The cryo-thermal therapy-induced IL-6-rich acute pro-inflammatory response promoted DCs phenotypic maturation as the prerequisite to CD4⁺ T cell differentiation

Abstract

In our previous studies, a novel tumour therapeutic modality of the cryo-thermal therapy has been developed leading to long-term survival in 4T1 murine mammary carcinoma model. The cryo-thermal therapy induced the strong acute inflammatory response and IL-6 was identified in an acute profile. In this study, we found that the cryo-thermal therapy triggered robust acute inflammatory response with high expression of IL-6 locally and systemically. The phenotypic maturation of dendritic cells (DCs) was induced by acute IL-6 following the treatment. The mature DCs promoted CD4⁺ T cell differentiation. Moreover, the production of interferon γ (IFN γ) in the serum and CD4⁺ T cells were both abrogated by IL-6 neutralisation following the treatment. Our findings revealed that the cryo-thermal therapy-induced acute IL-6 played an important role in initiating the cascading innate and adaptive anti-tumour immune responses, resulting in CD4⁺ T cell differentiation. It would be interesting to investigate acute IL-6 as an early indicator in predicating tumour therapeutic effect.

Keywords:

• Acute inflammation
• interleukine-6
• DCs maturation
• anti-tumour immune response

Introduction

Suppression of the normal immune response is pivotal in tumour growth and metastasis. During tumour development, tumour cells attract suppressive immune cells, such as myeloid-derived suppressor cell (MDSCs) and regulatory T cells (Tregs), to inhibit antitumour T cells responses establishing an immunosuppressive microenvironment leading to tumour cells proliferation and spreading [1–4]. Tumour-induced immunosuppression develops through down-regulation of cells surface antigens, mutations within malignant cells, lack of costimulatory function, secretion of immunosuppressive cytokines and induction of tolerance [5]. Therefore, it is indispensable for effective cancer therapy to overcome the tumour-induced immunosuppression and elicit the durable systemic anti-tumour immunity [6].

It is known that immunosuppression is associated with chronic inflammation in the tumour microenvironment [7,8]. Inflammation is divided into two types: chronic inflammation or acute inflammation. Chronic inflammation mainly results from persistent infection or autoimmune diseases, leading to a prolonged abnormal immune response [9,10]. Chronic inflammatory response is associated with tumour progression through enhancing tumour immune suppression [8], and allowing tumour cells to escape from immune system by blocking the activation of CD4⁺ and CD8⁺ T cells, natural killer cells (NKs) cytotoxicity, macrophage polarisation, and maturation of dendritic cells (DCs) [11], as well as inducing MDSCs accumulation [12]. The acute inflammation caused by injury or pathogen lasts over a short period of time, while triggering innate and adaptive immune response [13,14]. As a result, neutrophils and other inflammatory cells migrate to the injured tissues within a large amount of acute inflammatory cytokines [7]. This acute inflammatory process is self-limiting response and resolves after tissue repair or elimination of pathogens. In resolution of inflammation, the level of pro-inflammatory mediators and infiltrated immune cells declines [9,10]. Moreover, the acute inflammation contributes to skew immune response toward Th1, which is a characteristic of immune cell-mediated cytotoxic killing [15,16].

Interleukin 6 (IL-6) is produced at the site of inflammation and has a dual effect in chronic inflammation or acute inflammation [17,18]. Though some reports reveal that IL-6 can promote tumour progression by supporting angiogenesis and tumour evasion of immune surveillance, accumulating evidences identify IL-6 as a key player in the activation, proliferation and survival of lymphocytes during the immune responses [18]. IL-6 plays a key role in regulating acute phase response, such as in the production of acute phase proteins [19]. Acute IL-6 signalling can restore the T cell immune response, shifting it from an immunosuppressive to an immunostimulatory state against tumour [20,21]. However, the role of acute IL-6 in promoting T cell immune response should be addressed.

In our previous study, a novel tumour therapeutic modality was developed for cryo-thermal therapy by alternating liquid nitrogen (LN2) cooling and radio frequency (RF) heating of tumour tissues [22–27]. Using a highly malignant murine 4T1 breast cancer xenograft model, we found that cryo-thermal therapy remodelled the tumour microenvironment from chronic inflammatory to an acute phenotype. Furthermore, IL-6 was significantly up-regulated during the acute phase after the cryo-thermal therapy [27]. However, whether acute IL-6 induced is essential to the enhancement of anti-tumour immunity is still unclear. To elucidate the role of acute IL-6 in the initiation of anti-tumour immune response, in this study, IL-6 was neutralised in vivo during the acute phase triggered by the cryo-thermal therapy. We found that the cryo-thermal therapy-induced acute IL-6 promoted phenotypic maturation of DCs, which was the prerequisite to CD4⁺ T cell differentiation.

Materials and methods

4T1 cell culture and animal experiment

BALB/c mice were obtained from Shanghai Slaccas Experimental Animal Co., Ltd. (China) and used for experimental study at the age of 6–8 weeks (∼20 g). They were housed in isolated cages and a 12 h light/dark cycle environment, feeding with the sterile food and acidified water. All animal experiments were approved by the Animal Welfare Committee of Shanghai Jiao Tong University, and experimental methods were performed in accordance with the guidelines of Shanghai Jiao Tong University Animal Care (approved by Shanghai Jiao Tong University Scientific Ethics Committee). 4T1 mouse mammary tumour cell line was obtained from Shanghai First People’s Hospital (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT) supplemented with 10% FBS, plus 100 U/mL penicillin, and 100 g/Ml streptomycin (Shanghai Sangon, China) at 37 °C in a humidified 5% CO₂ incubator. To prepare the tumour-bearing mice, approximately 5 × 10⁵ cells were injected subcutaneously into the right femoral region of each mouse. Tumour sizes were measured every 2–3 days and its volume was estimated using the following formula: V (cm³) = π × L (major axis) × W (minor axis) × H (vertical axis)/6.

The thermal treatment procedures

The system developed in our laboratory was composed of liquid nitrogen for cooling and radiofrequency (RF) for heating [24]. Three weeks after tumour inoculation when the tumour volume size reached about 0.2 cm³, mice were divided into three groups: (1) tumour-bearing group without the treatment (control); (2) hyperthermia group with RF heating at the temperature of 50 °C for 15 min on primary tumour (hyperthermia); and (3) The cryo-thermal group with freezing at the temperature of −20 °C for 5 min followed by RF heating at the temperature of 50 °C for 10 min on primary tumour (in cryo-thermal treatment, a total treatment duration was 15 min to be consistent with treatment duration of hyperthermia). The mice were anaesthetised with intraperitoneal (i.p.) injection of 1.6% pentobarbital sodium (0.5 ml/100 g, Sigma-Aldrich). The tumour site was sanitised with alcohol and iodine tincture before the treatment. All of the procedures were performed aseptically.

Flow cytometry analysis

Single-cell suspension of splenocytes were prepared using GentleMACS™ dissociator (Miltenyi Biotec), and then splenocyte suspension was treated with erythrocyte-lysing reagent containing 0.15 M NH₄Cl, 1.0 M KHCO₃, and 0.1 mM Na₂EDTA to remove red blood cells. These cells were then stained to identify and characterise immune cell populations using fluorescence conjugated antibodies binding cell specific surface marker for 30 min at 4 °C. Staining antibodies including FITC-anti-mouse-CD3, PE-anti-mouse-CD4, APC-anti-mouse-CD8, Alexfluor488-antimouse-CD11c, PerCP-Cy5.5-anti-mouse-MHCII, PE-anti-mouse-CD86, were purchased from eBioscience (Santiago, CA). 1 × 10⁴ events were collected for all analyses using the BD FACS Aria II cytometer (BD Biosciences) and the data were analysed using FlowJo software. Four mice per group were tested at each time point.

In vivo IL-6 neutralisation

4T1 tumour bearing mice were treated by the cryo-thermal therapy. The treated mice were divided into two groups: IL-6 neutralisation group or the isotype IgG group. 100 μg of mouse anti-IL6 antibody or the isotype IgG (Sanjian, Tianjin, China) was intraperitoneally injected to the treated mice at 3 and 24 h post the cryo-thermal therapy. The untreated 4T1 tumour-bearing mice were used as the control group (n = 4 mice per group).

Isolation of DCs, CD4⁺ T cells and CD8⁺ T cells

For isolation of CD4⁺, CD8⁺ T cells and DCs, spleens from the treated mice and 4T1 tumour-bearing mice were harvested at 48 h after the treatment, and single cell suspensions were prepared using GentleMACS™ dissociator (Miltenyi Biotec). DCs were isolated by DC isolation micro-bread kit (EasysepTM CD11c positive selection kit, STEM CELL) according to the manufacturer’s instructions. CD4⁺ and CD8⁺ T cells were isolated by Easysep™ CD4⁺ T cell negative selection kit and Easysep™ CD8⁺ T cell negative selection kit (StemCell technologies, Vancouver, BC, Canada), respectively. CD4⁺ and CD8⁺ T cells with a purity of >90% were used for experiments.

ELISA analysis

Tumour tissues were surgically removed at 6 or 24 h after the cryo-thermal therapy, hyperthermia, or from 4T1 tumour-bearing mice control group, and tissue homogenate was prepared using 1× PBS (0.1 g/ml). Then IL-6 concentration in the supernatant of tumour tissue homogenate was analysed using commercial ELISA kits (Boster Biotech, China). Six mice were used at each time point from each group.

In the cryo-thermal therapy or hyperthermia group, one millilitre of blood was obtained at different time points (6, 24, 48 and 72 h) after the treatment. The untreated 4T1 tumour-bearing mice were used as the control. Six mice were used at each time point from each group. The samples were centrifuged and the serum was collected. Then IL-6 concentration in the serum samples was analysed using commercial ELISA kits (Boster Biotech, China). A standard curve was established according to the manufacture’s instruction. Experimental values were computed with the use of regression analysis.

The serum from the treated mice following rejection of IL-6 neutralisation or the isotype IgG was collected for IFN-γ assay by using commercial ELISA kits (Boster Biotech, China). The untreated 4T1 tumour-bearing mice were used as the control. On day 2 after the cryo-thermal therapy, the CD4⁺ T cells from the treated mice following neutralising IL-6 or the isotype IgG were isolated using Easysep™ CD4⁺ T cell negative selection kit (StemCell technologies, Vancouver, BC, Canada). Then the isolated CD4⁺ T cells were lysed in RIPA lysis buffer (with cocktail proteinase inhibitor, 0.1% SDS and added PMSF to 1 mM before use) and the extracted protein was subject to for the examination of IFN-γ concentration. Furthermore, CD4⁺ T cells from naïve mice were co-cultured with isolated DCs from the treated mice receiving neutralising IL-6 antibody or isotype IgG for 24 h, and the culture supernatant was collected for IFN-γ detection.

RNA isolation and real-time PCR

Total freshly exercised tumours and spleen RNA were isolated from three groups (control, hyperthermia and the cryo-thermal) using TRIzol Reagent (TaKaRa, Dalian, China). Total RNA was also isolated from purified splenic DCs, CD4⁺ and CD8⁺ T cells in IL-6 neutralisation and the isotype IgG groups. DCs, CD4⁺ and CD8⁺ T cells were selected using EasysepTM selection kit (StemCell technologies, Vancouver, BC, Canada). The untreated 4T1 tumour-bearing mice were used as the control group. Absorbance at 260/280 nm for mRNA purity at a ratio above 1.9 was achieved for all samples used. cDNA was made using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). Quantitative real-time PCR was performed on ABI 7900HT sequence detection system, and SDS software (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq (TaKaRa) and samples were amplified in 384-well plates. The primer sequences of mouse genes presented in Supplementary Table 1. Relative expression level of mRNA for each gene was normalised to GAPDH determined by using the Ct value and assessed using relative quantification (delta–delta Ct method). All experiments were performed in triplicates.

Western blotting analysis

DCs were isolated using isolation kit from different groups (IL-6 neutralisation, the isotype IgG group, and the untreated 4T1 tumour-bearing control group), and then the protein of DCs were subjected to 12% SDS-PAGE gel. The protein concentration was evaluated with Ponceau S (Beyotime, Shanghai, China). Targeted protein was immunoblotted with specific Abs as the following: anti-IDO2 (1:500, Absci, Baltimore, MD) and anti-CXCL12 (1:500, Proteintech, Rosemont, IL). Each protein was detected in four independent mice with three technical replicates. Blots were evaluated with Quantity One 1-D (Bio-Rad, Hercules, CA). Results were expressed as relative pixel intensity normalised with that of the control group.

In vitro cell co-culture

The isolated DCs from each group (n = 4 per group) as described above were co-cultured with CD4⁺ T cells from the spleen of healthy mice at a ratio of 1:5 in six-well plate for 24 h, and the supernatants were collected for IFN-γ analysis by using ELSIA kit (Boster Biotech, China) according to the manufacture’s instruction. The co-cultured CD4⁺ T cells were purified and used for quantitative real-time PCR analysis.

Statistical analysis

The Student’s t-test and one or two-way ANOVA were used for statistical comparisons using Graph Pad Prism 6. Figures denoted statistical significance of p < .05 as *, p < .01 as **, p < .001 as ***. A p value <.05 was considered to be statistically significant. Results were expressed as mean ± SD.

Results

The cryo-thermal therapy-induced IL-6-rich acute pro-inflammatory response

Previously, we found that the cryo-thermal therapy triggered strong acute pro-inflammatory response with up-regulated expression of IL-6 [27] along with a good therapeutic effect. It was demonstrated that the cryo-thermal therapy was superior to hyperthermia in reducing MDSCs and increasing CD4⁺ and CD8⁺ T cells, and the prolonged survival rate [26,27]. To verify if significant up-regulated expression of IL-6 following the treatment is a characteristic hallmark of anti-tumour immunity triggered by the cryo-thermal therapy, the level of IL-6 was analysed in local tumour tissues after the cryo-thermal therapy (pre-freezing at the temperature of −20 °C for 5 min followed by RF heating at the temperature of 50 °C for 10 min), and hyperthermia (50 °C for 15 min) in comparison with that in the tumour-bearing control group.

The real-time PCR analysis of tumour extracts harvested at 6 h and 24 h after the treatment was performed. The results showed that the mRNA expression level of IL-6 was elevated at 6 h after the cryo-thermal therapy and hyperthermia (Figure 1(A)). However, at 24 h following the treatment, the expression level of IL-6 in the cryo-thermal group was 6-fold higher than that in the control group (p < .001), whereas the expression level of IL-6 in the treated mice of hyperthermia group was markedly diminished (Figure 1(A)). Then, the protein level of IL-6 was detected in local tumour tissues via ELSIA. The concentration of IL-6 in tumour tissues was significantly increased at 6 or 24 h after the cryo-thermal therapy compared with that in hyperthermia or the tumour-bearing control group (Figure 1(B)). In hyperthermia group, the up-regulation of IL-6 was only shown at 24 h after the treatment compared with that in the tumour-bearing control group, but it was markedly lower than that in the cryo-thermal therapy.

Figure 1. The cryo-thermal therapy-induced IL-6-rich acute pro-inflammatory response. (A) Relative mRNA expression level of IL-6 in primary tumour at 6 or 24 h after the cryo-thermal therapy and hyperthermia was analysed by real-time PCR, compared with the tumour-bearing control group. n = 6 mice per group; (B) The protein level of IL-6 in primary tumour at 6 or 24 h after the cryo-thermal therapy and hyperthermia was analysed by ELSIA, compared with the tumour-bearing control group. n = 6 mice per group; (C) The serum level of IL-6 in treated mice from hyperthermia, and the cryo-thermal group at 6, 24, 48 and 72 h after the treatment was analysed by ELISA, compared with the tumour-bearing control group. n = 6 mice at each time point for each group. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

To evaluate the systemic release of IL-6 by the cryo-thermal therapy, IL-6 level in serum was examined via ELISA. The serum IL-6 gradually stabilised at a higher level between 6 and 48 h following the cryo-thermal therapy, and then decreased to a lower level with no significant difference in comparison to that of the control group at 72 h post the treatment. However, the serum IL-6 in hyperthermia group was not significantly increased as compared to that in the control group (Figure 1(C)). The results indicated that IL-6 level was up-regulated systemically in the early stage after the cryo-thermal therapy. In summary, acute phase response with stronger expression of IL-6 was induced locally and systemically after the therapy.

The cryo-thermal therapy-induced acute IL-6 promoted the phenotypic maturation of DCs

We observed that the cryo-thermal therapy-induced strong systemic acute pro-inflammatory cytokine, IL-6, in 48 h after the treatment. As the spleen is the largest secondary immune organ and is responsible for initiating systemic immune responses, we analysed DC maturation in the spleen to observe the cryo-thermal-induced systemic anti-tumour immunity. DCs provide an essential link between the innate and adaptive immune responses [28]. To investigate if DCs could be responsive to this acute pro-inflammatory cytokine, the phenotypic maturation of DCs in spleen after the cryo-thermal therapy was analysed using the flow cytometry. The data showed that the percentage of mature DCs (CD11c⁺ MHCII⁺CD86⁺) was significantly increased at 48 h and 72 h, and it was much higher at 120 h after the treatment, whereas it remained at a relative low level in the control group (Figure 2). In Figure 2 from our previous work [27], we added one time point (48 h after the treatment) in this study at which DC phenotypic maturation was found. Moreover, the cryo-thermal therapy induced strong systemic acute pro-inflammatory cytokine IL-6 in 48 h after the treatment. Considering that acute pro-inflammatory cytokine IL-6 induced and DC phenotypic maturation occurred in the same time window, we supposed that the cryo-thermal therapy induced-strong, acute pro-inflammatory IL-6 would affect DCs phenotypic maturation.

Figure 2. The cryo-thermal therapy promoted the phenotypic maturation of DCs. The tumour-bearing mice were treated by the cryo-thermal therapy and hyperthermia three weeks after 4T1 breast cancer cells inoculation. Mice spleen were then harvested for preparation of single cell suspension. (A&B&C).Flow-cytometry analysis of the dynamic change of mature DCs (CD11c⁺CD86⁺MHCII⁺) was performed at different time points after the treatment (24, 48, 72 and 120 h after the treatment), compared with the tumour-bearing control group. n = 4 mice at each time point per group. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by student t test.

It has been reported that chronically induced immune cells in the tumour microenvironment including immunosuppressive DCs have lost its ability to activate anti-tumour immune response [29]. However, those immune cells in the acute inflammatory response are shown of capability to fight against the tumour [30,31]. To identify whether acute IL-6 induced by the cryo-thermal therapy was critical for DCs maturation, IL-6 neutralising antibody was injected intraperitoneally (i.p.) into tumour-bearing BALB/c mice at 3 and 24 h after the therapy. The efficacy of IL-6 neutralisation in vivo was confirmed via ELISA, on day 2 after the cryo-thermal therapy, serum IL-6 level in IL-6 neutralisation group was markedly down-regulated in comparison to that in the isotype IgG group (Figure 3(A)). The mature DCs (CD11c⁺ MHCII⁺CD86⁺) were abrogated by IL-6 neutralisation on day 2 after the treatment (Figure 3(B)). The data suggested the cryo-thermal therapy-induced acute IL-6 promoted the phenotypic maturation of DCs.

Figure 3. The cryo-thermal therapy-induced high-level transient IL-6 promoted the phenotypic maturation of DCs. Neutralizing anti-IL-6 antibody was injected i.p. into tumour-bearing BALB/c mice at 3 and 24 h after the cryo-thermal therapy. (A) The neutralisation efficacy of IL-6 was confirmed by ELISA assay in IL-6 neutralisation group, the isotype IgG injection group and tumour-bearing mice were as the control; (B) The percentage of mature DCs (MHCII⁺CD86⁺CD11c⁺) in spleen was significantly abrogated by IL-6 neutralisation on day 2 after the treatment, compared with that in the isotype IgG group (n = 4 per group); (C) The relative mRNA expression of FOXO3, IDO1, IDO2, PDL1 and STAT3 in the isolated DCs from the treated mice on day 2 after the cryo-thermal therapy, in comparison to that in the isotype IgG group; (D) The relative mRNA expression of CD40, CXCL12, IL-1β, IL-12, IL-15 and IL-7 in the isolated DCs from the treated mice on day 2 after the cryo-thermal therapy, in comparison to that in the isotype IgG group; (E&F) The expression of IDO2 and CXCL12 in the isolated DCs from spleen on day 2 after the treatment with in vivo IL-6 neutralisation or the isotype IgG was confirmed by western blotting analysis. n = 4 mice per group for mRNA analysis. Each protein was detected on four independent mice with three technical replicates. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

It is reported that the mature DCs exclusively promote T cells activation, however, this paradigm is challenged by the immuno-inhibitory molecules associated with immunosuppressive DCs [32]. Forkhead box O3 (FOXO3), indoleamine 2, 3-dioxygenase 1 (IDO1), indoleamine 2, 3-dioxygenase 2 (IDO2), programmed death-ligand 1 (PDL1) and signal transducer and activator of transcription 3 (STAT3) are known to be associated with immunosuppressive function of DCs [33–35]. The expression level of these immunosuppressive factors in the isolated DCs on day 2 after the treatment with IL-6 neutralisation was evaluated using the real-time PCR analysis. The relative mRNA expression of FOXO3, IDO1, PDL1 and STAT3 in the isolated DCs from the treated mice with in vivo IL-6 neutralisation was not significantly changed in comparison to that in the isotype IgG group. However, much higher level of IDO2 was expressed in the isolated DCs on day 2 after the treatment with in vivo IL-6 neutralisation than that in the isotype IgG group (Figure 3(C)). The up-regulated expression of IDO2 in the isolated DCs with in vivo IL-6 neutralisation was confirmed by western blot analysis (Figure 3(D)). These results indicated that the cryo-thermal therapy-induced acute IL-6 could affect the phenotype of DCs.

To verify whether acute IL-6 following the cryo-thermal therapy could affect DCs-secreted pro-inflammatory cytokines, co-stimulatory molecules and chemokines, the expression of the normal profile of pro-inflammatory cytokines, co-stimulatory molecules and chemokines in the isolated DCs on day 2 after the treatment with IL-6 neutralisation was assessed using the real-time PCR analysis. Chemoattractants, CXC chemokine ligand (CXCL) 12, exerts chemotactic effects on T cells, B cells, monocytes and dendritic cells to help direct lymphocyte traffic into sites of inflammation and into lymphoid and non-lymphoid tissues [36]. The data showed that the expression of CXCL12 in the isolated DCs on day 2 after the treatment with IL-6 neutralisation was significantly abrogated in comparison to that after the treatment with the isotype IgG, whereas other pro-inflammatory cytokines (IL-1β, IL-12, IL-5 and IL-7) were not significantly affected after the treatment with in vivo neutralising IL-6 antibody (Figure 3(E)). Moreover, the down-regulated expression of CXCL12 with in vivo IL-6 neutralisation was confirmed by western blot analysis (Figure 3(F)). These data suggested that the cryo-thermal therapy affect the phenotype of DCs, at least in part, in an IL-6-dependent manner. The enhanced IL-6 production in the early stage after the cryo-thermal therapy affected the phenotype of DCs.

The cryo-thermal therapy-induced acute IL-6 boosted polyfunctional polarised CD4⁺ T cells

The mature DCs could launch the differentiation of antigen-specific T cells into effector T cells with unique functions and cytokine profiles. Once interacting with mature DCs, naive CD4⁺ T cells and CD8⁺ T cells could differentiate into antigen-specific effector T cells with different functions. CD8⁺ T cells exert cytotoxic effector function against tumour cells, and CD4⁺ T cells differentiate into multiple sublineages with unique cytokine profiles that induce and maintain destructive immune response to tumour antigens [6,28,37,38]. The above results revealed that the cryo-thermal therapy-induced acute IL-6 affected the phenotype of DCs, whether the therapy-induced acute IL-6 boosted the expansion and differentiation of CD8⁺ and CD4⁺ T cells was investigated. On day 2 after the cryo-thermal therapy, flow cytometry analysis was performed to examine the percentages of CD8⁺ and CD4⁺ T cells in the treated mice with IL-6 neutralisation. IL-6 neutralisation in vivo led to markedly decrease CD3⁺CD8⁺ T cells in the spleen (Figure 4(A)), which could be due to the decrease of cell recruitment and cell proliferation, or the increase of cell death. As compared to the control group, IFN-γ, granzyme B, and perforin levels in CD8⁺ T cells drastically reduced on day 2 after the cryo-thermal therapy. With IL-6 neutralisation, IFN-γ and granzyme B levels in CD8⁺ T cells trended to be up-regulated, but were not significantly different compared with that in the cryo-thermal and control group. Only Perforin level in CD8⁺ T cells was drastically increased upon anti-IL-6 antibody treatment compared with the cryo-thermal therapy (Figure 4(B)). The data suggested that the function of CD8⁺ T cells would be impaired under the acute pro-inflammatory microenvironment at the early stage induced by the cryo-thermal therapy. IL-6 would reduce perforin level in CD8⁺ T cells during the acute phase triggered by the therapy.

Figure 4. The cryo-thermal therapy-induced acute IL-6 did not affect the cytotoxic function of CD8⁺ T cells in the early stage after the therapy. (A) The flow cytometry analysis was performed to examine the percentages of CD8⁺ T cells in the treated mice received injection of neutralising IL-6 antibody. The percentage of the CD3⁺CD8⁺ T cells was significantly decreased on day 2 after the cryo-thermal therapy with IL-6 neutralisation in comparison to that with the isotype IgG antibody; (B) The mRNA expression of IFN-γ, perforin, granzyme B in the isolated CD8⁺ T cells from the treated mice on day 2 after the cryo-thermal therapy with IL-6 neutralisation in comparison to that with the isotype IgG group, tumour-bearing mice were used in other control. n = 4 mice per group. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

Similarly, on day 2 after the cryo-thermal therapy, the proportion of CD3⁺CD4⁺ T cells in the spleen of the treated mice receiving injection of neutralising IL-6 antibody was significantly decreased (Figure 5(A)), which also could be due to the decrease of cell recruitment and cell proliferation, or the increase of cell death. Our previous study showed that the cryo-thermal therapy skewed to a Th1 adaptive anti-tumour immunity that mediated tumour therapy through CD8⁺ CTLs and Th1-skewed CD4⁺ T helper cells, but we did not investigate the changes of CD4⁺ sub-lineages. There is increasing evidence to support a direct role for CD4⁺ T cells in anti-tumour immunity, independent of CD8⁺ T cells [39]. In CD4⁺ T cell lineages, Th1 cells have superior anti-tumour response with generating large amounts of cytotoxic cytokines and chemokines to promote activation of innate immune cells and CD8⁺ T cells [39–41]. Before regulating anti-tumour CTL responses, both Th1 and Th2 subsets could activate innate immune that may have activity against tumour cells. Th2 cells recruit eosinophils and stimulate production of eosinophil cationic protein (ECP) and major basic protein (MBP) resulted in the elimination of tumour cells [41]. CD4⁺ CTL, a type of CD4⁺ effector cells, that is distinct from known conventional CD4⁺ T cell subsets, could exert potent anti-tumour immunity with secretion of granzyme B, perforin and IFN-γ [42]. Th17 cells, another subset of regulatory CD4⁺ T cells, stimulate recruit CD8⁺T cells via CCL20 chemoattraction, and directly stimulate tumour-specific CD8⁺ T cell responses exerting direct killing activity [43]. Tregs are crucial for the maintenance of immunologic homeostasis, and its contribution to the prevention of excessive immune response and inhibition of anti-tumour response are well recognised [44,45]. Tfh survive as long-term memory cells by regulating its signature genes and effective cytokines contributing to CD4⁺ T cells memory formation [42,46].

Figure 5. The cryo-thermal therapy-induced acute IL-6 boosted polyfunctional polarised CD4⁺ T cells with cytotoxic function. (A) The flow cytometry analysis was performed to examine the percentages of CD4⁺ T cells in the treated mice received injection of neutralising IL-6 antibody. The percentage of CD3⁺CD4⁺ T cells was significantly decreased on day 2 after the treatment with IL-6 neutralisation in comparison to that with the isotype IgG; The mRNA expression of marker profiles in splenic CD4⁺ T cells from the treated mice on day 2 after the cryo-thermal therapy with IL-6 neutralisation in comparison to that with the isotype IgG, tumour-bearing mice were used in other control. The expression of IFN-γ, T-bet, TNF-α (for Th1 cells); IL-5, IL-4, IL-13, GATA3 (for Th2 cells); TGF-β, IL-10, FoxP3 (for Treg cells); RORγt, CCL20 (for Th17 cells); Perforin, GzmB, IFN-γ, Eomes (for CD4⁺ CTL cells); Bcl-6 and IL-21 (for Tfh cells) in splenic CD4⁺ T cells from the treated mice was examined by real-time PCR. The expression of marker profiles for CD4⁺ CTL (B), T-helper 1(Th1) (C), T-helper 17 (Th17) (D), follicular helper T cells (Tfh) (E), regulator cells (Treg) (F), T-helper 2(Th2) (G) in CD4⁺ T cells on day 2 after the treatment with IL-6 neutralisation or the isotype IgG. n = 4 mice per group. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

In this study, we performed to analyse the differentiation of CD4⁺ sub-lineages following the cryo-thermal therapy. Neutralizing IL-6 antibody or the isotype IgG was intraperitoneally injected to the treated mice 3 and 24 h post cryo-thermal therapy. At 48 h, CD4⁺ T cells from the spleens of treated mice and 4T1 tumour-bearing mice were isolated, and genes associated with CD4⁺ T sub-lineages differentiation were analysed by RT-PCR. As shown in Figure 5(B–D), CTL, Th1, Th17-related genes in CD4⁺ T cells after the cryo-thermal therapy were all up-regulated compared with that in control group, IL-6 neutralisation seemed to inhibit these gene expressions. Only GATA3 genes in Th2 after the cryo-thermal therapy was up-regulated compared with that in control group, IL-6 neutralisation inhibited this gene expression (Figure 5(G)). Treg related genes in the cryo-thermal therapy and IL-6 neutralisation group were up-regulated compared with that in control group, but there was no significant change in the cryo-thermal therapy and IL-6 neutralisation group (Figure 5(F)), respectively. No significant changes were found in Tfh related genes among the cryo-thermal therapy, IL-6 neutralisation and control group (Figure 5(E)). These data suggested that acute IL-6 at the early stage induced by the cryo-thermal therapy would promote CD4⁺ T cells differentiation into CTL, Th1, and especially Th17 [47,48].

IFN-γ is a cytokine that plays a pivotal role in anti-tumour immunity. IFN-γ elicits potent anti-tumour immunity by inducing Th1 polarisation, CTL activation, and dendritic cell tumouricidal activity. The ability to secrete IFN-γ is a hallmark of Th1 cells, and IFN-γ is one of the mainly effector molecules of cytotoxic CD4⁺ T lymphocytes. To further identify the cytotoxic function of CD4⁺ T cells, on day 2 after the cryo-thermal therapy, the concentration of IFN-γ in isolated CD4⁺ T cells from the treated mice following neutralising IL-6 antibody or the isotype IgG was detected for ELSIA assay. After the treatment following IL-6 neutralisation, IFN-γ concentration in CD4⁺ T cells was a remarkably lower level than that in the isotype group. At the same time, IFN-γ in the serum of the treated mice following IL-6 neutralisation was markedly decreased to the level of the tumour-bearing mice as compared with that in the isotype group (Figure 6(A,B)). These results suggested that the cryo-thermal therapy-induced acute IL-6 could increase CD4⁺ T cells and enhance secretion of IFN-γ.

Figure 6. The cryo-thermal therapy-induced acute IL-6 promoted secretion of IFN-γ in mice serum and spleen CD4⁺T cells. (A) The IFN-γ level in the isolated CD4⁺ T cells from the treated mice on day 2 after the cryo-thermal therapy with IL-6 neutralisation or the isotype IgG was tested by ELSIA; (B) The serum concentration of IFN-γ from the treated mice on day 2 after the cryo-thermal therapy with IL-6 neutralisation or the isotype IgG was tested by ELSIA. n = 6 mice per group. Data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

The phenotypic mature DCs promoted by acute IL-6 regulated CD4⁺ T cell differentiation

It was reported that the mature DCs could stimulate the activation of naïve T cells through direct or indirect interaction [49,50]. Considering with the above results that the cryo-thermal therapy-induced acute IL-6 was the key mediator of DCs phenotypic maturation and CD4⁺ T cell differentiation, whether the differentiation of CD4⁺ T cells was regulated through interacting with phenotypic mature DCs would be addressed. At 48 h after cryo-thermal, DCs from spleens of the treated mice administrated with neutralising IL-6 antibody or the isotype IgG were isolated. Naïve CD4⁺ T cells isolated from the healthy mice were co-cultured with the above harvested DCs in the DMEM medium supplemented with 10% FBS for 24 h in vitro, respectively. The results showed that the proportion of CD4⁺ T cells was not changed after the co-culture with isolated DCs from the treated mice receiving neutralising IL-6 antibody as comparison to that in the isotype IgG group (Figure 7(A)). However, the ELSIA assay showed that the secretion of IFN-γ in naïve CD4⁺ T cells co-cultured with isolated DCs from the treated mice receiving neutralising IL-6 antibody was significantly abrogated as comparison to that in the isotype IgG group (Figure 7(B)), which indicated that the phenotypic mature DCs mediated by acute IL-6 activated naïve CD4⁺ T cells. The issue whether the phenotypic mature DCs mediated by acute IL-6 modulated differentiation of CD4⁺ T cells was determined. CD4⁺ Th1, Th2, CTL, Th17, Tregs and Tfh related genes were analysed by real-time PCR. In the co-culture DMEM medium supplemented with 10% FBS, certain genes in CD4⁺ sub-lineages were inhibited when naïve CD4⁺ cells were co-culture with isolated DCs from the treated mice receiving neutralising IL-6 antibody, as shown in Figure 7(C–H). These results indicated that phenotypic mature DCs medicated by the cryo-thermal therapy-induced acute IL-6 could regulate naïve CD4⁺ T cell differentiation.

Figure 7. The phenotypic mature DCs promoted by acute IL-6 activated naïve CD4⁺ T cells and regulated their differentiation under normal culture condition. At 48 h after the cryo-thermal, DCs from the spleens of the treated mice receiving neutralising IL-6 antibody or the isotype IgG were isolated. Naïve CD4⁺ T cells isolated from the healthy mice were co-cultured with the above harvested DCs in the DMEM medium supplemented with 10% FBS for 24 h in vitro, respectively. The naïve CD4⁺ T cells isolated from the healthy mice were co-cultured with DCs isolated from the treated mice receiving neutralising IL-6 antibody or the isotype IgG for 24 h, respectively. (A) The percentage of CD4⁺ T cells was analysed by flow-cytometry; (B) The secretion of IFN-γ in supernatants of naïve CD4⁺ T cells co-cultured with isolated DCs from the treated mice receiving neutralising IL-6 antibody or the isotype IgG was determined using ELISA; The expression of marker profiles for CD4⁺ CTL (C), T-helper 1(Th1) (D), T-helper 17 (Th17) (E), follicular helper T cells (Tfh) (F), regulator cells (Treg) (G), T-helper 2(Th2) (H) in CD4⁺ T cells was determined by RT-PCR on day 2 after the treatment with IL-6 neutralisation or the isotype IgG. n = 4 mice per group. Data are shown as mean ± SD. *p < .05, **p < .01, ***p < .001 by two-way ANOVA with the Bonferroni correction.

Discussion

In our previous study, a novel tumour therapeutic modality of the cryo-thermal therapy was developed, and we presented that the local cryo-thermal therapy improved long-term survival along with significant reduction of distant lung metastasis in 4T1 murine mammary carcinoma model [27]. Furthermore, our studies reported that the cryo-thermal therapy reset the tumour chronic inflammation to an “acute” profile, with up-regulation of acute phase factors including IL-6 [26,27]. In this study, the role of acute IL-6 induced by the cryo-thermal therapy in the innate immunity and adaptive immunity was determined. We found that the cryo-thermal therapy induced up-regulation of IL-6 in the early stage after the treatment. The high-level transient (“acute”) IL-6 induced by the treatment promoted DCs phenotypic maturation. In the early stage after the therapy, acute IL-6 impaired the cytotoxic function of CD8⁺ T cells, but promoted CD4⁺ T cell differentiation which was more permissive for tumour eradication mediated by systemic immune response. Our results revealed that the cryo-thermal therapy-induced high level of IL-6 in the early stage initiated a cascading innate and adaptive immune response. The acute IL-6 promoted DCs phenotypic maturation, which was required to drive the differentiation of CD4⁺ T cells with ability to protective anti-tumour immune response.

In cancer, chronic inflammation promotes cancer growth and progression, IL-6 with its ability to drive tumour progression and metastasis in the tumour chronic inflammatory microenvironment is reported widely [28,51–53]. Chronic IL-6 signalling is related to tumourigenesis in numerous mouse models as well as in human disease. However, acute inflammation may induce potent anti-tumour immunity [54,55]. In this study, the cryo-thermal therapy promoted IL-6 production in the early stage leading to induced strong acute IL-6 locally and systemically. Considering that the cryo-thermal therapy improved long-term survival in 4T1 murine mammary carcinoma model [27], we suggested that the time phase, duration and magnitude of pro-inflammatory cytokine IL-6 in the tumour microenvironment was the key factor to determine the effectiveness of IL-6 in anti-tumour immunity.

Despite that recent evidence implicating that some therapeutic modalities can switch the activities of IL-6 to a predominantly anti-tumorigenic function that promotes anti-tumour immunity [18,32,56–58], the role of IL-6 in anti-tumour immunity was not well defined. We highlighted the pivotal features of IL-6 induced by the cryo-thermal therapy was exhibited to transient, high-level expression (“acute”) pattern, as opposed to the continuous, low-level (“chronic”) pattern of IL-6 in the tumour microenvironment. The acute IL-6 induced by the cryo-thermal therapy would be a characteristic hallmark of anti-tumour immunity enhanced by the treatment. The role of acute IL-6 in anti-tumour immunity boosted by the cryo-thermal therapy would be involved in: (1) IL-6-rich acute inflammatory response shifted the tumour microenvironment from an immunosuppressive state to an immunostimulatory state; (2) IL-6-rich acute inflammatory response was required to endow DCs phenotypic maturation leading to the functional differentiation of CD4⁺ T cells. Therefore, our studies suggested that increased plasma IL-6 in acute inflammatory response following the cryo-thermal therapy affected the phenotype of DCs, which was essential for efficacious T cells priming and enhancing anti-tumour immune response.

Dendritic cells (DCs) have been found to be the most potent professional antigen-presenting cells (APCs) and are initiators of the immune response. DCs play central roles in the induction, regulation and maintenance of anti-tumour immunity and can trigger the T cells activation, which is required for the activation and modulation of immune response [59,60]. The differentiation of DCs has two distinct stages: immature DCs and mature DCs. The immature DCs have weak ability to stimulate the proliferation of naïve T cells and may induce the disability of T cells [61]. The maturation of DCs is critical for the function of DCs. Only mature DCs can activate an immune response [62]. However, The phenotype and function of DCs depends on the local microenvironment and can be directly modulated by the tumour microenvironment resulting in the differentiation of DCs with tolerogenic and immunosuppressive status [63]. Therefore, optimisation of approaches that restore or reverse DCs immunosuppressive activities, and promote activation and modulation of T cells subsequently will be useful in improving tumour therapy [64]. In this study, we found that the cryo-thermal therapy-induced acute IL-6 could mobilise DCs into DCs phenotypic maturation with high level of co-stimulatory surface markers (MHC II and CD86). IL-6 has been demonstrated to affect the activation, expansion, survival, and polarisation of T cells during an immune response [65], under IL-6-rich acute inflammatory microenvironment, DCs activated by IL-6 promoted differentiation of CD4⁺ T cells, which could be clarified the role of acute IL-6 in the development of T cell responses. Taken together, all of these data indicated that the cryo-thermal therapy-induced acute IL-6 could promote effective innate and adaptive immune responses. Although the number of DC maturation was decreased upon IL-6 neutralisation on day 2, it remained at higher level with no significant difference as compared to that in the cryo-thermal therapy on day 5 (Supplementary Figure 1). This could be explained in part by the results from our previous study that on day 5 after the cryo-thermal therapy, all acute phase proteins induced by the cryo-thermal therapy were decreased to the control level, and the time frame of "acute" inflammatory response was over [27]. Also, the results suggested that there would be other cytokine responsible for DC maturation at later time points. But, it is worth noting that under the "acute" phase response occurred on day 2 after the cryo-thermal therapy, IL-6 not only affected DCs maturation (Figure 2), but also inhibited phenotype of tolerogenic DCs through decreasing IDO2 expression (Figure 3), which might play important roles at early time points.

From some references, IDO2 expression confers to DCs tolerogenic features [66]. IDO expression in DCs is inducible after exposure with IFN-γ [67], and IL-6 could block the stimulatory activity of IFN-γ on IDO production in DCs [68]. In this study, high expression of IFN-γ was found, thus, we speculated that acute IL-6 could inhibit IDO expression of DCs under exposure with IFN-γ. It would be interesting to study how IL-6 regulates IDO2 expression in DCs in the near future.

In this study, we also found that the function of CD8⁺ T cells could be impaired by acute IL-6 in the early stage induced by the cryo-thermal therapy. In line with our results, some studies report that IL-6 can directly act on CD8⁺ T cells failing to effectively differentiate from a naive CD8⁺ T cell into an effector or memory T cells [69–71], but the process which acute IL-6 impaired the function of CD8⁺ T cells was transient, when IL-6 level was reduced to the level of healthy mice after the cryo-thermal therapy [27], IFN-γ, granzyme b, and perforin levels in CD8⁺ T cells were significantly up-regulated compared with that in control group (Data not shown). To identify anti-tumour response in CD8⁺ T cells with high IFN-γ, granzyme B, and perforin levels after “acute” phase, the cytotoxic activity of CD8⁺ T cells was measured by using CCK8 assay. The results showed that the ability of killing 4T1 tumour cells of CD8⁺ T cells on day 14 after the cryo-thermal therapy was obviously enhanced compared with CD8⁺ T cells from the tumour-bearing mice (Supplementary Figure 2). Although IL-6 is classically defined as a central regulator of the acute phase response, it is increasingly evident that IL-6 performs a pivotal role in influencing T cell responses. The transcription factor T-bet profoundly influences CD8⁺ T cell differentiation into effector CTLs through regulating IFN-γ, perforin, and granzyme B [69,70,72]. Wu reports that T-bet expression in CD8⁺ T cells is significantly decreased after IL-6 treatment [71]. It could explain why down-regulated IFN-γ, perforin, and granzyme B in CD8⁺ T cell was observed in the cryo-thermal-induced acute IL-6 in our study. On the other hand, Longhi MP finds that CD4⁺ but not CD8⁺ T cell memory is critically dependent upon IL-6 [73]. IL-6 facilitates the activity of specific memory CD4⁺ T cells. In this study, T-bet expression in CD4⁺ T cells was significantly increased in the cryo-thermal-induced acute IL-6, along with significantly up-regulated IFN-γ, perforin, and granzyme B in CD4⁺ T cell. Effect of acute IL-6 on the function of CD8⁺ T cells deserves to be investigated in the near future.

In conclusion, we demonstrated that the cryo-thermal therapy-induced acute IL-6 promoted the phenotypic maturation of DC was the prerequisite to CD4⁺ T cell differentiation. It was clear that acute inflammatory response induced by the cryo-thermal therapy was required for modulating anti-tumour response. To continue the investigation on acute IL-6 as early indicator in predicating tumour therapeutic effect is desirable. Further studies are necessary to investigate the role of CD4⁺ T cell subtypes in the cryo-thermal therapy-induced anti-tumour immune response.

Acknowledgements

This work was supported by National Key Research and Development Program (2016YFC0106201), National Natural Science Foundation of China (U1532116), the Science and Technology Commission of Shanghai Municipality (11DZ2211000).

Disclosure statement

The authors declare no competing financial interests.

Additional information

Funding

This work was supported by National Key Research and Development Program (2016YFC0106201), National Natural Science Foundation of China (U1532116), the Science and Technology Commission of Shanghai Municipality (11DZ2211000).