BAFF is increased in renal transplant patients following treatment with alemtuzumab

Abstract

Alemtuzumab is a monoclonal antibody that depletes T and B cells and is used as induction therapy for renal transplant recipients. Without long-term calcineurin inhibitor (CNI) therapy, alemtuzumab-treated patients have a propensity to develop alloantibody and may undergo antibody-mediated rejection (AMR). In pursuit of a mechanistic explanation, we analyzed peripheral B cells and serum of these patients for BAFF (Blys) and BAFF-R, factors known to be integral for B-cell activation, survival, and homeostasis. Serum BAFF levels of 22/24 alemtuzumab-treated patients were above normal range, with average levels of 1967 pg/mL compared to 775 pg/mL in healthy controls (p = 0.006). BAFF remained elevated 2 years posttransplant in 78% of these patients. BAFF-R on CD19(+) B cells was significantly downregulated, suggesting ligand/receptor engagement. BAFF mRNA expression was increased 2-7-fold in CD14(+) cells of depleted patients, possibly linking monocytes to the BAFF dysregulation. Addition of recombinant BAFF to mixed lymphocyte cultures increased B-cell activation to alloantigen, as measured by CD25 and CD69 coexpression on CD19(+) cells. Of note, addition of sirolimus (SRL) augmented BAFF-enhanced B-cell activation whereas CNIs blocked it. These data suggest associations between BAFF/BAFF-R and AMR in alemtuzumab-treated patients.

Figure 1. BAFF but not APRIL levels are elevated in alemtuzumab-treated transplant patients

(A) Serum BAFF levels measured by ELISA for the following groups: healthy control individuals, renal transplant patients pretransplant, nondepleted transplant patients treated with anti-CD25 induction therapy (measured 6 months posttransplant), depleted transplant patients treated with alemtuzumab (measured at 6 months posttransplant). Statistics performed via two-tailed t-test. (B) Long-term BAFF levels of alemtuzumab treated renal transplant cohorts 2–4 months posttransplant and 24 months later. Absolute CD20 counts (X-axis) at the 2–4 month time point and 24 months later. Pearson correlation coefficients were generated to test whether an association exists between BAFF levels and CD20⁺ B-cell levels. (D) Serum APRIL levels measured by ELISA for nondepleted transplant patients treated with anti-CD25 induction therapy (measured 6 months posttransplant) and depleted transplant patients treated with alemtuzumab (measured at 6 months posttransplant). Statistics performed via unpaired two-tailed t-test.

Figure 2. BAFF mRNA is increased in monocytes of alemtuzumab patients

(A) RT-QPCR performed for eight patients at 2–4 months posttransplant and four patients 26–28 months post-transplant. (B) Correlation between BAFF mRNA levels and serum BAFF levels in the eight patients measured at the 2–4 month time point. Pearson correlation coefficients were generated to test whether an association exists between BAFF levels and monocyte BAFF mRNA levels. (C) MCF of BAFF on the monocytes of normal, BAFF^med and BAFF^high alemtuzumab patients at early and late posttransplant time points. Statistics performed via unpaired two-tailed t-test.

Figure 3. BAFF-R is downregulated on peripheral CD19^± lymphocytes

(A) Representative dot plots of BAFF-R expression (Y-axis) in two healthy control individuals and two alemtuzumab (campath-1H)-treated patients at M2–4 posttransplant. CD19⁺ cells (X-axis) are derived from the lymphocyte gate. Composite histograms are also shown: gray lines are healthy controls and black lines are depleted patients. (B) BAFF-R expression on CD19⁺ cells of patients that are BAFF^med (900–1000 pg/mL) versus BAFF^high (>2500 pg/mL) at the M2–4 and M26–28 posttransplant time points. (C) The percent of CD19⁺ cells in the lymphocyte gate which is BAFF⁺, at both the early and late time points posttransplant. All statistics performed via an unpaired two-tailed t-test.

Figure 4. Exogenous rhBAFF increases the percentage activated CD19^± lymphocytes in MLR; SRL augments this effect

(A) Percent of activated lymphocytes after 4-day one-way MLR with increasing concentrations of exogenous recombinant human BAFF. Activation is measured as the percentage CD25⁺CD69⁺ double positives within the CD19⁺ cells of the lymphocyte gate. These data are the combination of four responder/stimulator pairs. Statistics were performed via a two-tailed t-test. (B) Comparison of self (autologous) versus allogeneic B-cell stimulation with exogenous rhBAFF and immunosuppressive agents SRL or TAC. Activation is measured by percentage CD25⁺CD69⁺ double positives within the CD19⁺ cells of the lymphocyte gate. Statistics were performed via an unpaired two-tailed t-test. (C) IL-2 T-cell cytokine analysis of MLR supernatants of experiments from (B). Both cytokines, as well as a host of others, are effectively downregulated by both immunosuppressants.