Autoregulatory function of interleukin-10-producing pre-naïve B cells is defective in systemic lupus erythematosus

Abstract

Introduction: Pre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis.

Methods: Pre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation.

Results: CD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4(+) T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4(+) T-cell proliferation and induction of CD4(+)FoxP3(+) T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4(+) T-cell proliferation.

Conclusions: There is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4(+) T-cell activation and may thereby enhance the development of autoimmunity.

Fig. 1

Identification of CD40-activated human B cells which produce high levels of interleukin-10 (IL-10). a Peripheral blood CD20⁺ B cells were stained for CD20, CD27, and CD38 and gated as previously shown [3]. A representative dot plot shows the CD27⁻CD38^lo naïve (N), CD27⁻CD38^Int pre-naïve (PN), CD27⁻CD38^hi transitional (T), and CD27⁺ memory (M) B-cell subsets (n = 50). b Representative contour plot showing the frequencies of IL-10-producing B cells measured by intracellular cytokine staining of naïve, pre-naïve, and memory B-cell populations following stimulation with CD154-L cells for 72 hours. c The frequencies of IL-10-producing B cells in B-cell subsets as described in (b) (n = 10, cumulative data from nine independent experiments). d Supernatants from cultures as described in (b) and (c) were tested for the presence of IL-10 by enzyme-linked immunosorbent assay (ELISA). e Frequencies of IL-10-producing pre-naïve B cells following stimulation with L cells with and without CD154 expression as described in (b) (n = 3, cumulative data from three independent experiments). f IL-10 production by pre-naïve B cells following stimulation with L cells with and without CD154 expression was measured from culture supernatants by ELISA as described in (e). **P < 0.01, ***P < 0.001. IFN-γ interferon-gamma, IL interleukin

Fig. 2

Activated pre-naïve B cells show IL-10-mediated ineffective promotion of CD4⁺ T-cell proliferation. a Representative contour plot showing the frequencies of CD4⁺INF-γ⁺ T cells and CD4⁺TNF-α⁺ T cells measured by intracellular cytokine staining after co-culture with B cells and CD154-L cells for 72 hours. b The frequencies of CD4⁺INF-γ⁺ T cells and CD4⁺TNF-α⁺ T cells measured as described in (a) (n = 4, cumulative data from three independent experiments). c Effect of total B cells and each B-cell subset on CD4⁺ T-cell proliferation in co-culture with B cells and CD154-L cells for 5 days (n = 10, cumulative data from eight independent experiments). d IFN-γ and TNF-α secretion was measured by enzyme-linked immunosorbent assay in the culture supernatants from experiments described in (c). e The effect of each B-cell subset on T-cell proliferation cells in co-culture of CD4⁺ T cells, B cells, and CD154-L cells for 5 days cultured in medium alone or in the presence of anti-IL-10 mAb (n = 6, cumulative data from five independent experiments). f IFN-γ and TNF-α secretion tested in supernatants from cultures as described in (e). g Representative histogram showing frequencies of CD4⁺FoxP3⁺ T cells in co-culture with CD4⁺ T cells, B cells, and CD154-L cells for 5 days. h The effect of each B-cell subset on frequencies of CD4⁺FoxP3⁺ T cells from experiments described in (g) (n = 3, cumulative data from three independent experiments). i The effect of pre-naïve B cells on frequencies of CD4⁺FoxP3⁺ T cells from experiments described in (g) cultured in medium alone or in the presence of anti-IL-10 mAb (n = 3, cumulative data from three independent experiments). *P < 0.05, **P < 0.01. IFN-γ interferon-gamma, IL interleukin, mAb monoclonal antibody, TNF-α tumor necrosis factor-alpha

Fig. 3

Impaired expression of co-stimulatory molecules and secretion of lower amounts of cytokine involved in T-cell activation by pre-naïve B cells. a Mean fluorescence intensity (MFI) of CD80, CD86, CD54, and HLA-DR expression of naïve, pre-naïve, and memory B-cell population on days 0–2 after stimulation with CD154-L cells (n = 18, cumulative data from 10 independent experiments). Data are expressed as mean MFI ± standard error (SE). b MFI of CD80 and CD86 expression of each B-cell population on day 1 of cultures described in (a) in medium alone or in the presence of anti-IL-10+ anti-IL-10Ra, rhIL-10, and rhIL-10+ anti-IL-10+ anti-IL-10Ra (n = 18, cumulative data from 10 independent experiments). Data are expressed as mean ± SE fold change over non-treated samples. c IL-6 and TNF-α levels in culture supernatants on day 1 of culture as described in (b). IL-6 and TNF-α secretion in the culture supernatants was measured by multiplex assay (n = 18, cumulative data from 10 independent experiments). Data are expressed as mean ± SE. *P < 0.05, **P < 0.01. ***P <0.001. IL-10 interleukin-10, TNF-α tumor necrosis factor-alpha, rhIL-10 recombinant IL-10, anti-IL-10Ra anti-IL-10 receptor antibody

Fig. 4

Antibodies produced by pre-naïve B cells are autoreactive. a Sorted pre-naïve and naïve B cells were cultured with irradiated CD4⁺ T cells in anti-CD3 pre-coated microtiter wells and, after 11 days of culture, were assessed for CD38^highCD27⁺IgD⁻ plasma cells. Numbers represent percentage of CD27⁺CD38^high B cells and CD27⁺CD38^highIgD⁻ B cells. b Reactivity of antibodies produced by pre-naïve and naïve B cells. Culture supernatants from experiment described in (a) from pre-naïve B cells (●) and naïve B cells (○) were analyzed for reactivity with indicated antigens, histone, dsDNA, and ssDNA by enzyme-linked immunosorbent assay. Values indicate results from individual donors of a total of 15 tested. c Sorted pre-naïve and naïve B cells were stimulated with anti-CD40 2 μg/ml and interleukin-21 (IL-21) 200 ng/ml in the presence and absence of anti-IL-10 monoclonal antibodies 10 μg/ml and assessed for frequencies of CD27⁺CD38^highIgD⁻ plasma cells at days 3, 6, and 8. Numbers represent percentage of cells. A.U. arbitary units, dsDNA double-stranded DNA, PC plasma cells, ssDNA, single-stranded DNA

Fig. 5

Systemic lupus erythematosus (SLE) pre-naïve B cells were defective in producing interleukin-10 (IL-10) and promoted robust CD4⁺ T-cell activation. a Representative contour plot showing the frequencies of IL-10-producing B cells from an SLE patient, measured as described in Fig. 1b. b Comparison of the frequencies of IL-10-producing B cells between six patients with SLE and 10 healthy donors (cumulative data from 6 independent experiments). c Supernatants from cultures as described in (b) were tested for the presence of IL-10 by enzyme-linked immunosorbent assay. d Representative histogram comparing CD80, CD86, CD54, and HLA-DR expression on pre-naïve B cells from a healthy individual and an SLE patient, obtained as described in Fig. 3a. e Comparison of mean fluorescence intensity of CD80, CD86, CD54, and HLA-DR expression of pre-naïve B cells from six healthy individuals and three SLE patients on days 0–2 (cumulative data from three independent experiments). Data are expressed as mean ± standard error. f Representative histogram showing proliferation of T cells in co-culture with B cells from two SLE patients and two healthy donors. Proliferation of T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) dilution. *P < 0.05, **P < 0.01, ***P < 0.001. IFN-γ interferon-gamma, MFI mean fluorescence intensity