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. 2023 Dec 5;24(24):17141. doi: 10.3390/ijms242417141

Figure 4.

Figure 4

Inhibition of the interaction between sialic acids and SIGLEC-5/14 increases the activation of CD11b/CD18 in the context of neutrophil ADCC. (a) Conjugate formation measured as the percentage of gated population (neutrophils/calcein violet AM- and tumor cells/cell tracker red- positive cells) between neutrophils and A431 tumor cells. Neutrophils were pre-incubated with SIGLEC-5/14 blocking antibody and tumor cells were opsonized with cetuximab, where indicated (9 donors from 5 independent experiments). (b) Adhesion of unstimulated or Phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. Neutrophils were pre-incubated with intact IgG antibody as an isotype control, or SIGLEC-5/14, FcγRIIa F(ab)2, FcγRIIa F(ab)2 and SIGLEC-5/14, CD18 F(ab)2 and SIGLEC 5-14 or CD18 F(ab)2 blocking antibodies. (c) Flow cytometry analysis displaying the ratio of activated epitope of CD11b (CBRM1/5) towards the total CD18 expression. Neutrophils were pre-incubated with SIGLEC-5/14 blocking antibody, and tumor cells were opsonized with cetuximab, where indicated (12 donors from 6 independent experiments). (d) Avidity curve shows the percentage of neutrophils bound to A431 in the presence or absence of cetuximab after application of increasing acoustic force (pN). (e) Bar graph presentation of the neutrophils bound to A431 after force application of 1000 pN. Statistics: (a) paired t-test; (b,c) one-way ANOVA with Tukey correction; (e) one-way ANOVA with Sidak correction; ns, nonsignificant; *, p < 0.05; **, p < 0.01, **** p < 0.0001. Cmab, cetuximab.