Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death

Abstract

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD8(+) CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1(+), CD133(+)) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Figure 1

Tumor cells treated with docetaxel show increased surface expression of CRT, but do not undergo ICD. Four human tumor cell lines were treated with 2.5–250 ng/ml (black bars), or 3500 ng/ml docetaxel (open bars). Mitoxantrone (1 μM) was used as a positive control (crosshatched bars). After 72 h of incubation, cells were examined for cardinal signs of ICD. (A) Surface expression of CRT. (B) HMGB1 secretion. (C) ATP secretion. (D) Percentage of dying cells (7AAD⁺). * = statistical significance relative to untreated cells. This experiment was repeated 2 times with similar results.

Figure 2

Tumor cells treated with a chemotherapeutic agent undergo immunogenic modulation and demonstrate significantly increased sensitivity to antigen-specific CTL killing. (A) Human tumor cells were treated in vitro for 72 h with 2.5, 25, or 250 ng/mL of docetaxel, or left untreated. Cells were analyzed after each treatment for surface expression Fas, ICAM-1, CEA, MUC-1, MHC-I, and CRT. Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. Bold type indicates marked upregulation (≥ 10% increase in percent of cells or 30% increase in MFI not observed in isotype control vs. untreated cells). (B) Human tumor cells treated in vitro for 72 h with 25 (white bars) or 250 (black bars) ng/ml of docetaxel, or left untreated (gray bars), were used as targets in an 18-h CTL lysis assay. CEA-, PSA-, or MUC-1-specific CD8⁺ T cells were used as effector cells at an E:T ratio of 30:1. For controls, tumor cells were incubated with anti-HLA-A2 mAb or concanamycin A (CMA). CEA⁺HLA-A2⁻ LS174T cells were used to verify CTL specificity. ND; not determined. * = statistical significance relative to untreated cells. This experiment was repeated 4 times with similar results.

Figure 3

In vivo treatment with docetaxel modulates tumor phenotype. Nude mice bearing LNCaP xenografts were treated with docetaxel or vehicle (PBS). One week later, tumors were surgically removed, stained, and evaluated by immunohistochemistry for expression of the tumor antigens CEA or MUC-1 (A, 40X, inset: isotype control), or CRT (B, 20X, inset 40X). Numbers indicate percentage of positive cells as determined by pixel analysis (n = 2 mice/treatment group). Arrows indicate CRT membrane staining. (C) Flow cytometry of tumors treated with docetaxel (open histograms) or PBS (shaded histograms). Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. * = statistical significance relative to untreated cells.

Figure 4

Docetaxel-resistant tumor cells undergo immunogenic modulation and are killed by antigen-specific CTLs at significantly greater levels after treatment with docetaxel. SW620 cells and SW620 cells passaged in increasing concentrations of docetaxel (designated docetaxel-resistant) were treated with docetaxel for up to 96 h. (A) Tumor cell proliferation was measured after treatment with 0 (closed circles) or 250 (open squares) ng/ml of docetaxel. Viable cells as determined by trypan blue exclusion werereported after 1, 2, 3, and 4 days of continuous exposure to docetaxel. * = statistical significance. (B) Expression of multidrug-resistance pump MDR-1 and stem cell marker CD133 on SW620 and docetaxel-resistant SW620 cells. (C) SW620 and docetaxel-resistant SW620 cells were treated in vitro for 72 h with 250 ng/mL of docetaxel (open histograms) or left untreated (shaded histograms), then analyzed by flow cytometry. Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. * = statistical significance relative to untreated cells. (D) CEA- and MUC-1-specific CTL lysis. Cells incubated for 72 h with 0 (gray bars) or 250 (black bars) ng/mL of docetaxel were used as targets in an 18-h CTL lysis assay, using either CEA-specific or MUC-1-specific CD8⁺ T cells as effector cells. (E) Target tumor cells were pulsed with CEA peptide at an E:T ratio of 30:1. * = statistical significance. This experiment was repeated 3 times with similar results.

Figure 5

Tumor cells modulate components of the APM chain after treatment with docetaxel. (A) Schematic of antigen-processing components from cytoplasm to cell surface. Arrows indicate functional checkpoints for CRT and PERK. (B) Human tumor cells were treated in vitro for 72 h with 2.5–250 ng/mL of docetaxel or left untreated, then analyzed for key intracellular components of the APM chain by flow cytometry. Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. Bold type indicates marked upregulation (≥ 10% increase in percent of cells or 30% increase in MFI not observed in isotype control vs. untreated cells).

Figure 6

Docetaxel-mediated immunogenic modulation is reduced in the absence of CRT or PERK, and by CRT blocking peptide. (A) Knockdown of CRT or PERK in LNCaP tumor cells. (B) LNCaP tumor cells lacking CRT or PERK were treated with 0 (white bars) or 250 (black bars) ng/mL of docetaxel and used as targets in an 18-h CTL lysis assay using CEA-specific CD8⁺ T cells. CRT expression was confirmed by flow cytometry. Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. Bold type indicates marked upregulation (≥ 10% increase in percent of cells or 30% increase in MFI not observed in isotype control vs. untreated cells). (C) LNCaP tumor cells were treated with 0 (white bars) or 250 (black bars) ng/mL of docetaxel and used as targets in an 18-h CTL lysis assay using either CEA-specific or PSA-specific CD8⁺ T cells, in the presence or absence of CRT blocking peptide (0.17 μM). All assays were done at an E:T ratio of 30:1. * = statistical significance. This experiment was repeated 2 times with similar results.