Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8

Abstract

The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.

Figure 1

Impact of the yeast α factor on Cne1p exposure and mating. (a) Relative mating efficiency of WT Saccharomyces cerevisiae MATα BY4742 cells co-cultured with WT, Δcne1 or Δura1 MATa BY4741 cells, as determined via clonogenic mating assay. Mating efficiency of WT BY4741 cells was set to 100%. Data are means±S.E.M. (n⩾3; **P<0.01, as compared with WT BY4741 cells). (b and c) Exponentially growing WT, Δmfa1/Δmfa2, Δgcn2, Δyet3 or Δire1 BY4741 cells expressing Cne1-GFP were left untreated or treated with 10 μM MTX or 100 μM α factor for 4 h, followed by the assessment of Cne1-GFP localization by fluorescence microscopy. Representative pictures (scale bar=10 μm) and quantitative data (means±S.E.M.; n=3, with⩾1000 cells per sample; *P<0.05, **P<0.01, as compared with untreated WT cells; ^##P<0.01, NS=non significant, as compared with equally-treated WT cells) are reported in panels (b) and (c), respectively

Figure 2

Contribution of chemokine signaling to CRT exposure as triggered by ICD inducers. (a and b) Human cervical carcinoma HeLa cells were treated with 100 ng/ml PTX for 30 min and washed extensively (a and b). Alternatively, HeLa cells were transfected with a control siRNA (UNR) or with siRNA targeting the indicated chemokines and chemokine receptors for 48 h (c). Thereafter, cells were treated with either 1 μM MTX or 100 J/cm² UVC light and – 4 h later – processed for the confocal microscopy-assisted (a) or flow cytometry-assisted (b and c) detection of surface-exposed CRT. Representative pictures (scale bar=10 μm) and quantitative data on the percentage of live (excluding PI, PI⁻) cells exposing CRT on their surface (means±S.E.M.; n=3; **P<0.01, as compared with untransfected or UNR-transfected untreated cells; ^##P<0.01, NS=non significant, as compared with untransfected cells treated with MTX or UV only, or to equally treated UNR-transfected cells) are reported in panels (a) and (b and c) respectively

Figure 3

Autocrine/paracrine CXCL8 signaling and CRT exposure. (a and b) Human osteosarcoma U2OS cells stably expressing CRT-GFP were reverse-transfected with a control siRNA (UNR) as well as with a panel of siRNA targeting 250 proteins involved in cell stress and cell death signaling for 48 h, followed by the administration of 1 μM MTX for additional 4 h and the automated fluorescence microscopy-assisted acquisition of images for the assessment of CRT-GFP exposure. The modulation of CRT-GFP exposure is depicted in a color code (green=reduction, red=increase), and the siRNAs mediating inhibitory effects in the top 10 percentile are listed. (c–e). Human cervical carcinoma HeLa cells were treated with 80 ng/ml rCXCL8 or rCCL2 for the indicated time (c) or with the indicated concentrations of rCXCL8 for 8 h (d). Alternatively, HeLa cells were left untreated or treated with 50 μM z-VAD-fmk for 15 min, and then either kept in control (Co) culture conditions or administered with 1 μM MTX or 80 ng/ml rCXCL8 for additional 8 h (e). Subsequently, cells were processed for the flow cytometry-assisted detection of surface-exposed CRT. Quantitative data on the percentage of live (excluding PI, PI^–) cells exposing CRT on their surface (means±S.E.M.; n=3; **P<0.01, NS=non significant, as compared with untreated cells; ^##P<0.01, as compared with cells receiving MTX or rCXCL8 only) are reported. (f) HeLa cells were transfected with either a control siRNA (UNR) or with siRNAs targeting the indicated proteins for 48 h, and then left untreated or administered with 80 ng/ml rCXCL8 for 8 h. Finally, CRT exposure was assessed by surface immunostaining and flow cytometry. Quantitative data on the percentage of live (excluding PI, PI⁻) cells exposing CRT on their surface (means±S.E.M.; n=2; **P<0.01, as compared with untreated UNR-transfected cells; ^#P<0.05, ^##P<0.01, as compared with equally treated UNR-transfected cells) are reported

Figure 4

Impact of CXCL8 on anticancer immune responses elicited in vivo by ICD. (a and b) Immunocompetent BALB/c (n=10 per group) or C57Bl/6 (n=5 per group) mice were inoculated s.c. with CT26 or MCA205 cells, respectively, which had previously been left untransfected or transfected with siRNAs targeting Cxcr1 or Cxcr2 for 48 h and then treated with 2 μM MTX for 20 h. PBS was used as a negative control condition. Seven days later, all mice were re-challenged with live CT26 or MCA205 cells and tumor incidence was routinely monitored. Kaplan–Meier curves depict the percentage of tumor-free mice over time

Figure 5

Release and transcriptional regulation of CXCL8 and Cxcl2 in the course of ICD. (a–d) Human cervical carcinoma HeLa cells (a), human colorectal carcinoma HCT 116 cells (b), murine fibrosarcoma MCA205 cells (c) and mouse embryonic fibroblasts (d) were treated with 75 (a, b and d) or 150 (c) μM cisplatin (CDDP) or with 2 (a, b and d) or 4 (c) μM MTX for the indicated time, followed by the analysis of culture supernatants for CXCL8 (a and b) and Cxcl2 (c and d) levels. Quantitative data (means±S.E.M.; n=3; *P<0.05, **P<0.01, as compared with CDDP-treated cells) are reported. (e) Immunocompetent BALB/c or C57Bl/6 mice bearing established CT26 colon carcinomas and MCA205 fibrosarcomas, respectively, were treated with 5.17 mg/kg MTX, 10 mg/kg oxaliplatin (OXA) or 0.25 mg/kg cisplatin (CDDP) for 6 or 12 h, followed by the recovery of tumors and their processing for quantitative RT-PCR analyses. Average fold changes as determined on n=3 independent experiments are depicted

Figure 6

ICD as induced by non-immunogenic chemotherapeutics plus Cxcl2. (a–f) Immunocompetent C57Bl/6 mice (n=5 per group) were inoculated s.c. with PBS or murine MCA205 fibrosarcoma cells that had previously been transfected with a control siRNA (siUNR) or with a siRNA targeting CRT (siCRT) and then treated with 2 μM MTX, 200 μM mitomycin C (MitoC), 150 μM cisplatin (CDDP), alone or combined with 80 ng/ml rCxcl2, for 20 h. Panels (a–c) report Kaplan–Meier curves depicting the percentage of tumor-free mice over time. Panels (d–f) depict the percentage of tumor free animals at the end of the experiment. The numbers of mice allocated to each group is indicated. **P<0.01, as compared with animals receiving PBS only (d), siUNR-transfected cells treated with MitoC (e) or siUNR-transfected cells treated with CDDP (f); ^##P<0.01, as compared with animals receiving siUNR-transfected cells treated with MTX (d), MitoC plus rCxcl2 (e) or CDDP plus rCxcl2 (f)