Fig. 6 .

Zymosan-modulated neutrophils suppress CD4⁺ T cell proliferation via a cell contact–dependent mechanism. A Antigen-specific suppression assays were performed by co-culturing CFSE-labeled MOG₃₅–₅₅–specific 2D2 CD4⁺ T cells with MOG₃₅–₅₅–pulsed, T cell–depleted splenocytes in the absence of neutrophils (black circles) or in the presence of bone marrow–derived neutrophils (BMN; green triangles) or zymosan-modulated neutrophils (ZMN; blue squares) for 4 days. T cell proliferation was assessed by CFSE dilution using flow cytometry. Representative flow cytometry plots showing CFSE dilution and CD44 expression in CD4⁺ T cells are shown, along with quantification of the percentage of CFSElow dividing cells among total CD4⁺ T cells (gated on viable CD45⁺CD11b⁻CD4⁺ cells). B CFSE-labeled naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 and cultured alone or with BMN or ZMN for 4 days. C To assess contact dependence, CFSE-labeled naïve CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 were co-cultured with the indicated neutrophil populations using transwell inserts to prevent direct cell–cell contact. Each data point represents an individual biological replicate. Statistical significance was determined using a two-tailed Student’s t test. Error bars represent mean ± SEM. p < 0.05; **p < 0.001