Volume 85, Issue 3, Pages 590-599 (March 2014) B cells display an abnormal distribution and an impaired suppressive function in patients with chronic antibody–mediated rejection  Alexandre Nouël, Isabelle Ségalen, Christophe Jamin, Laurent Doucet, Sophie Caillard, Yves Renaudineau, Jacques-Olivier Pers, Yannick Le Meur, Sophie Hillion  Kidney International  Volume 85, Issue 3, Pages 590-599 (March 2014) DOI: 10.1038/ki.2013.457 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Abnormal distribution of B cells in chronic antibody–mediated rejection (cAbMR) subjects. Freshly isolated peripheral blood mononuclear cells (PBMCs) from 35 healthy volunteers (HVs, ▪), 27 patients with a stable graft function (ST, •), and 17 cAbMR (▴) patients were analyzed by flow cytometry. (a) Absolute numbers of peripheral blood B cells/μl in the different groups. (b) Frequency of CD19+ B cells in circulating lymphocytes depicted as individual values. (c) Individual values of activated (Bm2+Bm2’)/memory (eBm5+Bm5) ratio. (d) Representative histograms of immunoglobulin D (IgD) and CD38 expression on CD19+ gated B cells in each group. The B-cell distribution (mean of percentage) of the five mature subpopulations (Bm1 to Bm5) and the frequency of activated and memory B cells in each group are shown (mean±s.e.m.). (e) Frequency and absolute numbers/μl of CD19+CD27+ B cells and CD19+IgD-CD27+ B cells represented as individual values in each group. (f) Frequency and absolute numbers of CD24++CD38++ transitional cells gated on CD19+ B cells. Differences were evaluated with Mann–Whitney t-tests. *P<0.05; **P<0.01; ***P<0.001; NS, not significant. Kidney International 2014 85, 590-599DOI: (10.1038/ki.2013.457) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 B cells from chronic antibody–mediated rejection (cAbMR) patients are defective in regulatory functions. Peripheral blood B cells and T cells were isolated from 18 healthy volunteers (HVs, ▪), 12 patients with a stable graft function (ST, •), and 16 cAbMR patients (▴). Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled T cells were stimulated with anti-CD3 and anti-CD28 antibodies (Abs) for 5 days with or without B cells added at day 0 and stimulated with 0.25μmol/l CpG oligodeoxynucleotides (ODNs). (a) Representative flow cytometric analysis of diluted CFSE at day 4 in the presence or absence of B cells to evaluate the T-cell proliferation in the three groups (left panel). The B-cell suppression of T-cell proliferation is expressed as the percentage inhibition of proliferation relative to T cells cultured alone (right panel). (b) Secretion of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay (ELISA) at day 4 in supernatants of T cells cultured alone or in the presence of B cells. (c) Activated (Bm2+Bm2’) and memory (eBm5+Bm5) B cells from the peripheral blood of HVs were sorted by flow cytometry according to the expression of immunoglobulin D (IgD) and CD38 gated on CD19+ cells. Sorted B cells were added to autologous purified T cells and cocultured for 4 days. Significant differences were estimated by Mann–Whitney and paired t-tests. **P<0.01; ***P<0.001; NS, not significant. Kidney International 2014 85, 590-599DOI: (10.1038/ki.2013.457) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 B cells from chronic antibody–mediated rejection (cAbMR) patients showed an altered phenotype after 4 days of coculture with autologous T cells, and cAbMR sera display autoreactivity. B cells from healthy volunteers (HVs), stable graft function (ST), and cAbMR patients were stained with (a) anti-CD19/anti-CD24/anti-CD38, (b) anti-CD19/anti-CD38/anti-IgD Ab combinations on freshly isolated cells (d0) or after a 4-day period of coculture (d4) with autologous T cells stimulated with anti-CD3 and anti-CD28 antibodies (Abs). Representative flow cytometry histograms were depicted on CD19+ cells in the different groups (left panels). Frequencies (mean±s.e.m.) of (a) CD24highCD38high, (b) IgD+CD38+/high, and IgD-CD38-/low B cells were determined in the three groups (right panels). Significant differences were estimated by Mann–Whitney and paired t-test. *P<0.05; **P<0.01; ***P<0.001; NS, not significant. Kidney International 2014 85, 590-599DOI: (10.1038/ki.2013.457) Copyright © 2014 International Society of Nephrology Terms and Conditions