Modulation of T-cell activation by malignant melanoma initiating cells

Abstract

Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. Thus, only a restricted minority of tumorigenic malignant cells may possess the phenotypic and functional characteristics needed to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis. Tumorigenic ABCB5(+) malignant melanoma initiating cells (MMICs) possessed the capacity to preferentially inhibit IL-2-dependent T-cell activation and to support, in a B7.2-dependent manner, induction of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Compared with melanoma bulk cell populations, ABCB5(+) MMICs displayed lower levels of MHC class I, aberrant positivity for MHC class II, and lower expression levels of the melanoma-associated antigens MART-1, ML-IAP, NY-ESO-1, and MAGE-A. Additionally, these tumorigenic ABCB5(+) subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1, both in established melanoma xenografts and in clinical tumor specimens. In immune activation assays, MMICs inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5(-) melanoma cell populations. Moreover, coculture with ABCB5(+) MMICs increased the abundance of Tregs, in a B7.2 signaling-dependent manner, along with IL-10 production by mitogen-activated PBMCs. Consistent with these findings, MMICs also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T-cell modulatory functions of ABCB5(+) melanoma subpopulations and suggest specific roles for these MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance.

Figure 1. Identification of signal 1 and 2 members of immune activation on melanoma cells

(A) Representative flow cytometric analyses of melanoma cells stained for MHC class I or MHC class II antigens. Single-color flow cytometry analyses of melanoma specimens for expression of (B-C) costimulatory molecules and (D) MAAs. Horizontal bars indicate means. Bottom rows depict representative histogram plots showing marker-stained populations (red) compared to isotype-stained controls (shaded).

Figure 2. Immunophenotype of ABCB5⁺ MMICs

Expression of (A) MHC class I or MHC class II molecules and of (B-C) costimulatory molecules by ABCB5⁺ versus ABCB5⁻ malignant melanoma cells (MMC) as determined by dual-color flow cytometry. Illustrated are means±SEM (*, P<0.05; NS, not significant). Bottom rows depict representative histogram plots showing marker-stained populations (red) compared to isotype-stained controls (shaded). (D) Immunofluorescence double staining of clinical melanoma sections (left) and melanoma xenograft sections (right) for coexpression of ABCB5 (AF488, green) with B7.2 (AF594, red), PD-1 (AF594, red), or PD-L1 (AF594, red). Nuclei are visualized by staining with DAPI (blue).

Figure 3. Tumorigenicity of B7.2⁺ and PD-1⁺ melanoma subsets in human to NOD/SCID mouse xenotransplantation experiments

In vivo tumor formation capacity (%) (left panels) and limiting dilution analysis comparing log [fraction without tumors] of recipients as a function of the number of xenografted tumor cells (±95% confidence intervals(50)) (right panels) of (A) B7.2⁺ or B7.2⁻ and (B) PD-1⁺ or PD-1⁻ patient-derived melanoma cells following s.c. xenotransplantation of 10⁶, 10⁵, or 10⁴ cells/inoculum.

Figure 4. MAA expression of ABCB5⁺ MMICs compared to ABCB5⁻ tumor bulk populations

(A) Representative dual-color flow cytometry analysis of malignant melanoma cells (MMC) costained for ABCB5 expression (APC, Fl4 fluorescence) and the MAAs ML-IAP, NY-ESO-1, MAGE-A, and (B) MART-1 (FITC, Fl1 fluorescence) in clinical patient-derived melanoma cells (left panel) and established G3361 melanoma cells (right panel). (C) MAA expression by ABCB5⁺ versus ABCB5⁻ melanoma subpopulations (mean±SEM), as determined by dual-color flow cytometry (*: P<0.05; **: P<0.01). (D) Relative mRNA expression of TGF-β pathway members in ABCB5⁺ versus ABCB5⁻ melanoma cells.

Figure 5. Effects of human melanoma cells on T cell activation and cytokine secretion

(A) Left panel: ³H-thymidine uptake (mean cpm±SEM) of human PBMCs cultured in the presence or absence of PHA with or without addition of human melanoma cells (representative of n=4 independent experiments). Right panel: IL-2, (B) IL-10 (C) IFN-γ-, and (D) IL-5- (left), and IL-4 (right) production by PHA-stimulated PBMCs cultured in the presence or absence of melanoma cells as determined by ELISPOT analysis. Illustrated are mean spots per well±SEM of replicate wells representative of n=3-4 independent experiments (*, P<0.05, NS, not significant). Representative ELISPOT wells are shown.

Figure 6. ABCB5⁺ MMICs preferentially inhibit T cell activation

(A) Left panel: ³H-thymidine uptake (mean cpm±SD) and cell death (Annexin V-PE/7-AAD) staining (%, mean±SD; right panel) of human PBMCs cultured with or without PHA in the presence or absence of ABCB5⁺ or ABCB5⁻ malignant melanoma cells (MMC) (representative of n=3-6 independent experiments, respectively; *, P<0.05; NS, not significant). (B) Fold-differences (mean±SEM) of cytokine secretion by mitogen-stimulated PBMCs cultured in the presence of ABCB5⁺ versus ABCB5⁻ melanoma cells as determined by ELISPOT analysis (representative of n=4 independent experiments). Images of representative ELISPOT wells are shown. (C) Effects of B7.2 blockade in ABCB5⁺ or ABCB5⁻ melanoma cells on cocultured mitogen-stimulated PBMCs with regard to IL-10 production (mean spots per well±SEM, left panel) and Treg cell frequencies (% CD4⁺CD25⁺FoxP3⁺ triple-positive PBMCs±SEM, right panel). Results are representative of n=3-5 independent experiments, respectively. (D) IL-2 (left panel) and IL-10 (right panel) production by PBMCs cultured in the presence of patient-derived, syngeneic ABCB5⁺ versus ABCB5⁻ melanoma subpopulations, as determined by ELISA. Illustrated are mean cytokine concentrations (pg/ml) for n=3 replicate wells of n=6 independent experiments±SEM, respectively.