ELISA assays of IFN-γ, IL-17 and IL-4 in serum.

<p>Sera from collected blood at each time point (1, 3, 6, 11, 16, and 30 days) from xenotransplanted and sham control mice were used for ELISA analysis. Sera IFN-γ and IL-17 levels in the xenograft group were significantly higher than those in the serum in the control group (p<0.05) at day 1, day 3 and day 6, and decreased to control levels by day 11. There is no significant difference of IL-4 levels between recipients and control group. *, p<0.05; **, p<0.01; *** p<0.001. (We performed this experiment twice).</p

Xenotransplant after using IFN-γ and IL-17 neutralizing antibodies respectively.

<p>(A) Spleens were collected at each time point (1, 3, 6, 11, 16, and 30 days) from xenotransplanted mice with or without neutralizing antibodies (single used and mixed used) treatment, and used for flow cytometry analysis. The levels of IFN-γ producing CD3+CD8− T cells and IL-17 producing CD3+CD8− T cells from recipients with IL-17 neutralizing antibody and mixed used antibodies significantly decreased at day 3 compared with the control group. The levels of IFN-γ producing CD3+CD8+ T cells and IL-17 producing CD3+CD8+ T cells from recipients with IL-17 neutralizing antibody and mixed used antibodies significantly decreased at day 3 and day 6 compared with the control group. (B) Tissues surrounding the xenografts were used to do Immunohistochemical staining to measure the expression of IFN-γ+ and IL-17+ cells. We observed that mononuclear cells invasion significantly decreased at day 3 and day 6; and IFN-γ and IL-17 expression decreased in the recipients with IFN-γ and IL-17 neutralizing antibodies single used and mixed used from day 11 to day 30. *, p<0.05; **, p<0.01; *** p<0.001. (We performed this experiment twice).</p

H&E staining of nerve grafts.

<p>Sham control or xenotransplanted sciatic nerve from mice was collected at 1, 3, 6, 11, 16, and 30 days posttransplantation, sectioned, and stained with H&E. Staining demonstrated xenograft adhesion to the surrounding tissue, and infiltration of mononuclear cells into the peripheral nerve and surrounding tissue. There was no significant difference during 30 days after xenotransplantation in control group. The level of mononuclear cell infiltration, distension, and necrosis significantly increased in all xenografts at all time point after transplantation compared with control group. Magnification: 200×. (We performed animal experiment twice, and stained 2 sections for each animal.)</p

Flow cytometry analysis of IFN-γ, IL-17 and IL-4 in lymphocytes from spleen.

<p>Spleens were collected at each time point from xenotransplanted mice (day 1, 3, 6, 11, 16, and 30) and sham controls, and used for flow cytometry analysis (A) Representative data of IFN-γ, IL-17 and IL-4 expression in lymphocytes from spleen at day 1. Graph a, b, c and d are gated from R1; e, f, g, and h are gated from R2. (B) Percentage of CD3+CD8− T cells expressing IFN-γ, IL-17 and IL-4 at days 1–30 were averaged and graphed. IFN-γ expression increased from day 1 to day 6, and decreased to control levels by day 11. IL-17 expression increased at day 3 and lasted until day 6, and decreased to control levels by day 11. There is no significant difference of IL-4 producing CD3+CD8−T cells between recipients and control group. *, p<0.05; **, p<0.01; *** p<0.001. (C) In CD3+CD8+ T cells, IFN-γ and IL-17 expression increased from day 1 to day 6, and decreased to control levels by day 11. There is no significant difference of IL-4 producing CD3+CD8+T cells between recipients and control group. *, p<0.05; **, p<0.01; *** p<0.001. (We performed this experiment twice).</p

A Lower Proportion of Regulatory B Cells in Patients with Henoch–Schoenlein Purpura Nephritis

<div><p>Background</p><p>Henoch—Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.</p><p>Methods</p><p>In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.</p><p>Results</p><p>Compared to those in healthy controls, the numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and IL-10⁺CD5⁺CD1d⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38⁺CD19⁺ and CD86⁺CD19⁺ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL-10⁺CD19⁺, and IL-10⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38⁺CD19⁺ B cells but positively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺B cells but negatively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration.</p><p>Conclusion</p><p>Our results suggest that CD38⁺CD19⁺ and CD1d⁺CD5⁺CD19⁺ B cells (Bregs) contribute to the pathogenesis of HSPN.</p></div

Flow cytometric analysis of the numbers of different subsets of B cells.

<p>PBMCs were collected from HSPN patients and healthy controls. Then, they were stained with PerCP-anti-CD19, PE-anti-CD38, APC-anti-CD86, or PerCP-anti-CD19, PE-anti-CD27, and APC-anti-CD95 (Biolegend, San Diego, CA, USA), or isotype-matched control IgG (Beckton Dickinson, San Jose, CA, USA). For further analysis of different subsets of B cells, the cells were gated initially on living lymphocytes and then on CD19⁺ B cells. (A) Flow cytometric analysis results. (B) The numbers of CD19⁺ B cells. (C) The numbers of CD38⁺CD19⁺ plasma cells. (D) The numbers of CD86⁺CD19⁺ B cells. (E) The numbers of CD38⁺CD86⁺CD19⁺ B cells. (F)The numbers of CD95⁺CD19⁺ B cells. Data are expressed as the means for individual subjects included in two separate experiments. (G) Mean fluorescence intensity of CD86 on B cells. (H) Mean fluorescence intensity of CD95 on B cells. (I) Mean fluorescence intensity of CD95 on CD27⁺ B cell subsets. (J) Mean fluorescence intensity of CD95 on CD27^- B cell subsets. The horizontal lines represent the median values. Data were analyzed by Mann-Whitney U-test.</p

Correlations among different subsets of B cells and the serum concentration of IL-10 in HSPN patients.

<p>Potential correlations among the numbers of B cells of different subsets and the serum concentration of IL-10 were analyzed by the Spearman correlation tests. Data are expressed as the means or concentrations for individual subjects that participated in two separate experiments. (A-B) The number of CD38⁺CD19⁺ B cells was negatively correlated with the serum IL-10 level and the percentage of IL-10⁺CD19⁺ B cells. (C-D) The number of CD86⁺CD19⁺ B cells was negatively correlated with the serum IL-10 level and the number of IL-10⁺CD19⁺ B cells. (E-F) The serum IL-10 level was positively correlated with the numbers of CD1d⁺CD5⁺CD19⁺ and IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells.</p

The change in the frequency of B cell subtypes and the serum concentration of IL-10 in HSPN patients following treatment.

<p>Differences in patients pre- and post-treatment were analyzed by the Wilcoxon test. Data are expressed as the means or concentrations for individual subjects who participated in two separate experiments. (A) Serum level IL-10 in individual patients’ pre- and post-treatment. (B-F) The numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and CD5⁺CD1d⁺IL-10⁺CD19⁺ B cells of individual patients in the pre- and post-treatment stages.</p

The effect of treatment on the clinical parameters of HSPN patients during the follow-up period.

<p>The effect of treatment on the clinical parameters of HSPN patients during the follow-up period.</p

Flow cytometric analysis of Bregs isolated from PBMCs of HSPN patients and healthy controls.

<p>The cells were stained with anti-CD19, anti-CD5, anti-CD1d, and intracellular anti-IL-10 or isotype-matched IgG. The cells were characterized by flow cytometric analysis; living lymphocytes were gated initially, followed by gating on CD19⁺ B cells. Subsequently, we analyzed the numbers of CD5⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells among CD19⁺ B cells, and at least 30,000 events per sample were analyzed. (A) Flow cytometric analysis. (B) The numbers of CD5⁺CD19⁺ B cells. (C) The numbers of IL-10⁺CD19⁺ B cells. (D) The numbers of CD1d⁺CD5⁺CD19⁺ B cells. (E) The numbers of IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells. (F) Serum concentrations of IL-10. Data are expressed as the means or concentrations for individual subjects that participated in two separate experiments. The horizontal lines represent the median values of each group. Data were analyzed by the Mann-Whitney U-test.</p