IL-6 trans-signaling licenses mouse and human tumor microvascular gateways for trafficking of cytotoxic T cells

Abstract

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.

Figures

Figure 1. Homeostatic and thermally inducible trafficking of CD8+ T cells at organ sites in NT control and STT-treated mice.

(A) Photomicrographs of immunostained endogenous CD8+ T cells (arrowheads denote CD8+ T cells in melanin+ B16 tumors), and quantification of CD8+ T cell infiltration. (B) Infiltration of leukocyte subsets in B16-OVA tumors; CD8+ T cells (CD8+CD3+), CD4+ T cells (CD4+CD3+), Tregs (CD4+CD25+FoxP3+), granulocytic MDSCs (CD11b+Ly6G+Ly6Clo), monocytic MDSCs (CD11b+Ly6ChiLy6G–); polymorphonuclear cells (PMNs; CD11b+ Ly6G+ Ly6C–), and macrophages (MΦ, CD11b+Ly6C–Ly6G–). Data are pooled tumors (3 per group) from 5 independent experiments. (C and D) Short-term (1 hour) homing of TK1 cells (C) or activated OT-I T cells (D) to mesenteric LNs (MLNs), Peyer patches (PP), tumor, pancreas (Panc), and kidney. (C) Representative photomicrographs and quantification of TRITC-labeled TK1 CD8+ T cells (red) in tissues counterstained for CD31+ vessels (green). (D) Quantification of TRITC-labeled OT-I T cells in B16-OVA tumor–bearing mice by microscopy. Representative dot plots show the phenotype of TRITC-labeled OT-I T cells that trafficked into LNs and tumors. CD62L is L-selectin. Data are based on analysis of equivalent numbers of transferred CD8+ T cells in pooled samples (n = 3 mice); numbers denote percent positive cells. (A, C, and D) Data are representative of at least 3 independent experiments. *P < 0.01, #P < 0.05, NT versus STT. Scale bars: 100 μm.

Figure 2. T cell–EC interactions in normal skin vessels and tumor vessels.

(A) Representative images showing interactions between calcein-labeled TK1 CD8+ T cells and vessels. Rolling and sticking fractions of TK1 T cells and effector OT-I T cells in vessels are also shown. Scale bar: 100 μm. (B) Frequency of arrested T cells per 1,000 total cells. Data are from more than 3 independent experiments. *P < 0.001, #P < 0.04 versus NT. There was no statistical difference between NT and LPS treatment groups in the tumor (CT26, P < 0.08; B16-OVA, P < 0.4).

Figure 3. Contributions of trafficking molecules to CD8+ T cell extravasation.

Determination of rolling fractions and sticking fractions by intravital microscopy (A) or short-term (1 hour) homing (B) of fluorescently tagged TK1 CD8+ T cells in CT26 tumors after treatment with function-blocking reagents (i.e., antibodies or PTX). Data are pooled from 3 independent experiments (A) or are representative of at least 3 independent experiments (B). *P < 0.001, #P < 0.05 versus control antibody.

Figure 4. Thermal induction of ICAM-1 in tumor vessels.

Intravascular staining of ICAM-1 in pancreata from RIP-Tag5 mice (A) and CT26 tumors (B) by intravenous injection of anti–ICAM-1 antibody. Tissue sections were stained with TRITC-conjugated secondary antibody (red) and counterstained with anti-CD31 antibody (green) to visualize vascular structures. Dotted lines indicate tumor boundary; arrowheads indicate normal pancreatic islets of RIP-Tag5 mice. Scale bars: 100 μm. (C) Expression of vascular ICAM-1 and CD8+ T cells trafficking in the center (C) and periphery (P) of CT26 and B16-OVA tumors. Histograms depict quantification of immunofluorescence intensity of ICAM-1 or CD31 in all CD31+ vessels; numbers denote MFI. Bar graphs depict CD8+ T cell infiltration. All data are representative of at least 3 independent experiments. *P < 0.05 versus NT.

Figure 5. IL-6 trans-signaling mediates thermal stimulation of ICAM-1 and CD8+ T cell trafficking.

(A) Cytokine neutralizing antibody, isotype-matched control antibody, or recombinant sgp130 was administered intravenously in RIP-Tag5 mice 30 minutes before STT. Immunofluorescence staining of ICAM-1 (red) was performed in pancreatic cryosections. Photomicrographs depict representative regions. Dotted lines indicate tumor boundary; numbers denote MFI of ICAM-1 staining on all CD31+ vessels. Scale bar: 100 μm. (B) Representative photomicrographs showing thermal induction of gp130 (red) on CD31+ (green) B16-OVA tumor vessels. Numbers denote MFI of gp130 or CD31 staining on CD31+ vessels. Scale bar: 50 μm. (C) Rolling and sticking fractions of calcein-labeled TK1 CD8+ T cells in CT26 tumor vessels. n = 3 independent experiments. *P < 0.001, #P < 0.03 versus control. (D) Growth of B16-OVA tumors in NT mice measured after administration of IL-6 neutralizing antibody or sgp130 (arrows). Data are for at least 6 mice/group. *P < 0.04 versus isotype control.

Figure 6. Vascular response to H-IL-6 in murine and patient tumors.

(A) Immunofluorescence staining of pSTAT3 (red) in CD31+ (green) B16-OVA tumor vessels 15 minutes after intravenous administration of H-IL-6 fusion protein. Nuclei were stained with DAPI (blue). Microscopic quantification of pSTAT3 staining in CD31+ ECs is also shown; data are representative of 3 independent experiments. Scale bar: 50 μm. *P < 0.001 versus control. (B) OT-I interactions with B16-OVA tumor vessels 6 hours after H-IL-6 treatment; data are from 3 independent experiments. Intravascular staining of ICAM-1 (red) on CD31+ (green) B16-OVA tumor vessels 6 hours after H-IL-6 treatment is also shown. Scale bar: 100 μm. *P < 0.001, #P < 0.03 versus control. (C) Representative photomicrographs of immunofluorescence staining of ICAM-1 on CD31+ vessels in tumor explants of patient 1 (P1) after treatment with H-IL-6 for 6 hours. ICAM-1 and CD31 expression in 3 patient tumor explants is shown by histograms representing quantitative image analysis of the immunofluorescence intensity of adhesion molecules in CD31+ vessels. Numbers in photomicrographs and histograms are MFI. IL-6 concentration in patient tumor explants is also shown. Scale bar: 100 μm.

Figure 7. Nonhematopoietic stromal cell–derived IL-6 induces vascular ICAM-1 expression in the tumor microenvironment.

(A) IL-6 concentration in B16 tumor lysates from NT control or STT-treated WT and Il6–/– mice, determined by Luminex. Data are from at least 3 mice. n.d., not detected. P = 0.53, NT versus STT in WT mice. (B) Immunofluorescence staining of ICAM-1 (red) and CD31 (green) in WT and Il6–/– mice implanted with B16 tumors. Numbers denote MFI for CD31+ vessels. Scale bar: 100 μm. (C) Quantification of endogenous CD8+ T cells in B16 tumors. Data are from individual mice and are representative of at least 3 independent experiments. *P < 0.0001 versus NT. (D) Quantitative image analysis for ICAM-1 and CD31 staining of B16 tumor vessels in bone marrow chimeric mice. Numbers denote MFI for CD31+ vessels.

Figure 8. Antigen-restricted killing of tumor targets depends on CD8+ T cell trafficking across tumor vessels.

(A) Growth curves of B16-OVA tumors in mice treated with STT and adoptive cell transfer (ACT) of OT-I effector cells. Treatment times are denoted by arrows; numbers above arrows indicate tumor volume at the time of treatment. n = 5 mice per condition. *P < 0.04 versus NT plus ACT. (B) Representative photomicrographs of B16-OVA tumors 12 hours after adoptive transfer of TRITC-labeled OT-I cells (red). Apoptotic cells were detected by TUNEL assay (green), and nucleated cells were stained with DAPI (blue). Scale bar: 100 μm. Quantification of intratumoral TRITC+ OT-I cells and TUNEL+ apoptotic cells at 1 or 12 hours after adoptive transfer is also shown. (C) Neutralizing antibody specific for IL-6 or E/P-selectin was administered intravenously 30 minutes before STT treatment in B16-OVA tumor–bearing WT mice. Tumors were also implanted in Icam1–/– mice. (B and C) Data are from individual mice and are representative of at least 3 independent experiments. *P < 0.002 versus NT.

Figure 9. Proposed model for switch from predominantly protumorigenic to antitumorigenic activity of IL-6 trans-signaling via mobilization of CD8+ T cell trafficking across vascular EC gateways.