Preserving the B-cell compartment favors operational tolerance in human renal transplantation

Abstract

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-cell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT.

Figure 1

Renal function. One of the parameters used to evaluate the renal function was the median serum creatinine level. OT group, n = 5; CR group, n = 13; Sta group, n = 19; HI group, n = 9. *p < 0.05; ***p < 0.001.

Figure 2

Quantitative analysis of total circulating B cells in the study groups. CR individuals display lower frequencies (A) and absolute numbers (B) of B cells in relation to HI. The absolute cell numbers were calculated on the basis of the complete blood count of each individual performed at the same moment of study sample. OT group, n = 5; CR group, n = 10; Sta group, n = 19; HI group, n = 10. *p < 0.05.

Figure 3

OT display preserved numbers of circulating Bregs. (A) Representative gate strategy used to analyze the following B-cell subpopulations: naive B cells (CD19⁺CD24^intCD38^int), memory B cells (CD19⁺CD24^hiCD38⁻) and Bregs (CD19⁺CD24^hiCD38^hi). Percentage (B) and absolute number (C) of naive B cells (CD19⁺CD24^intCD38^int) are shown. Percentage (D) and absolute number (E) of memory B cells (CD19⁺CD24^hiCD38⁻) are shown. Percentage (F) and absolute number (G) of Bregs (CD19⁺CD24^hiCD38^hi) are shown. The absolute cell numbers were calculated on the basis of the complete blood count of each individual performed at the same time the blood was collected. OT group, n = 5; CR group, n = 10; Sta group, n = 19; HI group, n = 10. *p < 0.05; **p < 0.01.

Figure 4

OT displays higher representativeness of specific B-cell receptor CDR3 lengths. (A) Representative profiles of the BCR CDR3 region of the VH3 Ig family, for the IgM isotype, showing higher representativeness for the 16-aa CDR3 length. Analyses were performed using PBMC cDNA of study individuals, after fragment analysis of fluorescent PCR products on a 3730xl DNA Analyzer (Applied Biosystems). Profiles were obtained after analysis with GeneMapper software (Applied Biosystems). The IgM (B) and IgG (C) repertoire diversity was calculated by the sum of the number of different BCR CDR3 lengths expressed in each individual. The OT group exhibited expansion of clones expressing the BCR CDR3 lengths of the 16-aa VH3 family, for the IgM isotype (D), and 5-aa VH1 family, for the IgG isotype (E). The black dashed line corresponds to the point in which we considered higher gene expression than in healthy individuals (>2). The gray dashed line corresponds to the point at which we considered expression lower than in healthy individuals. OT group, n = 5; CR group, n = 11; Sta group, n = 17; HI group, n = 11. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

OT preserves the capacity to activate the CD40 signaling pathway. (A) Representative histograms of STAT3 phosphorylation in Bregs (CD19⁺CD24^hiCD38^hi), naive B cells (CD19⁺CD24^intCD38^int) and memory B cells (CD19⁺CD24^hiCD38⁻) at 0 and 5 min after stimulation with CD40 mAb. (B), (C) and (D) display the percentage of increase of the median of fluorescence intensity (MFI) of pSTAT3 in Bregs, naive B cells and memory B cells after stimulation, respectively. (E), (F) and (G) show the fold increase in the percentage of pSTAT3-positive cells after stimulation in Bregs, naive B cells and memory B cells, respectively. (H) Representative zebra plot for one healthy individual showing the frequency of IL-10–positive cells after stimulation within the B-cell subpopulations studied. Purified B cells were stimulated for 48 h with plate-bound anti-CD40 mAbs plus soluble CD40L. Phorbol myristic acid and ionomycin were added for the last 5 h of culture. CD19⁺IL-10⁺ B cells were measured by intracellular cytokine staining. (I) IL-10 production by the B-cell subpopulations in four healthy individuals. Bar graphs indicate mean ± SEM percentages of Breg, naive and memory B cells that produced IL-10. OT group, n = 5; CR group, n = 10; Sta group, n = 17; HI group, n = 10. *p < 0.05.