Arcangelo Nocera

Treatment of EBV-Related Post-Renal Transplant Lymphoproliferative Disease with a Tailored Regimen Including EBV-Specific T Cells

The treatment of EBV‐associated post‐transplant lymphoproliferative disease (PTLD) poses a considerable challenge. Efforts have been made to define regimens based on combination of the available therapeutic agents, chosen and tailored on a patient‐by‐patient basis, with the aim of augmenting event‐free patient and graft survival. Recently, autologous EBV‐specific cytotoxic T‐lymphocytes (CTL) have proved effective in enhancing EBV‐specific immune responses and reducing viral load in organ transplant recipients with active infection. We investigated the use of a tailored combined approach including autologous EBV‐specific CTL for the treatment of EBV‐related PTLD developing after pediatric kidney transplantation.

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.

Dendritic cells pulsed with polyomavirus BK antigen induce ex vivo polyoma BK virus-specific cytotoxic T-cell lines in seropositive healthy individuals and renal transplant recipients

Polyoma BK virus (BKV)-associated interstitial nephritis has emerged as a relevant complication of immunocompromise after kidney transplantation, leading to reduced survival of the renal allograft. The limitations of current antiviral treatment and the high probability of rejection in kidney graft recipients when control of viral replication is attempted by reduction of immunosuppression warrant further efforts to develop alternative therapeutic tools. Cellular immunotherapy has proved to be a successful approach for prevention and/or treatment of other viral complications in the immunocompromised host. For assessing the feasibility of translating this strategy to the prevention of BKV-associated disease, a procedure for ex vivo reactivation of BKV-specific cytotoxic T cells (CTL) was developed from BKV-seropositive healthy donors and allograft recipients through stimulation with dendritic cells pulsed with inactivated BKV. The CTL lines thus obtained showed BKV specificity, as an efficient lysis of BKV-infected targets was accompanied by little or no reactivity against mock-infected autologous or allogeneic targets. In vitro killing of allogeneic BKV-infected targets, likely as a result of populations of TCRgammadelta+/CD3+ displaying MHC class I unrestricted cytotoxicity, was also displayed. Application of this culture system may allow a preemptive therapy approach to BKV-related complications in transplant recipients, based on CTL treatment guided by BKV DNA levels.

A lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

This paper reports the case of a patient with an abnormally expanded population of circulating lymphoid cells displaying the features of the so-called large granular lymphocytes (LGL). These cells were peroxidase negative and nonphagocytic, formed rosettes with sheep erythrocytes, had receptors for IgG, and contained azurophilic (electron-dense) granules. Like normal LGL, the patient cells were positive for two acid hydrolases (acid phosphatase and β-glucuronidase) but did not stain for α-naphthyl acetate esterase (ANAE), which is present in normal LGL. Ultrastructural studies revealed that the patient cells were rich in Golgi-derived vesicles, coated vesicles, multivesicular bodies, and immature granules, indicating that, unlike normal LGL, they were engaged in granulogenesis. These features, together with the absence of ANAE activity, are suggestive of some degree of cell immaturity. The patient cells displayed natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities comparable to those of normal peripheral blood mononuclear cells, or even higher, and did not respond to T-cell mitogens or allogeneic cells. Furthermore, they were incapable of suppressing normal T-cell proliferation or pokeweed mitogen-induced B-cell differentiation. Analysis of the NK activity at the single-cell level revealed that a large proportion of the patient cells bound to the K562 target cells but could not accomplish the entire lytic process. This finding supports further the possibility that the patient cells were immature LGL. The surface phenotype of the patient cells (as defined by a battery of monoclonal antibodies) was somewhat different from that usually observed in the majority of the normal LGL because, in addition to the HNK-1 marker, the cells were OKT3+, aLeul+, aLeu4+, OKT8+, aLeu2a+, and 3A1+ but were OKM1− and 4F2−. This phenotype could correspond to that of maturing LGL.

HLA class-I-soluble antigen serum levels in liver transplantation : a predictor marker of acute rejection

The serum levels of sHLA-I have been determined in 16 patients following liver transplantation. sHLA-I levels did not show remarkable variations in six patients without evidence of transplant-related complications. sHLA-I levels strongly increased in 10 patients undergoing acute rejection episodes. In these patients, an average 20% daily increase of sHLA-I levels was detected on the 6 days preceding and on the 2 days following the rejection episode. A fast decrease of sHLA-I levels was observed in seven patients following treatment of acute rejection with anti-CD3 mAb. The serum level of sHLA-I antigens positively correlated with ALT serum level and inversely correlated with PT. The determination of sHLA-I in serum may therefore be proposed as a useful marker in the monitoring of patients following liver transplantation. The increase of sHLA-I antigens may predict the onset of acute rejection whereas their decrease may be related to a good response of acute rejection to immunosuppressive treatment.

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.

In vitro removal of anti-HLA IgG antibodies from highly sensitized transplant recipients by immunoadsorption with protein A and protein G sepharose columns: a comparison

Abstract. In the present study we compared the capabilities of sepharose‐bound protein A versus protein G columns to remove in vitro lymphocytotoxic anti‐HLA antibodies from sera of four highly sensitized renal transplant recipients (PRA≥70%). In none of the patients were protein A sepharose columns capable of completely removing anti‐HLA antibodies, as demonstrated by the presence of residual alloreactive lymphocytotoxic activity in IgG 3 antibodies containing fractions eluted at pH 7. In contrast, no residual anti‐HLA lymphocytotoxic antibody activity was found in fractions eluted at pH 7 from protein G columns. These data demonstrate that: (1) IgG 3 antibodies can be partly responsible for lymphocytotoxic anti‐HLA reactivity in some sensitized renal transplant recipients and (2) at least in this patient category, in vitro immunoadsorption with protein G is more efficient than protein A sepharose columns in completely removing anti‐HLA IgG antibodies from sera.

HLA matching in pediatric recipients of a first kidney graft. A single center analysis.

We retrospectively examined the effect of HLA-A, -B, and -DR serological matching on graft survival in 88 pediatric end-stage renal disease patients who underwent primary renal transplantation. Actuarial graft survivals (GS) at 2 and 6 years in patients with zero DR mismatches (MM) (12 patients) or 1 DR MM (58 patients) were significantly higher than those in patients with 2 DR MM (18 patients) (2-year GS: 100% vs. 90% vs. 59%; 6-year GS: 100% vs. 79% vs. 59%, respectively). Because of the low number of patients in the zero DR MM group, only the GS difference between 1 DR MM and 2 DR MM had a significant result at 1 year (92% vs. 68%). No clear HLA matching effect was obtained in the HLA-A and -B loci. When DR were combined with A or B antigens (0-2 MM vs. 3-4 MM), significantly higher GS at 1, 2, and 6 years persisted for patients with 0-2 MM only in the A, DR group (96%, 94%, and 85% vs. 68%, 63%, and 56%, respectively). It is suggested that avoidance of mismatching for DR alleles at the serological level, in the selection of pediatric recipients of first cadaveric renal transplantation, leads to an improvement of both short- and longterm graft outcome.