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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: J Immunol. 2022 Apr 13;208(9):2239–2245. doi: 10.4049/jimmunol.2100885

IL-27 induces CCL5 production by T lymphocytes which contributes to anti-tumor activity

Aiyan Hu 1,*, Jianmin Zhu 1,*, Chunxi Zeng 1, Cho-Hao Lin 1, Jianyu Yu 1, Jin-Qing Liu 1, Kimberly Lynch 1, Fatemeh Talebian 1, Xueliang Pan 2, Jingyue Yan 3, Yizhou Dong 3, Zihai Li 4, Xue-Feng Bai 1,4,#
PMCID: PMC9050872  NIHMSID: NIHMS1786143  PMID: 35418466

Abstract

Interleukin-27 (IL-27) is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this work, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27 induced CCL5 production was primarily dependent on Stat1 activation, while in CD8+ T cells, Stat1-deficiency does not abrogate CCL5 induction. Chromatin immunoprecipitation (ChIP) assay revealed that in the CCL5 promotor region, both putative Stat3 binding sites exhibit significant binding to Stat3, while only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to IL-27-mediated anti-tumor effect. This is signified by diminished tumor inhibition in anti-CCL5 and IL-27 treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to anti-tumor activity.

Keywords: IL-27, Recombinant adeno-associated virus, T lymphocytes, CCL5, cancer immunotherapy

Introduction

IL-27 is a member of the IL-12 cytokine family that consists of an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3), and a p35-related subunit, p28 (1). IL-27 signals through a heterodimeric receptor (IL-27R) composed of the WSX-1 (IL-27Rα) and the gp130 subunits in a variety of cell types, including T lymphocytes (2). IL-27R signaling enhances the recruitment of several JAK family kinases and activates STAT family transcription factors 1 and 3 (3, 4). IL-27 enhances Th1/Tc1 responses by activating the Stat1-T-bet axis and promotes T cell expression of T-bet, Eomes, IL-12Rβ2, granzyme B and Perforin (5–7). It also inhibits Th2 and Th17 responses by blocking the expression of transcription factors GATA-3 (Th2) and RoRγτ (Th17) (8, 9) and is an inducer of IL-10 production by T cells (10–12). Moreover, IL-27 can also induce PD-L1 expression in T cells which restrain T cell effector functions by interacting with PD-1 on T cells (13). These functional properties of IL-27 suggest that it enhances anti-tumor immunity and inhibits Th17/Th2 mediated autoimmunity.

We recently evaluated the therapeutic potential of IL-27 delivered by adeno-associated virus (AAV-IL-27) in mouse tumor models. Our studies (14, 15) revealed that first, AAV-IL-27 significantly inhibited the growth of a broad spectrum of tumor types in mice with low or no toxicity. Second, AAV-IL-27 treatment resulted in dramatic reduction of Tregs without causing autoimmunity. Third, AAV-IL-27 therapy shows strong synergy with PD-1 antibody or cancer vaccines in inhibiting tumor growth. In searching for IL-27 induced genes in T cells, we found that C-C motif chemokine ligand 5 (CCL5), also known as RANTES, was most significantly upregulated. CCL5 is a member of the CC chemokine family and is detected in multiple cell types including immune cells such as NK cells, T cells, dendritic cells (DCs) and macrophages (16). In tumor microenvironment (TME), cancer cells could also serve as a source of CCL5 (17). CCL5 works primarily through interaction with CCR5 receptor (18), but it can also engage other receptors such as CCR1 (18) and CCR3 (19). Functionally, CCL5 is a proinflammatory chemokine recruiting various leukocytes including T, NK, and DCs to the site of inflammation (19). However, the role of CCL5 in tumor immunity remains controversial. While some studies suggest that CCL5 production may lead to a more immune suppressive TME (20–22), other evidence suggests that CCL5 in TME is in favor of tumor immunity (23–25).

In this work, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent, and requires both Stat3 and Stat1 signaling. In CD4+ T cells, IL-27 induced CCL5 production is primarily dependent on Stat1 activation, while in CD8+ T cells, Stat1-deficiency does not abrogate CCL5 induction. Chromatin immunoprecipitation (ChIP) assay reveals that Stat3 may be the dominant mediator of IL-27-induced induction of CCL5 in CD8+ T cells, since both putative Stat3 binding sites exhibited significant binding of Stat3, while only one out of four Stat1 binding sites displayed moderate binding to Stat1. In IL-27 treated tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to IL-27-mediated anti-tumor effect, as significantly less tumor inhibition was observed in anti-CCL5 and IL-27 treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth.

Materials and Methods

Mice

CD45.1 congenic C57BL6, C57BL/6, IL27R−/−C57BL6 and BALB/c mice were originally purchased from The Jackson Laboratory. Stat1−/− BALB/c mice (14) and P1CTL TCR transgenic mice (26) were described previously. These mouse strains were maintained in the animal facilities of the Ohio State University (OSU). All animal protocols were approved by the OSU Animal Care and Use Committee (Approved IACUC protocol 2008A0093-R4) and mice were treated in accordance with institutional guidelines for animal care. The OSU Laboratory Animal Shared Resource is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited program that follows Public Health Service policy and guidelines.

Preparation of CD4+ and CD8+ T cells and in vitro activation

To prepare CD4+ and CD8+ T cells, mouse spleen and lymph nodes were processed into single-cell suspensions. CD4+ and CD8+ cells were purified by positive selection using anti-CD4 or anti-CD8-PE and anti-PE-magnetic microbeads on a MACS column (Miltenyi Biotech, Germany). Positive selection was also used to purify human CD8+ T cells from peripheral blood mononuclear cells. Purified CD4+ or CD8+ T cells were incubated with anti-CD3/CD28 beads (Dynabeads Mouse T-Activator CD3/28, Thermo Fisher) in click’s EHAA medium (Invitrogen) containing 100 μg/mL penicillin and 100 μg/mL streptomycin, 1 mM 2-ME, 5% fetal bovine serum, in the absence or presence of 50 ng/mL recombinant IL-27 (Biolegend) for up to 5 days. To activate P1CTL T cells, 0.3 × 106/mL spleen cells from P1CTL TCR transgenic mice were stimulated with 0.1 μg/mL P1A35–43 peptide in EHAA medium in the absence or presence of 50 ng/mL recombinant mouse IL-27 for up to 5 days.

Production of rAAV and treatment of mice

rAAV-mIL-27 and rAAV-ctrl viruses were produced as described previously (14). AAV viruses were diluted in PBS and injected intramuscularly (i.m.) into two sites of hind legs in a total volume of 100 μl, containing 2 × 1011 DNase-resistant particles (DRP) of AAV.

Flow cytometry

FITC-, PE-, PE-CY7, APC-, APC-CY7 or Percp-Cyanine5.5 labeled antibodies to mouse CCL5 (2E9/CCL5), CD45.1 (A20), CD4 (GK1.5), CD8α (53–6.7), CD11b (M1/70), CD11c (N418), NK1.1 (PK136) and isotype-matched control antibodies were purchased from Biolegend or BD Biosciences. APC-labeled antibody to human CD8 (RPA-T8) and CCL5 (VL1) were purchased from BD Biosciences. For identification of cellular phenotypes, disassociated cells from spleens or tumors were re-suspended in PBS containing 1% bovine serum albumin and incubated with the antibodies on ice for 30 minutes. Cells were fixed in 1% paraformaldehyde in PBS after washing. For intracellular CCL5 staining, viable cells were fixed and permeabilized with transcription staining buffer set (eBioscience) and stained with respective antibody. Stained cells were analyzed on a FACSCalibur or FACS Celesta flow cytometer, and data were analyzed using the Flowjo software.

Determination of IL-27 induced CCL5 mRNA expression in P1CTL cells

Spleen cells from P1CTL mice were cultured in Click’s EHAA medium containing 0.1 μg/mL of P1A35–43 in the presence or absence of mIL-27 (50 ng/ml). The total RNA was extracted at day 1, 2, 3, 4, and 5 after T cell culture using Qiagen RNeasy Mini kit following the manufacturer’s protocol. The expression levels of CCL5 mRNA were determined by qRT-PCR using primers specified in Supplementary Table 1.

Chromatin Immunoprecipitation (ChIP)

P1CTL cells were activated with P1A peptide with or without IL-27 for four days as described above. Then the resulting CTLs were fixed and sheared by sonication. ChIP was performed using ChIP-IT® Express Kits (Active Motif, 53008) according to the manufacturer’s protocol. The following antibodies, including anti-STAT1 (Cell Signaling #9172), anti-STAT3 (Santa Cruz, sc-428), anti-RNA polymerase II (Abcam, ab5153), and a control antibody (Abcam, ab171870) were used in precipitation. 25 μg sheared chromatin was used in each precipitation reaction. Selective immunoprecipitation of CCL5 promoter fragments was determined by qPCR using the high capacity cDNA reverse transcription kit (Applied Biosystems, 4368813) and the primers are specified in Supplementary Table 1. Input % was calculated by ChIP/Input × 100%.

Tumor establishment in mice and treatments with AAV-IL-27 and anti-CCL5 antibody

C57BL6 mice were injected with B16.F10 melanoma cells, which were originally obtained from ATCC, and maintained in RPMI1640 (Gibco) medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, and 10% FBS (Gibco). To establish tumors in mice, 1 × 105 B16.F10 cells were injected into each C57BL6 mouse s.c. in 100 μl of PBS. Four days after tumor inoculation, the mice received i.m. injection of AAV-IL-27 or AAV-ctrl virus at a dose of 2 × 1011 DRP per mouse. On day 13, 17, 22 and 26 mice were also treated with 100 μg/mouse of anti-CCL5 (Clone# 53405, R&D system) or a control IgG2a antibody (BioXcell) i.p. The length and width of tumors were measured using a digital caliper every 2 days. The tumor volume was calculated according to the formula volume (V) = ab2/2, where a represents length and b represents width of tumors.

CCL5 mRNA synthesis and encapsulation into nanoparticles

CCL5 expression plasmid was purchased from InvivoGen (San Diego, CA, USA) and was used as a template for in vitro transcription. mRNAs were synthesized with full substitution of UTP by pseudouridine-5’-triphosphate (TriLink, USA) using AmpliScribe T7-Flash Transcription Kit (Lucigen, USA). The resulting mRNA was purified by RNA Clean & Concentrator (Zymo, USA) and capped using Vaccinia Capping System (NEB, USA) and Cap 2´-O-Methyltransferase (NEB, USA). Purified CCL5 mRNA was quantified using a NanoDrop 2000 Spectrophotometer (ThermoFisher, USA), and was mixed with lipid nanoparticles (NP) to prepare NP-CCL5 mRNA using a method described before (27).

Treatment of mice via intratumoral injection of NP-CCL5 mRNA

C57BL6 mice were inoculated with B16.F10 melanoma cells (1 × 105 cells/mouse) s.c. When tumors were fully established on day 10, mice started to receive intratumor injection of either NP-CCL5 mRNA (2 μg/mouse in 50 μl PBS) or vehicle only every other day for 6 times. Tumor size was measured every two days and tumor volumes were calculated.

Statistical Analysis

One way ANOVA and student’s t test were used for comparison among multiple groups or between two groups. The GraphPad Prism software 8.0 was used for ANOVA and t-tests.

Results

1. IL-27 induces CCL5 production in mouse and human T cells.

We recently evaluated the potential of AAV-IL-27 as a therapeutic in mouse tumor models. Our studies (14, 15) revealed that AAV-IL-27 significantly inhibited the growth of a broad spectrum of tumor types in mice with low or no toxicity. To understand pathways induced by IL-27, we performed RNAseq analysis of CD8+ T cells from mice treated with AAV-IL-27 (GSE195736; https://www.ncbi.nlm.nih.gov/geo/info/linking.html). We found that one of the most significantly upregulated genes is CCL5 (Figure 1A). To validate this observation, we purified naïve CD4+ and CD8+ T cells from mouse spleen and lymph nodes, and activated T cells with anti-CD3/CD28 beads in the presence or absence of IL-27. As shown in Figure 1B, we found that IL-27 upregulated CCL5 production in both CD4+ and CD8+ T cells three or five days after culture. However, IL-27 most significantly upregulated CCL5 in CD8+ T cells five days after activation. We also activated P1CTL TCR transgenic T cells with P1A peptide with or without IL-27, then analyzed CCL5 production using flow cytometry. As shown in Figure 1C, IL-27 stimulation significantly upregulated CCL5 production in P1CTL T cells. Similarly, purified human CD8+ T cells were activated with anti-CD3/CD28 beads in the presence or absence of IL-27. We found IL-27 also upregulated CCL5 production in human CD8+ T cells (Figure 1D).

Figure 1. IL-27 induces CCL5 production by mouse and human T cells in vitro.

Figure 1.

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(A) C57BL6 mice were treated with AAV-IL-27 or AAV-ctrl virus (2×1011 DRP/mouse i.m.). Three weeks after AAV injection, mice were sacrificed, CD8+ T cells were purified from AAV-treated mice and submitted for RNAseq analysis. Major differentially expressed genes are shown. (B) Naïve CD4 or CD8 T cells were purified from spleen and lymph nodes from C57BL6 mice. Cells were activated with anti-CD3/CD28 beads with or without recombinant mouse IL-27 (50 ng/ml). Production of CCL5 was detected by flow cytometry on day 3 or day 5 after T cell culture. (C) Spleen cells from P1CTL TCR transgenic mice were activated with 0.1 ug/ml of P1A35–43 with or without recombinant IL-27 (50 ng/ml). Production of CCL5 by CD8+Vα8.3+ cells was detected by flow cytometry on day 3 or day 5 after T cell culture. (D) CD8+ T cells were purified from human PBMC. Cells were activated with anti-CD3/CD28 beads with or without recombinant hIL-27 (50 ng/ml). Production of CCL5 was detected by flow cytometry on day 3 or day 5 after T cell culture. Data are expressed as Mean ± SD of 3–6 samples in each group, and data represent 3–5 experiments with similar results. *P < 0.01, **P < 0.001, ***P < 0.0001 by student’s t-test.

To determine if IL-27 directly induces CCL5 production in T cells, we purified naïve CD4+ and CD8+ T cells from IL27Rα−/− and WT mice, and stimulated T cells with anti-CD3/CD28 with or without IL-27. As shown in Figure 2, IL-27 only induced CCL5 in T cells from WT but not IL-27Rα−/− mice. Additionally, when T cells from IL27Rα−/− mice were mixed with T cells from WT mice at 1:1 ratio followed by activation, IL-27 only induced CCL5 in CD4+ (Figure 2A) and CD8+ (Figure 2B) T cells from WT, but not CD27Rα−/− T cells. Thus, IL-27 induces CCL5 production in T cells through direct stimulation of IL27R on T cells, but not through bystander factors induced by IL-27 and T cell activation.

Figure 2. IL-27 induced CCL5 production in T cells is IL-27R-dependent.

Figure 2.

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Naïve CD4+ and CD8+ T cells were purified from spleen and lymph nodes from CD45.1+ C57BL6 mice or CD45.2+ IL-27Rα−/− mice. Cells were activated with anti-CD3/CD28 beads with or without recombinant mouse IL-27 (50 ng/ml) separately or in mixture (1:1 ratio). Production of CCL5 was detected by flow cytometry on day 5 after T cell culture. Data are expressed as Mean ± SD of 3 samples in each group, and data represent three experiments with similar results. *P < 0.01, **P < 0.001, ***P < 0.0001 by student’s t-test.

2. Differential involvement of Stat1 and Stat3 in IL-27-induced CCL5 production in T cells

IL-27 signals through IL-27R and activates Stat1 and Stat3 (3, 4). We therefore tested if IL-27 induces CCL5 through Stat1 or Stat3. As shown in Figure 3A, while IL-27 induced significant CCL5 on day 3 and day 5 in CD4+ T cells, Stat1-deficiency abrogated CCL5 induction in CD4+ T cells. For CD8+ T cells, we found that Stat1-deficiency also significantly diminished CCL5 induction on day 3 and day 5. However, significant CCL5 induction by IL-27 was still evident on day 5 after T cell activation (Figure 3B). Thus, it appears that IL-27-induced CCL4 production in CD4+ and CD8+ T cells is dependent on Stat1, while in CD8+ T cells, Stat3 is also likely involved.

Figure 3. IL-27 induced CCL5 production by T cells in the absence of Stat1.

Figure 3.

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Naïve CD4 or CD8 T cells were purified from spleen and lymph nodes from Stat1−/− and control BALB/c mice. Cells were activated with anti-CD3/CD28 beads with or without recombinant mouse IL-27 (50 ng/ml). Production of CCL5 in CD4+ (A) and CD8+ (B) T cells were detected by flow cytometry on day 3 or day 5 after T cell culture. Data are expressed as Mean ± SD of 3 samples in each group, and data represent four experiments with similar results. **P < 0.001, ***P < 0.0001 by student’s t-test.

To determine the peak of CCL5 mRNA expression in the presence of IL-27, primary mouse P1CTL cells were activated with P1A peptide in the presence or absence of IL-27 for 1 to 5 days. The relative expression of CCL5 mRNA was determined by RT-qPCR. As shown in Figure 4A, dramatic induction of IL-27 mRNA was detected through day 3–5, with day 4 as the peak. Recently, the global roles of STAT1 and STAT3 in IL-27-induced transcriptomic changes were revealed by ChIP-seq using anti-STAT1 and anti-STAT3 antibodies (28). Therefore, we examined the ChIP-seq data deposited to Gene Expression Omnibus (Accession Number GSE65621). Close examination of the region upstream of CCL5 transcription start site (TSS) revealed several putative STAT1 and STAT3 binding sites (Supplementary Figure 1). Based on the ChIP-seq data, STAT1 possibly binds to the region spanning from −4000bp to −4800bp relative to CCL5 TSS, while STAT3 may bind to a −200bp site and a −4700bp site. To confirm STAT1 and/or STAT3 binding, primary mouse P1CTL cells were activated with P1A peptide for 4 days with and without IL-27. Then ChIP assay was performed on P1CTL cells using qPCR primers designed to probe the putative binding sites (Supplementary Table 1). As expected in controls, IL-27 stimulation enhanced RNA polymerase II’s binding to the promoter of CCL5 (Figure 4B). Increased STAT3 binding were detected at both the −200bp and −4700bp sites upstream of CCL5 TSS (Figure 4C), while significantly increased STAT1 binding was detected in one of the four binding sites (−4700bp site) (Figure 4D). These results suggest that while both STAT1 and STAT3 are involved in IL-27 induced CCL5 induction, STAT3 activation is more evident in the CCL5 promotor of CD8+ T cells.

Figure 4. ChIP assay of Stat1 and Stat3 binding in CCL5 promotor.

Figure 4.

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P1CTL TCR transgenic T cells were activated with 0.1 ug/ml of P1A35–43 with or without recombinant IL-27 (50 ng/ml). (A) The relative expression of CCL5 mRNA were determined by RT-qPCR. Data are expressed as Mean ± SD of 3 samples in each group, and data represent two experiments with similar results. (B) Binding of RNA polymerase II to CCL5 promoter was confirmed by ChIP using an anti-RNA pol II antibody. (C) ChIP assay was performed on P1CTL cells using qPCR primers designed to probe the two putative binding sties of Stat3. Both the −200bp and −4700bp sites upstream of CCL5 TSS revealed significant binding of STAT3. *P < 0.05 by student’s t test. (D) ChIP assay was performed using qPCR primers designed to probe the binding sites of Stat1 in CCL5 promotor. Moderate binding of Stat1 to the −4700bp site but not to the other three putative sites were detected. *P < 0.05 by student’s t-test.

3. IL-27 treatment upregulates CCL5 in T cells in vivo

To determine if IL-27 also induces CCL5 induction in T cells in vivo, IL27Rα−/− and Stat1−/− mice and their relative control mice were treated with AAV-IL-27 or AAV-ctrl virus at a dose of 2 × 1011 DRP/mouse i.m. Three weeks after viral treatment, mice were sacrificed and expression of CCL5 in spleen T cells were analyzed by flow cytometry. As shown in Figure 5A, treatment with AAV-IL-27 significantly induced CCL5 in both CD4+ and CD8+ spleen T cells from WT, but not IL-27Rα−/− mice, while treatment with AAV-ctrl failed to induce CCL5 in both WT and IL27Rα−/− mice. Additionally, we found that STAT1-deficiency abrogated IL-27-induced CCL5 production in CD4+ T cells, but not in CD8+ T cells (Figure 5B). In AAV-IL-27 treated mice, we also evaluated whether AAV-IL-27 treatment induced CCL5 in other splenic leukocytes. We found that AAV-IL-27 treatment failed to induce CCL5 in cells including B cells, NK cells, CD11b+ monocytes and CD11c+ dendritic cells (Supplemental Figure 2), suggesting that IL-27 alone is insufficient to induce CCL5 in these cell types.

Figure 5. AAV–IL-27 treatment upregulates CCL5 in T cells in vivo.

Figure 5.

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AAV-IL-27 or AAV-ctrl virus were injected into IL-27Rα−/− and control C57BL6 (A), or Stat1−/−BALB/c and control BALB/c mice (B) i.m. at a dose of 2×1011 DRP. Three weeks later, mice were sacrificed and T cell production of CCL5 in spleens were analyzed by flow cytometry. Data shown represent 3–5 experiments with similar results. ns: no significant difference. **P < 0.01, ***P < 0.001 by student’s t-test.

To determine CCL5 induction by IL-27 in tumor-bearing mice, WT and IL27Rα−/− mice were injected with 1 × 105 cells/mouse of B16F10 cells s.c. Four days later, mice were treated with AAV-IL-27 or AAV-ctrl virus at a dose of 2 × 1011 DRP/mouse i.m. Three weeks after viral treatment, mice were sacrificed and expression of CCL5 in spleen T cells and tumors were analyzed by flow cytometry. As shown in Figure 6A, AAV-IL-27 treatment induced CCL5 in both spleen T cells and tumor-infiltrating T cells, while much higher CCL5 induction was observed in tumor-infiltrating T cells. In both spleens and tumors, AAV-IL-27 induction of CCL5 was strictly IL-27R-dependent (Figure 6B).

Figure 6. AAV–IL-27 therapy induces CCL5 production in tumor-infiltrating T cells.

Figure 6.

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(A) C57BL6 mice were first treated with AAV-IL-27 or AAV-ctrl virus (2×1011 DRP/mouse i.m.). Mice were also challenged with B16.F10 tumor cells (1 × 105 cells/mouse) s.c. Three weeks after AAV injection, T cell expression of CCL5 was evaluated by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 by student’s t test. (B) IL-27Rα−/− and control C57BL6 mice were first treated with AAV-IL-27 or AAV-ctrl virus (2×1011 DRP/mouse i.m.). Mice were also challenged with B16.F10 tumor cells (1 × 105 cells/mouse) s.c. Three weeks after AAV injection, T cell expression of CCL5 in tumors was evaluated by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 by student’s t test. Data shown represent three experiments with similar results.

4. IL-27-induced CCL5 inhibits tumor growth

Previous reports concerning the role of CCL5 in tumors were controversial. While some studies suggest that CCL5 production may lead to a more immune suppressive TME (20–22), other evidence suggests that CCL5 in TME is in favor of tumor immunity (23–25). To determine the role of IL-27 induced CCL5 in tumor immunity, we performed the following two sets of experiments. First, C57BL6 mice were initially inoculated with B16.F10 tumor cells s.c. followed by treatment with AAV-IL-27 or AAV-ctrl virus (2×1011 DRP/mouse i.m.) on day 4. Then, mice were also treated with anti-CCL5 (100 μg/mouse) or a control IgG2a antibody (100 μg/mouse) i.p. on days 13, 17, 22 and 26. As shown in Figure 7A, while mice treated with AAV-ctrl/anti-CCL5 and AAV-ctrl/ctrl Ab exhibited similar tumor growth kinetics, anti-CCL5 treatment significantly reduced the efficacy of AAV-IL-27-mediated tumor inhibition. This conclusion was the result of significantly larger tumor volumes measured on days 26, 28 and 30. Second, we generated lipid nanoparticles (NP) encapsulated with CCL5 mRNA (NP-CCL5 mRNA). In vitro cell culture experiments revealed that NP mediated delivery of CCL5 mRNA could be efficiently translated into CCL5 protein in B16.F10 cells (Figure 7B). As shown in Figure 7C, intratumoral injection of NP-CCL5 mRNA significantly inhibited tumor growth, as measured by significantly reduced mean tumor volume. Thus, neutralizing CCL5 compromised AAV-IL-27-mediated antitumor activity, while intratumor delivery of CCL5 mRNA directly inhibited tumor growth.

Figure 7. IL-27-induced CCL5 production contributes to anti-tumor immunity.

Figure 7.

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(A) C57BL6 mice were inoculated with B16.F10 melanoma cells (1 × 105 cells/mouse) s.c. Mice were treated with AAV-IL-27 or AAV-ctrl virus (2×1011 DRP/mouse i.m.) four days later. On day 13, 17, 22 and 26 mice were also treated with anti-CCL5 (100 μg/mouse) or a control IgG2a antibody (100 μg/mouse) i.p. Tumor growth was monitored and tumor volume was calculated. Five to seven mice were used for the experiment and data represents two experiments with similar results. *P < 0.05, **P < 0.01 by one way ANOVA. (B) B16.F10 cells were co-cultured with control NP or NP-CCL5 mRNA (50 ng mRNA/ml). Eighteen hours later, the supernatants of cell cultures were collected and ELISA was used to measure CCL5 concentrations in culture supernatants. (C) C57BL6 mice were inoculated with B16.F10 melanoma cells (1 × 105 cells/mouse) s.c. When tumors were fully established on day 10, mice received intratumor injection of either NP-CCL5 mRNA (2 μg/mouse; n=7) or vehicle control (n=6) every other day for 6 times. Tumor size was measured every two days and calculated as tumor volume. ***P<0.001; ****P<0.0001 by one way ANOVA.

Discussion

There is an increasing body of literature that suggest IL-27 regulates lymphocyte chemotaxis by modulating expression of chemokine/chemokine receptors. IL-27 was found to induce the release of CCL2, CXCL9 and CXCL10 in human primary fibroblast-like synoviocytes (29). We recently found (15) that IL-27 directly upregulates CXCR3 in T cells, which is the receptor for CXCL9 and CXCL10. Another study reported that IL-27 signaling suppresses splenic CD4+ T cell CCR5-dependent chemotactic responses during infection through restricting CCR5 expression (30). Interestingly, IL-27-treatment during influenza reduced secretion level of multiple chemokines including CXCL1, CCL4 and CCL5 by CD11b+ macrophages and CD11c+ myeloid cells (31). In contrast to this observation, it was reported that pretreatment with IL-27 followed by stimulation with TL8, R848, or CL075 significantly enhanced production of IL-8 and CCL5 in macrophages (32). In this work, we demonstrate that IL-27 directly induces CCL5 production in both mouse and human T cells, in particular CD8+ T cells in vitro and in vivo. Thus, our current study reveals a novel function of IL-27 in T cells.

In this work, we observed that IL-27 induced CCL5 production only in WT but not IL-27Rα−/− T cells even when IL-27Rα−/− and WT T cells were mixed, suggesting that no bystander effect is involved and IL-27 directly induces CCL5 production by T cells. We observed that in CD4+ T cells, IL-27 induced CCL5 production is primarily dependent on Stat1 activation in vitro (Figure 3) and in vivo (Figure 5). However, in CD8+ T cells, Stat1-deficiency reduced, but did not abrogate CCL5 induction in vitro (Figure 3). In vivo, IL-27-induced CCL5 production by CD8+ T cells was not significantly affected by Stat1-deficiency (Figure 5). ChIP assay also revealed both Stat1 and Stat3 involvement. STAT3 appears to be the dominant mediator of IL-27-induced activation of CCL5 in CD8+ T cells (Figure 4). Thus, our study reveals a mechanism of IL-27 induction of CCL5 in T cells: in CD4+ T cells, CCL5 induction is Stat1-dependent, while in CD8+ T cells, induction of CCL5 is mediated by Stat1 in early stages, while persistent induction of CCL5 requires activation of Stat3.

Previous reports concerning the role of CCL5 in tumors were controversial. While some studies suggest that CCL5 production may lead to a more immune suppressive TME (20–22), other evidence suggests that CCL5 in TME is in favor of tumor immunity. First, tumor expressed CCL5 positively correlates with CD8+ T cell infiltration in a variety of human tumors (23–25). In stage IV melanoma patients, it was found that the presence of the CCR5Δ32 polymorphism resulted in a decreased survival following immunotherapy (33). Second, in experimental studies, CCL5 produced by CD4+ T cells induced DC infiltration into tumors, which enhanced cross priming of tumor-specific CD8+ T cells (34). CCL5 was also shown to enhance NK cell infiltration into tumors (33, 35). Third, CCL5 was shown to enhance glucose uptake and ATP generation in T cells (36). Moreover, CCL5 in CD8+ T cells enhanced T cell memory (37), while CCL5-deficient CD8+ T cells exhibited exhausted phenotype (38). In this work, using two sets of experiments, we found that IL-27-induced CCL5 contributes to IL-27-mediated anti-tumor effect, as significantly less tumor inhibition was observed in anti-CCL5 and AAV-IL-27 treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth (Figure 7).

Taken together, our current study revealed a novel function of IL-27 in inducing robust CCL5 production by T cells. In CD4+ T cells, CCL5 induction is Stat1-dependent, while in CD8+ T cells, induction of CCL5 appears to require activation of Stat3. Moreover, we found that IL-27 induced CCL5 production enhances anti-tumor activity. Our results suggest that IL-27-mediated anti-tumor effect is partially achieved by induction of CCL5 in T cells, particularly in CD8+ T cells.

Supplementary Material

1

Key points.

  • IL-27 signaling directly induces CCL5 production by mouse and human T cells.

  • Stat3 and Stat1 bind to the CCL5 promoter in IL-27-stimulated CD8+ T cells.

  • IL-27 induced CCL5 production enhances anti-tumor activity.

Grant support

This work was supported by a grant (R01CA229254) from National Cancer Institute. This project was also supported by the OSUCCC Biostatistics and Analytical Cytometry supported in part by NCI grant P30 CA016058.

References

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