The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection

Nicole M van Besouw, Lin Yan, Ronella de Kuiper, Mariska Klepper, Derek Reijerkerk, Marjolein Dieterich, Dave L Roelen, Frans H J Claas, Marian C Clahsen-van Groningen, Dennis A Hesselink, Carla C Baan, Nicole M van Besouw, Lin Yan, Ronella de Kuiper, Mariska Klepper, Derek Reijerkerk, Marjolein Dieterich, Dave L Roelen, Frans H J Claas, Marian C Clahsen-van Groningen, Dennis A Hesselink, Carla C Baan

Abstract

Interleukin (IL)-21 supports induction and expansion of CD8+ T cells, and can also regulate the differentiation of B cells into antibody-producing plasma cells. We questioned whether the number of circulating donor-specific IL-21 producing cells (pc) can predict kidney transplant rejection, and evaluated this in two different patient cohorts. The first analysis was done on pre-transplantation samples of 35 kidney transplant recipients of whom 15 patients developed an early acute rejection. The second study concerned peripheral blood mononuclear cell (PBMC) samples from 46 patients obtained at 6 months after kidney transplantation of whom 13 developed late rejection. Significantly higher frequencies of donor-specific IL-21 pc were found by Elispot assay in both patients who developed early and late rejection compared to those without rejection. In addition, low frequencies of donor-specific IL-21 pc were associated with higher rejection-free survival. Moreover, low pre-transplant donor-specific IL-21 pc numbers were associated with the absence of anti-HLA antibodies. Donor-reactive IL-21 was mainly produced by CD4+ T cells, including CD4+ follicular T helper cells. In conclusion, the number of donor-specific IL-21 pc is associated with an increased risk of both early and late rejection, giving it the potential to be a new biomarker in kidney transplantation.

Keywords: Elispot; IL-21; biomarker; end-stage renal disease; kidney transplantation; rejection.

Figures

Figure 1
Figure 1
Number of post-transplant donor-specific IL-21 producing PBMC in patients who will or will not develop rejection in pre-transplant cohort (A: n = 20 without rejection, n = 15 with rejection) and 6 months post-transplant cohort (B: n = 33 without rejection, n = 13 with rejection).
Figure 2
Figure 2
Receiver operating characteristic (ROC) analysis was performed to define the cut-off number of donor-specific IL-21 producing cells (pc) (A: PBMC samples taken from patients prior to transplantation; C: PBMC samples taken from patient at 6 months after transplantation), and discriminated between patients with and without rejection. A cut-off of 18 spots per 300.000 PBMCs was determined with a specificity of 80% and a sensitivity of 73% in the pre-transplant cohort (A), and a cut-off of 62 spots per 300.000 PBMCs was determined with a specificity of 88% and a sensitivity of 54% in the 6 months post-transplant cohort (C). Thereafter, the percentage of patients with high and low numbers (cut-off values) of IL-21 pc free from rejection were determined in the pre-transplant (B) and post-transplant cohort (D). AUC, area under the curve.
Figure 3
Figure 3
The donor-specific IL-21 producing cell frequency determined in PBMC, CD4+ and CD8+ T cells in PBMCs of four transplant recipients. The IL-21 response in PBMC was defined as 100%. Mean with SEM is presented.
Figure 4
Figure 4
A typical example is depicted for intracellular IL-21 production after stimulation of patient's PBMCs with irradiated donor PBMCs depleted for CD3 (A). The proportion of IL-21 producing cells was determined after 3 days within the CD8+(B) and CD4+ T cells (C). Tfh cells (CXCR5+PD1+) were determined within the CD8+(D) and CD4+(E) T cell population. The percentage of CD8+ Tfh cells was too low to be analyzed for IL-21. Within the CD4+ Tfh cells 31.22% produced IL-21 (F). The proportion of IL-21 producing cells was determined in PBMC samples from 5 kidney transplant recipients (G). The percentage donor-reactive IL-21 was determined in CD4+ and CD8+ T cells, and Tfh cells (CD4+CXCR5+PD1+). IgG1 isotypes are presented of each T cell subpopulations.

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