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Synergistic effect of adoptive immunotherapy and docetaxel inhibits tumor growth in a mouse model
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Synergistic effect of adoptive immunotherapy and docetaxel inhibits tumor growth in a mouse model Yuefeng Hua,1, Jingwei Liub,1, Peilin Cuic,1, Tao Liud, Chunmei Piaoe, Xianghong Xuf, ⁎ Qike Zhangg, Man Xiaoh, Yongcheng Lui, Xuesong Liub, Yue Wangb, Xu Lub, a
Department of Interventional Radiology, Beijing Friendship Hospital, Capital Medical University, Beijing, China Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd, Beijing, China c Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China d Key Laboratory of Digestive System Tumors, Second Hospital of Lanzhou University, Lanzhou, China e Department of Oncology, Beijing Anzhen Hospital Affiliated to the Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China f Department of Biotherapy Center, Gansu Provincial Hospital, Lanzhou, China g Department of Hemotology, Gansu Provincial Hospital, Lanzhou, China h Department of Biochemistry and Molecular Biology, Hainan Medical College, Haikou, China i Department of Physiology, Michigan State University, East Lansing, MI, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adoptive T cell transfer therapy Docetaxel Immunosuppression Myeloid-derived suppressor cells
Adoptive T cell transfer therapy (ACT) has emerged as a promising approach to cancer immunotherapy; however, the efficacy of ACT is limited by the T-cell suppressive activity of myeloid-derived suppressor cells (MDSCs), which accumulate in the tumor microenvironment after ACT. We sought to determine whether the efficacy of ACT could be enhanced by co-treatment with docetaxel, a taxane chemotherapy agent that has been shown previously to inhibit MDSC function. Using a mouse tumor model, we demonstrated that ACT and docetaxel synergistically inhibit the growth either of engrafted CT26 colon cancer or 4T1 mammary carcinoma cells. While ACT mediated an increase in the recruitment of MDSCs to the site of the tumor, docetaxel reversed this increase. Furthermore, ex vivo cultures of tumor-associated MDSCs suppressed the cytotoxic activity of tumorspecific T cells, and this suppressive activity was abolished by docetaxel treatment. These results suggest that docetaxel inhibits both the tumor recruitment and T cell suppressive activity of MDSCs. Inhibitors of iNOS and arginase partially inhibited ex vivo MDSC activity, and combined inhibition of iNOS and arginase had a similar effect as docetaxel, which supports the possibility that docetaxel may function by inhibiting ACT-associated activation of these pathways. Furthermore, docetaxel mediated inhibition of the T cell suppressive activity of MDSCs from human blood, which supports the potential clinical applicability of these findings. On the basis of these findings, docetaxel treatment may represent an effective therapeutic approach for reversing immunosuppression by MDSCs subsequent to ACT-based therapy.
1. Introduction Immunotherapy based on adoptive T cell transfer therapy (ACT) could mediate tumor regression in patients with metastatic cancer [1,2]. To exert a measurable antitumor effect, ex vivo expanded immune cells are infused back into a patient and traffic to the tumor, where they mediate its destruction [3]. However, tumors often persist after ACT, despite having enriched for T cells that are specific for antigens expressed by the tumor. There is a growing body of work
suggesting that tumor-specific T cells are restrained in vivo by the immunosuppressive tumor environment [4]. These immunosuppressive mechanisms are recapitulated in transplantable mouse tumor models, which are valuable for identifying cellular and molecular immunosuppressive pathways and screening for immunomodulatory drugs as adjuncts to the adoptive transfer of tumor-specific T cells [5,6]. Most commonly, tumor progression is accompanied by local and systemic immunosuppression. This may be mediated by increased secretion of specific cytokines, such as interleukin-10 (IL-10); or increased
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Corresponding author at: Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd, FL2, Building 3, Park B, Shunyi District Airport High Tech Zoon, Beijing 101300, China. E-mail address: [email protected] (X. Lu). 1 These authors have contributed equally to this study. https://doi.org/10.1016/j.cellimm.2019.104036 Received 6 November 2019; Received in revised form 20 December 2019; Accepted 29 December 2019 0008-8749/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Yuefeng Hu, et al., Cellular Immunology, https://doi.org/10.1016/j.cellimm.2019.104036
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tumor accumulation of immunosuppressive cells, such as FOXP3 + regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSCs), and M2 macrophages [7]. In particular, MDSCs are known to support tumor progression by contributing to the immunosuppressive local microenvironment [8–10]. MDSCs suppress tumor immunity by a variety of mechanisms, which include increased production of nitric oxide (NO) and reactive oxygen species (ROS), L-arginine metabolism, and immunosuppressive cytokines such as IL-10 [11–13]. Moreover, MDSC repressive anti-tumor immunity has been shown to be correlated with early recurrence and poor prognosis [11,14]. Indeed, successful anticancer therapies are able to increase the level of immunosurveillance by reactivating pre-existing antitumor immune responses [15,16]. Several chemotherapeutic agents appear to promote antitumor responses by inhibiting MDSCs. A recent study has shown that members of the taxane family, including docetaxel and paclitaxel (Taxol), are able to limit the accumulation and immunosuppressive activity of tumor-infiltrating MDSCs in mice bearing mammary tumors [8], transgene-induced melanomas, and spontaneous melanomas [17,18], Thus, chemotherapeutic agents that target MDSCs may provide a potent approach to relieving the immunosuppressive tumor, which could potentially improve the response to ACT and other forms of immunotherapy. In this study, we investigated the cellular effects of docetaxel in response to ACT in a mouse model. Our findings demonstrate that administering docetaxel down-regulates MDSC suppressive pathways, restores antitumor immunity and results in enhancement of the therapeutic efficacy of ACT. These results suggest docetaxel treatment should be considered as a therapeutic approach for reversing immunosuppression in the tumor microenvironment subsequent to ACTbased therapy. 2. Results 2.1. ACT synergizes with docetaxel to delay tumor outgrowth Fig. 1. Increased antitumor activity of adoptive immunotherapy plus docetaxel in BALB/c mice engrafted with CT26 colon carcinoma cells. (A) Male BALB/c mice were inoculated subcutaneously in the flank with 1 × 106 CT26 colon carcinoma cells. After 14 days, mice bearing CT26 tumors were left untreated (Control), injected with adoptive T cells (ACT), treated with docetaxel (Docetaxel) or treated with both ACT and docetaxel (Combination). Docetaxel was administered at a weekly interval on days 14 and 21. Tumor volumes were measured using a caliper every 3 days up to day 27 after tumor implantation. (B) The mice were euthanized on day 27 after tumor implantation, and the tumors were excised and weighed. The results represent three independent experiments (n = 5 per group). Results are expressed as the mean tumor volume ± standard error (SE). *P < 0.05, **P < 0.01 vs Combination.
We sought to determine whether docetaxel could enhance the therapeutic efficacy of ACT in the transplantable mouse tumor model. Balb/c mice were engrafted with mouse CT26 colon carcinoma cells, and then 14 days after tumor challenge, groups of mice were left untreated or were administered docetaxel and/or purified syngeneic T lymphocytes generated from splenocytes of tumor-free mice. As shown in Fig. 1, ACT alone compared with no treatment caused only a marginal antitumor effect. Docetaxel alone also had a minimal effect; however, coupling docetaxel with adoptive transfer resulted in a significant antitumor effect (P < 0.01; Fig. 1(A, B)). To extend this finding to another tumor type, the experiment was repeated using 4T1 mammary carcinoma cells. As observed for CT26 colon carcinoma cells, adoptive immunotherapy alone had a marginal effect, whereas docetaxel + adoptive transfer imparted a statistically significant antitumor benefit (P < 0.01; Fig. 2(A, B)). These data suggest that docetaxel is able to augment the therapeutic efficacy of ACT.
(Fig. 3(A, B)). Similar results were observed for 4T1 tumors (Fig. 3(C, D)). These results suggest the docetaxel’s ability to protect against tumor development after ACT may be explained in part by its function in preventing MDSC recruitment to the site of tumors. To further examine the antitumor effect of docetaxel, we isolated murine T lymphocytes from splenocytes of tumor-free mice and murine MDSCs from CT26 or 4T1 tumor-bearing mice. Lymphocyte lytic activity was tested against CT26 or 4T1 tumor cells in the presence or absence of MDSCs. Naïve splenocytes were used as a control for MDSCs. Our results showed a dose-dependent suppression of T lymphocyte cytotoxicity when lymphocytes were co-cultured with MDSCs, whereas no suppression was observed when lymphocytes were co-cultured with splenocytes (Fig. 4(A, C)). As a control, MDSCs did not lyse lymphocytes or CT26 or 4T1 cells at any ratio (data not shown). Next, we assessed whether administration of docetaxel could rescue the cytotoxicity of T lymphocytes. As shown in Fig. 4(B, D), MDSCs isolated from docetaxel-treated tumors did not exert suppressive effects relative to those of naïve splenocytes on syngeneic T cells. These data confirm the immunosuppressive properties of MDSCs in impairing the
2.2. Docetaxel treatment restores ACT-mediated antitumor activity Emerging data reveal populations of MDSCs isolated from different organs vary in their immunosuppressive ability, with intratumoral MDSCs being the most highly immunosuppressive [19–21], Since members of the taxane family augment antitumor immunity [8,17,18], we asked whether docetaxel treatment may affect the ability of intratumoral MDSCs to alter the activation state of T lymphocytes. To test this hypothesis, we performed flow cytometry of tumor-infiltrating immune cells. After ACT, Gr-1+CD11b+ MDSCs accumulated in the CT26 tumor microenvironment, with a reduction in the frequency of the MDSC population in the spleen. Administration of docetaxel reversed the intratumoral accumulation of MDSCs in response to ACT 2
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combined with nor-NOHA and/or L-NMA (Fig. 5(A, B)). Furthermore, docetaxel had no effect on CD11b+Gr-1+ cell-depleted cell populations, which is consistent with the specificity of docetaxel function (data not shown). These results support the role of ARG1 and iNOS in mediating the pathway of MDSC suppression and are consistent with the possibility that docetaxel functions through the blockade of ARG1 and iNOS. 2.4. Restoration of the anti-tumor activity of human T lymphocytes upon the treatment with docetaxel The above results were obtained using a mouse model. To confirm that the findings are also applicable to humans, we collected blood from healthy donors. We isolated human MDSCs (CD14+HLA-DR−/low) and generated human lymphocytes from peripheral blood lymphocytes. As shown in Fig. 6(A), CD14+HLA-DR−/low MDSCs significantly suppressed the cytotoxicity of T cells against the human MCF-7 cell line, whereas control CD14+HLA-DR+ monocytes showed no significant effect. As observed in the mouse model, docetaxel reversed the suppression. Furthermore, nor-NOHA and L-NMA each mediated partial reversal of the CD14+HLA-DR−/low MDSC inhibitory effect, with high level inhibition by the combination of nor-NOHA and L-NMA (Fig. 6(B)). These results confirm the function of docetaxel in suppressing the activity of human MDSCs and demonstrate a role for ARG1 and iNOS in MDSCs-mediated immunosuppression. Based on our results, docetaxel may provide a novel pharmacologic approach for regulating MDSC-mediated immunosuppressive pathways. 3. Discussion The development of the anti-tumor immune response is associated with the accumulation and activation of T cells that move through tissues, while tumor-infiltrating MDSCs are known to suppress T cell activity. ACT after lymphodepletion has emerged as a promising approach to cancer immunotherapy in which tumor-specific T cells are expanded and re-introduced into the patient. This approach has been especially useful in maintenance treatment, for which it is able to reduce cancer recurrence and metastasis [1,24,25], Despite the success of ACT and other forms of cancer immunotherapy, the efficacy is limited by tumor-associated immune suppression, mediated in large part by tumor-associated MDSCs [19–21], Functionally, MDSCs suppress the tumoricidal activity of activated T-cells, through ARG-1 and iNOS, each of which have been shown to induce the anergy of reactive T cells [5,26], Given these immunosuppressive effects of MDSCs, the elimination of MDSCs may significantly improve antitumor responses and enhance the efficacy of immunotherapy [23,27,28], In this study, we examined the immune-mediated mechanisms that suppress the function of ACT-based therapies and provided an approach by which the immunosuppressive mechanism could be overcome using docetaxel, a chemotherapeutic reagent that has already been approved in previous studies for its ability to alter important immunologic parameters. Docetaxel, and its related compound, paclitaxel, are taxanes, a class of chemotherapy drugs that shows great promise due to its mechanism of action and relatively low toxicity. The present study shows that ACT and docetaxel each reduce tumor progression in a mouse cancer model. Furthermore, the reduction was greatest when ACT and docetaxel were applied in combination, which supports the combined use of docetaxel and ACT. Inasmuch as docetaxel and other chemotherapeutic drugs have been shown to directly eliminate MDSCs in pathologic settings [8,29–31], we determined whether docetaxel might enhance the effects of ACT by suppressing the activity of MDSCs. In support of this possibility, our results demonstrate that adoptively transferred T cell therapy caused increased recruitment of MDSCs to the site of the tumor, but that the recruitment could be reversed by concurrent docetaxel treatment. We further observed that docetaxel could inhibit the T cell suppressive
Fig. 2. Increased antitumor activity of adoptive immunotherapy plus docetaxel in BALB/c mice engrafted with 4T1 mammary carcinoma cells. (A) Male BALB/ c mice were inoculated subcutaneously in the flank with 1 × 106 4T1 mammary carcinoma cells. After 14 days, mice bearing 4T1 mammary tumors were left untreated (Control), injected with adoptive T cells (ACT), treated with docetaxel (Docetaxel) or treated with both ACT and docetaxel (Combination). Docetaxel was administered at a weekly interval at days 14 and 21. Tumor volumes were measured using a caliper every 3 days up to day 27 after tumor implantation. (B) The mice were sacrificed on day 27 after tumor implantation, and the tumors were excised and weighed. The results represent three independent experiments (n = 5 per group). Results are expressed as the mean tumor volume ± standard error (SE). *P < 0.05, **P < 0.01 vs Combination.
cytotoxicity of T lymphocytes against tumor cells and suggest that MDSC inhibition by docetaxel may provide a useful approach for enhancing tumor specific immunotherapy. 2.3. Down-regulation of tumor-associated MDSC activity is mediated by ARG-1 and iNOS pathway inhibition MDSCs derived from tumors are known to express elevated levels of arginase 1 (ARG 1) and inducible nitric oxide synthase (iNOS), which are each major suppression pathways [22,23], To determine whether modulation of these pathways may contribute to the inhibitory effect of docetaxel on MDSC-mediated suppressive activity, MDSCs were admixed with syngeneic T cells in the presence or absence of nor-NOHA (an ARG1-specific inhibitor), L-NMA (an iNOS inhibitor) or docetaxel. As previously demonstrated, docetaxel alone could restore the tumor lytic activity of syngeneic T cells that were suppressed by incubation with MDSCs derived from CT26 (Fig. 5(A)) or 4T1 (Fig. 5(B)) tumors. Nor-NOHA and L-NMA also each mediated a partial inhibition of MDSCsuppressive function, and these two inhibitors had a greater effect when combined. However, no additional effect was detected for docetaxel 3
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Fig. 3. Accumulation of intratumoral and splenic myeloid-derived suppressor cells (MDSCs) in tumor-engrafted BALB/c after adoptive T cells (ACT) and/or docetaxel treatment. Mice were engrafted with CT26 colon carcinoma cells (A and B) or 4T1 mammary carcinoma cells (C and D). Tumor bearing mice (n = 5) were treated with ACT and/or docetaxel as described in Figs. 1 and 2. Accumulation of intratumoral (A or C) and splenic (B or D) CD11b+Gr-1+ cells was determined by flow cytometry. *P < 0.05 compared with the ACT-treated group.
combined clinical use of ACT-based immunotherapies and chemotherapeutic agents.
function of MDSCs from tumor-bearing mice. Therefore, docetaxel may function synergistically with ACT in reducing tumor progression by inhibiting MDSCs both at the level of recruitment and activity. To examine pathways that contribute to MDSC activation during ACT, we assessed the effect of inhibitors of ARG-1 and iNOS on the activity of ex vivo MDSC cultures. Each of these inhibitors mediated a partial reversal of MDSC-mediated T cell suppression, and the combination of ARG-1 and iNOS inhibitors caused an effect that was similar to that of docetaxel. Furthermore, no additional effect was observed when docetaxel was combined with ARG-1/iNOS inhibitors. These results are consistent with the possibility that docetaxel may restore the tumor lytic activity of ACT cell function by simultaneous ARG-1 and iNOS inhibition, though additional assessment will be needed to verify the role of these pathways in docetaxel-mediated MDSC suppression. Importantly, docetaxel had similar effects on MDSCs isolated from human blood, which supports the potential use of docetaxel and ACT as a therapeutic combination for cancer patients. Our results suggest that docetaxel is able to augment ACT, though additional studies are needed to determine whether adoptive T cells may also affect MDSCs in patients with metastatic cancer. Though chemotherapy resistance is a common deterrent to metastatic cancer treatment, our findings raise the possibility that patients with tumors that are unresponsive to docetaxel alone may benefit from the drug’s ability to enhance immune responses in the context of immunotherapy. Thus, these findings have potentially important implications for
4. Materials and methods 4.1. Tumor cell lines and mice Two murine cell lines (CT26 colon cancer and 4T1 mammary tumor) and a human breast adenocarcinoma cell line (MCF-7) were purchased from ATCC. Cells were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone). All cells were grown at 37 °C in a humidified atmosphere of 5% CO2. Six- to 8-week-old male Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were maintained under specific pathogen-free conditions and treated according to the institutional protocols approved by the Animal Care Committee of Peking University Health Science Center. 4.2. Reagents Docetaxel (Taxotere, Sanofi-Aventis) was dissolved in 80% ethanol at 10 mg/mL according to the manufacturer’s instructions and then further diluted to 5 mg/mL in sterile PBS. For each experiment, docetaxel was administered intraperitoneally at 33 mg/kg body weight 4
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Fig. 4. Myeloid-derived suppressor cells (MDSCs) isolated from docetaxel-treated mice are less suppressive than those from control-treated mice. CD11b+Gr-1+ cells were purified from the spleens of CT26 (panels A and B) or 4T1 (panels C and D) tumor-bearing mice that were untreated or treated with docetaxel. Different ratios of CD11b+Gr-1+ cells or naïve splenocytes were incubated with syngeneic total T cells as demonstrated. After 24 h, CT26 colon carcinoma cells or 4T1 mammary carcinoma cells were added at a ratio of 10:1 (E:T). Naive splenocytes were used as a control for MDSCs. The % lysis was determined as a measure of tumor cellspecific T-cell-mediated toxicity. **P < 0.01, compared with the naïve splenocyte-treated group.
manufacturer’s protocol. The purity of Gr-1+CD11b+ MDSCs in isolated cell populations was approximately 85%. Positive and negative fractions were sorted with LS columns. CD4+ or CD8+ T lymphocytes were negatively selected from the CD11b-depleted splenocytes using the CD4+ or CD8+ T cell isolation kit (Miltenyi Biotec). For ACT experiments, mice were administered 3 × 106 T lymphocytes (supplemental Figure) in the tail vein on day 14. For isolation of CD14+HLADR−/low and CD14+HLA-DR+ cells, human peripheral blood mononuclear cells (PBMCs) were purified using CD14 Microbeads and the AutoMACS separation unit (Miltenyi Biotech) according to the manufacturer’s instructions. Human peripheral blood lymphocytes were collected from healthy donors after obtaining informed consent according to an Institutional Review Board-approved protocol. PBMCs were isolated on a Ficoll gradient and then stimulated with anti-CD3/CD28 antibody-coated Dynal beads (bead:cell ratio 3:1). Isolated and activated T cells were expanded in complete media (RPMI1640 + 100 U/mL IL-2) for 7 days. Dynabeads were magnetically removed at day 3 post stimulation and analyzed by flow cytometry. Docetaxel was added as indicated. Single cell suspensions from spleens or tumors were treated with Fcblock and mAbs for 30 min at 4 °C, with isotype-matched antibodies as control. Live cells were gated based on 7-amino-actinomycin D, annexin V staining. Samples were run on a flow cytometer (FACSCalibur; BD Biosciences), and the data were analyzed using FCS Express software.
[32] on the indicated days. Docetaxel was used in vitro at a concentration of 11 nM [8]. N-omega-hydroxy-nor-L-arginine (nor-NOHA; Calbiochem) and NG-monomethyl-L-arginine (L-NMA; Calbiochem) were used at 10 μM in vitro. 4.3. Assessment of anti-tumor activity Tumors were established in Balb/c mice by injecting 1 × 106 CT26 colon carcinoma or 4T1 mammary carcinoma cells. On day 14, when the tumors had reached a mean area of 10 mm2, the mice were randomized into four groups with 5 mice per group as follows: (I) tumor bearers, (II) tumor bearers treated with ACT, (III) tumor bearers treated with docetaxel, and (IV) tumor bears treated with ACT and docetaxel. On the day of ACT transfer and 2 days after, mice received intraperitoneal injections of docetaxel. Tumor measurements were performed with a caliper by measuring the largest diameter and its perpendicular length in a blind fashion. The tumor size index was determined as the average of the product of these diameters and was measured independently by two operators. The tumor volume was calculated using the modified formula: 0.5 × (length × width2). Results are expressed as the average tumor volume ± standard error (SE). 4.4. Cell preparation and flow cytometry Single cell suspensions prepared from the spleens of three mice group were depleted of RBCs using RBC lysis buffer as previously scribed [33]. CD11b+ cells were isolated from tumors using CD11b+ MicroBeads isolation kit (Miltenyi Biotec) according to
4.5. Cytotoxicity assessment
per dethe the
The T cell-suppressive activity of MDSCs was evaluated by the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according 5
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Fig. 6. Docetaxel reverses myeloid-derived suppressor cell (MDSC)-mediated suppression of human activated T-cells function. (A) Human PBMCs from healthy donors were collected as a source of human MDSCs and T cells. Activated T-cells were cultured and incubated with different ratios of HLADRlow/−/CD14+ cells or HLA-DR+/CD14+ as indicated. After 24 h, MCF-7 cells were added at a ratio of 10:1 (E:T) and the % lysis was calculated. ** P < 0.01, compared with the HLA-DR+/CD14+ treated group. (B) Human activated T-cells and HLA-DRlow/−/CD14+ were co-cultured with in the presence or absence of nor-NOHA (10 μmol/L), L-NMA (10 μmol/L), and/or docetaxel (11 nmol/L). After 36 h, MCF-7 cells were added at a ratio of 10:1 (E:T). The activated T-cells, purified human MDSCs (HLA-DRlow/−/CD14+), and target tumor cells were co-cultured at a ratio of 10:1:1. *P < 0.05, **P < 0.01 compared with control.
Fig. 5. Myeloid-derived suppressor cell (MDSC) T cell suppression is mediated through a pathway that involves nitric oxide (NO) and arginase. Syngeneic total T cells were cocultured with CD11b+Gr-1+ cells purified from the spleens of CT26 tumor-bearing mice (A) or 4T1 tumor-bearing mice (B) at a 10:1 ratio in the presence or absence of nor-NOHA (10 μmol/L), L-NMA (10 μmol/L), and/or docetaxel (11 nmol/L). After 36 h, the corresponding target tumor cells were added at a ratio of 10:1 (E:T). The % lysis was determined as a measure of tumor cell-specific T-cell-mediated toxicity. *P < 0.05, **P < 0.01 compared with the control.
to the manufacturer’s instructions. Briefly, target tumor cell dilutions starting at 5000 cells per well were plated in triplicate in 96-well roundbottomed microplates. T lymphocytes co-cultured with MDSCs, or splenocytes depleted of CD11b+ cells were added to the target tumor cells at different ratios of effector to MDSCs. Specific lysis for each effector-to-target (E:T) cell ratio was calculated with the following formula:
P ≤ 0.05 were considered statistically significant. Funding This work was supported by the National Natural Science Foundation of China (81770468), Beijing Municipal Natural Science Foundation (7162030) and the Beijing Science and Technology Plan special issue (Z14010101101).
% Cytotoxicity = [(Experimental − Effector Spontaneous − Target Spontaneous )
CRediT authorship contribution statement
/ (Target Maximum − Target Spontaneous)] × 100 Yuefeng Hu: Conceptualization, Writing - original draft. Jingwei Liu: Data curation, Writing - review & editing. Peilin Cui: Methodology, Validation. Tao Liu: Formal analysis. Chunmei Piao: Funding acquisition. Xianghong Xu: Resources. Qike Zhang: Resources. Man Xiao: Investigation. Yongcheng Lu: Methodology. Xuesong Liu: Validation. Yue Wang: Validation. Xu Lu: Writing - review & editing.
All determinations were done in triplicate. 4.6. Statistical analysis The statistical significance of differences between values of the control and treatment groups were determined via Student’s t test. For all experiments, the graphs represent the mean of three separate experiments and the error bars represent the SE. Differences with 6
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Declaration of Competing Interest [17]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellimm.2019.104036.
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Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Synergistic effect of adoptive immunotherapy and docetaxel inhibits tumor growth in a mouse model Yuefeng Hua,1, Jingwei Liub,1, Peilin Cuic,1, Tao Liud, Chunmei Piaoe, Xianghong Xuf, ⁎ Qike Zhangg, Man Xiaoh, Yongcheng Lui, Xuesong Liub, Yue Wangb, Xu Lub, a
Department of Interventional Radiology, Beijing Friendship Hospital, Capital Medical University, Beijing, China Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd, Beijing, China c Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China d Key Laboratory of Digestive System Tumors, Second Hospital of Lanzhou University, Lanzhou, China e Department of Oncology, Beijing Anzhen Hospital Affiliated to the Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China f Department of Biotherapy Center, Gansu Provincial Hospital, Lanzhou, China g Department of Hemotology, Gansu Provincial Hospital, Lanzhou, China h Department of Biochemistry and Molecular Biology, Hainan Medical College, Haikou, China i Department of Physiology, Michigan State University, East Lansing, MI, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adoptive T cell transfer therapy Docetaxel Immunosuppression Myeloid-derived suppressor cells
Adoptive T cell transfer therapy (ACT) has emerged as a promising approach to cancer immunotherapy; however, the efficacy of ACT is limited by the T-cell suppressive activity of myeloid-derived suppressor cells (MDSCs), which accumulate in the tumor microenvironment after ACT. We sought to determine whether the efficacy of ACT could be enhanced by co-treatment with docetaxel, a taxane chemotherapy agent that has been shown previously to inhibit MDSC function. Using a mouse tumor model, we demonstrated that ACT and docetaxel synergistically inhibit the growth either of engrafted CT26 colon cancer or 4T1 mammary carcinoma cells. While ACT mediated an increase in the recruitment of MDSCs to the site of the tumor, docetaxel reversed this increase. Furthermore, ex vivo cultures of tumor-associated MDSCs suppressed the cytotoxic activity of tumorspecific T cells, and this suppressive activity was abolished by docetaxel treatment. These results suggest that docetaxel inhibits both the tumor recruitment and T cell suppressive activity of MDSCs. Inhibitors of iNOS and arginase partially inhibited ex vivo MDSC activity, and combined inhibition of iNOS and arginase had a similar effect as docetaxel, which supports the possibility that docetaxel may function by inhibiting ACT-associated activation of these pathways. Furthermore, docetaxel mediated inhibition of the T cell suppressive activity of MDSCs from human blood, which supports the potential clinical applicability of these findings. On the basis of these findings, docetaxel treatment may represent an effective therapeutic approach for reversing immunosuppression by MDSCs subsequent to ACT-based therapy.
1. Introduction Immunotherapy based on adoptive T cell transfer therapy (ACT) could mediate tumor regression in patients with metastatic cancer [1,2]. To exert a measurable antitumor effect, ex vivo expanded immune cells are infused back into a patient and traffic to the tumor, where they mediate its destruction [3]. However, tumors often persist after ACT, despite having enriched for T cells that are specific for antigens expressed by the tumor. There is a growing body of work
suggesting that tumor-specific T cells are restrained in vivo by the immunosuppressive tumor environment [4]. These immunosuppressive mechanisms are recapitulated in transplantable mouse tumor models, which are valuable for identifying cellular and molecular immunosuppressive pathways and screening for immunomodulatory drugs as adjuncts to the adoptive transfer of tumor-specific T cells [5,6]. Most commonly, tumor progression is accompanied by local and systemic immunosuppression. This may be mediated by increased secretion of specific cytokines, such as interleukin-10 (IL-10); or increased
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Corresponding author at: Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd, FL2, Building 3, Park B, Shunyi District Airport High Tech Zoon, Beijing 101300, China. E-mail address: [email protected] (X. Lu). 1 These authors have contributed equally to this study. https://doi.org/10.1016/j.cellimm.2019.104036 Received 6 November 2019; Received in revised form 20 December 2019; Accepted 29 December 2019 0008-8749/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Yuefeng Hu, et al., Cellular Immunology, https://doi.org/10.1016/j.cellimm.2019.104036
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tumor accumulation of immunosuppressive cells, such as FOXP3 + regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSCs), and M2 macrophages [7]. In particular, MDSCs are known to support tumor progression by contributing to the immunosuppressive local microenvironment [8–10]. MDSCs suppress tumor immunity by a variety of mechanisms, which include increased production of nitric oxide (NO) and reactive oxygen species (ROS), L-arginine metabolism, and immunosuppressive cytokines such as IL-10 [11–13]. Moreover, MDSC repressive anti-tumor immunity has been shown to be correlated with early recurrence and poor prognosis [11,14]. Indeed, successful anticancer therapies are able to increase the level of immunosurveillance by reactivating pre-existing antitumor immune responses [15,16]. Several chemotherapeutic agents appear to promote antitumor responses by inhibiting MDSCs. A recent study has shown that members of the taxane family, including docetaxel and paclitaxel (Taxol), are able to limit the accumulation and immunosuppressive activity of tumor-infiltrating MDSCs in mice bearing mammary tumors [8], transgene-induced melanomas, and spontaneous melanomas [17,18], Thus, chemotherapeutic agents that target MDSCs may provide a potent approach to relieving the immunosuppressive tumor, which could potentially improve the response to ACT and other forms of immunotherapy. In this study, we investigated the cellular effects of docetaxel in response to ACT in a mouse model. Our findings demonstrate that administering docetaxel down-regulates MDSC suppressive pathways, restores antitumor immunity and results in enhancement of the therapeutic efficacy of ACT. These results suggest docetaxel treatment should be considered as a therapeutic approach for reversing immunosuppression in the tumor microenvironment subsequent to ACTbased therapy. 2. Results 2.1. ACT synergizes with docetaxel to delay tumor outgrowth Fig. 1. Increased antitumor activity of adoptive immunotherapy plus docetaxel in BALB/c mice engrafted with CT26 colon carcinoma cells. (A) Male BALB/c mice were inoculated subcutaneously in the flank with 1 × 106 CT26 colon carcinoma cells. After 14 days, mice bearing CT26 tumors were left untreated (Control), injected with adoptive T cells (ACT), treated with docetaxel (Docetaxel) or treated with both ACT and docetaxel (Combination). Docetaxel was administered at a weekly interval on days 14 and 21. Tumor volumes were measured using a caliper every 3 days up to day 27 after tumor implantation. (B) The mice were euthanized on day 27 after tumor implantation, and the tumors were excised and weighed. The results represent three independent experiments (n = 5 per group). Results are expressed as the mean tumor volume ± standard error (SE). *P < 0.05, **P < 0.01 vs Combination.
We sought to determine whether docetaxel could enhance the therapeutic efficacy of ACT in the transplantable mouse tumor model. Balb/c mice were engrafted with mouse CT26 colon carcinoma cells, and then 14 days after tumor challenge, groups of mice were left untreated or were administered docetaxel and/or purified syngeneic T lymphocytes generated from splenocytes of tumor-free mice. As shown in Fig. 1, ACT alone compared with no treatment caused only a marginal antitumor effect. Docetaxel alone also had a minimal effect; however, coupling docetaxel with adoptive transfer resulted in a significant antitumor effect (P < 0.01; Fig. 1(A, B)). To extend this finding to another tumor type, the experiment was repeated using 4T1 mammary carcinoma cells. As observed for CT26 colon carcinoma cells, adoptive immunotherapy alone had a marginal effect, whereas docetaxel + adoptive transfer imparted a statistically significant antitumor benefit (P < 0.01; Fig. 2(A, B)). These data suggest that docetaxel is able to augment the therapeutic efficacy of ACT.
(Fig. 3(A, B)). Similar results were observed for 4T1 tumors (Fig. 3(C, D)). These results suggest the docetaxel’s ability to protect against tumor development after ACT may be explained in part by its function in preventing MDSC recruitment to the site of tumors. To further examine the antitumor effect of docetaxel, we isolated murine T lymphocytes from splenocytes of tumor-free mice and murine MDSCs from CT26 or 4T1 tumor-bearing mice. Lymphocyte lytic activity was tested against CT26 or 4T1 tumor cells in the presence or absence of MDSCs. Naïve splenocytes were used as a control for MDSCs. Our results showed a dose-dependent suppression of T lymphocyte cytotoxicity when lymphocytes were co-cultured with MDSCs, whereas no suppression was observed when lymphocytes were co-cultured with splenocytes (Fig. 4(A, C)). As a control, MDSCs did not lyse lymphocytes or CT26 or 4T1 cells at any ratio (data not shown). Next, we assessed whether administration of docetaxel could rescue the cytotoxicity of T lymphocytes. As shown in Fig. 4(B, D), MDSCs isolated from docetaxel-treated tumors did not exert suppressive effects relative to those of naïve splenocytes on syngeneic T cells. These data confirm the immunosuppressive properties of MDSCs in impairing the
2.2. Docetaxel treatment restores ACT-mediated antitumor activity Emerging data reveal populations of MDSCs isolated from different organs vary in their immunosuppressive ability, with intratumoral MDSCs being the most highly immunosuppressive [19–21], Since members of the taxane family augment antitumor immunity [8,17,18], we asked whether docetaxel treatment may affect the ability of intratumoral MDSCs to alter the activation state of T lymphocytes. To test this hypothesis, we performed flow cytometry of tumor-infiltrating immune cells. After ACT, Gr-1+CD11b+ MDSCs accumulated in the CT26 tumor microenvironment, with a reduction in the frequency of the MDSC population in the spleen. Administration of docetaxel reversed the intratumoral accumulation of MDSCs in response to ACT 2
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combined with nor-NOHA and/or L-NMA (Fig. 5(A, B)). Furthermore, docetaxel had no effect on CD11b+Gr-1+ cell-depleted cell populations, which is consistent with the specificity of docetaxel function (data not shown). These results support the role of ARG1 and iNOS in mediating the pathway of MDSC suppression and are consistent with the possibility that docetaxel functions through the blockade of ARG1 and iNOS. 2.4. Restoration of the anti-tumor activity of human T lymphocytes upon the treatment with docetaxel The above results were obtained using a mouse model. To confirm that the findings are also applicable to humans, we collected blood from healthy donors. We isolated human MDSCs (CD14+HLA-DR−/low) and generated human lymphocytes from peripheral blood lymphocytes. As shown in Fig. 6(A), CD14+HLA-DR−/low MDSCs significantly suppressed the cytotoxicity of T cells against the human MCF-7 cell line, whereas control CD14+HLA-DR+ monocytes showed no significant effect. As observed in the mouse model, docetaxel reversed the suppression. Furthermore, nor-NOHA and L-NMA each mediated partial reversal of the CD14+HLA-DR−/low MDSC inhibitory effect, with high level inhibition by the combination of nor-NOHA and L-NMA (Fig. 6(B)). These results confirm the function of docetaxel in suppressing the activity of human MDSCs and demonstrate a role for ARG1 and iNOS in MDSCs-mediated immunosuppression. Based on our results, docetaxel may provide a novel pharmacologic approach for regulating MDSC-mediated immunosuppressive pathways. 3. Discussion The development of the anti-tumor immune response is associated with the accumulation and activation of T cells that move through tissues, while tumor-infiltrating MDSCs are known to suppress T cell activity. ACT after lymphodepletion has emerged as a promising approach to cancer immunotherapy in which tumor-specific T cells are expanded and re-introduced into the patient. This approach has been especially useful in maintenance treatment, for which it is able to reduce cancer recurrence and metastasis [1,24,25], Despite the success of ACT and other forms of cancer immunotherapy, the efficacy is limited by tumor-associated immune suppression, mediated in large part by tumor-associated MDSCs [19–21], Functionally, MDSCs suppress the tumoricidal activity of activated T-cells, through ARG-1 and iNOS, each of which have been shown to induce the anergy of reactive T cells [5,26], Given these immunosuppressive effects of MDSCs, the elimination of MDSCs may significantly improve antitumor responses and enhance the efficacy of immunotherapy [23,27,28], In this study, we examined the immune-mediated mechanisms that suppress the function of ACT-based therapies and provided an approach by which the immunosuppressive mechanism could be overcome using docetaxel, a chemotherapeutic reagent that has already been approved in previous studies for its ability to alter important immunologic parameters. Docetaxel, and its related compound, paclitaxel, are taxanes, a class of chemotherapy drugs that shows great promise due to its mechanism of action and relatively low toxicity. The present study shows that ACT and docetaxel each reduce tumor progression in a mouse cancer model. Furthermore, the reduction was greatest when ACT and docetaxel were applied in combination, which supports the combined use of docetaxel and ACT. Inasmuch as docetaxel and other chemotherapeutic drugs have been shown to directly eliminate MDSCs in pathologic settings [8,29–31], we determined whether docetaxel might enhance the effects of ACT by suppressing the activity of MDSCs. In support of this possibility, our results demonstrate that adoptively transferred T cell therapy caused increased recruitment of MDSCs to the site of the tumor, but that the recruitment could be reversed by concurrent docetaxel treatment. We further observed that docetaxel could inhibit the T cell suppressive
Fig. 2. Increased antitumor activity of adoptive immunotherapy plus docetaxel in BALB/c mice engrafted with 4T1 mammary carcinoma cells. (A) Male BALB/ c mice were inoculated subcutaneously in the flank with 1 × 106 4T1 mammary carcinoma cells. After 14 days, mice bearing 4T1 mammary tumors were left untreated (Control), injected with adoptive T cells (ACT), treated with docetaxel (Docetaxel) or treated with both ACT and docetaxel (Combination). Docetaxel was administered at a weekly interval at days 14 and 21. Tumor volumes were measured using a caliper every 3 days up to day 27 after tumor implantation. (B) The mice were sacrificed on day 27 after tumor implantation, and the tumors were excised and weighed. The results represent three independent experiments (n = 5 per group). Results are expressed as the mean tumor volume ± standard error (SE). *P < 0.05, **P < 0.01 vs Combination.
cytotoxicity of T lymphocytes against tumor cells and suggest that MDSC inhibition by docetaxel may provide a useful approach for enhancing tumor specific immunotherapy. 2.3. Down-regulation of tumor-associated MDSC activity is mediated by ARG-1 and iNOS pathway inhibition MDSCs derived from tumors are known to express elevated levels of arginase 1 (ARG 1) and inducible nitric oxide synthase (iNOS), which are each major suppression pathways [22,23], To determine whether modulation of these pathways may contribute to the inhibitory effect of docetaxel on MDSC-mediated suppressive activity, MDSCs were admixed with syngeneic T cells in the presence or absence of nor-NOHA (an ARG1-specific inhibitor), L-NMA (an iNOS inhibitor) or docetaxel. As previously demonstrated, docetaxel alone could restore the tumor lytic activity of syngeneic T cells that were suppressed by incubation with MDSCs derived from CT26 (Fig. 5(A)) or 4T1 (Fig. 5(B)) tumors. Nor-NOHA and L-NMA also each mediated a partial inhibition of MDSCsuppressive function, and these two inhibitors had a greater effect when combined. However, no additional effect was detected for docetaxel 3
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Fig. 3. Accumulation of intratumoral and splenic myeloid-derived suppressor cells (MDSCs) in tumor-engrafted BALB/c after adoptive T cells (ACT) and/or docetaxel treatment. Mice were engrafted with CT26 colon carcinoma cells (A and B) or 4T1 mammary carcinoma cells (C and D). Tumor bearing mice (n = 5) were treated with ACT and/or docetaxel as described in Figs. 1 and 2. Accumulation of intratumoral (A or C) and splenic (B or D) CD11b+Gr-1+ cells was determined by flow cytometry. *P < 0.05 compared with the ACT-treated group.
combined clinical use of ACT-based immunotherapies and chemotherapeutic agents.
function of MDSCs from tumor-bearing mice. Therefore, docetaxel may function synergistically with ACT in reducing tumor progression by inhibiting MDSCs both at the level of recruitment and activity. To examine pathways that contribute to MDSC activation during ACT, we assessed the effect of inhibitors of ARG-1 and iNOS on the activity of ex vivo MDSC cultures. Each of these inhibitors mediated a partial reversal of MDSC-mediated T cell suppression, and the combination of ARG-1 and iNOS inhibitors caused an effect that was similar to that of docetaxel. Furthermore, no additional effect was observed when docetaxel was combined with ARG-1/iNOS inhibitors. These results are consistent with the possibility that docetaxel may restore the tumor lytic activity of ACT cell function by simultaneous ARG-1 and iNOS inhibition, though additional assessment will be needed to verify the role of these pathways in docetaxel-mediated MDSC suppression. Importantly, docetaxel had similar effects on MDSCs isolated from human blood, which supports the potential use of docetaxel and ACT as a therapeutic combination for cancer patients. Our results suggest that docetaxel is able to augment ACT, though additional studies are needed to determine whether adoptive T cells may also affect MDSCs in patients with metastatic cancer. Though chemotherapy resistance is a common deterrent to metastatic cancer treatment, our findings raise the possibility that patients with tumors that are unresponsive to docetaxel alone may benefit from the drug’s ability to enhance immune responses in the context of immunotherapy. Thus, these findings have potentially important implications for
4. Materials and methods 4.1. Tumor cell lines and mice Two murine cell lines (CT26 colon cancer and 4T1 mammary tumor) and a human breast adenocarcinoma cell line (MCF-7) were purchased from ATCC. Cells were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone). All cells were grown at 37 °C in a humidified atmosphere of 5% CO2. Six- to 8-week-old male Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were maintained under specific pathogen-free conditions and treated according to the institutional protocols approved by the Animal Care Committee of Peking University Health Science Center. 4.2. Reagents Docetaxel (Taxotere, Sanofi-Aventis) was dissolved in 80% ethanol at 10 mg/mL according to the manufacturer’s instructions and then further diluted to 5 mg/mL in sterile PBS. For each experiment, docetaxel was administered intraperitoneally at 33 mg/kg body weight 4
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Fig. 4. Myeloid-derived suppressor cells (MDSCs) isolated from docetaxel-treated mice are less suppressive than those from control-treated mice. CD11b+Gr-1+ cells were purified from the spleens of CT26 (panels A and B) or 4T1 (panels C and D) tumor-bearing mice that were untreated or treated with docetaxel. Different ratios of CD11b+Gr-1+ cells or naïve splenocytes were incubated with syngeneic total T cells as demonstrated. After 24 h, CT26 colon carcinoma cells or 4T1 mammary carcinoma cells were added at a ratio of 10:1 (E:T). Naive splenocytes were used as a control for MDSCs. The % lysis was determined as a measure of tumor cellspecific T-cell-mediated toxicity. **P < 0.01, compared with the naïve splenocyte-treated group.
manufacturer’s protocol. The purity of Gr-1+CD11b+ MDSCs in isolated cell populations was approximately 85%. Positive and negative fractions were sorted with LS columns. CD4+ or CD8+ T lymphocytes were negatively selected from the CD11b-depleted splenocytes using the CD4+ or CD8+ T cell isolation kit (Miltenyi Biotec). For ACT experiments, mice were administered 3 × 106 T lymphocytes (supplemental Figure) in the tail vein on day 14. For isolation of CD14+HLADR−/low and CD14+HLA-DR+ cells, human peripheral blood mononuclear cells (PBMCs) were purified using CD14 Microbeads and the AutoMACS separation unit (Miltenyi Biotech) according to the manufacturer’s instructions. Human peripheral blood lymphocytes were collected from healthy donors after obtaining informed consent according to an Institutional Review Board-approved protocol. PBMCs were isolated on a Ficoll gradient and then stimulated with anti-CD3/CD28 antibody-coated Dynal beads (bead:cell ratio 3:1). Isolated and activated T cells were expanded in complete media (RPMI1640 + 100 U/mL IL-2) for 7 days. Dynabeads were magnetically removed at day 3 post stimulation and analyzed by flow cytometry. Docetaxel was added as indicated. Single cell suspensions from spleens or tumors were treated with Fcblock and mAbs for 30 min at 4 °C, with isotype-matched antibodies as control. Live cells were gated based on 7-amino-actinomycin D, annexin V staining. Samples were run on a flow cytometer (FACSCalibur; BD Biosciences), and the data were analyzed using FCS Express software.
[32] on the indicated days. Docetaxel was used in vitro at a concentration of 11 nM [8]. N-omega-hydroxy-nor-L-arginine (nor-NOHA; Calbiochem) and NG-monomethyl-L-arginine (L-NMA; Calbiochem) were used at 10 μM in vitro. 4.3. Assessment of anti-tumor activity Tumors were established in Balb/c mice by injecting 1 × 106 CT26 colon carcinoma or 4T1 mammary carcinoma cells. On day 14, when the tumors had reached a mean area of 10 mm2, the mice were randomized into four groups with 5 mice per group as follows: (I) tumor bearers, (II) tumor bearers treated with ACT, (III) tumor bearers treated with docetaxel, and (IV) tumor bears treated with ACT and docetaxel. On the day of ACT transfer and 2 days after, mice received intraperitoneal injections of docetaxel. Tumor measurements were performed with a caliper by measuring the largest diameter and its perpendicular length in a blind fashion. The tumor size index was determined as the average of the product of these diameters and was measured independently by two operators. The tumor volume was calculated using the modified formula: 0.5 × (length × width2). Results are expressed as the average tumor volume ± standard error (SE). 4.4. Cell preparation and flow cytometry Single cell suspensions prepared from the spleens of three mice group were depleted of RBCs using RBC lysis buffer as previously scribed [33]. CD11b+ cells were isolated from tumors using CD11b+ MicroBeads isolation kit (Miltenyi Biotec) according to
4.5. Cytotoxicity assessment
per dethe the
The T cell-suppressive activity of MDSCs was evaluated by the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according 5
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Fig. 6. Docetaxel reverses myeloid-derived suppressor cell (MDSC)-mediated suppression of human activated T-cells function. (A) Human PBMCs from healthy donors were collected as a source of human MDSCs and T cells. Activated T-cells were cultured and incubated with different ratios of HLADRlow/−/CD14+ cells or HLA-DR+/CD14+ as indicated. After 24 h, MCF-7 cells were added at a ratio of 10:1 (E:T) and the % lysis was calculated. ** P < 0.01, compared with the HLA-DR+/CD14+ treated group. (B) Human activated T-cells and HLA-DRlow/−/CD14+ were co-cultured with in the presence or absence of nor-NOHA (10 μmol/L), L-NMA (10 μmol/L), and/or docetaxel (11 nmol/L). After 36 h, MCF-7 cells were added at a ratio of 10:1 (E:T). The activated T-cells, purified human MDSCs (HLA-DRlow/−/CD14+), and target tumor cells were co-cultured at a ratio of 10:1:1. *P < 0.05, **P < 0.01 compared with control.
Fig. 5. Myeloid-derived suppressor cell (MDSC) T cell suppression is mediated through a pathway that involves nitric oxide (NO) and arginase. Syngeneic total T cells were cocultured with CD11b+Gr-1+ cells purified from the spleens of CT26 tumor-bearing mice (A) or 4T1 tumor-bearing mice (B) at a 10:1 ratio in the presence or absence of nor-NOHA (10 μmol/L), L-NMA (10 μmol/L), and/or docetaxel (11 nmol/L). After 36 h, the corresponding target tumor cells were added at a ratio of 10:1 (E:T). The % lysis was determined as a measure of tumor cell-specific T-cell-mediated toxicity. *P < 0.05, **P < 0.01 compared with the control.
to the manufacturer’s instructions. Briefly, target tumor cell dilutions starting at 5000 cells per well were plated in triplicate in 96-well roundbottomed microplates. T lymphocytes co-cultured with MDSCs, or splenocytes depleted of CD11b+ cells were added to the target tumor cells at different ratios of effector to MDSCs. Specific lysis for each effector-to-target (E:T) cell ratio was calculated with the following formula:
P ≤ 0.05 were considered statistically significant. Funding This work was supported by the National Natural Science Foundation of China (81770468), Beijing Municipal Natural Science Foundation (7162030) and the Beijing Science and Technology Plan special issue (Z14010101101).
% Cytotoxicity = [(Experimental − Effector Spontaneous − Target Spontaneous )
CRediT authorship contribution statement
/ (Target Maximum − Target Spontaneous)] × 100 Yuefeng Hu: Conceptualization, Writing - original draft. Jingwei Liu: Data curation, Writing - review & editing. Peilin Cui: Methodology, Validation. Tao Liu: Formal analysis. Chunmei Piao: Funding acquisition. Xianghong Xu: Resources. Qike Zhang: Resources. Man Xiao: Investigation. Yongcheng Lu: Methodology. Xuesong Liu: Validation. Yue Wang: Validation. Xu Lu: Writing - review & editing.
All determinations were done in triplicate. 4.6. Statistical analysis The statistical significance of differences between values of the control and treatment groups were determined via Student’s t test. For all experiments, the graphs represent the mean of three separate experiments and the error bars represent the SE. Differences with 6
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Declaration of Competing Interest [17]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[18]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellimm.2019.104036.
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