The novel C24D synthetic polypeptide inhibits binding of placenta immunosuppressive ferritin to human T cells and elicits anti-breast cancer immunity in vitro and in vivo

Abstract

Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain-like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti-breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2(+)) or T47D (HLA-A2(-)) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti-MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2(+)) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.

Figure 1

C24D inhibits the binding of C48-PLIF protein to T lymphocytes. Representative histograms for FACS analysis of binding of C24D (A) and C48 (B) to HD-MAR T cells detected by anti-C24D IgG and anti-C48 mAb, respectively. C48 binding to HD-MAR cells is inhibited by 0.2 μg per sample of C24D (C). C48 binding to HD-MAR cells is inhibited by 1 μg per sample of C24D (D). The above results are further demonstrated as MFI value ± SD (n = 3) of C24D binding (E) and binding of C48 to HD-MAR cells and its inhibition by C24D (F).

Figure 2

In vitro development of anti-tumor cytotoxic T cells following C24D treatment. (A) A schematic illustration describing the basic concept of this study, i.e., blocking C48-PLIF by C24D on T cells results in the development of specific anti-tumor CTLs. The two stages of the experiments are exhibited: In primary cultures, human PBMCs are co-cultured with human breast cancer cell lines at an E:T ratio of 10:1. The mixed cultures are either untreated (control) or treated with C24D at 0, 24 and 48 hours and incubated for 7 days. The culture medium is replaced on day 7 with growth medium supplemented with human IL-2. The cultures are further incubated for 4 weeks and harvested. Highly cytotoxic cells were obtained from PBMCs incubated with tumor cells in presence of C24D. In contrast, there is no anti-tumor cytotoxic activity from PBMCs incubated with tumor cells without C24D. (B and C) Giemsa staining of MCF-7 tumor cells co-cultured for 7 days with control PBMCs (B) or with C24D-treated PBMCs (C). (D) Alamar Blue cytotoxicity assay performed on day 7 on MCF-7 and T47D tumor cells cultured with their respective control or C24D-treated PBMCs (E:T ratio of 10:1). Histograms represent mean value ± SD (n = 6). (E) Flow cytometry analysis of T cells in secondary culture shows a predominance of CD8⁺ over CD4⁺ T cells. PBMCs from three different donors are presented.

Figure 3

C24D-induced CTLs are highly cytotoxic to their specific tumor cells. (A–F) Representative light microscopy images of MCF-7 and T47D tumor cells co-cultured for 5 hours with control T lymphocytes or induced CTLs. (G and H) E:T cell ratio–dependent cytotoxicity detected by MTT cytotoxicity assay (5-hour incubation). Data represent mean value ± SD (n = 3).

Figure 4

Activated CTLs secrete IFN-γ and initiate apoptosis of their specific targets. (A and B) IFN-γ concentrations in the supernatants obtained from 5-hour co-cultures of MCF-7 and T47D tumor cells with increasing CTL ratios (0.5:1 and 1:1). (A) Anti–MCF-7 CTLs. (B) Anti-T47D CTLs. (C) IFN-γ secreted by anti–MCF-7 CTLs and anti-T47D CTLs (E:T ratio of 1:1) determined after extended incubation (24 hours). All values are expressed as mean ± SD (n = 3). (D and E) Enhanced apoptotic death of MCF-7 and T47D tumor cells co-cultured with (D) anti–MCF-7 CTLs or (E) anti-T47D CTLs determined by FACS analysis (Annexin V and PI labeling).

Figure 5

Anti–MCF-7 CTLs cross-activated by HLA-A2⁺ MDA-BM-231 cells induced target cell apoptosis and TNF-α and IFN-γ secretion. (A) Representative light microscopy images of MDA-MB-231 tumor cells co-cultured for 7 hours with anti–MCF-7 CTLs or anti-T47D CTLs. (B) Stages of apoptotic death of MDA-MB-231 tumor cells co-cultured for 5 hours with anti–MCF-7 CTLs, determined by imaging flow cytometry: lower left—viable cells; lower right—early apoptosis; upper right—late apoptosis; upper left—dead cells (Annexin V and PI labeling). (C) Representative time course development of early and late apoptosis of MDA-MB-231 tumor cells by anti–MCF-7 CTLs. (D and E) Cytokine concentrations in the supernatants obtained from co-cultures of MDA-MB-231 and MCF-7 tumor cells with anti–MCF-7 CTLs or anti-T47D CTLs at 3 hours and 5 hours for (D) TNF-α and (E) IFN-γ. All values are expressed as mean ± SD (n = 3).

Figure 6

In vivo C24D treatment inhibits MCF-7 tumor growth in human PBMC transfused nude mice. (A–D) Immune-compromised nude mice engrafted with MCF-7 tumor cells were treated with C24D or PBS (control) for 18 days (five mice in each group). (A) Mean value of mice weight ± SD. (B) Mean volumes ± SD of tumors removed on day 19. (C) Photographs of removed tumors. (D) H&E staining (top panels) and anti-CD45⁺ staining (bottom panels) of tumor sections. CD45⁺ cell infiltration (stained brown, right bottom panel) is exhibited only in the tumor removed from C24D-treated mice. No infiltration of CD45⁺ cells was seen in the tumor removed from control mice (left bottom panel).