Progressive loss of anti-HER2 CD4+ T-helper type 1 response in breast tumorigenesis and the potential for immune restoration

Abstract

Genomic profiling has identified several molecular oncodrivers in breast tumorigenesis. A thorough understanding of endogenous immune responses to these oncodrivers may provide insights into immune interventions for breast cancer (BC). We investigated systemic anti-HER2/neu CD4⁺ T-helper type-1 (Th1) responses in HER2-driven breast tumorigenesis. A highly significant stepwise Th1 response loss extending from healthy donors (HD), through HER2^pos-DCIS, and ultimately to early stage HER2^pos-invasive BC patients was detected by IFNγ ELISPOT. The anti-HER2 Th1 deficit was not attributable to host-level T-cell anergy, loss of immune competence, or increase in immunosuppressive phenotypes (T_reg/MDSCs), but rather associated with a functional shift in IFNγ:IL-10-producing phenotypes. HER2^high, but not HER2^low, BC cells expressing IFNγ/TNF-α receptors were susceptible to Th1 cytokine-mediated apoptosis in vitro, which could be significantly rescued by neutralizing IFNγ and TNF-α, suggesting that abrogation of HER2-specific Th1 may reflect a mechanism of immune evasion in HER2-driven tumorigenesis. While largely unaffected by cytotoxic or HER2-targeted (trastuzumab) therapies, depressed Th1 responses in HER2^pos-BC patients were significantly restored following HER2-pulsed dendritic cell (DC) vaccinations, suggesting that this Th1 defect is not "fixed" and can be corrected by immunologic interventions. Importantly, preserved anti-HER2 Th1 responses were associated with pathologic complete response to neoadjuvant trastuzumab/chemotherapy, while depressed responses were observed in patients incurring locoregional/systemic recurrence following trastuzumab/chemotherapy. Monitoring anti-HER2 Th1 reactivity following HER2-directed therapies may identify vulnerable subgroups at risk of clinicopathologic failure. In such patients, combinations of existing HER2-targeted therapies with strategies to boost anti-HER2 CD4⁺ Th1 immunity may decrease the risk of recurrence and thus warrant further investigation.

Keywords: CD4+ T-helper immunity; HER2/neu; breast cancer; dendritic cell; immune monitoring; immune restoration; vaccination.

Figure 1.

Study-eligible patient and donor cohorts. Hierarchy diagram representing patient/donor groups included in study. Cohorts are labeled A–H for ease of comparison (of immune responses), and are referred to in Results. Treatment schedules in cohorts G and H, as well as time-points at which blood was drawn are indicated in red callout boxes. Specifically, in the T/C-treated HER2^pos-IBC cohort (G), patients received either neoadjuvant T/C, followed by surgery and completion of adjuvant trastuzumab; patients selected for a surgery-first approach completed adjuvant T/C. Blood was drawn either <6 mo or ≥6 mo from completion of adjuvant trastuzumab.

Figure 2.

Anti-HER2 CD4⁺ Th1 responses and IgG1/IgG4 reactivity are progressively lost in HER2^pos breast tumorigenesis. (A) IFNγ ELISPOT analysis of PBMCs demonstrated a progressive loss of anti-HER2 CD4⁺ Th1 response in HER2^pos breast tumorigenesis (i.e., HD/BDHER2^pos-DCIS HER2^pos-IBC), illustrated by anti-HER2 responsivity, response repertoire, and cumulative response (all ANOVA p < 0.001). No differences in Th1 responses were observed between HER2^neg-DCIS and HER2^neg-IBC (IHC 0/1+) and HD/BD subjects. Post-hoc Scheffé p value comparisons between groups are shown alongside histograms. (B) Anti-CD3/ CD28 stimulus served as positive control for all donors in experiments shown in (A) above; corresponding IFNγ production by ELISPOT in respective patient groups is shown. Results presented as median ± interquartile range (IQR) IFNγ SFC per 2 × 10⁵ cells in box-and-whiskers plots; (C) Variations in anti-HER2 Th1 cumulative responses in HD/BDs donors stratified by donor age (<50 vs. ≥50 years), menopausal status (pre-menopausal vs. post-menopausal) (upper panel); and race (white vs. other), or gravidity (zero vs. ≥1 pregnancies) (bottom panel). Within each Th1 metric, results are expressed as proportion or mean (±SEM). (D) ELISA of serum reactivity against recombinant HER2 extracellular domain revealed significantly elevated anti-HER2 IgG1 and IgG4 antibody levels in HER2^pos-DCIS compared with HDs that decayed in HER2^pos-IBC patients. ELISA measurements are shown as optical density (OD) at 1:100 sera dilutions (grouped scatter plot, with horizontal line indicating mean OD); (***p < 0.001 by unpaired t-test or ANOVA with post-hoc Scheffé testing, as applicable).

Figure 3.

Anti-HER2 Th1 deficit in HER2^pos-IBC is not attributable to lack of immunocompetence or increase in immunosuppressive phenotypes, but associated with a functional shift in IFNγ:IL-10-producing phenotypes. PBMCs from HER2^pos-IBC patients, both treatment-naive and T/C-treated, did not differ significantly from HDs in (A) IFNγ production to recall stimuli tetanus toxoid or Candida albicans by ELISPOT. Results presented as median ± interquartile range (IQR) IFNγ SFC per 2 × 10⁵ cells in box-and-whiskers plots; (B and C) Relative proportions of CD4⁺ (CD3⁺CD4⁺) (B top) or CD8⁺ (CD3⁺CD8⁺) T-cells (B bottom), T_reg (CD4⁺CD25⁺FoxP3⁺) (C top) or MDSCs (CD11b⁺CD33⁺HLA-DR⁻CD83⁻) (C bottom) by flow cytometry. Representative stainings within groups are shown; results in adjoining histograms expressed as mean proportions (%) ± SEM as indicated. (D) Donor-matched cumulative IFNγ and IL-10 production (SFC per 10⁶ cells) across six HER2 Class II peptides compared in HD, treatment-naive HER2^pos-IBC, and T/C-treated HER2^pos-IBC patients. Relative HER2-specific IFNγ to IL-10 proportions (% depicted in graph) decreased significantly from HDs [IFNγ/(IFNγ + IL-10) = 86.9%; IL-10/(IFNγ + IL-10) = 13.1%] to HER2^pos-IBC patients with (49.3%; 50.7%) or without (42.5%; 57.5%) T/C treatment. Absolute IFNγ:IL-10 production ratio changed from 6.6:1 (HDs) to 0.97:1 (T/C-treated) and 0.74:1 (HER2^pos-IBC), respectively (top panel). No significant relative shifts in IFNγ:IL-10 production were observed to positive controls (anti-CD3/anti-CD3/CD28) (bottom panel).

Figure 4.

CD4⁺ Th1 induces apoptosis of HER2^high, but not HER2^low, human and murine breast cancer cells. (A) Using a transwell system, 50 × 10³ SK-BR-3 cells were co-cultured with medium alone (complete medium), 10⁶ CD4⁺ T-cells alone (CD4⁺ only), 10⁶ CD4⁺ T-cells + 10⁵ each of HER2 Class II peptide (iDC H)- or irrelevant Class II BRAF peptide (iDC B)-pulsed iDCs, and 10⁶ CD4⁺ T-cells + 10⁵ each HER2 (DC1 H)- or BRAF (DC1 B)-pulsed DC1s. Increased caspase-3 cleavage indicated dose-dependent apoptosis of SK-BR-3 cells when co-cultured with DC1 H:CD4⁺ T-cells, but not DC1 B, iDC H, or iDC B groups. Vinculin was used as loading control. The displayed protein gel blot is representative of three experiments (top panel), and results are expressed as mean caspase-3/vinculin ratios ± SEM indicating fold induction of apoptosis (quantified using ImageJ software). Compared with IgG isotype control, CD4⁺:DC1 H-induced SK-BR-3 apoptosis was significantly rescued by neutralizing IFNγ and TNF-α. Bars represent % rescue of mean caspase-3/vinculin ratio ±SEM (31.4 ±5.3% IFNγ/TNF-α neutralization vs. control). Results are representative of three experiments (middle panel). Corresponding production of IFNγ (left y-axis) and TNF-α (right y-axis) in respective co-cultures by ELISA is shown. Results are expressed in pg/mL, and are representative of three experiments (bottom panel). (B) Significantly greater proportion of apoptotic SK-BR-3 cells (asterisks) were observed by DAPI staining in the DC1 H:CD4⁺ group, correlating with a 25-fold increase in apoptosis, compared with CD4⁺ only or DC1 B:CD4⁺ groups. Results are representative of three experiments, and expressed as mean % apoptotic cells ± SEM. (C) HER2^high SK-BR-3, HER2^intermediate MCF-7, and HER2^low MDA-MB-231 human BC cells uniformly maintained expression of IFNγ-Rα and TNF-α-R1 receptors. (D) In transgenic murine HER2^high mammary carcinoma TUBO and MMC15 cells, combination treatment with recombinant murine (rm) Th1 cytokines rmIFNγ and rmTNF-α resulted in significantly greater apoptosis compared with untreated controls or treatment with either cytokine alone. This effect was not reproduced with dual rmIFNγ + rmTNF-α treatment in murine HER2^low/neg cells 4T1. Results are representative of three experiments, and expressed as mean % apoptotic cells ± SEM, detected by proportion of PI^pos/Annexin V^pos cells by flow cytomety. (* p ≤ 0.05, **p < 0.01, *** p < 0.001).

Figure 5 (See previous page).

Anti-HER2 CD4⁺ Th1 immunity is differentially restored following HER2-pulsed DC immunization, but not after HER2-targeted therapies. (A) Compared with treatment-naive Stage I/II HER2^pos-IBC patients (no tx), anti-HER2 Th1 responses were not globally augmented following T/C treatment in stage I-III HER2^pos-IBC patients (T/C-treated), illustrated by anti-HER2 responsivity, repertoire, or cumulative response. Relative proportion of IFNγ:IL-10 reactive cells (% depicted in histogram) following HER2-specific and tetanus (positive control) stimuli did not improve in T/C-treated (n = 5) compared with no tx (n = 5) patients; (B) Significant improvements in all anti-HER2 Th1 immune metrics were observed in 11 Stage I HER2^pos-IBC (PRE vax) patients immediately following HER2 pulsed-DC1 immunization (POST vax). While relative proportion of IFNγ to IL:10 reactive cells (% depicted in histogram) did not change appreciably following tetanus stimulation, HER2-pulsed vaccination significantly increased the relative proportion of IFNγ to IL:10 reactive cells in POST vax (n = 5) compared with PRE vax (n = 5) patients; (C) The differential Th1 restoration following HER2-pulsed DC immunization, but not T/C treatment, persisted on stage-matched comparisons in stage I HER2^pos-IBC patients. Results are expressed as proportion or mean ± SEM; (**p < 0.01, ***p < 0.001). (D and E) Beyond the immediate post-vaccination period, anti-HER2 CD4⁺ Th1 immunity remained durably augmented in 9 of 11 evaluable patients ≥6 mo following vaccination, despite initiation/completion of systemic chemotherapy in all patients by this time-point (broken arrows). Scatter plots demonstrate CD4⁺ Th1 reactivity profiles by response repertoire (D) and cumulative response (E) for individual vaccinated subjects.

Figure 6.

Depressed anti-HER2 Th1 responses following T/C treatment correlate with adverse clinical and pathologic outcomes. (A–D) Subgroup analysis of T/C-treated HER2^pos-IBC patients demonstrated no appreciable differences in anti-HER2 responsivity, repertoire, or cumulative response when stratified by (A) sequencing of chemotherapy (neoadjuvant vs. adjuvant); (B) time from completion of trastuzumab to enrollment in study (<6 vs. ≥6 mo); (C) estrogen-receptor status (ER^pos vs. ER^neg) and (D) pathologic stage (I vs. II vs. III). (E) Compared with HER2^pos-IBC patients who did not incur breast events (no BE) following completion of T/C, patients incurring BEs (+BE) had significantly depressed anti-HER2 responsivity and cumulative Th1 responses (D left). In HER2^pos-IBC patients achieving pathologic complete response (pCR) following neoadjuvant T/C, anti-HER2 Th1 response repertoire and cumulative response was significantly greater compared to non-pCR patients (D right).