Supplementary Figure 8: Effect of ACKR1 on the expression of cell surface markers and cell numbers in BM and blood.

(a) CD62L, CD11a, CD16/32, CD45 and CD11b expression in BM neutrophils (CD11b⁺Ly6G⁺) from wild-type (WT, blue) and ACKR1-deficient (KO, red) mice; NC (white), negative control staining. Top, representative flow cytometry histograms; bottom, quantitative analysis. (b) Representative flow cytometry histograms of staining blood neutrophils (CD11b⁺Ly6G⁺) from WT and KO mice with antibodies against CD62L and CD11a and (c) antibodies against CXCR2, CCR1, CCR2, CCR3 and CCR5. (d) Representative flow cytometry histograms of CXCR1, CXCR2 and CCR2 antibodies staining of blood neutrophils from Duffy-positive and Duffy-negative individuals, grey and black histograms, respectively, negative control staining, NC (white). (e) PMN (CD11b⁺Ly6G⁺CD115^–), monocyte (CD11b⁺Ly6G^–CD115⁺) and B lineage cell (B220⁺CD19⁺) counts in BM of WT and ACKR1-deficient mice (n=6). (f) Monocyte (CD45⁺CD11b⁺Ly6G^– CD115⁺) and lymphoid cell (CD45⁺ and B220⁺ or CD3⁺) counts in blood of WT and ACKR1-deficient mice (n=6). (g) Platelet counts in blood of WT and ACKR1-deficient mice (n=16). (h) Expression of CD16/32 on blood neutrophils (CD11b⁺Ly6G⁺CD115^–) in reciprocal BM chimeric mice; WT BM cells reconstituted into WT mice (blue), WT BM cells reconstituted into KO mice (light blue), KO BM cells reconstituted into KO mice (red) and KO BM cells reconstituted into WT mice (pink); n=4 from two independent experiments. (i) Representative immunofluorescence micrograph of human spleen stained with monoclonal anti-ACKR1 (Fy6, red) antibody. All data show Mean±SEM. (a and e-g) two-tailed Student’s t-test; *P < 0.05, **P < 0.01. (h) one-way ANOVA; **P < 0.01.