A novel dendritic cell-based immunization approach for the induction of durable Th1-polarized anti-HER-2/neu responses in women with early breast cancer

Abstract

Twenty-seven patients with HER-2/neu overexpressing ductal carcinoma in situ of the breast were enrolled in a neoadjuvant immunization trial for safety and immunogenicity of DC1-polarized dendritic cells (DC1) pulsed with 6 HER-2/neu promiscuous major histocompatibility complex class II-binding peptides and 2 additional human leukocyte antigen (HLA)-A2.1 class I-binding peptides. DC1 were generated with interferon-γ and a special clinical-grade bacterial endotoxin (lipopolysaccharide) and administered directly into groin lymph nodes 4 times at weekly intervals before scheduled surgical resection of ductal carcinoma in situ. Patients were monitored for the induction of new or enhanced antipeptide reactivity by interferon-γ ELISPOT and enzyme-linked immunosorbentassays performed on Th cells obtained from peripheral blood or excised sentinel lymph nodes. Responses by cytotoxic T lymphocyte against HLA-A2.1-binding peptides were measured using peptide-pulsed T2 target cells or HER-2/neu-expressing or nonexpressing tumor cell lines. DC1 showed surface phenotype indistinct from "gold standard" inflammatory cocktail-activated DC, but displayed a number of distinguishing functional characteristics including the secretion of soluble factors and enhanced "killer DC" capacity against tumor cells in vitro. Postimmunization, we observed sensitization of Th cells to at least 1 class II peptide in 22 of 25 (88%; 95% exact confidence interval, 68.8%-97.5%) evaluable patients, whereas 11 of 13 (84.6%; 95% exact confidence interval, 64%-99.8%) HLA-A2.1 patients were successfully sensitized to class I peptides. Perhaps most importantly, anti-HER-2/neu peptide responses were observed up to 52-month postimmunization. These data show that even in the presence of early breast cancer such DC1 are potent inducers of durable type I-polarized immunity, suggesting potential clinical value for development of cancer immunotherapy.

Figures

Figure 1

CONSORT diagram of trial charting subject recruitment, and analysis of various available lymphocytes and tissues.

Figure 2

Dendritic cell characteristics. (A) Flow cytometry analysis of surface marker expression. ICAIT DC were compared to immature monocyte-derived DC (iDC), and “gold standard” inflammatory cytokine-matured DC (CMC-DC) for the expression of CD80, CD86 and CD83. Open traces indicate specific antibody-stained cells and filled traces represent isotype-matched controls. (B) Secreted factors. Production of IL-12, IL-6, IP-10, RANTES, MIP-1α, TARC and MDC were measured in 8–24h post-activation supernatants by ELISA. Data shown are the average of 4 representative experiments including standard error of mean. (C) Assessment of killer DC function. ICAIT-DC were compared with cytokine matured DC for their ability to induce apoptosis of breast cancer tumor cell line T47D. Results shown are representative of 7 different experiments with similar results.

Figure 3

Assessment of CD8pos T cell sensitization to peptide following ICAIT immunization. There were 13 HLA A2pos subjects that received ICAIT DC pulsed with additional HLA-A2 binding peptides (369–377 and 689–697). CD8pos T cells obtained before and after ICAIT-DC immunization were separately co-cultured for one week with immature autologous DC pulsed with either HER-2/neu 369 or 689 peptides prior to testing against monocyte pulsed with either HER-2/neu peptides or irrelevant controls. *Denotes those 2 subjects that did not have evidence of 369 or 689 peptide specificity. A summary of the number of subjects displaying positive peptide reactivity is displayed in the figure inset, with positive responses defined as at least 2-fold greater specific IFN-γ secretion post-vaccination compared to pre-vaccination. Note the single subject demonstrating pre-existing peptide reactivity.

Figure 4

Assessment of direct tumor recognition by immune CTL. Purified peripheral CD8pos T cells were expanded by co-culture with autologous DCs pulsed with HER-2/neu 369 peptide. Seven -10 days later the cells were harvested and re-cultured with a variety of tumor lines selected for their divergent expression of HER-2/neu and the HLA-A2.1 restriction element (see materials and methods). Culture supernatants were harvested 24h later and analyzed by ELISA for IFN-γ production. Data shown is from one representative subject.

Figure 5

ELISPOT analysis of peripheral blood CD4pos T cells pre- and post- ICAIT immunization. Unexpanded purified CD4pos T cells were co-cultured overnight with individual HER-2/neu-pulsed immature dendritic cells. Analysis enumerated IFN-γ secreting lymphocytes per 400,000 total cells. Solid lines connect pre- and post-vaccination IFN-γ spots for individual peptides.

Figure 6

Immunohistochemical analysis of pre-ICAIT immunization biopsies and post-ICAIT surgical specimens. (A) Slides stained for CD4, CD8 (T lymphocytes) or CD20 (B lymphocytes). (B) Increased magnification of CD8-stained slides demonstrating in post-ICAIT immunization tissues the enhanced migration of T cells across the epithelial border (EB) and into ducts containing DCIS.

Figure 7

Assessment of CD4pos anti-HER-2/neu T cells in draining sentinel lymph nodes. Single cell suspensions were made from surgically-excised axillary lymph nodes for all ICAIT immunized subjects from whom these tissues were available. Unexpanded cells were stimulated with the indicated individual HER-2/neu synthetic peptides pulsed onto immature dendritic cells and subjected to ELISPOT analysis to determine the number of IFN-γ-secreting T cells per 400,000 total cells.

Figure 8

ICAIT immunization induces long-lasting T cell immunity. Peripheral blood mononuclear cells (PBMC) were obtained from representative subjects ranging from 6 to 52 months post vaccination. Unstimulated PBL were tested against each of the six HLA Class II-restricted HER-2/neu peptides. ELISPOT analysis enumerated IFN-γ secreting T cells per 300,000 total cells with a minimum limit for positivity of 10 or more spots. Tetanus toxoid acted as the positive control.