Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients

Abstract

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Keywords: ADCC; Adaptive NK cells; HER2 + breast cancer; Trastuzumab.

Fig. 1

Presence of adaptive NK cells in BC patients and HD. a Percentage of FcεRIγ- (above) or NKG2C + (bellow) within CD3-CD56dimCD16+ NK cells in BC patients (left) or HD (right) seronegative or seropositive for HCMV (Mann–Whitney test). b Venn diagrams, showing the prevalence of the adaptive NK cell markers in BC patients (left) or HD (right). c Gating strategy for NK cell subsets and examples of the population`s distribution obtained from three different patients (Pt 1–3). d Proportion of FcεRIγ+NKG2C– , FcεRIγ+NKG2C+, FcεRIγ– NKG2C– and FcεRIγ– NKG2C+ CD56dim NK cells, between BC patients and HD (left), different subtypes (center) and stages (right) (two-way ANOVA with Sidak’s post-tests). Graph shows median bar. *p < 0.05; ****p < 0.0001

Fig. 2

Phenotypic characterization of FcεRIγ− and FcεRIγ+ NK cells in BC patients and HD seropositive for HCMV. a Pearson correlation matrix for the expression of the CD56dim NK cell receptors FcεRIγ, NKG2C, CD16, NKp30 and CD85j in BC patients (left) and HD (right). b Principal component analysis of NK cells receptors in FcεRIγ− (red) and FcεRIγ+ (blue) NK cells in BC patients (■) and HD (●). c Percentage of NKp30, CD161 and CD85j and CD16 rMFI in FcεRIγ+ and FcεRIγ− NK cells (Wilcoxon test). ****p < 0.0001, ***p < 0.001, *p < 0.05

Fig. 3

Degranulation assay in FcεRIγ+ and FcεRIγ− NK cells. a Schematic representation of the assay workflow. b Representative example of FcεRIγ+ and FcεRIγ− NK cells degranulation of a HD. c Percentage of CD107a+ in FcεRIγ+ NK cells in the presence of SKBR3 cells opsonized or not with TRZ (n = 7). d Percentage of CD107a+ in FcεRIγ− NK cells in the presence of SKBR3 cells opsonized or not with TRZ (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001

Fig. 4

IFN-γ production by adaptive NK and conventional NK cells of BC patients and HD. a Percentage of IFN-γ in CD56dim NK cells from BC patients and HD (Mann–Whitney test). b Representative density plot of IFN-γ production in FcεRIγ+ and FcεRIγ− subsets in CD56dim NK cells. IFN-γ production in FcεRIγ− versus FcεRIγ+ NK cells in BC patients c and HD d (Wilcoxon test). e Representative density plot of IFN-γ production in NKG2C+ and NKG2C− subsets in CD56dim NK cells. IFN-γ production in NKG2C− versus NKG2C + NK cells in BC patients (f) and HD (g) (Wilcoxon test). **p < 0.01 ***p < 0.005 ****p < 0.001

Fig. 5

Percentage of IFN-γ+ in the four NK cell subsets against SKBR3 cells coated with TRZ, using PBMCs of BC patients a or HD b as effector cells (ANOVA with Dunns post-tests). Differences in IFN-γ production between NK cell subpopulations in BC patients and HD characterized as ΔIFN-γBC-HD = IFN-γ (CD56dimNKG2C+ FcεRIγ−) — IFN-γ (CD56dimNKG2C−FcεRIγ+) c, ΔIFN-γBC-HD = IFN-γ (CD56dimNKG2C+ FcεRIγ+) — IFN-γ (CD56dimNKG2C−FcεRIγ+) d and ΔIFN-γBC-HD = IFN-γ (CD56dimNKG2C−FcεRIγ−) — IFN-γ (CD56dimNKG2C−FcεRIγ+) e, (Mann–Whitney test). n = 10 *** p < 0.001, *p < 0.05

Fig. 6

Changes in NK cells after neoadjuvant treatment. a Percentage of NK cells (left) and absolute NK cell number (right) before (PRE) and after (POST) neoadjuvant therapy (Paired t test). b Percentage of FcεRIγ− NK cells PRE and POST neoadjuvant therapy (left) and fold change calculated as the absolute number after therapy divided by absolute number before treatment (right) (Paired t test). c Percentage of NKG2C+ NK cells PRE and POST neoadjuvant therapy (left) and fold change calculated as the absolute number after therapy divided by absolute number before treatment (right) (Wilcoxon test). d Percentage of FcεRIγ−NKG2C+ (Paired t test), FcεRIγ−NKG2C− (Wilcoxon test), FcεRIγ+ NKG2C+ (Paired t test) and FcεRIγ+ NKG2C− (Paired t test) NK cell subsets PRE and POST neoadjuvant therapy, n = 11. *p < 0.05 **p < 0.01. In b and c, the dot shows an outlier value

Fig. 7

Changes in the degranulation and IFN-γ production with the neoadjuvant treatment. a ΔCD107a calculated as the percentage of CD107a positive NK cells POST treatment minus CD107a positive NK cells PRE treatment. b ΔIFN-γ calculated as the percentage of IFN-γ positive NK cells POST treatment minus IFN-γ positive NK cells PRE treatment. *p < 0.05 **p < 0.01