Neutrophils co-localize in T cell zones and influence CD4⁺ T cell responses in a BAFF-dependent manner.

<p>(A) Confocal microscopy of spleen sections demonstrating the <i>in </i><i>situ</i> localization of neutrophils. CD11b – white, Ly6G – red, IgD – blue, PNA – green. Scale bar indicates 100 µm. (B) Confocal microscopy of spleen sections demonstrating the co-localization of neutrophils and CD4⁺ T cells. Ly6G – white, CD4– red, IgD – blue, PNA – green. Scale bar indicates 100 µm. Scale bar of inset indicates 20 µm. Images are representative of at least three images from 4 mice/genotype. (C) Representative histograms showing BR3 expression on CD4⁺ T cells from spleens of three mice/genotype. Total B cells (B220⁺) and a FMO stain served as controls. (D) Purified neutrophils from spleens of lupus-prone mice were co-cultured with CD4⁺ T cells from WT mice at a 1∶1 ratio in the presence or absence of BR3 blocking antibody. After 3 days of culture, CD4⁺ T cell proliferation was measured by CellTrace Violet dilution and cytokine concentrations in culture supernatant were measured by ELISA. Combined data from three mice/genotype. One of three independent experiments with similar results is shown. (E) Gene expression levels of <i>Tnfrsf13C</i> (BR3) in purified neutrophils from mice of the indicated genotype. Samples were normalized to <i>HPRT</i> and the expression in WT animals was set to one. Total B cells are shown as a positive control. Combined data from 3 mice/genotype. (F) Serum IFNγ levels from 6- and 25-week-old mice of the indicated genotype were measured by ELISA. Combined data from 5–9 mice/genotype. (G) Total splenocytes from mice of the indicated genotype were cultured in the presence or absence of PMA/ionomycin with GolgiStop. The frequency of IFNγ-producing CD4⁺ T cells was assessed following 5 hour <i>in </i><i>vitro</i> stimulation. Combined data from 4 mice/genotype at 6 and 25weeks of age. Error bars indicate mean ± SEM. Statistics determined with a two-way ANOVA using a Bonferroni post test (D), or a one-way ANOVA with a Tukey post test (F, G), and denoted as follows: *p<0.05, **p<0.01, ***p<0.001, ns – not significant.