Chimeric HLA antibody receptor T cells for targeted therapy of antibody-mediated rejection in transplantation.

The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation.

Extracorporeal Photopheresis Improves Graft Survival in a Full-Mismatch Rat Model of Kidney Transplantation.

Extracorporeal photopheresis (ECP) is an immunomodulatory therapy based on the infusion of autologous cellular products exposed to ultraviolet light (UV) in the presence of a photosensitizer. The study evaluates the ECP efficacy as induction therapy in a full-mismatch kidney transplant rat model. Dark Agouti to Lewis (DA-L) kidney transplant model has been established. ECP product was obtained from Lewis rat recipients after DA kidney graft transplantation (LewDA). Leukocytes of those LewDA rats were exposed to 8-methoxy psoralen, and illuminated with UV-A. The ECP doses assessed were 10 × 106 and 100 × 106 cells/time point. Lewis recipients received seven ECP infusions. DA-L model was characterized by the appearance of donor-specific antibodies (DSA) and kidney function deterioration from day three after kidney transplant. The dysfunction progressed rapidly until graft loss (6.1 ± 0.5 days). Tacrolimus at 0.25 mg/kg prolonged rat survival until 11.4 ± 0.7 days (p = 0.0004). In this context, the application of leukocytes from LewDA sensitized rats accelerated the rejection (8.7 ± 0.45, p = 0.0012), whereas ECP product at high dose extended kidney graft survival until 26.3 ± 7.3 days, reducing class I and II DSA in surviving rats. ECP treatment increases kidney graft survival in full-mismatch rat model of acute rejection and is a suitable immunomodulatory therapy to be explored in kidney transplantation.

Cellular and humoral response after MRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients.

According to preliminary data, seroconversion after mRNA SARS-CoV-2 vaccination might be unsatisfactory in Kidney Transplant Recipients (KTRs). However, it is unknown if seronegative patients develop at least a cellular response that could offer a certain grade of protection against SARS-CoV-2. To answer this question, we prospectively studied 148 recipients of either kidney (133) or kidney-pancreas (15) grafts with assessment of IgM/IgG spike (S) antibodies and ELISpot against the nucleocapside (N) and the S protein at baseline and 2 weeks after receiving the second dose of the mRNA-1273 (Moderna) vaccine. At baseline, 31 patients (20.9%) had either IgM/IgG or ELISpot positivity and were considered to be SARS-CoV-2-pre-immunized, while 117 (79.1%) patients had no signs of either cellular or humoral response and were considered SARS-CoV-2-naïve. After vaccination, naïve patients who developed either humoral or cellular response were finally 65.0%, of which 29.9% developed either IgG or IgM and 35.0% S-ELISpot positivity. Factors associated with vaccine unresponsiveness were diabetes and treatment with antithymocytes globulins during the last year. Side effects were consistent with that of the pivotal trial and no DSAs developed after vaccination. In conclusion, mRNA-1273 SARS-CoV-2 vaccine elicits either cellular or humoral response in almost two thirds of KTRs.

B Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in Kidney Graft Recipients Undergoing Pre-Transplant Desensitization.

Background: Living-donor kidney transplant (LDKT) recipients undergoing desensitization for Human Leukocyte Antigen (HLA)-incompatibility have a high risk of developing antibody-mediated rejection (ABMR). The purpose of the study is to evaluate if residual B cell activity after desensitization could be estimated by the presence of circulating B cell-derived extracellular vesicles (BEVs). Methods: BEVs were isolated by Sepharose-based size exclusion chromatography and defined as CD19+ and HLA-II+ extracellular vesicles. We analyzed stored serum samples from positive crossmatch LDKT recipients before and after desensitization at first post-transplant biopsy and at 12-month protocol biopsy (n = 11). Control groups were formed by hypersensitized patients who were not submitted to desensitization (n = 10) and by low-risk recipients (n = 9). A prospective validation cohort of 11 patients also included the analysis of B cells subpopulations in recipients' blood and lymph nodes recovered upon graft implantation, along with BEVs analysis before and after desensitization. Results: We found out that CD19+ and HLA-II+BEVs dropped significantly after desensitization and relapse in patients who later developed ABMR was evident. We validated these findings in a proof-of-concept prospective cohort of 6 patients who received the same desensitization protocol and also in a control group of 5 LDKT recipients. In these patients, B cell subpopulations were also studied in recipients' blood and lymph nodes that were recovered before the graft implantation. We confirmed the significant drop in BEVs after desensitization and that this paralleled the reduction in CD19+cells in lymph nodes, while in peripheral blood B cells, this change was almost undetectable. Conclusions: BEVs reflected B cell residual activity after desensitization and this could be a valid surrogate of humoral alloreactivity in this setting.

Two-dimensional difference gel electrophoresis urinary proteomic profile in the search of nonimmune chronic allograft dysfunction biomarkers.

INTRODUCTION: Despite advances in therapeutics, graft loss associated with chronic allograft dysfunction (CAD) remains high. Urinary proteomic analysis is a noninvasive method that could be used to detect and evaluate CAD in renal transplant recipients. This study was aimed to establish the normal proteome map of stable transplant patients and to validate the utility of two-dimensional difference gel electrophoresis (2DE-DIGE) in identifying new candidates as urinary biomarkers of CAD. METHODS: Morning spot urine samples that were collected from kidney transplant recipients with biopsy-proven interstitial fibrosis and tubular atrophy (IFTA) stages 0-I-II/III (n=8/group) under immunosuppressive treatment with tacrolimus plus mycophenolate with or without prednisone. 2DE silver staining and mass spectrometry analyses were used to establish the normal proteome map, and 2DE-DIGE and mass spectrometry were used to identify proteins exhibiting differential abundance. RESULTS AND CONCLUSIONS: This study defines the normal proteome of stable renal transplant patients, which is composed of several plasma proteins, as well as of immunologic proteins that are probably specific to transplant recipients. The 2DE-DIGE study showed 19 proteins with differential concentrations, depending on the IFTA histologic score. These 19 proteins could be used as urinary biomarkers of the severity of IFTA in renal transplant recipients.

Effect of mTOR inhibitor on body weight: from an experimental rat model to human transplant patients.

UNLABELLED: The aim was to study the influence of sirolimus (SRL) on body weight in a rat model and in kidney transplant patients. Wistar rats (15 weeks old) were either treated with vehicle (VEH; n = 8) or SRL (n = 7) 1.0 mg/kg three times per week for 12 weeks. Body mass and food intake were measured weekly. Adipocyte diameter was determined in hematoxylin-eosin stains. The body mass index (BMI) obtained from clinical kidney transplant trials comparing SRL-based with cyclosporine-based therapy was analyzed. ANIMALS: SRL produced a decrease of the weight gain curve. At the end of the study, mean body weight in the SRL group was lower than in the VEH group (356 vs. 507 g, P < 0.01) in spite of comparable food intake normalized for body weight was not different. Mean adipocyte diameter was 36 mum in VEH and 25 mum in SRL rats (P = 0.009). Mean SRL blood trough concentration was 38 ng/ml. Kidney transplant patients: Two years after transplantation, BMI was significantly lower in the SRL-based treatment arm compared to cyclosporine (24.17 +/- 2.99 vs. 25.97 +/- 5.01 kg/m(2), P = 0.031). SRL treatment leads to less body mass. Adipocyte cell diameter was reduced in SRL-treated animals. A possible explanation may be the effects of SRL on metabolic regulation and cell growth.