Myelin Basic Protein-Induced Production of Tumor Necrosis Factor-α and Interleukin-6, and Presentation of the Immunodominant Peptide MBP85-99 by B Cells from Patients with Relapsing-Remitting Multiple Sclerosis

B cells are involved in driving relapsing-remitting multiple sclerosis (RRMS), as demonstrated by the positive effect of therapeutic B-cell depletion. Aside from producing antibodies, B cells are efficient antigen-presenting and cytokine-secreting cells. Diverse polyclonal stimuli have been used to study cytokine production by B cells, but here we used the physiologically relevant self-antigen myelin basic protein (MBP) to stimulate B cells from untreated patients with RRMS and healthy donors. Moreover, we took advantage of the unique ability of the monoclonal antibody MK16 to recognize the immunodominant peptide MBP85-99 presented on HLA-DR15, and used it as a probe to directly study B-cell presentation of self-antigenic peptide. The proportions of B cells producing TNF-α or IL-6 after stimulation with MBP were higher in RRMS patients than in healthy donors, indicating a pro-inflammatory profile for self-reactive patient B cells. In contrast, polyclonal stimulation with PMA + ionomycin and MBP revealed no difference in cytokine profile between B cells from RRMS patients and healthy donors. Expanded disability status scale (EDSS) as well as multiple sclerosis severity score (MSSS) correlated with reduced ability of B cells to produce IL-10 after stimulation with MBP, indicative of diminished B-cell immune regulatory function in patients with the most severe disease. Moreover, EDSS correlated positively with the frequencies of TNF-α, IL-6 and IL-10 producing B cells after polyclonal stimulation. Patient-derived, IL-10-producing B cells presented MBP85-99 poorly, as did IL-6-producing B cells, particulary in the healthy donor group. B cells from MS patients thus present antigen to T cells in a pro-inflammatory context. These findings contribute to understanding the therapeutic effects of B-cell depletion in human autoimmune diseases, including MS

Influenza Virus–induced Dendritic Cell Maturation Is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein

We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-γ, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-γ. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2–3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently

Characterization of Regulatory B Cells in Graves' Disease and Hashimoto's Thyroiditis

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24hiCD38hi and CD27+CD43+, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10+ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10+ B cells from patients and healthy donors are discussed

Proportions of cytokine-producing B cells after polyclonal stimulation.

<p>Mononuclear cells from 12 healthy donors (HD) and 13 relapsing-remitting multiple sclerosis (RRMS) patients were stimulated with myelin basic protein (MBP) for 24 hours, and with PMA + ionomycin for the last 4 hours of incubation. Cells were stained intracellularly with antibodies against (A) TNF-α, (B) IL-6 and (C) IL-10, and assessed by flow cytometry. Shown are the proportions of CD19+ B cells producing these cytokines; the corresponding values for unstimulated cell cultures have been subtracted. Box plots indicate median, interquartile range (box) and range (whiskers).</p

Association between MBP-induced cytokine production by B cells and disease severity.

<p>Mononuclear cells from 12 relapsing-remitting multiple sclerosis patients were stimulated with myelin basic protein (MBP) for 24 hours, stained for content of (A and D) TNF-α, (B and E) IL-6, or (C and F) IL-10, and assessed by flow cytometry. The proportion of cytokine-producing B cells adjusted for background (un-stimulated cells) is shown as a function of the Expanded Disability Status Scale (EDSS; upper row) and the Multiple Sclerosis Severity Score (MSSS; lower row)(both missing for one patient). Spearman’s correlation coefficient (R_S) and the corresponding <i>p</i>-values are also shown.</p

MBP-induced cytokine production by B cells.

<p>Mononuclear cells from 12 healthy donors (HD) and 13 patients with relapsing-remitting multiple sclerosis (RRMS) were stimulated with whole myelin basic protein (MBP) for 24 hours and stained intracellularly for (A) TNF-α, (B) IL-6, and (C) IL-10 before assessment by flow cytometry. The proportions of CD19+ B cells producing these cytokines are shown as median, interquartile range (box) and range (whiskers), adjusted for background (positive events in unstimulated cell cultures). In some cases these numbers were larger than in MBP-stimulated cultures, hence negative values. <i>p</i>-values indicate the probability of no difference between the groups (two-tailed Mann Whitney U-test) or from zero (Wilcoxon signed-rank test).</p

Presentation of MBP85-99 and cytokine production by HLA-DR15+ B cells.

<p>Mononuclear cells from 7 healthy donors (HD; all heterozygous for HLA-DR15) and 7 RRMS patients (2 homozygous, and 5 heterozygous for HLA-DR15) were either left unstimulated (-Stim), or were stimulated with whole myelin basic protein (MBP) for 24 hours. Cells were then stained with the mAb MK16, recognizing the MBP-derived peptide MBP85-99 presented on HLA-DR15. (A and C) Representative histogram plots showing MK16 binding to the total CD19+ B-cell pool (bulk) and the subsets of B cells producing IL-6, TNF-α, and IL-10. (B) Median fluorescence intensity (MFI) values of MK16 binding to bulk B cells and B cells producing IL-6, TNF-α, and (D) IL-10 after MBP stimulation are shown as median, interquartile range (box) and range (whiskers). The corresponding values for unstimulated cell cultures have been subtracted. Values from donors homozygous for HLA-DR15 were halved to obtain the MFI value per allele. <i>p</i>-values indicate probabilities for no difference between cytokine-producing B-cell subsets and the total B-cell pool (Wilcoxon matched-pairs signed rank test), or between study groups (two-tailed Mann Whitney U-test). NS: Not significant.</p