High Numbers of Circulating CD57⁺ NK Cells Associate with Resistance to HER2-Specific Therapeutic Antibodies in HER2⁺ Primary Breast Cancer

pCR achievement, trastuzumab-induced NK-cell activation, and CD57⁺ NK cells in breast cancer patients with distinct CD16A 158V/F genotype. The CD16A 158V/F genotype was analyzed in DNA samples from patients with primary HER2⁺ breast cancer (n = 60) by PCR. A, pCR rates in patients categorized according to their CD16A 158F/F or CD16A 158V/V and V/F genotypes. B, NK-cell degranulation analyzed in baseline PBMC samples from patients with HER2⁺ breast cancer upon coculture with trastuzumab-coated SKBR3 cells by flow cytometry. Percentage of CD107a⁺ CD56^dim NK cells in response to trastuzumab-coated SKBR3 cells in patients with CD16 158 F/F (n = 20) as compared with patients harboring CD16 158 V/F or V/V genotype (n = 21). P values calculated by Mann–Whitney U test. C and D, Relative and absolute numbers of circulating CD57⁺ NK cells in baseline samples from patients stratified by their CD16 158V/F genotype (*P < 0.05; Mann–Whitney test).

Reduced presence of CD57⁺ lymphocytes in tumor-associated as compared with circulating paired samples from patients with breast cancer. Tumor-associated immune cells, isolated by mechanical and enzymatic digestion, and paired PBMC samples, isolated by Ficoll gradient, were analyzed by multiparametric flow cytometry. CD45⁺ DAPI⁻ cells with lymphocyte morphology were gated for further analysis. A, t-SNE plot showing lymphocyte clusters defined by CD3, CD8, CD4, CD56, CD16, and CD57 expression in circulating and tumor-associated lymphocyte samples. Color scale represents high expression (red) or low expression (blue) of the indicated marker. Numbers indicate the CD57⁺CD16⁺ NK-cell cluster (1), the CD57⁺ CD8⁺ T-cell cluster (2), and the CD57⁺CD4⁺ T-cell cluster (3). B, Density maps in the t-SNE field displaying peripheral blood-derived (top) or tumor-associated (bottom) samples compiled separately. C–F, Proportions of NK cells (CD56⁺ CD3⁻), CD16⁺ NK cells, CD16⁺ CD57⁺, and CD16⁺CD57⁻ NK cells in paired circulating and tumor-associated samples from patients with breast cancer (n = 7). Statistical significance by Mann–Whitney U test.

Circulating CD57⁺ NK cells show reduced CXCR3 expression and CD16-induced proliferation. Surface expression of CXCR1, CXCR3, CX3CR1, CCR7, and CD62L was analyzed by multiparametric flow cytometry in CD57⁺ and CD57⁻ CD56^dim NK cells from patients with HER2⁺ breast cancer. A and B, Dot plots showing CXCR1, CX3CR1, CXCR3, CCR7, CD62L, and CD57 coexpression in circulating NK cells. Data from four representative patients. C, Proportions of CD57⁺ and CD57⁻ NK cells positive for each molecule. Each dot represents a single determination in individual patients (n = 6). Statistical comparison by Wilcoxon paired test. D–F, CFSE dilution assays by flow cytometry using purified NK cells from healthy volunteers cultured for 6 days with plate bound anti-CD16 agonist at 0.5 or 5 μg/mL in the presence of IL2. D, Dot plots and histograms showing CFSE labeling in CD57⁺ and CD57⁻ NK cells in the indicated conditions. Data from a representative experiment out of 6 performed are shown. E, Percentage of CD57⁺ and CD57⁻ NK cells ≥ 4 divisions at day 6 (mean ± SEM; n = 6). F, Numbers of total and CD57⁺ NK cells recovered at day 6 (mean ± SEM; n = 6).

Comparable responses of CD57⁺ and CD57⁻ circulating NK cells from HER2⁺ breast cancer patients in trastuzumab-induced ADCC in vitro assays. A–C, NK-cell activation as analyzed in baseline PBMC samples from patients with HER2⁺ breast cancer by flow cytometry upon coculture with trastuzumab-coated SKBR3 cells. A, Frequencies of CD57⁺ NK cells in PBMC samples from patients categorized as CD57^high (≥65% CD57⁺ NK cells, n = 18) or CD57^low (<65% of CD57⁺ NK cells, n = 16). B and C, Percentage of CD107a⁺ or TNFα⁺ CD56^dim NK cells in patients with high or low proportions of CD57⁺ NK cells. P values calculated by Mann–Whitney U test. D–G, PBMCs from patients with HER2⁺ breast cancer and healthy controls paired by their CD16 genotype and NKG2C profile were cocultured with trastuzumab-coated SKBR3 for 4 hours and degranulation was analyzed by CD107a mobilization assay by flow cytometry (D). Dot plots showing the proportions of CD107a⁺ NK cells according to CD57 expression in NK cells from patients and controls paired by their CD16 genotype. Data from two representative examples are shown. E–G, Percentage of total as well as CD57⁺ and CD57⁻ NK cells degranulating against trastuzumab-coated SKBR3 cells in PBMC samples from healthy controls and patients with HER2⁺ breast cancer. Control and patient samples with the same CD16 genotype were assayed in parallel.