Longitudinal analysis of T and B cell phenotype and function in renal transplant recipients with or without rituximab induction therapy

Abstract

Background: Prevention of rejection after renal transplantation requires treatment with immunosuppressive drugs. Data on their in vivo effects on T- and B-cell phenotype and function are limited.

Methods: In a randomized double-blind placebo-controlled study to prevent renal allograft rejection, patients were treated with tacrolimus, mycophenolate mofetil (MMF), steroids, and a single dose of rituximab or placebo during transplant surgery. In a subset of patients, we analyzed the number and phenotype of peripheral T and B cells by multiparameter flow cytometry before transplantation, and at 3, 6, 12, and 24 months after transplantation.

Results: In patients treated with tacrolimus/MMF/steroids the proportion of central memory CD4+ and CD8+ T cells was higher at 3 months post-transplant compared to pre-transplant levels. In addition, the ratio between the percentage of central memory CD4+ and CD4+ regulatory T cells was significantly higher up to 24 months post-transplant compared to pre-transplant levels. Interestingly, treatment with tacrolimus/MMF/steroids resulted in a shift toward a more memory-like B-cell phenotype post-transplant. Addition of a single dose of rituximab resulted in a long-lasting B-cell depletion. At 12 months post-transplant, the small fraction of repopulated B cells consisted of a high percentage of transitional B cells. Rituximab treatment had no effect on the T-cell phenotype and function post-transplant.

Conclusions: Renal transplant recipients treated with tacrolimus/MMF/steroids show an altered memory T and B-cell compartment post-transplant. Additional B-cell depletion by rituximab leads to a relative increase of transitional and memory-like B cells, without affecting T-cell phenotype and function.

Trial registration: ClinicalTrials.gov NCT00565331.

Figure 1. Subset distribution of circulating T cells in renal transplant recipients after treatment with tacrolimus, MMF and steroids over time.

(A) Representative dot plots for a renal transplant recipient showing CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells within the CD45⁺ lymphocyte population. Circulating CD4⁺ and CD8⁺ T cells can be characterized as naive (T_N; CD27⁺CD45RO⁻), central memory (T_CM; CD27⁺CD45RO⁺), effector memory (T_EM; CD27⁻CD45RO⁺) and highly differentiated memory (T_EMRA; CD27⁺CD45RO⁻) cells. Furthermore, CD4⁺ T cells can be characterized as regulatory T cells (T_REGS; CD25^hiFOXP3⁺). (B) Shown are the absolute numbers of CD4⁺ T cells and the percentages of T_N, T_CM and T_EM within the CD4⁺ T-cell population for 14 triple immunosuppression-treated patients before transplantation (t = 0) and at 3, 6, 12 and 24 months after transplantation (n = 10 at 24 m). (C) As described under B, for CD8⁺ T cells. (D) The ratio between the percentage of CD4⁺ T_CM or T_EM and the percentage of T_REGS. (E) Longitudinal analysis of the percentages of CXCR3⁺, CCR4⁺, and CCR6⁺ cells within the CD4⁺ T-cell population of 14 triple immunosuppression-treated patients (n = 10 at 24 m). Results are shown as box plots displaying the median, 25^th and 75^th percentiles as the box, and the 5^th and 95^th percentiles as whiskers. Significant differences are indicated compared to pre-transplant levels: *P<0.05, **P<0.01.

Figure 2. Ex vivo cytokine production by circulating T cells in renal transplant recipients after treatment with tacrolimus, MMF and steroids.

Peripheral blood mononuclear cells (PBMCs) were stimulated for 4 hours in the presence of PMA, ionomycin and Brefeldin A. Shown are the percentages IL-2, IL-4, IL-17, IFNγ or TNFα-producing cells within the CD4⁺ or CD8⁺ T-cell population of 14 triple immunosuppression-treated patients before transplantation (t = 0) and at 3, 6, 12, and 24 months after transplantation (n = 10 at 24 m). Results are shown as box plots displaying the median, 25^th and 75^th percentiles as the box, and the 5^th and 95^th percentiles as whiskers. Significant differences are indicated compared to pre-transplant levels: *P<0.05, **P<0.01, ***P<0.001.

Figure 3. Longitudinal analysis of circulating B cells in renal transplant recipients after treatment with tacrolimus, MMF and steroids.

(A) Shown are the absolute numbers of CD19⁺ B cells of 14 triple immunosuppression-treated patients before transplantation (t = 0) and at 3, 6, 12, and 24 months after transplantation (n = 10 at 24 m). (B) Representative dot plots for a renal transplant recipient over time using the Bm1-Bm5 classification: Bm1 (IgD⁺CD38⁻), Bm2 (IgD⁺CD38⁺), Bm2’ (IgD⁺CD38⁺⁺), Bm3+4 (IgD⁻CD38⁺⁺), Early Bm5 (IgD⁻CD38⁺) and Late Bm5 (IgD⁻CD38⁻) cells within the CD19⁺ B-cell population. (C) Shown are the percentages of the different B-cell subsets using the Bm1-Bm5 classification over time. (D) Shown are the percentages of CD80⁺, CD95⁺, and BAFF-receptor⁺ (BAFF-R) cells within the CD19⁺ B-cell population. (E) Overlay plot of the BAFF-R expression (MFI: median fluorescence intensity) within the CD19⁺ B-cell population of one patient before transplantation (pre-Tx) and 24 months (24 m) after transplantation under treatment with tacrolimus, MMF and steroids. Gray line shows unstained cells. (F) Summary graph showing the BAFF-R MFI of 14 triple immunosuppression-treated patients before over time (n = 10 at 24 m). Results are shown as box plots displaying the median, 25^th and 75^th percentiles as the box, and the 5^th and 95^th percentiles as whiskers. Significant differences are indicated compared to pre-transplant (pre-Tx; t = 0) levels: *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Longitudinal analysis of circulating B cells in renal transplant recipients after treatment with tacrolimus, MMF and steroids, and a single dose of rituximab (RTX) during transplant surgery.

(A) Representative dot plots of CD19⁺ B cells within the CD45⁺ lymphocyte population for a RTX-treated and a triple immunosuppression (IS)-treated patient before transplantation (pre-Tx) and at 3, 12, and 24 months after transplantation. (B) Shown are the absolute numbers of CD19⁺ B cells for RTX-(gray, n = 12) and triple IS-treated (white, n = 14) patients before transplantation (t = 0) and up to 24 months after transplantation (n = 10 and n = 9 at t = 24 m, respectively). (C) Pie charts depicting the distribution the different B cells subsets over time using the Bm1-Bm5 classification as depicted in Figure 3B: Bm1 (IgD⁺CD38⁻), Bm2 (IgD⁺CD38⁺), Bm2’ (IgD⁺CD38⁺⁺), Bm3+4 (IgD⁻CD38⁺⁺), Early Bm5 (IgD⁻CD38⁺) and Late Bm5 (IgD⁻CD38⁻) cells within the CD19⁺ B-cell population. Data are represented as means of 14 triple IS+RTX-treated and 12 IS-treated patients before transplantation (pre-Tx) and at 3, 12, and 24 months after transplantation (n = 10 and n = 9 at t = 24 m, respectively). (D) Shown are the percentages of CD80⁺, CD95⁺ and BAFF-R⁺ cells within the CD19⁺ B-cell population for RTX- (gray, n = 12) and triple IS-treated (white, n = 14) patients before transplantation (t = 0) and up to 24 months after transplantation. Results are shown as box plots displaying the median, 25^th and 75^th percentiles as the box, and the 5^th and 95^th percentiles as whiskers. Significant differences are indicated by asterisks: **P<0.01.