Pre-transplant donor-reactive IL-21 producing T cells as a tool to identify an increased risk for acute rejection

Abstract

Pre-transplant screening focuses on the detection of anti-HLA alloantibodies. Previous studies have shown that IFN-γ and IL-21 producing T cells are associated with the development of acute rejection (AR). The aim of this study, was to assess whether pre-transplant donor-reactive T cells and/or B cells are associated with increased rejection risk. Samples from 114 kidney transplant recipients (transplanted between 2010 and 2013) were obtained pre-transplantation. The number of donor-reactive IFN-γ and IL-21 producing cells was analyzed by ELISPOT assay. The presence of donor specific antibodies (DSA) was also determined before transplantation. Numbers of donor-reactive IFN-γ producing cells were similar in patients with or without AR whereas those of IL-21 producing cells were higher in patients with AR (p = 0.03). Significantly more patients with AR [6/30(20%)] had detectable DSA compared to patients without AR [5/84(5.9%), p = 0.03]. Multivariate logistic regression showed that donor age (OR 1.06), pre-transplant DSA (OR 5.61) and positive IL-21 ELISPOT assay (OR 2.77) were independent predictors of an increased risk for the development of AR. Aside from an advanced donor-age and pre-transplant DSA, also pre-transplant donor-reactive IL-21 producing cells are associated with the development of AR after transplantation.

Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients

“…ELISPOT assays were performed as described in a previous study. 9 In brief, patient PBMCs were incubated on plates coated with anti-human IFN-γ or IL-21 mAb (U-CyTech Biosciences, Utrecht, the Netherlands) with irradiated (40 Gy) PBMCs or spleen cells derived from the donor or irradiated third-party cells, which were completely HLA-mismatched with donor and recipient to determine an overall HLA response. The incubation period of the IFN-γ and IL-21 assays was 20 h and 44 h, respectively.…”

High Tacrolimus Intrapatient Variability and Subtherapeutic Immunosuppression are Associated With Adverse Kidney Transplant Outcomes

“…The presence of DSA was determined by comparing the measured HLA specificities with donor HLA typing. As described in a previous study, 25 polyvinylidene fluoride (PVDF) plates (Millipore, Darmstadt, Germany) were coated with antihuman IFN-γ or IL-21 mAb (U-CyTech Biosciences, Utrecht, Netherlands). Patient's PBMCs were incubated for 5–7 years after transplantation (IFN-γ assay: 20 hours incubation after resting all samples overnight, and IL-21: 44 hours incubation) with irradiated (40 Gy) PBMCs or spleen cells derived from the donor or irradiated third-party cells, which were completely HLA-mismatched with donor and recipient, in a culture medium [RPMI-1640 with GlutaMAX (Life Technologies/Gibco) + 10% heat-inactivated FBS (Biowest, Haarlem, The Netherlands) + penicillin + streptomycin (100 IU/mL penicillin, 100 IU/mL streptomycin; Lonza)].…”

“…The presence of DSA was determined by comparing the measured HLA specificities with donor HLA typing. As described in a previous study, 25 was added. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) followed by AEC substrate (U-CyTech Biosciences) until distinct spots were formed; color development was stopped by washing with water, and spots were counted automatically using a Bioreader 6000 ELISPOT-reader (Bio-Sys GmbH, Karben, Germany).…”

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients

“…M any studies in transplantation have used peripheral blood mononuclear cells (PBMCs) to assess immunological risk and to unravel the mechanisms of rejection and tolerance. [1][2][3] Examples include functional T-and B-lymphocyte studies by ELISPOT, 1,4,5 T-lymphocyte proliferation studies, 2 gene expression profiling, 3 and intracellular tacrolimus concentration measurements. 6 However, the PBMC fraction consists of various leukocyte subpopulations, including T, B, and NK lymphocytes and monocytes, with potential contamination by granulocytes.…”

“… 11 PBMCs were stored in liquid nitrogen at –190 °C until use. 1 , 12 All participants gave written informed consent. The study was performed in accordance with the declaration of Helsinki (2013) and approved by the institutional review board of the Erasmus MC (No.…”