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Cellular and Molecular Immunology logoLink to Cellular and Molecular Immunology
. 2014 Apr 28;12(1):66–76. doi: 10.1038/cmi.2014.21

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling

Fei Yang 1,2,4, Yinxiang Wei 1,4, Zhijian Cai 1,4, Lei Yu 1, Lingling Jiang 1, Chengyan Zhang 1, Huanmiao Yan 1, Qingqing Wang 1, Xuetao Cao 1,3, Tingbo Liang 2, Jianli Wang 1
PMCID: PMC4654365  PMID: 24769795

Abstract

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8+ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.

Keywords: activated cytotoxic T lymphocytes, Fas/FasL, myeloid-derived suppressor cells, prostaglandin E2, tumor escape

Introduction

Some tumors can escape attack from the immune system of the body, a process that is called tumor escape. Active T cells play a key role in antitumor escape. In particular, cytotoxic T lymphocytes (CTLs) are important effector cells involved in eliminating tumor cells.1 However, some tumor tissues are infiltrated by terminally differentiated cytotoxic T lymphocytes that exist in an unresponsive state.1,2 The role of these infiltrating CTLs in tumor progression is unclear. During T-cell activation, increased FasL expression on activated CTL cell surfaces renders CTLs able to kill tumor cells and induce auto-apoptosis. In the presence of extensive lung metastases, Fas/FasL signaling plays an important role in CTL-prompted tumor regression.3 However, in CTL-based immunotherapy, tumor cells are unresponsive to tumor infiltrating lymphocytes (TILs) and thus, the susceptibility of tumor cells to Fas-mediated cytotoxicity is diminished, ultimately leading to tumor escape. Our previous work showed that exosomes derived from activated T cells promoted tumor invasion via the Fas signaling pathway.4 In the present study, we investigated whether Fas signaling initiated by FasL expressed on infiltrating CTLs has a negative effect on the immune response of Fas-resistant tumor cells, thus causing tumor escape during tumor development and progression.

The death receptor Fas (CD95/APO-1) is a member of a tumor growth factor receptor superfamily. After Fas is triggered by its natural ligand, FasL, Fas signaling transmits intracellular apoptotic signals and leads to the apoptosis of cells to maintain systematic homeostasis.5 However, under certain conditions, Fas signaling can exert non-apoptotic effects, including inflammatory responses, liver regeneration, increased branching of developing neurons, migration of cells, angiogenesis, fibrosis, proliferation and differentiation of cells and advancement of the cell cycle.6,7,8,9,10,11 Therefore, although almost all tumor cells express the Fas receptor, the Fas pathway may also be beneficial to tumor cell survival rather than apoptosis.6,8,9,10 Activation of Fas signaling in the Lewis lung cancer cell line (3LL cells) does not cause apoptosis but induces 3LL cells to secrete more prostaglandin E2(PGE2).12 High levels of PGE2 aid 3LL cells in recruiting myeloid-derived suppressor cells (MDSCs), leading to tumor cell escape.13

CTLs (antigen-specific CD8+ T cells) together with natural killer cells are key defenders of host organismsagainst viruses and tumors.14 CTLs exist as inactive precursor cells in vivo, and they must be activated by specific antigens before differentiation into effector cells. Similar to other T-cell subsets that possess immune activation functions, activated CTLs induce and express high levels of FasL and mediate auto-apoptosis. These mechanisms can prevent the excessive activation of the immune system to sustain systematic immune balance.15 In Fas-sensitive tumor cells, CTLs can induce tumor cell apoptosis by FasL, which is one of the mechanisms by which CTLs kill tumor cells.16 It is not clear whether the activation of Fas signaling initiated by FasL on CTLs affects apoptosis-resistant tumor cells. We questioned whether CTLs could improve tumor escape via the activation of Fas-induced non-apoptotic signaling in Fas-resistant tumor cells.

Heterogeneous-population MDSCs comprise granulocytes, macrophages, dendritic cell precursors and myeloid cell precursors in the early differentiation phase.17 MDSCs inhibit the activation and proliferation of T and natural killer cells, promote the metastasis of tumors, advance the cell cycle and increase the invasive capacity of tumors to mediate tumor escape.17,18,19,20,21,22,23 A study of tumor patients over the course of clinical therapy revealed that there are large amounts of MDSCs in the peripheral blood and tumor-infiltrating tissues of patients suffering from head and neck cancers, squamous-cell epithelioma, mammary cancer and small-cell lung cancer. After tumor tissues are surgically removed, the number of MDSCs in the peripheral blood of tumor patients decreased.24 Moreover, after being transferred into tumor tissues, MDSCs differentiated into microvessel tumor endotheliocytes, which can form an environment that is favorable for tumor growth by promoting the generation of tumor neovascularity.25 These results suggest that the accumulation of MDSCs in tumor tissues is closely related to tumor growth and escape.

However, it remains unknown whether CTLs promote tumor cells to secrete PGE2, increasing tumor cell chemoattraction of MDSCs and thereby leading to tumor escape via Fas signaling. We obtained CTLs expressing high levels of FasL in vitro by stimulating CD8+ T cells from OT-I mice with the OVA257–264 peptide and evaluated the functions of Fas signaling activated by FasL-expressing CTLs in tumor tissues. We found that CTLs increased tumor cell chemoattraction of MDSCs by promoting tumor cells to secrete PGE2, which is associated with the activation of the ERK and p38 signaling pathways. This study suggests that activation of tumor Fas signaling driven by FasL on CTLs probably contributes to the accumulation of MDSCs in tumor tissues and promotes the progression of tumor growth.

Material and methods

Mice

C57BL/6J mice (6–8 weeks) were obtained from Joint Ventures Sipper BK Experimental Animal Co. (Shanghai, China). OVA257–264-specific TCR-transgenic OT-I mice were generously provided by Professor Yizhi Yu (the National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China). Female mice at 6–8 weeks of age were bred in a specific pathogen-free facility. The experimental protocols were approved by the Animal Care and Use Committee of the School of Medicine, Zhejiang University (Hangzhou, China).

Reagents

Phospho-antibodies (Abs) against ERK p44/p42 (ERK1/2, Thr202/Tyr204), JNK/stress-activated protein kinase (JNK/SAPK, Thr183/Tyr185), p38 (Thr180/Tyr182), NF-κB (Ser536), Stat3 (Tyr705) and corresponding Abs against non-phosphorylated signaling proteins were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-PGE2 Abs (2B5) and cyclooxygenase-2 (COX-2) inhibitor were purchased from Cayman Chemical (Ann Arbor, MI, USA). HRP-coupled secondary Abs and Stat3 inhibitor were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PE-conjugated FasL (CD178) was obtained from eBioscience (San Diego, CA, USA). OVA257–264 peptides were synthesized by Chinese Peptide Company (Hangzhou, China). NF-κB-, JNK-, p38- and ERK-specific inhibitors were purchased from Calbiochem (San Diego, CA, USA).

Plasmid construction

Full-length mouse wild-type (WT) Fas and OVA (Fas-WT/OVA), dominant negative Fas that did not contain the intracellular signaling domain and OVA (Fas-DN/OVA) or OVA alone were amplified by PCR and subcloned with internal ribosome entry sites (IRES) in the plasmid pMIR-REPORT. Fas-WT and Fas-DN were confirmed by sequencing. The primers are as follows: Fas WT: sense, 5′-CGGGATCCGCAGACATGCTGTGGATCTGGGCTGTCCT-3′, antisense, 5′-CTTAAGTCACTCCAGACATTGTCCTTCATTT-3′ Fas DN: sense, 5′-CGGGATCCGCAGACATGCTGTGGATCTGGGCTGTCCT-3′, antisense, 5′-CTTAAGCTAGTTTTCAGGTTGGCATGGTTGACAGC-3′ OVA: sense,5′-AGGCGCGCCTTCACCATGGGCTCCATCGGCGCA-3′, antisense, 5′-GACTAGTTTAAGGGGAAACACATCTGCCAAAG-3′ IRES: sense, 5′-CGGGATCCAGCGCTTAAGGCCCCTCTCCCTCCCC-3′, antisense, 5′-GGACTAGTGAGCTCGGCGCGCCATTATCATCGTGTTTTTCAAAGGAA-3′.

Preparation of WT and dominant negative Fas-overexpressing lung cancer cells

Fas-WT/OVA, Fas-DN/OVA or OVA plasmid DNA was transfected into 3LL lung cancer cells that were cultured in RPMI 1640 complete culture medium supplemented with 50 mM 2-ME, 1 mM sodium pyruvate, 10 mM HEPES, 10 U/ml IL-2 and 500 µg/ml geneticin for 7 days. Then, resistant 3LL cell clones that expressed high levels of Fas-WT or Fas-DN were selected by limiting dilutions, confirmed and characterized by real-time PCR. The 3LL cell clones expressing high levels of Fas-WT/OVA, Fas-DN/OVA and OVA alone were designated as 3LL/Fas-WT/OVA, 3LL/Fas-DN/OVA and 3LL/OVA, respectively.

Preparation and specific cytotoxicity assay of CD8+ T cells

CD8+ T cells were generated as described previously.4 Briefly, 2×106 cells/ml lymphocytes from OT-I mice were cultured in RPMI 1640 complete medium and stimulated with or without OVA257–264 peptide (2 µg/ml) (defined as CTLs and Ctrl T, respectively) for 72 h, and CD8+ T cells were purified using CD8+ Dynabeads (Invitrogen, Carlsbad, Cal, USA).

The specific cytotoxicity of CD8+ T cells was assayed as described previously26,27 with minor modifications. Briefly, splenocytes from OT-I mice were primed with 10 µg/ml OVA257–264 peptide as target cells. After 2 h of incubation, the cells were harvested and labeled with 2 µM CFSE. Then, the target cells were mix-cultured with active CD8+ T cells at different ratios (1∶0, 1∶5, 1∶10 or 1∶20). The cell mixtures were incubated at 37 °C for 4 h and then labeled with propidium iodide (PI). Finally, the cells were analyzed by FACS, and CFSE+ cells were gated and collected in equal numbers for each group. CFSE+PI− cells were counted as living cells, and the specific lysis rate was calculated according to the following formula:

Specific lysis rate (%)=[(negative control group cell count−treatment group cell count)/negative control group cell count]×100%.

Assay for cell chemoattraction

Cell chemoattractionassays were performed in 3.0-µm pore-size polyethylene terephthalate track-etched membrane cell-culture inserts. The supernatants of 3LL cells co-cultured with CD8+ T cells were placed below the inserts. Then, CD11b+Gr1+ MDSCs (2×105) purified from the spleens of tumor-bearing mice using magnetic beads (MiltenyiBiotec Inc., Auburn, CA, USA) were placed into the transwell inserts. Four hours after incubation at 37 °C, cells that had migrated into the lower chambers were harvested and suspended in 200 µl of phosphate-buffered saline (PBS). PI− cells were counted with a FACS Caliburmachine at low rate over 5 min and analyzed with CellQuest software.

Detection of 3LL cell apoptosis and proliferation

Cell apoptosis and proliferation assays were performed to detect whether increased FasL expression on activated CTLs influenced the survival of 3LL cells. Briefly, 50 µg/ml of mitomycin C was used to eliminate the activity of activated CTLs. The inactivated CTLs were cocultured with 3LL cells at indicated ratios (3LL to CTL: 1∶0.3, 1∶1 or 1∶3) for 24 h and were removed with immunomagnetic beads. 3LL cells administered different treatments were then used in the cell apoptosis assay and the proliferation assay.

Immunofluorescence staining and confocal microscopy

The distribution of FasL+ and CD8+ cells in lymph nodes and tumor tissues was detected by immunofluorescence staining. Frozen sections (8 µm) of tissues from healthy or tumor-bearing mice were fixed in acetone and blocked with PBS containing 3% bovine serum albumin. The samples were sequentially immunostained with Abs against FasL and CD8 and then with DyLight549- and DyLight488-conjugated secondary Abs, respectively. Slides were finally examined under an Olympus FluoView FV1000 confocal microscope and imaged using the Olympus Fluoview version 1.4a viewer (Olympus, Metamora, IL, USA).

RNA interference of Fas and antibody neutralization of FasL

To confirm the chemoattraction of MDSCs to 3LL tumor cells via Fas signaling, we knocked down Fas on 3LL cells with RNA interference and neutralized FasL on CTLs with FasL-blocking Abs. These two cell types were cultured together at the indicated ratios (3LL to CTL: 1∶0.3, 1∶1 or 1∶3), and suspensions of the mixed cultures were collected for the chemoattraction assay. The specific siRNA for Fas or scrambled siRNA were synthesized as follows: Fas siRNA: 5′-CUGCUCAGAAGGAUUAUAUTT-3′ and 5′-AUAUAAUCCUUCUGAGCAGTT-3′, siRNA scramble control: 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. A standard lipofectamine transfection was performed according to the manufacturer's manual. Twenty-four hours later, the interferential effects were confirmed with real-time PCR. For FasL neutralization, different concentrations of FasL-blocking Abs (5 µg/ml, 10 µ g/ml or 20 µg/ml) were added followed by incubation at 37 °C for 2 h to inactivate CTLs.

Cytokine assay

PGE2 in the supernatants of cocultured 3LL and CTL cells with different treatments was assayed using ELISA kits according to the manufacturer's instructions (R&D Systems, San Diego, CA, USA).

Reverse transcription PCR and real-time PCR

Total cellular RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription and real-time PCR were performed as described previously.3 The real-time PCR primers were as follows: COX-2: sense, 5′-ACCCGAGGACTGGGCCATGG-3′, antisense, 5′-TGCCCCACAGCAAACTGCAGG-3′ β-actin: sense, 5′-CGTTGACATCCGTAAAGACC-3′, antisense, 5′-AACAGTCCGCCTAGAAGCAC-3′ Fas: sense, 5′-AGGAGGCGGGTTCGTGAAACT-3′, antisense, 5′-TTTGGCTTCTTTACCCACCCCACC-3′.

Western blots

Activation of ERK, JNK, p38, NF-κB and Stat-3 and the expression of COX-2 in 3LL lung cancer cells with different treatments were detected using western blotting. Blots were probed with corresponding primary Abs above for 1 h, washed with Tris-buffered saline plus 0.05% Tween-20 and incubated with corresponding HRP-conjugated secondary Abs. Proteins were visualized using SuperSignal West Femto Maximum.

Flow cytometry analysis

To detect FasL on the cell membrane, activated CTLs were incubated with Abs against CD16/CD32 to block the receptor for Fc. Then, the cells were washed twice with PBS and stained with fluorescently-labeled Abs against FasL on ice for 30 min. Flow cytometry was performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

Preparation and observation of tumor-bearing mice

To induce tumor formation, 5×105 3LL, 3LL/Fas-WT/OVA, 3LL/Fas-DN/OVA or 3LL/OVA cells were inoculated s.c. into the flanks of C57BL/6J mice. On day 10, 5×105 activated CD8+ T cells from OT-I mice were injected i.p. Then, tumor masses were measured with a caliper after tumor inoculation. The survival of tumor-bearing mice was monitored daily. Mice were sacrificed when the transplanted tumors reached 3 cm in diameter or severe ulceration developed. Fourteen days after CTL injection, tumor-infiltrating lymphocytes were isolated to analyze MDSCs by FACS. Experiments were performed three times with eight mice in each group.

Statistical analysis

Experimental data were analyzed with one-way ANOVA and t-tests using SPSS 11.5. Differences were considered to be significant when the P value was <0.05.

Results

CTLs have no effect on 3LL tumor cell apoptosis and proliferation

To evaluate the expression of FasL on activated CTLs, we isolated lymphocytes from OT-I mice, purified CD8+ T lymphocytes and stimulated them with OVA257–264 peptides. After stimulation for 3 days, we found that the CD8+ T lymphocytes specifically killed splenocytes pulsed with OVA257–264, but exhibited no cytotoxic effects against unpulsed splenocytes (Figure 1a, P<0.01). This result demonstrated that we successfully obtained antigen-specific CTLs by stimulating lymphocytes from OT-I mice with OVA257–264 peptides in vitro. Additionally, we detected the expression of FasL on CTLs by FACS and found that the CTLs were induced to express high levels of FasL (Figure 1b) and acquired specific cytotoxic functions. We verified that 3LL cells constitutively expressed Fas at the mRNA and protein levels (Figure 1c). Subsequently, we determined whether CTLs expressing high levels of FasL could induce the apoptosis of 3LL cells by Fas signaling, and we found that these CTLs did not induce the apoptosis of 3LL cells (Figure 1d). It was reported that Fas signaling can promote tumor cell proliferation.10,28 To exclude the possibility that the CTL-induced apoptosis resistance of 3LL cells was not caused by increased proliferation mediated by Fas signaling, we examined 3LL cell proliferation and determined that CTLs did not have an effect (Figure 1e).

Figure 1.

Figure 1

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CTLs had no effect on 3LL tumor cell apoptosis and proliferation. (a) Specific cytotoxicity of activated CD8+ T cells from OT-I mice to OVA257–264-pulsed target cells (**P<0.01). (b) Splenocytes from OT-I mice stimulated with or without 1 µg/ml of OVA256–267 for 72 h (defined as CTL and Ctrl T); gated CD8+ T cells and FasL expression were detected by FACS. (c) 3LL tumor cells were examined for expression of Fas at the mRNA (left) and protein (right) levels. (d) Inactivated CTLs were cocultured with 3LL cells at serial dilutions for 24 h, and apoptosis of 3LL cells was detected by FACS. (e) Inactivated CTLs were cocultured with 3LL cells by serial dilution for 24 h. 3LL cells were isolated via CD8− sorting. Proliferation of 3LL cells was detected at 24 h, 48 h or 72 h. Data are representative of three independent experiments. CTL, cytotoxic T lymphocyte.

CTLs promote the chemoattraction of MDSCs by 3LL tumor cells via Fas signaling

As Fas signaling was shown to promote the recruitment of MDSCs by tumor cells,12 we examined whether 3LL cells treated with CTLs would result in the chemoattraction of more MDSCs. We cocultured activated CTLs and 3LL cells for 24 h, collected the supernatant and tested its ability to induce the chemoattraction of MDSCs from tumor-bearing mice. We found that CTLs clearly promoted the chemoattraction of MDSCs by 3LL cells (Figure 2a, *P<0.05 versus 3LL). To further confirm whether FasL expressed on CTLs mediated the chemoattraction effect, we used Fas siRNA to knock down Fas expression on 3LL cells. Compared with nonspecific siRNA, Fas siRNA clearly inhibited the chemoattraction effect of 3LL cells on MDSCs (Figure 2b and c; Figure 2c, *P<0.05 versus 3LL cells ). Similarly, we neutralized FasL on CTLs with FasL blocking Abs to prevent the activation of Fas signaling by CTLs. We found that the chemoattraction effect of 3LL cells on MDSCs was clearly inhibited (Figure 2d, *P<0.05 versus 3LL cells). These results suggest that CTL promotion ofMDSC chemoattraction by 3LL cells was Fas/FasL-dependent.

Figure 2.

Figure 2

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CTLs promoted the chemoattraction of MDSCs through 3LL tumor cells via Fas signaling. (a) Four hours after incubation at 37 °C, MDSCs migrating into lower chambers were harvested and suspended in 200 µl of PBS. PI− MDSCs were counted with a FACSCalibur machine at a low rate over 90 s and were analyzed with CellQuest software. (b) The effects of Fas RNA interference. (c) Chemoattraction of MDSCs by 3LL cells was evaluated by FACS after Fas RNA interference. (d) Chemoattraction of MDSCs by 3LL cells was evaluated by FACS after pre-treatment of CTLs with FasL neutralizing Abs. Data are representative of three independent experiments. *P<0.05 versus 3LL cells. Ab, antibody; CTL, cytotoxic T lymphocyte; MDSC, myeloid-derived suppressor cell; PBS, phosphate-buffered saline; PI, propidium iodide.

PGE2-derived 3LL tumor cells treated with CTLs contribute to the enhanced chemoattraction of MDSCs

It has been reported that 3LL cells promote the chemoattraction of MDSCs by secreting higher levels of PGE2.12 Therefore, we detected changes to COX-2,which is the rate-limiting enzyme in PGE2 synthesis.29 We found that COX-2 expression was significantly up-regulated at both the mRNA (Figure 3a and b; Figure 3a, **P<0.01 versus 3LL cells) and protein levels (Figure 3c). Similarly, in an ELISA assay, we confirmed that the PGE2 levels secreted by 3LL cells treated with CTLs were significantly higher than for non-CTL-treated 3LL cells (Figure 3d, **P<0.001 versus 3LL cells). To confirm the roles of COX-2 and PGE2 in the chemoattraction of MDSCs, we performed experiments to evaluate MDSC chemoattraction by 3LL tumor cells induced by CTLs after COX-2 inhibition or PGE2 blocking, and we found that the COX-2 inhibitor reduced the percentage of MDSCs that were recruited by 3LL tumor cell-mediated chemoattraction (Figure 3e, *P<0.05 and **P<0.01 versus untreated 3LL cells stimulated CTL). Similarly, the increased chemoattraction of MDSCs by CTL-treated 3LL tumor cells was abrogated after PGE2 blockage (Figure 3f, *P<0.05 and **P<0.01 versus untreated 3LL stimulated CTLs). These results demonstrated that CTLs promoted the chemoattraction of MDSCs by 3LL cells via the upregulation of COX-2 and subsequently, PGE2.

Figure 3.

Figure 3

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PGE2-derived 3LL tumor cells treated with CTLs contributed to the enhanced chemoattraction of MDSCs. (a–d) For all experiments, 5×105/ml 3LL cells were stimulated with CTLs or Ctrl T with serial dilution for 24 h. (a) Real-time PCR analysis of COX-2 expression (**P<0.01 versus 3LL cells). (b) Reverse transcription PCR analysis of COX-2 expression. (c) Western blot analysis of COX-2 expression. (d) PGE2 concentrations in the supernatants of 3LL cells cocultured with CTLs were detected by ELISA (**P<0.001 versus 3LL cells). (e) The COX-2 selective inhibitor SC-58125 (70 nM and 700 nM) was used to treat 3LL cells for 2 h after 3LL cells were treated with CTLs. The chemoattraction of MDSCs by 3LL cells was evaluated by FACS. (f) PGE2 neutralizing Abs (2B5) were used to treat 3LL cells for 2 h after 3LL cells were stimulated with CTLs. The chemoattraction of MDSCs by 3LL cells was determined by FACS. 3LL cells were isolated with CD8− sorting. *P<0.05, **P<0.01 versus CTL-treated 3LL cells. Data are representative of three independent experiments. Ab, antibody; COX-2, cyclooxygenase-2; CTL, cytotoxic T lymphocyte; MDSC, myeloid-derived suppressor cell; PGE2, prostaglandin E2.

The increased activation of the p38 and ERK signaling pathways is responsible for the chemoattraction of MDSCs by CTL-treated 3LL tumor cells

Next, we investigated which pathway was responsible for the enhanced chemoattraction of MDSCs. Activation of Fas signaling is accompanied by the activation of the MAPK and NF-κB pathways9,28,30,31,32,33,34,35 over the course of cell proliferation, tumor metastasis and induction of the inflammatory response, and the activation of Stat-3 in tumor cells is very important in tumor-induced immune tolerance.18,36 Thus, we detected the expression of phosphorylated ERK, JNK, p38, NF-κB and Stat-3 in 3LL cells after treatment with CTLs. The western blot results demonstrated that CTLs promoted the phosphorylation of ERK, p38, NF-κB and Stat-3, suggesting that CTLs activated the ERK, p38 MAPK, NF-κB and Stat-3 pathways (Figure 4a and b). To confirm the signaling pathway responsible for the chemoattraction of MDSCs by CTL-treated 3LL tumor cells, 3LL tumor cells were pretreated with specific inhibitors for signaling pathways before CTL treatment. Consistent with previous reports,12 we found that the p38-specific inhibitor SB203580 and the ERK1/2-specific inhibitor U0126 decreased the number of MDSCs recruited via CTL-treated 3LL tumor cell-mediated chemoattraction (Figure 4c, **P<0.01 versus untreated 3LL cells), indicating that CTL activation of the p38 and ERK MAPK signaling pathways was responsible for the increased chemoattraction of MDSCs by CTL-treated 3LL tumor cells.

Figure 4.

Figure 4

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Increased activation of the p38 and ERK signaling pathways was responsible for the chemoattraction of MDSCs by CTL-treated 3LL tumor cells. Western blot analysis of (a) the MAPK pathway, (b) the NF-κB pathway and the Stat-3 pathway in 3LL cells cultured alone in medium or stimulated with Ctrl T or CTLs for the indicated times. (c) 3LL cells were pre-treated with the NF-κB inhibitor PDTC, the Stat-3 inhibitor AG490, the p38 MAPK inhibitor SB203580, the JNK/SAPK inhibitor SP600125 or the eERK1/2 inhibitor U0126 for 30 min before CTL stimulation. The chemoattraction of MDSCs by 3LL cells was evaluatedwith FACS. 3LL cells were isolated with CD8− sorting. **P<0.01 versus CTL-treated 3LL cells. Data are representative of three independent experiments. CTL, cytotoxic T lymphocyte; MDSC, myeloid-derived suppressor cell.

CTLs promote increased numbers of MDSCs in Fas-overexpressing tumor tissues and tumor growth in vivo

To further confirm the existence of CD8+ and FasL+ T cells in tumor-bearing mice, we detected the localization of FasL and CD8 double-positive cells in tumor tissues with an immunofluorescence assay. We confirmed that there was a group of CD8+ T cells expressing FasL in 3LL tumor tissues (Figure 5), suggesting that FasL+CD8+ T cells may affect tumor development in vivo.

Figure 5.

Figure 5

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Existence of FasL+CD8+ T cells in tumor-bearing mice. The localization of FasL+CD8+ T cells in lymph nodes and tumor tissues was detected with an immunofluorescence assay. Green represents CD8 and red represents FasL (magnification: ×40). Data are representative of three independent experiments.

To decipher the effects of FasL-expressing CTLs on the chemoattraction of MDSCs and lung cancer growth in vivo, we generated expression vectors containing WT Fas and full-length OVA (Fas-WT/OVA), dominant negative Fas that did not contain the intracellular signaling domain and full-length OVA (Fas-DN/OVA) or full-length OVA alone. 3LL tumor cells were transfected with each of these vectors and then screened for the stable overexpression of Fas and/or OVA. The effects of 3LL tumor cell overexpression of Fas and OVA were determined using real-time PCR. 3LL/Fas-WT/OVA, 3LL/Fas-DN/OVA and 3LL/OVA all expressed OVA, while only 3LL/Fas-WT/OVA expressed the Fas intracellular segment at high levels, and the expression of the Fas extracellular domain for 3LL/Fas-WT/OVA and 3LL/Fas-DN was higher than for 3LL/OVA (Figure 6a, **P<0.01 versus 3LL). 3LL/Fas-WT/OVA, 3LL/Fas-DN and 3LL/OVA showed no obvious differences in proliferation (Figure 6b). Next, we detected the accumulation of MDSCs in tumor tissues after the inoculation of activated CD8+ T cells 10 days post-inoculation of 3LL/Fas-WT/OVA, 3LL/Fas-DN/OVA or 3LL/OVA in C57BL/6J mice. Fourteen days after CTL injection, TILs were isolated, and the MDSC ratio was analyzed by FACS. The ratio of Gr1+CD11b+ MDSCs in TILs derived from 3LL/Fas-WT/OVA tumor-bearing mice was higher than that from 3LL/Fas-DN/OVA or 3LL/OVA; the MDSC ratios were 36.82% for 3LL/Fas-WT/OVA, 15.6% for 3LL/Fas-DN/OVA and 19.52% for 3LL/OVA (Figure 6c, **P<0.01 versus 3LL/OVA or 3LL/Fas-DN/OVA). We found that tumor growth accelerated when activated CD8+ T cells were transferred into C57BL/6J mice 10 days post-inoculation of 3LL/Fas-WT/OVA compared with the inoculation of 3LL/Fas-DN/OVA or 3LL/OVA (Figure 6d, *P<0.05, **P<0.01 versus 3LL/OVA or 3LL Fas-DN/OVA). Accordingly, the survival rate of the mice bearing 3LL/Fas-WT/OVA was significantly lower than the mice bearing 3LL/Fas-DN/OVA or 3LL/OVA (Figure 6e, *P<0.05, **P<0.01 versus 3LL/OVA or 3LL/Fas-DN/OVA). These results suggest that activated CD8+ T cells promoted tumor growth via the upregulation of MDSC chemoattraction through Fas signaling in 3LL tumor cells.

Figure 6.

Figure 6

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CTLs prompted increased numbers of MDSCs in Fas-overexpressing tumor tissues and tumor growth in vivo. (a) Quantification of Fas and OVA expression in 3LL cells transfected with a plasmid encoding WT-Fas and OVA (3LL/Fas-WT/OVA), dominant negative Fas and OVA (3LL/Fas-DN/OVA) or OVA (3LL/OVA) by real-time PCR (left, OVA; middle, Fas extracellular domain; right, Fas intracellular segment. RQ indicates relative mRNA expression). **P<0.01 versus 3LL. (b) In vitro proliferation of 3LL/Fas-WT/OVA, 3LL/Fas-DN/OVA or 3LL/OVA was measured by CCK8. **P<0.01 versus 3LL/OVA. (c–e) Cytotoxic activated CD8+ T cells from OT-I mice specific to OVA257–264 were injected intravenously 10 days after 3LL tumor cells were inoculated s.c. into C57BL/6J mice. Fourteen days after CTL injection. (c) TILs were isolated from tumor tissues, and the ratio of MDSCs in TILs was analyzed by FACS. *P<0.05, **P<0.01 versus 3LL/OVA or 3LL/Fas-DN/OVA. Tumor size and the survival of tumor-bearing mice were monitored and analyzed. (d) Tumor size, *P<0.05, **P<0.01 versus 3LL/Fas-DN/OVA. ▾P<0.05, ▾▾P<0.01 versus 3LL/OVA. (e) Survival (n=8). Data are representative of three independent experiments. Mice were sacrificed when tumor sizes reached 30×30×30 mm3. CTL, cytotoxic T lymphocyte; MDSC, myeloid-derived suppressor cell; TIL, tumor-infiltrating lymphocyte.

Discussion

Lung cancer is one of the largest causes of cancer mortality despite current therapy.37 To eliminate tumor cells, some scientists have attempted to apply immunotherapy.38 As one of the major components of the antitumor response, adoptive CTL therapy draws much attention; however, its effects are limited, and some malignant cells can survive anticancer therapy.2,39 In the tumor microenvironment, complex interactions between CTLs and multiple cell types contribute to immunosuppression. It was found that CTLs that infiltrated tumors recruited MDSC sand triggered a counter-regulatory immunosuppressive mechanism after adoptive CTL lymphocyte therapy for malignancy.40 The accumulation of MDSCs is caused by IFN-γ produced by CTLs.40 However, it remains unknown whether Fas signaling in tumor cells initiated by FasL on CTLs participates in the CTL recruitment of MDSCs.

In this study, we demonstrated that CTLs expressed high levels of FasL and acquired specific killing functions. The expression of FasL on CTLs mediated the activation of Fas signaling in 3LL tumor cells and induced tumor cells to secrete PGE2, which subsequently increased the ability of 3LL tumor cells to mediate MDSC chemoattraction. Meanwhile, Fas signaling activated the downstream MAPK, NF-κB and Stat-3 pathways in 3LL cells, and the activation of the ERK and p38 pathways was associated with increased MDSC chemoattraction by 3LL tumor cells. These results suggested that CTLs performed tumor killing functions, but the expression of FasL on CTLs induced greater 3LL tumor cell-mediated MDSC chemoattraction through the activation of Fas signaling, thus probably inhibiting CTL tumor killing functions.

CTLs are important effector cells in the elimination of tumor cells. In general, CTLs that infiltrate tumor tissues are in an unresponsive state.1 During T-cell activation, FasL expression increases on activated CTLs, and this increased FasL expression mediates CTL tumor cell killing and auto-apoptosis. Our previous work demonstrated that exosomes derived from activated T cells promoted tumor invasion via the Fas signaling pathway,4 indicating that FasL on CTLs possessed the ability to mediate tumor escape. In this study, we found that CTLs induced 3LL tumor cell-mediated MDSC chemoattraction via Fas signaling.

We found that 3LL tumor cells resisted apoptosis induced by high levels of FasL on CTLs, and CTL-treated 3LL tumor cells upregulated COX-2 expression to increase PGE2 secretion, thus resulting in the chemoattraction of more MDSCs. MDSCs are critical mediators of tumor immune suppression and inhibit antitumor immunity through multiple pathways, including the regulation of arginine and tryptophan metabolism through arginase, inducible nitric oxide synthase and indoleamine-2,3-dioxygenase, leading to the suppression of proliferation and CTL functions.13 COX-2 and PGE2 play an important role in tumor progression. COX-2 is the key regulator of PGE2 synthesis41 and can depress the immune response and facilitate tumor growth.42 COX-2 overexpression increases the production of PGE2 to enhance the recruitment of regulatory T cells.43 PGE2 can promote MDSC recruitment into tumor environments by inducing chemokines such as CXCL12/SDF-1 and CXCR4 and stabilizing the CXCL12 receptor. In addition, PGE2 also induces suppressive factors and directly inhibits T-cell immune responses.13 Indeed, although Fas induces apoptosis triggered by FasL to maintain systemic homeostasis, as a classic death receptor Fas signaling also exerts non-apoptotic functions under certain conditions.28 In addition, Fas signaling not only fails to induce tumor cell apoptosis, but also promotes tumor growth by inducing tumor cells to release more PGE2 and enhancing tumor cell recruitment of MDSCs to form an immunosuppressive tumor microenvironment.12

In this study, we found that CTLs activated tumor cell Fas signaling, which in turn activated MAPK, NF-κB and Stat-3 intracellular signal transduction pathways, leading to 3LL tumor cell resistance to apoptosis induced by CTLs. Our previous work on activated T-cell exosomes showed that FasL+ exosomes derived from CD8+ T cells promoted the accumulation of c-FLIP, which was induced by the NF-κB and ERK signaling pathways in B16 tumor cells.4 Therefore, the difference between these signaling pathways must be further investigated, and it will be worth discerning the molecules that may be involved upstream of MAPK, NF-κB and Stat-3 signaling for this purpose. For example, FAP-1 or c-FLIP (FLICE-inhibitory protein) in 3LL tumor cells may play a role in tumor escape mediated by CTLs via Fas signaling. The transmission of Fas apoptosis signaling is interrupted by truncated Fas receptors or increased anti-apoptosis proteins, such as FAP-1, in tumor cells,44,45 and high levels of c-FLIP inhibit caspase 8 from binding and cleaving the death-inducing signaling complex, leading to Fas-apoptosis resistance in some tumor cells. In addition to inducing tumor cells to release more PGE2 for MDSC chemoattraction, activated CTLs also increase tumor cell invasiveness by upregulating MMP9 via Fas signaling.7,9 Therefore, the effects of CTL FasL on tumor escape may be ascribed to multiple functions instead of one single mechanism.

The development of tumors resembles a war between the tumor and the host immune system. At the beginning of tumor development, antitumor-specific CTLs may be induced by tumor-specific antigens or tumor-associated antigens to kill or eliminate tumors.46 However, FasL expression on CTLs may promote greater MDSC chemoattraction by tumor cells via the activation of Fas signaling to inhibit CTL tumor killing functions. In this study, we learned that in addition to antitumor functions, CTLs facilitate tumor cell evasion of attacks by the immune system. In the tumor microenvironment, tumor cells induce CTLs to lose their tumor killing functions. Higher levels of FasL expression are not only found on activated T cells but also on cancer-associated dendritic cells and macrophages.47,48 Similar to CTLs, dendritic cells and macrophages are also important effector cells against tumors. However, based on our discoveries, it can be speculated that dendritic cells and macrophages may be accomplices of CTLs in facilitating tumor escape.

In conclusion, we found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell chemoattraction of MDSCs via Fas signaling, suggesting that CTLs may participate in the process of tumor immune evasion. To our best knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. At the beginning of tumor growth, Fas signaling can recruit MDSCs, but CTLs can still kill tumors. As tumor growth progresses, tumor microenvironments are favorable for tumor escape via the recruitment of more MDSCs to suppress the antitumor functions of CTLs through Fas signaling. Our findings suggest the utility of a strategy to enhance the antitumor immune response by reducing negative tumor immune responses that are promoted by CTLs via Fas signaling.

Acknowledgments

The work was supported by the Specialized Research Fund for the Chinese National 973 Project (2013CB530502), the Doctoral Program of Higher Education of China (20110101110105), the Project of the Chinese National Nature Science Foundation (31370902, 31070795, 31270944), the Projects in Science and Technology Plan of Zhejiang Province (013C33G2010434) of China, the National Key Science and Technology Specific Project of China (2012ZX10002006), the National High Technology Research and Development Program (2012AA020900), and the Project of the Chinese National Natural Science Fund Committee for Talent Cultivation (J1103603).

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