Fig. 3.

Alternatively activated neutrophils isolated from EAE spinal cords suppress T cell proliferation ex vivo. CFSE-labeled naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 and cultured either alone or with the indicated Ly6G⁺ neutrophil populations at a 1:1 neutrophil–to–T cell ratio for 4 days. T cell proliferation was assessed by CFSE dilution using flow cytometry. Representative flow cytometry plots (left) show CD44 versus CFSE expression among gated viable CD45⁺CD11b⁻CD4⁺ cells, illustrating activation and proliferation under each condition. CFSE dilution among CD44⁺ activated T cells was used to quantify proliferating cells. Quantification of the percentage of CFSE^low dividing CD4⁺ T cells is shown on the right. Splenic neutrophils (SPL) were isolated from naïve mice by Ly6G magnetic bead separation. Arg1⁺ and Arg1⁻ neutrophils were FACS-isolated from the spinal cords of Arg1^YFP mice with EAE and defined as CD45⁺CD11b⁺Ly6G⁺Arg1-YFP⁺ and CD45⁺CD11b⁺Ly6G⁺Arg1-YFP⁻ cells, respectively. Each data point represents an individual biological replicate (CNS neutrophil subsets pooled from 1–9 mice). Data are pooled from three independent experiments. Bars indicate mean ± SEM. Statistical significance was determined using an unpaired, two-tailed Student’s t test. ns, not significant. *p < 0.05; ****p < 0.0001