Fig. 2

Partial SNX27 deficiency in CD4-Cre-SNX27^fl/+ mice dysregulates circulating lymphocyte populations. A-H Aliquots of whole blood from SNX27^fl/fl and CD4-Cre-SNX27^fl/+ were incubated with a mix of LIVE/DEAD violet fixable cell stain and the following surface markers: CD45, CD3, B220, CD8, CD4, Ly6C, and Ly6G (20 min, RT). Versalyte solution was then added to lyse red cells (10 min, RT, darkness). Afterwards, flow cytometry analysis of the distinct populations was performed. Histograms depicting percentage of (A) B cells (CD45⁺ CD3⁻ B220⁺), (B) T cells (CD45⁺ CD3⁺ B220⁻), (C) CD4⁺ and (D) CD8⁺ (CD45⁺ B220⁻ CD3⁺), (E) the ratio between CD8⁺ and CD4⁺ cells, (F) monocytes (CD45⁺ CD3⁻ B220⁻ Ly6C⁺), (G) inflammatory monocytes (CD45⁺ CD3⁻ B220⁻ Ly6C⁺ high), and (H) granulocytes (CD45⁺ CD3⁻ B220⁻ Ly6G⁺) are shown. Gating strategy is shown in Suppl Fig. 3. Data shown as mean ± SEM; *p < 0.05; unpaired t-test; n = 12 mice in (A-E), and n = 7 mice in (F–H). I, J CD8⁺/CD4⁺ ratio in bone marrow (I) and spleen (J) is shown. Gating strategies used are depicted in Suppl Fig. 4. Data shown as mean ± SEM; *p < 0.05; ***p < 0.001 unpaired t-test; n = 11 mice in (I) and n = 14 mice in (J). K Representative maximum intensity projections from confocal planes (z) of SNX27^fl/fl and CD4-Cre-SNX27^fl/+ mice lymph nodes stained for CD4 in basal conditions. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. L Quantification of CD4 expression measured as the signal intensity of the whole cell. Data are shown as ± SEM of an experiment (SNX27^fl/fl = 211 cells; CD4-Cre-SNX27^fl/+ = 179 cells); unpaired t-test; ****p < 0.0001. SNX Sorting nexin, RT Room temperature, SEM Standard error of the mean