Poly-ICLC promotes the infiltration of effector T cells into intracranial gliomas via induction of CXCL10 in IFN-alpha and IFN-gamma dependent manners

Abstract

Stimulation of double-stranded (ds)RNA receptors can increase the effectiveness of cancer vaccines, but the underlying mechanisms are not completely elucidated. In this study, we sought to determine critical roles of host IFN-alpha and IFN-gamma pathways in the enhanced therapeutic efficacy mediated by peptide vaccines and polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in the murine central nervous system (CNS) GL261 glioma. C57BL/6-background wild type (WT), IFN-alpha receptor-1 (IFN-alphaR1)(-/-) or IFN-gamma(-/-) mice bearing syngeneic CNS GL261 glioma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes with or without intramuscular (i.m.) injections of poly-ICLC. The combinational treatment induced a robust transcription of CXCL10 in the glioma site. Blockade of CXCL10 with a specific monoclonal antibody (mAb) abrogated the efficient CNS homing of antigen-specific type-1 CTL (Tc1). Both IFN-alphaR(-/-) and IFN-gamma(-/-) hosts failed to up-regulate the CXCL10 mRNA and recruit Tc1 cells to the tumor site, indicating non-redundant roles of type-1 and type-2 IFNs in the effects of poly-ICLC-assisted vaccines. The efficient trafficking of Tc1 also required Tc1-derived IFN-gamma. Our data point to critical roles of the host-IFN-alpha and IFN-gamma pathways in the modulation of CNS glioma microenvironment, and the therapeutic effectiveness of poly-ICLC-assisted glioma vaccines.

Figures

Fig.1. Poly-ICLC administration significantly improves the therapeutic effects of GAA-vaccines in mice bearing GL261 glioma, associated with robust induction of type-1 chemokine transcripts in the CNS glioma microenvironment

(a), WT C57BL/6 mice bearing i.c. GL261 glioma received s.c. immunizations with each of GAA peptides and HBVcore128 T-helper epitope peptide and/or i.m. poly-ICLC injections as described in Materials and Methods. The mice were stratified into 4 treatment groups: 1) GAA-vaccine and poly-ICLC (solid squares) (n=17); 2) GAA-vaccine and i.m. PBS (solid circles) (n=14); 3) Mock-vaccines and poly-ICLC (hollow circles) (n=11) and 4) Mock-vaccines and i.m. PBS (hollow squares) (n=12). Symptom-free survival of mice was monitored. ** p < 0.0001 for mice treated with GAA-vaccine and poly-ICLC compared with mice treated with mock-treatments only or mock-vaccine plus poly-ICLC, * p < 0.0001 for mice treated with GAA-vaccine and PBS compared with the mice treated with mock-treatments only or mock-vaccine plus poly-ICLC, *** p = 0.0124 for mice treated with GAA-vaccine and poly-ICLC compared with the mice treated with GAA-vaccine alone. (b), On day 23 following tumor inoculation (one day following the third s.c and i.m. treatments), brain tissues were collected from WT C57BL/6 mice bearing i.c. GL261 glioma treated with either mock-treatments, mock-vaccines plus poly-ICLC, GAA-vaccines plus PBS or GAA-vaccines plus poly-ICLC. ISH of CXCL10 mRNA in the brain bearing GL261 glioma. Parallel hybridization of tissue sections with the cognate sense control probe provided no non-specific ISH signals (data not shown). Original magnification, 100 ×. Results from one of two independent experiments with highly similar results are shown.

Fig.2. CXCL10 plays a critical role in CNS glioma homing of Tc1 cells

WT C57BL/6 mice bearing i.c. GL261 tumors received i.m. injections of poly-ICLC (20µg/dose) or control PBS on days 20 and 23. Mice treated with poly-ICLC received i.p. injections of anti-mouse CXCL10 mAb (250µg/dose) or the same amounts of hamster IgG on day 20 and 100ug on days 21, 22, 23, and 24. All mice received a single i.v. infusion of 3 × 106 pmel-1 derived Tc1 cells on day 21. Mice were sacrificed on day 25 and BILs were analyzed by flow cytometry. (a), Numbers in each dot plot indicate the percentage of CD8+/gp100-tetramer+ T-cells in BILs. (b), Total numbers of CD8+/GP100-tetramer+ T-cells in BILs per mouse. Results from one of two experiments with similar results are shown. Due to the small number of BILs obtained per mouse, BILs obtained from all mice in a given group (5 mice/group) were pooled and then evaluated for the relative number/mouse and phenotype of the BILs between groups.

Fig.3. Host IFN-α pathway is critical for induction of CXCL10 and Tc1 homing in CNS gliomas

(a), WT or IFN-αR1−/− mice bearing i.c. GL261 glioma were treated with GAA-vaccines plus poly-ICLC. Control WT mice received mock-vaccines plus PBS. On day 23, brain tissues were collected and ISH for CXCL10 mRNA was conducted (Original magnification, 200 ×). (b and c), WT or IFN-αR1−/− mice bearing i.c. GL261 gliomas received i.v. infusion of pmel-1-derived CD8+ Tc1 cells on day 18 and i.m. injections of poly-ICLC on days 17, 18 and 20. Control WT mice received i.v. infusion of pmel-1-derived CD8+ Tc1 cells on day 18 and i.m. injections of PBS. On day 22, BILs were harvested and evaluated. (b), Numbers in each dot plot indicate the percentage of CD8+/hgp10025–33 tetramer+ cells in lymphocyte-gated BILs. (c), Total numbers of CD8+/hgp10025–33 tetramer+ cells per mouse brain. Results from one of two independent experiments with similar results are shown (see Supplementary Fig. 3 for reproducible results from another experiment). Due to the small number of BILs obtained per mouse, BILs obtained from all mice in a given group (5 mice/group) were pooled and then evaluated for the relative number/mouse and phenotype of the BILs between groups.

Fig.4. IFN-γ serves as a critical mediator for induction of CXCL10 and Tc1 homing in CNS gliomas

(a), WT or IFN-γ−/− mice bearing i.c. GL261 glioma were treated with GAA-vaccines plus poly-ICLC. Control WT mice received mock treatments (i.e. mock vaccines and PBS) only. On day 23, brain tissues were collected and ISH for CXCL10 mRNA was conducted (Original magnification, 200 ×). (b), WT mice bearing i.c. GL261 gliomas received i.v. infusion of IFN-γ+/+/pmel-1- or IFN-γ−/−/pmel-1-derived CD8+ Tc1 cells on day 18 and i.m. injections of poly-ICLC or PBS on days 17, 18 and 20. On day 22, BILs were harvested, counted and enumerated by flow cytometry for the total numbers of CD8+/hgp10025–33 tetramer+ cells per mouse brain. Results from one of two independent experiments with similar results are shown (see Supplementary Fig. 4 for reproducible results from another experiment). Due to the small number of BILs obtained per mouse, BILs obtained from all mice in a given group (5 mice/group) were pooled and then evaluated for the relative number/mouse and phenotype of the BILs between groups.