attending of clinical visits and evidence of appropriate drug levels. Patient graft function progressed to failure in the following 22 months due to AMR not responsive to three courses of plasmapheresis associated to low-dose iv immunoglobulins and rituximab infusions. Despite these treatments, DSA MFI levels, but HLA-A*03, remained unchanged (Table 1b). All sera were retrospectively tested with C1q binding single-antigen bead (SAB) technology [6]. The results showed that, during pregnancy, in addition to the DQB1*0201 DSA that tested positive for C1q binding, other DSA directed to DQB1*06 (reacting with both DQB1*0603 and DQB1*0602) and B*08 were detected by this assay. DQB1*0603 and B*08 antibodies were positive also in prepregnancy sera. On the first postpregnancy serum, collected before any antibody removal procedure, all DSA showed C1q binding ability (Table 1b).

Gene expression in highly sensitized dialysis patients waiting for a kidney transplant: A real-time PCR analysis.

• We investigated gene expression in highly sensitized patients waiting for a kidney graft.

Failure to remove de novo donor-specific HLA antibodies is influenced by antibody properties and identifies kidney recipients with late antibody-mediated rejection destined to graft loss - a retrospective study

Current research is focusing on identifying bioclinical parameters for risk stratification of renal allograft loss, largely due to antibody‐mediated rejection (AMR). We retrospectively investigated graft outcome predictors in 24 unsensitized pediatric kidney recipients developing HLA de novo donor‐specific antibodies (dnDSAs), and treated for late AMR with plasmapheresis + low‐dose IVIG + Rituximab or high‐dose IVIG + Rituximab. Renal function and DSA properties were assessed before and longitudinally post treatment. The estimated GFR (eGFR) decline after treatment was dependent on a negative % eGFR variation in the year preceding treatment (P = 0.021) but not on eGFR at treatment (P = 0.74). At a median follow‐up of 36 months from AMR diagnosis, 10 patients lost their graft. Altered eGFR (P < 0.001) and presence of C3d‐binding DSAs (P = 0.005) at treatment, and failure to remove DSAs (P = 0.01) were negatively associated with graft survival in the univariable analysis. Given the relevance of DSA removal for therapeutic success, we analyzed antibody properties dictating resistance to anti‐humoral treatment. In the multivariable analysis, C3d‐binding ability (P < 0.05), but not C1q‐binding, and high mean fluorescence intensity (P < 0.05) were independent factors characterizing DSAs scarcely susceptible to removal. The poor prognosis of late AMR is related to deterioration of graft function prior to treatment and failure to remove C3d binding and/or high‐MFI DSAs.