Article Figures & Data
Figures
- Figure 1.
Downregulation of CD47 expression promoted IgA-mediated ADCC by neutrophils. A, Representative histograms (n = 2) showing the expression of CD47 and HER2/neu or EGFR in SKBR3 and A431 cells (top) and their respective CD47KD or CD47KO variants (bottom). B and C, Genetic disruption of CD47–SIRPα interactions led to increased ADCC of tumor cells (SKBR3 and A431) by freshly isolated neutrophils using increasing concentrations of the indicated IgG1 (black lines) or their IgA2 variants (gray lines). Data shown are means ± SEM pooled from two to three experiments with 2 to 3 donor samples per experiment with n = 2–5 (B, SKBR3) and n = 3–4 (C, A431) individual donors. Statistics shown in B and C are for the highest antibody concentrations or highest E:T ratio using two-way ANOVA with Tukey correction for multiple tests. ***, P < 0.001. Cmab, cetuximab; Tmab, trastuzumab.
- Figure 2.
Different approaches to inhibit CD47–SIRPα interactions enhanced IgA-mediated ADCC. ADCC of cancer cells by freshly isolated neutrophils without (white background) and with inhibition of CD47–SIRPα interactions (gray background) by either using cell lines with no (A431-CD47KO) or reduced (SKBR3-CD47KD) expression of CD47 (A), inhibition of CD47 with B6H12 F(ab′)2 antibodies (B), or inhibition of SIRPα using antibody 12C4 (C). Used are either anti-HER2-IgA2 antibody against SKBR3 or anti-EGFR-IgA2 antibody (either at 5 μg/mL) against A431 cells, respectively. Data shown are means ± SEM pooled from two to three experiments with 2 to 3 individual donor samples per experiment with n = 8 (A), n = 7–8 (B), and n = 6–8 (C). Statistics were performed by a paired t test (**, P < 0.01; ***, P < 0.001).
- Figure 3.
IgA-mediated trogocytosis of cancer cells by neutrophils. A–F, Representative live-cell imaging stills at different time points of a neutrophil binding to an anti-HER2-IgA2–opsonized cancer cell and trogocytosing pieces of fluorescent cancer cell membrane. Scale bar, 10 μm. G, Trogocytosis of SKBR3 cells opsonized with increasing concentrations of trastuzumab or anti-HER2-IgA2. Tmab, trastuzumab. ADCC (H) and trogocytosis (I) of anti-EGFR-IgA2–opsonized A431 cancer cells by freshly isolated neutrophils isolated from healthy controls or FHL5 patients (for patient characteristics, see patients B and C described in ref. 32) combined without (white background) or with CD47–SIRPα inhibition by knockout of CD47 in the target cells (gray background). ADCC (J) and trogocytosis (K) of anti-HER2-IgA2–opsonized SKBR3 cells by freshly isolated neutrophils isolated from healthy controls or CGD patients (for patient characteristics, see Materials and Methods), combined without (white background) or with CD47–SIRPα inhibition by knockdown of CD47 in the target cells (gray background). Data shown are means ± SEM pooled from two separate experiments with two replicates of 2 individual FHL5 patient samples and 11 total healthy controls (H and I), three separate experiments with 3 (K) or 4 (J) individual CGD patient samples and 4-day controls (J and K), or pooled from two experiments with 1 to 2 donor samples per experiment with n = 4 (G) individual donors. Statistics shown in G are for the highest antibody concentrations using two-way ANOVA with Tukey correction for multiple tests. No statistics could be performed on FHL5 patient data in H and I due to low patient count (n = 2). Statistics shown in J and K using one-way ANOVA with Sidak correction for multiple tests (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). MFI, mean fluorescence intensity.
- Figure 4.
Inhibition of CD47–SIRPα did not result in alternative FcαR-mediated ADCC. Neutrophil-mediated trogocytosis (A) and ADCC (B) of SKBR3 (white background) and SKBR3-CD47KD (gray background) cells using IgA2-Her2 antibody with inhibition of Syk, PI3K, myosin light chain kinase, CD11b/CD18 integrins and calcium mobilization. Data shown are means ± SEM pooled from two experiments with 2 donor samples per experiment with n = 4 individual donors. Statistics are shown using one-way ANOVA with Sidak correction for multiple tests (*, P < 0.05; ****, P < 0.001).
- Figure 5.
Interference with CD47–SIRPα interactions enhanced tumor eradication in a xenogeneic long-term in vivo model. A, Schematic overview of the in vivo xenograft model and the injection scheme. Long-term in vivo tumor growth comparing the maternal A431-SCR cell line (B) with one with no expression of CD47 (A431-CD47KO; C). Treatment was started when mice had palpable tumors (day 6) with either PBS (light gray line), a single intravenous injection of 50 μg cetuximab (black line), or an intravenous injection of 250 μg anti-EGFR-IgA2 followed by four intraperitoneal injections of 250 μg (dark gray line) to compensate for the shorter half-life of IgA compared with IgG. Tumor outgrowth was measured with calipers, and volume was calculated as length × width × height. Data shown are means ± SEM from one experiment with 8 mice per group in B and C. Statistics performed are for day 17 using two-way ANOVA with Tukey correction for multiple tests (ns, nonsignificant; *, P < 0.05). Cmab, cetuximab.
- Figure 6.
Inhibition of SIRPα effectively triggered syngeneic tumor cell killing by neutrophils. A, Expression of CD47 and HER2 or EGFR in Ba/F3 cells transfected with human HER2 or EGFR, respectively. B, ADCC of trastuzumab or anti-HER2-IgA2 (either at 5 μg/mL)–opsonized Ba/F3-HER2 cells by mouse neutrophils isolated from wild-type (NTg) or FcαR-transgenic (FcαR-Tg) mice combined without (white background) or with inhibition of SIRPα (MY-1; gray background). C, Same as B but with cetuximab or anti-EGFR-IgA2–opsonized Ba/F3-EGFR cells. D, In vivo inhibition of SIRPα (MY-1) was combined with trastuzumab or anti-HER2-IgA2 treatment in a short intraperitoneal model. The ratio of Ba/F3-HER2 and Ba/F3 cells in wild-type mice [PBS, isotype + Tmab, anti-SIRPα (MY-1) + Tmab] or FcαR-transgenic mice [isotype + anti-HER2-IgA2, anti-SIRPα (MY-1) + anti-HER2-IgA2] in the peritoneal washes was determined by flow cytometry. E, Number of granulocytes (Ly6G+/CD11b+) present in the peritoneal cavity at the end of the experiment in D. F, Overview of all cells present in the peritoneal cavity during the experiment shown in D. G, Inhibition of SIRPα (MY-1) is combined with anti-HER2-IgA2 and neutrophil depletion (Ly6G) in an in vivo mouse experiment, showing the ratio of Ba/F3-HER2 and Ba/F3 cells in wild-type mice [isotype, anti-SIRPα (MY-1), anti-SIRPα (MY-1) + anti-HER2-IgA2 (NTg)] or FcαR-transgenic mice [isotype + anti-HER2-IgA2, anti-SIRPα (MY-1) + anti-HER2-IgA2, anti-SIRPα (MY-1) + anti-HER2-IgA2 + Ly6G]. Data shown for B and C are means ± SEM pooled from two experiments with 2 mice per experiment with a total of n = 4 individual mice. Data shown for D–F are means ± SEM from one experiment with 6 mice per group with A representative of n = 2, B n = 3–6, C n = 6, and D n = 6 individual mice. Statistics shown for B and C are calculated by one-way ANOVA with Dunnett correction for multiple tests. Statistics shown for D and G are calculated by paired one-way ANOVA with Sidak correction for multiple tests. Statistics performed for E are calculated by one-way ANOVA with Sidak correction for multiple tests (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Cmab, cetuximab; Tmab, trastuzumab.
Additional Files
Supplementary Data
- Supplementary Figures - Supplementary Figures
- Supplementary Data - Supplementary Materials and Methods
Volume 8, Issue 1
