Figure 5

CD15⁺ neutrophils react with magnetic beads. CD15⁺ cells were isolated from PBMC of three lung transplant patients (LT50 was shipped overnight, while LT48 and LT49 were processed within two hours of blood draw). (a) Aliquots of the CD15⁺ cells were used for suppression assays, using anti-CD3 microbeads for stimulation (3.5 beads/cell). proliferation of CD4⁺ (top) and CD8⁺ (bottom) responders are shown. (b,c) CD15⁺ cells were incubated with anti-CD3 microbeads (2 beads/cell) on microscopic slides for 1 hour. (b) Cells were then fixed, stained and evaluated by microscopy (×160); demonstrating CD15⁺ cells aggregating with the microbeads. (c) Quantification of neutrophil-bead binding (ANOVA). (d,e) CD15⁺, or CD15⁻ control cells, were incubated overnight with anti-CD3 microbeads and healthy donor PBMC at a 1:1 ratio. (d) Microscopic evaluation showed lymphocytes and neutrophils connected with anti-CD3 microbeads (x100). (e) Quantification of lymphocyte-bead binding (ANOVA). (f–i) CD15⁺ and CD15⁻ cells were separated from PBMC and incubated overnight with anti-CD3 microbeads (2 beads/cell) ± Fc-blocking antibodies overnight. Then beads were dissociated from cells, shown by flow cytometry as (f) % microbeads positive for anti-mouse IgG or (g) mean fluorescence of anti-mouse IgG. Unmodified anti-mouse IgG-stained CD3 beads served as a control. (h,i) Quantification of (f,g) pooled from 2 experiments with 4 samples. (j,k) Rescue of CD3-microbeads from antibody shedding. CD15⁺ cells were incubated overnight with anti-CD3 microbeads (2 beads/cell) in presence or absence of Marimastat. Next day beads were dissociated from cells, and evaluated as (j) % microbeads positive for anti-mouse IgG or (k) as mean fluorescence of anti-mouse IgG. Unmodified stained anti-CD3 beads served as positive control. Beads co-incubated with CD15⁺ cells without Marimastat served as a negative control. Data combined from 2 experiments with 3 samples. Data shown as mean ± SEM (h–k) with *, **, ***, and **** indicating p < 0.05, p < 0.01, p < 0.001, and p < 0.001, respectively. (h,j) Kruskal-Wallis test; (i,k) ANOVA. Presented histograms consist of 2,146 ± 235 cells (Mean ± SEM), and dot plots analyzing microbeads consist of 32,313 ± 3,712 (Mean ± SEM) events.