Immunologic human renal allograft injury associates with an altered IL-10/TNF-α expression ratio in regulatory B cells

Abstract

Human B cells with immunoregulatory properties in vitro (Bregs) have been defined by the expression of IL-10 and are enriched in various B-cell subsets. However, proinflammatory cytokine expression in B-cell subsets is largely unexplored. We examined the cytokine profiles of human PBMCs and found that subsets of CD24(hi)CD38(hi) transitional B cells (TrBs), CD24(hi)CD27(+) memory B cells, and naïve B cells express IL-10 and the proinflammatory cytokine TNF-α simultaneously. TrBs had the highest IL-10/TNF-α ratio and suppressed proinflammatory helper T cell 1 (Th1) cytokine expression by autologous T cells in vitro more potently than memory B cells did, despite similar IL-10 expression. Whereas neutralization of IL-10 significantly inhibited TrB-mediated suppression of autologous Th1 cytokine expression, blocking TNF-α increased the suppressive capacity of both memory and naïve B-cell subsets. Thus, the ratio of IL-10/TNF-α expression, a measure of cytokine polarization, may be a better indicator of regulatory function than IL-10 expression alone. Indeed, compared with TrB cells from patients with stable kidney graft function, TrBs from patients with graft rejection displayed similar IL-10 expression levels but increased TNF-α expression (i.e., reduced IL-10/TNF-α ratio), did not inhibit in vitro expression of Th1 cytokines by T cells, and abnormally suppressed expression of Th2 cytokines. In patients with graft dysfunction, a low IL-10/TNF-α ratio in TrBs associated with poor graft outcomes after 3 years of follow-up. In summary, these results indicate that B cell-mediated immune regulation is best characterized by the cytokine polarization profile, a finding that was confirmed in renal transplant patients.

Keywords: chronic allograft rejection; immunology; renal function decline.

Figure 1.

Characterization of transitional, memory, and naïve B subsets. (A–C) Definition of human B subsets including CD19⁺CD24^hiCD38^hiCD27⁻ TrB cells, CD19⁺CD24⁺CD38⁺ CD27⁻ naïve B cells, and CD19⁺CD24^hiCD27⁺ memory B cells. (D) Representative overlay histograms comparing the strength of expression of CD20, CD10, and IgM between the TrB and mature naïve B cells (n=5).

Figure 2.

Analysis of IL-10 and TNF-α expression by B subsets. Magnetic bead–enriched CD19⁺ B cells are stimulated with CpG and CD40L for 48 hours plus phorbol ester, ionomycin, and brefeldin A (last 5 hours). (A and C) Each representative dot plot shows the frequencies of IL-10⁺ or TNF-α⁺ B cells within the respective B subsets. (B and D) Cumulative frequency (mean±SEM) of IL-10 (*P<0.001, TrB versus memory or naive) and TNF-α producing cells (*^#P=0.001, TrB versus memory; P=0.01, TrB versus naïve) within each B cell subset in 15 healthy volunteers. (E) Analysis of the ratio of IL-10⁺/TNF-α⁺ cells within B cell subsets analyzed by flow cytometry (^¥P=0.002, TrB versus memory; *P<0.001, TrB versus naïve). (F) Cumulative results expressed as mean±SEM for the IL-10/TNF-α ratio obtained from purified B cell subsets and analyzed by ELISA in 11 healthy volunteers (*P<0.001, TrB versus memory or naive). (G) Representative dot plot for the combined analysis of IL-10 and TNF-α by dual staining in the B subsets. (H) Cumulative results (mean±SEM) from six healthy volunteers of the distribution of either IL-10⁺ or TNF-α⁺ or mixed cytokine-positive B cells (*P<0.001; ^#P=0.01; **P=0.001; ^##P<0.001, in comparison with TrBs). (I) Analysis of IL-10⁺ and TNF-α⁺ B subsets at 12, 24, and 48 hours in five independent controls after stimulation with CpG and CD40L. The graphs represent the IL-10/TNF-α ratio obtained for the respective B-cell subset at the three different time points in five healthy volunteers. Statistical analysis is performed by ANOVA using Tukey post hoc correction for multiple comparisons.

Figure 3.

TrB cells with a high IL-10/TNF-α ratio selectively suppress inflammatory cytokines by conventional T cells. Magnetic bead–enriched Tconv cells (CD4⁺CD25⁻) are stimulated for 72 hours with plate-bound anti-human CD-3 (0.5 µg/ml) in the presence of TrB (CD24^hiCD38^hi) B cells, CD24^hiCD27⁺ memory B cells, CD24⁺CD38⁺ naïve B cells, or CD4⁺CD25^hi Treg cells. Phorbol ester, ionomycin, and brefeldin A are added for the last 6 hours of cell culture. Data are derived from six healthy volunteers. (A) Representative dot plots showing frequency of TNF-α, IFN-γ, and IL-4 expression among Tconvs in the respective cell cultures. (B) Cumulative percentage stimulation/inhibition of the frequency of TNF-α⁺, IFN-γ⁺, or IL-4⁺cells (mean±SEM) after culture with Tregs or the respective B subsets. (*P<0.05, TrB versus CD24^hiCD27⁺; **P=0.03, TrB versus CD24⁺CD38⁺; ^#P=0.02, TrB versus CD24^hiCD27⁺; ^##P=0.01, TrB versus CD24⁺CD38⁺).

Figure 4.

The IL-10/TNF-α ratio predicts the capacity of the B subsets to suppress autologous Th1 cytokines. Magnetic bead-enriched Tconvs (CD4⁺CD25⁻) are stimulated for 72 hours with plate-bound anti-human CD-3 (0.5 µg/ml) in the presence of TrB (CD24^hiCD38^hi) B cells, CD24^hiCD27⁺ memory B cells, CD24⁺CD38⁺ naïve B cells, or CD4⁺CD25^hi Treg cells with either an isotype control or neutralizing antibodies to IL-10, TNFR1, TNFR2, or a combination of TNFR1 and TNFR2. Phorbol ester, ionomycin, and brefeldin A are added for the last 6 hours of cell culture. Data are derived from three healthy volunteers. (A and C) Representative scatter plots showing the detection of TNF-α and IFN-γ by intracellular staining when Tconvs are cultured alone or in the presence of TrB, memory, and naïve B cells with matched isotype controls or neutralizing antibodies to IL-10, TNFR1, or TNFR2. Appropriate isotype controls (negative controls) for cytokine staining labeled as “neg” are included for defining the cytokine positive gates for respective cell subsets. (B and D) Cumulative percentage stimulation/inhibition of the frequency of TNF-α⁺ (^$P=0.01; *P=0.02; **P=0.10; ^#P=0.20; ^##P=0.80) and IFN-γ⁺ (^©P=0.03; ^ΔP=0.01; ^@P=0.04; ^φP=0.2; ^®P=0.1) T cells (mean±SEM) after culture with the respective B subsets and neutralizing antibodies. Statistical analysis is performed by a paired t test.

Figure 5.

Details of the Banff classification of the biopsies for patients in the GD-R group. Classification is as follows: 0, absent; 1, mild; and >1, moderate to severe. These findings show that the biopsies have a significant degree of chronic damage as suggested by ct, cv, and ci scores. aah, arteriolar hyalinosis; c4d, C4d deposition in the peritubular capillaries; cg, chronic transplant glomerulopathy with basement membrane multilayering; ci, interstitial fibrosis; ct, tubular atrophy; cv, fibrointimal thickening; g, glomerulitis; i, interstitial inflammation; mm, mesangial expansion; ptc, peritubular capillaritis; t, tubulitis; ti, total inflammation; v, endothelitis.

Figure 6.

CD19⁺CD24^hiCD38^hi TrB cells are altered qualitatively and quantitatively in renal allograft rejection. Bar graphs (mean±SEM) comparing healthy volunteers and patient groups for the percentage (A) and absolute number (B) of CD24^hiCD38^hi TrB cells (^#P=0.03, S versus GD-R; *P=0.05, GDR versus GD-NR; **P<0.001, S versus GD-NR; ^##P<0.001, S versus GD-R). (C–E) The percentage of IL-10 for TrBs, IL-10/TNF-α ratio for TrBs, and whole B cells (^¥P<0.001, S versus GD-R; ^ɸP=0.03, GD-NR versus GD-R). (F) Representative dot plots for one of five patients in the GD-R group for the TNF-α, IFN-γ, and IL-4 expression by Tconvs cultured alone or in the presence of TrBs or Tregs. Numbers in each plot indicate the frequency of Tconvs expressing each cytokine under respective culture conditions. (G) Graphical representation (mean±SEM) of the cumulative results for the suppressive/stimulatory effect of the TrB cells or Tregs on the Tconv cytokine expression in healthy volunteers (n=6), stable (n=7), and GD-R (n=5) groups (^$P<0.001, S versus GD-R: TNF-α; ^$$P=0.002, IFN-γ; ^$$$P<0.001, IL-4,) Statistical analysis is performed by ANOVA using Tukey post hoc correction. GD-NR, patients with graft dysfunction-no rejection; GD-R, patients with graft dysfunction-rejection; HV, healthy volunteers; S, patients with stable graft function.

Figure 7.

Clinical significance of TrB IL-10/TNF-α in patients with graft dysfunction. (A) Receiver operating characteristic curve showing the ability of TrB-IL-10/TNF-α to distinguish stable grafts and rejection. All patients who are biopsied are divided into those with a high or low IL-10/TNF-α based on the mean IL-10/TNF-α for the group. (B) Bar chart showing the change in eGFR (Δ eGFR) over 3 years from the time of the biopsy in patients with graft dysfunction (mean±SEM) based on their IL-10/TNF-α (TrB). (C) Kaplan–Meier survival analysis for the high and low IL-10/TNF-α (TrB) for 100% increase in serum creatinine or graft loss over 3 years in patients with graft dysfunction. (D) Kaplan–Meier survival analysis for the high and low IL-10/TNF-α (TrB) for a 100% increase in serum creatinine or graft loss over 3 years in the GD-R group. Statistical analysis is performed by a Mann–Whitney U test and survival analysis is performed by the Kaplan–Meier method. Groups are compared using the log-rank test.