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Abstract
Background Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells.
Methods The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors.
Results The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4+ and CD8+ T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors.
Conclusions In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy.
- Non-Small Cell Lung Cancer
- Drug Evaluation, Preclinical
- T-Lymphocytes
Data availability statement
Data are available upon reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Tetracyclines exhibit anti-inflammatory activities via various mechanisms. However, the effect of tetracyclines on antitumor T-cell immunity remains to be clarified.
WHAT THIS STUDY ADDS
Tetracyclines enhanced antitumor T-cell immunity via T-cell receptor signal transduction in human peripheral T cells and the lung tumor tissues of patients with non-small cell lung cancer. These effects on T-cell signaling were not observed for anti-programmed cell death-1 antibodies under the same conditions.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
These results will contribute to the development of cancer immunotherapy based on novel mechanisms.
Background
Cancer immunotherapy including immune checkpoint inhibitors (ICIs) is only beneficial for a limited population of patients with cancer. Anti-programmed cell death-1 (PD-1) therapy is not effective for patients with non-small cell lung cancer (NSCLC) who do not express PD-ligand 1 (PD-L1) on tumor cells.1 Therefore, the development of novel immunotherapies based on different mechanisms from current cancer immunotherapies is desired.
Attempts have been made to develop cancer immunotherapy agents based on a drug-repurposing approach. Metformin, a drug prescribed for the treatment of type 2 diabetes, was previously reported to exert immune-mediated antitumor effects by stimulating the production of mitochondrial reactive oxygen species in the tumor-infiltrating CD8+ T cells of murine tumors.2 3 Bezafibrate, a drug prescribed to treat hyperlipidemia, also enhanced antitumor immunity during PD-1 blockade by increasing and maintaining the number of functional cytotoxic T lymphocytes (CTLs) of murine tumors via the activation of mitochondrial and cellular metabolism.4 5
We previously reported that tetracyclines, classical antibiotics, enhanced T-cell immunity in vitro using a bispecific T-cell engager (BiTE) that was specific for CD3 expressed on T cells and an antigen expressed on the surface of tumor cells.6 The pattern of T-cell cytotoxicity to tumor cells induced by BiTE showed similarities to tumor cell killing by the endogenous tumor antigen-specific T cells of patients with cancer.7 The BiTE-mediated cytotoxicity assay of human T cells is a useful method for evaluating human T-cell immunity. Previous studies assessed T-cell function in the tumor microenvironment using BiTE technology.8–10 Based on our findings on the effects of tetracyclines on T-cell immunity, we conducted a randomized clinical trial to evaluate the efficacy of demeclocycline for patients with mild-to-moderate coronavirus disease 2019 (COVID-19) with a focus on T-cell responses, and found a significant increase in the number of peripheral CD4+ T cells in the tetracycline-treated group, which negatively correlated with plasma interleukin-6 levels.11 We also investigated the effects of minocycline on the outcomes of patients with epidermal growth factor receptor (EGFR)-mutant NSCLC treated with first-line EGFR-tyrosine kinase inhibitors (TKIs) based on a retrospective analysis and showed that the administration of minocycline correlated with good progression-free survival and overall survival (OS) independently of skin rash as an adverse event of EGFR-TKIs.12
However, the rationale for and the mechanism of action of tetracyclines on T-cell immunity remain unclear. In the present study, we investigated the efficacy of tetracyclines in antitumor T-cell immunity focusing on T-cell signaling with a comparison of anti-PD-1 antibodies by human peripheral T cells, murine models, and the lung tumor tissues of patients with NSCLC.
Methods
Chemical reagents
Demeclocycline hydrochloride was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Minocycline hydrochloride and doxycycline hydrochloride were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Nivolumab was provided by Ono Pharmaceutical (Osaka, Japan). A human IgG4 isotype antibody (BioLegend, San Diego, California, USA) was used under control conditions.
Human sample preparation
The peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated from peripheral blood by gradient density centrifugation using Lymphoprep (Axis Shield, Dundee, UK) and then subjected to T-cell assays or natural killer (NK) cell assays. CD8+ T cells, CD4+ T cells, and NK cells were isolated from PBMCs using the CD4+, CD8+ T Cell Isolation Kit or NK cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
The surgically resected fresh tumors of patients with NSCLC were minced in a 6 cm dish and digested to a single cell suspension using a Tumor Dissociation Kit for humans (Miltenyi Biotec) and gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. The cell suspension was applied to a 70 µm nylon cell strainer (BD Biosciences, Franklin Lakes, New Jersey, USA) with the lysis of red blood cells by BD Pharm Lyse. Dead cells and debris were removed by centrifugation in isodensity Percoll solution (Pharmacia Biotech, Uppsala, Sweden), followed by T-cell assays using freshly isolated cells. The present study was conducted in accordance with the Declaration of Helsinki.
T-cell cytotoxicity assay using the EphA2/CD3 bispecific T-cell engager (BiTE-assay system)
The construction of EphA2/CD3 BiTE is described in our previous study.6 10 13 The U251 cell line was kindly provided by Dr Yasuko Mori (Kobe University, Japan). Cell line authentication by short tandem repeat profiling and Mycoplasma testing were performed in the JCRB Cell Bank (Osaka, Japan). U251 cells were plated on 96-well flat-bottomed cell culture plates (Corning, Corning, New York, USA) at a density of 1×104 cells per well with RPMI medium 1640 (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS). After a 24-hour culture at 37°C with 5% CO2, 5×104 PBMCs, isolated CD8+ or CD4+ T cells, or freshly isolated cells from lung tumor tissues were added to plates with 100 ng/mL of EphA2/CD3 BiTE±tetracyclines or nivolumab. After a 48-hour co-culture at 37°C with 5% CO2, culture supernatants were cryopreserved for the Cytometric Bead Array (BD Biosciences). Non-adherent cells were removed by gentle washing four times with RPMI medium 1640 containing 10% FBS, and the remaining adherent viable tumor cells were detected using the 3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium (MTS) assay (CellTiter 96 aqueous one solution cell proliferation assay, Promega, Madison, Wisconsin, USA), which was performed in triplicate. The calculation of EphA2/CD3 BiTE-mediated killing was based on the degree of the reduction in viable target cells using the following formula:
% EphA2/CD3 BiTE-mediated killing = ((absorbance of non-treated wells) − (absorbance of treated wells))/(absorbance of non-treated wells) × 100.
Each non-treated well consisted of 1×104 U251 cells without EphA2/CD3 BiTE. T cells collected after the co-culture in the BiTE-assay system were analyzed by flow cytometry or interferon-gamma (IFN-γ) secretion assay (Miltenyi Biotec).
Human CTL assay
Melanoma antigen recognized by T cells-1 (MART-1) tetramer-positive CD8+ T cells were induced by a co-culture with the MART-1 peptide as described in our previous study.14 PBMCs from an HLA-02:01-positive donor (Cellular Technology Limited, CLE, Ohio, USA) were co-cultured with 1 µg/mL of the 02;01-MART-1 peptide (Medical & Biological Laboratories, Tokyo, Japan) and 50 U/mL of IL-2 for 14 days. MART-1 tetramer-positive CD8+ T cells were then isolated using MACS Quant Tyto (Miltenyi Biotec) after being stained with CD8a and HLA-A*02:01 MART-1 Tetramer-ELAGIGILTV (Medical & Biological Laboratories). MART-1 tetramer-positive CD8+ T cells were sorted again after a 14-day culture with 100 U/mL of IL-15 and stored in an N2 bank. SK-MEL-5 cells were plated on 96-well flat-bottomed cell culture plates at a density of 5×103 cells per well with RPMI medium 1640 containing 10% FBS. After a 24-hour culture at 37°C with 5% CO2, 2×103 MART-1 tetramer-positive CD8+ T cells were added to each well±minocycline, which was performed in triplicate. After a 48-hour co-culture at 37℃ with 5% CO2, non-adherent cells were removed by gentle washing four times with RPMI medium 1640 containing 10% FBS, and the remaining adherent viable tumor cells were detected using the MTS assay. MART-1 tetramer-positive CD8+ T cell-mediated killing was calculated based on the degree of the reduction in viable target cells using the following formula:
% MART-1 tetramer-positive CD8+ T cell-mediated killing = ((absorbance of non-treated wells) − (absorbance of treated wells))/(absorbance of non-treated wells) × 100.
Each non-treated well consisted of 5×103 SK-MEL-5 cells.
NK cell cytotoxicity assay
U251 cells were plated on 96-well flat-bottomed cell culture plates at a density of 1×104 cells per well with RPMI medium 1640 containing 10% FBS. After a 24-hour culture at 37°C with 5% CO2, 1×104 isolated NK cells were added to plates±minocycline. After a 48-hour co-culture at 37°C with 5% CO2, the MTS assay was performed in a similar manner to the T-cell cytotoxicity assay. NK cell-mediated killing was calculated based on the degree of the reduction in viable target cells using the following formula:
% NK cell-mediated killing = ((absorbance of non-treated wells) − (absorbance of treated wells))/(absorbance of non-treated wells) × 100.
Each non-treated well consisted of 1×104 U251 cells. NK cells collected after the co-culture in the NK cell assay system were analyzed by flow cytometry.
Flow cytometry analysis
Surface marker staining was performed after dead cell staining (fixable viability dye; eBioscience, Waltham, Massachusetts, USA) and the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend) for human samples and an anti-CD16/32 Ab (clone 93, BioLegend) for mouse samples. The Foxp3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific) was used for intracellular staining. In the analysis of IFN-γ producing murine tumor-infiltrating cells, cell staining was performed after stimulation by 40 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, Missouri, USA) and 4 µg/mL of ionomycin (Sigma-Aldrich) in the presence of BD GolgiStop (BD Bioscience) at 37°C for 4 hours. Stained cells were analyzed using the NovoCyte Quanteon Flow Cytometer with NovoExpress Software (Agilent, Santa Clara, California, USA). Staining antibodies are described in online supplemental table S1. The gating strategy is shown in online supplemental figure S1.
Supplemental material
Supplemental material
Data measured by flow cytometry were further analyzed using Cytobank software (Beckman Coulter, Brea, California, USA). viSNE, FlowSOM algorithms, and a CITRUS analysis were performed for dimensionality reduction, a PhenoGraph clustering analysis, and the fully automated discovery of significant stratifying biological signatures, respectively.15–17
IFN-γ secretion assay
The percentage of CD4+, CD8+ T cells, CD14+ monocytes, or NK cells with the ability to produce IFN-γ was measured by the IFN-γ Secretion Assay Detection Kit (Miltenyi Biotec) after a 72-hour co-culture in the BiTE-assay system with and without minocycline. The IFN-γ secretion assay was performed according to the manufacturer’s instructions and analyzed using the NovoCyte 3000 Flow Cytometer with NovoExpress software (Agilent). PBMCs used in the BiTE-assay system were collected from healthy donors.
Cytokine bead array
BD human cytokine bead array kits (granzyme B, tumor necrosis factor-α, and Fas ligand; BD Biosciences, San Diego, California, USA) were used to quantitatively measure cytokine levels in the supernatant of the BiTE-assay system co-cultured with and without minocycline for 48 hours. The assay was performed according to the manufacturer’s instructions and analyzed on a NovoCyte 3000 Flow Cytometer with NovoExpress software (Agilent).
ELISA for the quantification of Zap70 phosphorylation
The phospho-Zap-70 (Tyr319) Sandwich ELISA Kit (Cell Signaling, Danvers, Massachusetts, USA) was used for the protein quantification of phosphorylated Zap70. PBMCs cultured in the BiTE-assay system for 5 min with and without minocycline were lysed and analyzed according to the manufacturer’s instructions. The absorbance ratio of Zap70 phosphorylation was calculated using the following formula: the absorbance ratio of Zap70 phosphorylation = ((absorbance of the PBMC lysate after a co-culture) − (absorbance of the PBMC lysate before a co-culture))/(absorbance of the PBMC lysate before a co-culture) × 100.
Quantification of IFN-γ and CD69 messenger RNA expression
Healthy donor PBMCs cultured in the BiTE-assay system for 6 hours with and without minocycline were subjected to a quantitative reverse transcription PCR (qRT-PCR). Whole PBMCs or CD14+ monocytes after their isolation from PBMCs using CD14 MicroBeads (Miltenyi Biotec) were used for qRT-PCR. RNA was extracted using 2-mercaptoethanol (Sigma-Aldrich) and an miRNeasy Micro Kit (Qiagen, Germany) and reverse transcribed into complementary DNA (cDNA) using the QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific) with the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Messenger RNA (mRNA) levels were assessed with SYBR Green Realtime PCR Master Mix-Plus- (Toyobo, Osaka, Japan) for an SYBR Green real-time PCR analysis. Specific primer pairs (IFN-G forward TCG CCA GCA ACC TGA ATC TC, reverse GCA CGA AGC TCT TAG CGT CA; CD69 forward CAA GTT CCT GTC CTG TGT GC and reverse GAG AAT GTG TAT TGG CCT GGA) were synthesized by FASMAC (Kanagawa, Japan). The detection of amplified products was performed with StepOne (Thermo Fisher Scientific). Data were analyzed with StepOne Software V.2.3 (Thermo Fisher Scientific). The PCR protocol consisted of a first denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 10 s and at 60°C for 60 s for an annealing/extension step and at 95°C for 10 s before the melting curve was achieved. Real-time qPCR was performed in duplicate for all targets. Relative mRNA levels were assessed using the comparative threshold method after checking primer efficiency. Normalization to β-actin (forward TTG TTA CAG GAA GTC CCT TG and reverse CAC GAA GGC ACA TCA TTC AA) levels were performed. Relative mRNA levels were expressed as fold changes from untreated samples.
RNA sequencing and data processing
The CD8+ T Cell Isolation Kit (Miltenyi Biotec) was used to isolate CD8+ T cells from healthy donor PBMCs co-cultured with and without minocycline for 6 hours in the BiTE-assay system. The RNA of CD8+ T cells was extracted using the miRNeasy Micro Kit (Qiagen, Hilden, Germany). An RNA library was prepared with the TruSeq stranded mRNA Library prep kit (Illumina, San Diego, California, USA) according to the manufacturer’s instructions. Sequencing was performed on a NovaSeq 6000 platform (Illumina) in the 151 bp paired-end mode. Sequenced reads were mapped to human reference genome sequences using TopHat V.2.1.1 in combination with Trimmomatic V.0.38. The number of fragments per kilobase of exon per million mapped fragments was calculated using Cufflinks V.2.2.1. Count data are shown in online supplemental table S2. We analyzed RNA sequencing (RNA-seq) data using an integrated differential expression and pathway analysis.18 Upregulated or downregulated pathways of Gene Ontology (GO) biological processes in the presence of minocycline were analyzed using Generally Applicable Gene-set Enrichment (GAGE).
Supplemental material
Generation of galectin-1 knockout U251 cells
Wild-type U251 cells were transfected with a galectin-1 CRISPR/Cas9 KO plasmid or control CRISPR/Cas9 plasmid (Santa Cruz Biotechnology, Santa Cruz, California, USA) to generate galectin-1 (LGALS1 gene) knockout U251 cells or mock-transfected control U251 cells. The process of transfection with the plasmid was performed according to the manufacturer’s instructions using 1 µg of plasmid DNA and 5 µL of UltraCruz Transfection Reagent (Santa Cruz Biotechnology). Transfection of the plasmid was confirmed by the measurement of green fluorescence protein expression using flow cytometry. After transfection of the plasmid, single-cell cloning was conducted by limiting dilutions. U251 cells were plated for single-cell cloning on 96-well flat-bottomed cell culture plates at a density of one cell per well with RPMI medium 1640 containing 10% FBS (100 µL of 10 cells/mL per well). Clonal colonies grown from each single cell were confirmed by microscopy. Galectin-1 protein expression by each cell colony after single-cell cloning and expansion was measured using galectin-1 ELISA and immunofluorescence staining.
ELISA for the quantification of galectin-1 concentrations
The Human Galectin-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota, USA) was used to quantitatively measure the concentration of galectin-1 in the supernatant of 1×104 U251 cells cultured on 96-well flat-bottomed plates for 48 hours. The assay was performed according to the manufacturer’s instructions.
Immunofluorescence staining
A total of 3×104 U251 cells cultured on 8-well slides were fixed with 100% methanol at 4°C for 15 min. After washing with phosphate-buffered saline (PBS), slides were incubated with 5 µg/mL of a goat anti-human galectin-1 polyclonal antibody or normal goat IgG control (R&D Systems) at 4°C overnight. After washing again with PBS, slides were incubated with 200 µg/mL of a goat anti-rabbit IgG highly cross-adsorbed secondary antibody (Invitrogen) at room temperature for 3 hours. Images were obtained with an all-in-one fluorescence microscope (BZ-X800L; Keyence, Tokyo, Japan) after slides had been washed with PBS following 15 min of staining with 4,6-diamidino-2-phenylindole solution (FUJIFILM Wako).
Surface plasmon resonance microscopy
Cell-based surface plasmon resonance microscopy (SPRM) was performed to analyze the binding of minocycline to proteins on cell surfaces.19 Galectin-1 knockout and wild-type U251 cells were used for SPRM. Measurement chips were coated with poly-D-lysine before 3×104 galectin-1 knockout or wild-type U251 cells were plated. After a 24-hour culture at 37°C with 5% CO2, cells were fixed for 10 min with 4% formaldehyde. Cells were washed with PBS three times and chips were stored at 4°C until SPRM was performed. Minocycline binding to galectin-1 knockout or wild-type U251 cells was measured using the SPRm 200AP system (Biosensing Instrument, Tempe, Arizona, USA).
Co-culture experiment of Jurkat cells and galectin-1 knockout U251 cells
The Jurkat T-cell line was obtained from the American Type Culture Collection (Manassas, Virginia, USA). Galectin-1 knockout or control U251 cells were plated on 96-well flat-bottomed cell culture plates at a density of 5×104 cells per well with RPMI medium 1640 containing 10% FBS. After a 24-hour culture at 37°C with 5% CO2, 1×104 Jurkat cells were added to plates with 100 ng/mL of EphA2/CD3 BiTE±tetracyclines. After a 24-hour co-culture at 37°C with 5% CO2, non-adherent cells were collected by gentle washing four times with RPMI medium 1640 containing 10% FBS. Jurkat cells collected after the co-culture were analyzed by flow cytometry.
Recombinant galectin-1 protein assay
PBMCs from healthy donors were cultured on plates coated with 1 µg/mL of an anti-human CD3 antibody (clone OKT3, BioLegend) ± recombinant galectin-1 (R&D Systems) ± minocycline. After a 48-hour co-culture at 37°C with 5% CO2, T cells were analyzed by flow cytometry.
In vivo antitumor study
6–8-week-old female wild-type BALB/c mice were obtained from CLEA Japan (Tokyo, Japan). Mice were kept under specific pathogen-free conditions and provided with food and water. Experiments were conducted in accordance with the protocol approved by the Institute of Experimental Animal Sciences Faculty of Medicine, Osaka University.
The EMT6 cell line and CT26 cell line were obtained from the American Type Culture Collection. EMT6 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FBS (Gibco, Life Technologies Corporation, Grand Island, New York, USA), and CT26 cells were cultured in RPMI medium 1640 (Nacalai Tesque, Kyoto, Japan) containing 10% FBS at 37°C with 5% CO2. EMT6 cells (5×105 cells) or CT26 cells (1×105 cells) were intradermally inoculated into BALB/c mice (day 0). Between days 5 and 18, 1–30 mg/kg of demeclocycline or minocycline was orally administered twice a day. Tumor volumes were monitored and calculated (mm3) on day 19 as follows: major axis (mm) × minor axis (mm) × minor axis (mm)/2. All tumor-bearing mice were euthanized according to institutional animal care guidelines based on tumor size, body weight, or general condition. Euthanasia was performed by CO2 inhalation.
An anti-mouse CD8a or CD4 inhibitor (Bio X Cell, Lebanon, New Hampshire, USA) was used to establish whether the effects of tetracyclines were mediated by T-cell immunity. Between days 5 and 18, 3 mg/kg of demeclocycline was administered orally twice a day to CT26-bearing mice with 400 µg of the anti-mouse CD8a, a CD4 inhibitor, rat IgG2a, or IgG2b isotype control antibody (Bio X Cell) being intraperitoneally injected on day 4.
Mouse sample preparation
White blood cells were extracted from peripheral blood on day 19 by erythrocyte lysis with BD Pharm Lyse buffer (BD Biosciences) and then subjected to a flow cytometry analysis after the CT26 inoculation (day 0) and administration of demeclocycline from days 5 to 18.
Excised tumor tissues on day 12 were minced and digested to a single cell suspension using the Tumor Dissociation Kit, mouse (Miltenyi Biotec) and gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions after the CT26 inoculation (day 0) and administration of demeclocycline from days 5 to 11. The cell suspension was applied to a 70 µm nylon cell strainer (BD Biosciences) with the lysis of red blood cells by BD Pharm Lyse and subjected to the flow cytometry analysis.
Measurement of blood demeclocycline concentrations in vivo
10 microliters of murine plasma were collected from mice 0 to 8 hours after a single oral dose of demeclocycline. The plasma concentration of demeclocycline was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS data were obtained by MS (Xevo TQ-S, Waters, Milford, Massachusetts, USA) connected to UPLC (ACQUITY UPLC, Waters) using the BEH C18 column (1.7 µm, 2.1×50 mm, Waters). Mobile phase A=0.1% formic acid/water, B=0.1% formic acid/acetonitrile, and the gradient system is as follows: 0 min-2% B, 1.8 min-98% B. The flow rate was 0.5 mL/min. Aliquots (5 µL) of plasma obtained from each blood sample were treated with 50 µL of acetonitrile and the organic layer was injected into the LC-MS/MS system (Waters). Pharmacokinetic parameters were obtained by fitting plasma concentration-time data to a non-compartmental model with PK-Plus software (Northern Science Consulting, Sapporo, Japan).
Retrospective cohort study of patients with NSCLC
To investigate the effects of minocycline on the outcomes of patients with NSCLC treated with ICIs, we analyzed a data set from our previous study.12 In the data set of patients with EGFR-mutant NSCLC who received first-line EGFR-TKIs, we extracted patients who were subsequently treated with ICIs (N=17). Patients who received minocycline for 30 days or longer before the ICI treatment were grouped into the “MINO+group” (N=5) and the remainder into the “MINO− group” (N=12). We compared OS with ICIs between the MINO+ and MINO− groups. OS was defined as the time from the initiation of the ICI treatment to the date of death.
Statistical analysis
A two-tailed Student’s t-test was used to examine the significance of differences between samples. A one-way analysis of variance with Tukey’s post hoc test was employed for multiple comparisons to compare differences with respective values for the control, with a p value<0.05 indicating a significant difference. GraphPad Prism was used for graphing and statistical analyses (GraphPad Software, San Diego, California, USA).
Results
T-cell cytotoxicity against tumor cells was enhanced by tetracyclines in vitro
To demonstrate that tetracyclines enhanced T-cell cytotoxicity against tumor cells, we used the BiTE-assay system described in our previous study (figure 1A).6 10 13 PBMCs from healthy donors were co-cultured with U251 cells and BiTE with and without tetracyclines, and the cytotoxicity of T cells against U251 cells was then measured using the MTS assay. Two types of tetracyclines, minocycline and demeclocycline, exhibited the same ability to increase antitumor T-cell cytotoxicity at concentrations of 0.1 and 1 µM (figure 1B), but did not show direct antitumor cytotoxicity without T cells at these concentrations (online supplemental figure S2). Since no significant differences were observed in enhancements in T-cell cytotoxicity by the different types of tetracyclines, which is consistent with our previous findings, we conducted subsequent experiments in vitro using minocycline.6 The cytotoxicity of CD8+ T cells after isolation by negative selection was similarly enhanced by minocycline (figure 1C), whereas that of CD4+ T cells was not (figure 1C), which is in agreement with previous findings showing that CD8+ T cells, but not CD4+ T cells, were primarily responsible for cytotoxicity in tumor immunity. Other than the BiTE-assay system, we examined the cytotoxic activity of Mart-1 tetramer-positive human CTLs against Mart-1-expressing target cells (SK-MEL-5 cells) and observed minocycline-induced increases in cytotoxic activity (figure 1D and online supplemental figure S3).
T-cell cytotoxicity against tumor cells enhanced by tetracyclines in vitro. (A) The BiTE-assay system that measures T-cell cytotoxicity against tumor cells using BiTE. PBMCs or lung tumor-infiltrating cells (5×104 cells per well of 96-well flat-bottomed cell culture plates) were co-cultured with U251 cells (1×104 cells per well of 96-well flat-bottomed cell culture plates) and BiTE (100 ng/mL) with and without tetracyclines or nivolumab. The cytotoxicity of T cells against U251 cells was measured using the MTS assay. (B) The cytotoxicity of T cells enhanced by tetracyclines was measured by the MTS assay after a 48-hour co-culture in the BiTE-assay system using PBMCs from healthy donors (N=4). (C) The cytotoxicity of CD4+ or CD8+ T cells isolated by negative selection using the CD4+ or CD8+ T Cell Isolation Kit (Miltenyi Biotec) was measured by the MTS assay after a 48-hour co-culture with and without 1 µM of minocycline in the BiTE-assay system (N=4). CD4+ or CD8+ T cells were collected from healthy donors. (D) The human CTL-assay system that measures antigen-specific cytotoxic T-cell cytotoxicity against tumor cells. MART-1 tetramer-positive CD8+ T cells (2×103 cells per well of 96-well flat-bottomed cell culture plates) were co-cultured with SK-MEL-5 cells (5×103 cells per well of 96-well flat-bottomed cell culture plates) ±1 µM of minocycline for 48 hours. The cytotoxicity of CTLs against SK-MEL-5 cells was measured in triplicate using the MTS assay. (E) The percentage of IFN-γ-secreting CD4+, CD8+ T cells, or CD14+ monocytes with and without 1 µM of minocycline was measured by the IFN-γ secretion assay (Miltenyi Biotec) after a 72-hour co-culture of PBMCs in the BiTE-assay system (N=5 or 3). PBMCs used in the BiTE-assay system were collected from healthy donors. (F) IFN-γ (IFN-G) mRNA expression by PBMCs or CD14+ monocytes from healthy donors after a 6-hour co-culture of PBMCs with and without 1 µM of minocycline in the BiTE-assay system was assessed using qRT-PCR (N=4). CD14 + monocytes were isolated from PBMCs prior to the qRT-PCR analysis. Data were shown as a relative ratio against a control condition modified by β-actin. (G) The percentage of GzmB+ CD8+ T cells cultured in the BiTE-assay system for 72 hours with and without 1 µM of minocycline was measured by a flow cytometry analysis (N=5). PBMCs used in the BiTE-assay system were collected from healthy donors. (H) The concentrations of GzmB, FasL, and TNF-α in the supernatant of the BiTE-assay system co-cultured with and without 1 µM of minocycline for 48 hours (N=5). PBMCs used in the BiTE-assay system were collected from healthy donors. (I) The cytotoxicity of T cells enhanced by the combination of 1 µM of minocycline and 1 µg/mL of nivolumab was measured by the MTS assay after a 48-hour co-culture in the assay system with BiTE using PBMCs from healthy donors (N=6). (J) The cytotoxicity of T cells enhanced by the combination of 1 µM of minocycline and 1 µg/mL of nivolumab was measured by the MTS assay after a 72-hour co-culture in the assay system with BiTE using lung tumor-infiltrating cells collected from the surgically resected lung tumor tissues of patients with NSCLC (N=3). (K) Kaplan-Meier curves of the overall survival of patients with NSCLC treated with ICIs in groups that received minocycline (N=5) and did not (N=12) before the ICI treatment. Data are shown as the mean±SEM or the mean±SD. A one-way analysis of variance with Tukey’s post hoc test, a paired two-tailed Student’s t-test, or a two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). Overall survival curves were generated by the Kaplan-Meier method and compared using the log-rank test. BiTE, bispecific T-cell engager; CTL, cytotoxic T-lymphocyte; DMC, demeclocycline; FasL, Fas ligand; GzmB, granzyme B; ICI, immune checkpoint inhibitor; IFN-γ, interferon gamma; MART-1, melanoma antigen recognized by T cells-1; MINO, minocycline; mRNA, messenger RNA; MTS, 3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium; NSCLC, non-small cell lung cancer; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse transcription PCR; TNF-α, tumor necrosis factor-α.
We also examined T-cell functions related to cytotoxicity using healthy donor PBMCs co-cultured with minocycline in the BiTE-assay system. IFN-γ-secreting CD4+ or CD8+ T cells increased in the IFN-γ secretion assay after the co-culture with minocycline (figure 1E). On the other hand, the secretion of IFN-γ from CD14+ monocytes did not significantly increase under the same conditions (figure 1E). Minocycline increased IFN-γ mRNA expression in PBMCs, but not in CD14+ monocytes (figure 1F). In addition, the number of granzyme B (GzmB)+ CD8+ T cells increased under co-culture conditions with minocycline (figure 1G). The protein concentrations of GzmB, Fas ligand (FasL), and tumor necrosis factor-α (TNF-α) in the supernatant of the BiTE-assay system co-cultured with minocycline were higher than those without minocycline (figure 1H).
We then validated the synergistic effects of minocycline and the anti-PD-1 inhibitor, nivolumab in vitro. T-cell cytotoxicity enhanced by nivolumab was confirmed in the BiTE-assay system (online supplemental figure S4). Antitumor cytotoxicity mediated by peripheral T cells from healthy donors was stronger under co-culture conditions with minocycline and nivolumab than with minocycline alone (figure 1I). Similar antitumor cytotoxicity was observed in human lung tumor-infiltrating lymphocytes from the surgically resected lung tumor tissues of patients with NSCLC (figure 1J). To assess the effects of minocycline on patients with NSCLC receiving anti-PD-1 therapy, we analyzed the OS of patients with EGFR-mutant NSCLC treated with anti-PD-1 antibodies after the first-line treatment with EGFR-TKIs±minocycline based on a data set from our previous study.12 The results of the retrospective analysis showed that OS was longer in patients administered minocycline with anti-PD-1 therapy than in those not administered minocycline (median OS, 37.8 months (95% CI, 12.6 to not reached (NR)) vs 18.6 months (95% CI, 8.0 to NR), p=0.041) (figure 1K).
These results show that minocycline exhibited the ability to enhance antitumor T-cell cytotoxicity, with increases in the production of GzmB, FasL, TNF-α, and IFN-γ. The enhancement in T-cell cytotoxicity by minocycline was achieved not only by human peripheral T cells, but also by the lung tumor-infiltrating lymphocytes of patients with NSCLC.
CD69 expression was upregulated by minocycline
To further examine the T-cell immunostimulatory effects of tetracyclines, T-cell markers of each T-cell subset were analyzed in a flow cytometry analysis of PBMCs from healthy donors under co-culture conditions with minocycline in the BiTE-assay system. We also compared minocycline with nivolumab in each experiment. Data from the flow cytometry analysis were further analyzed using Cytobank software. A FlowSOM clustering analysis was performed using data from the flow cytometry analysis after gating CD3+ T cells (figure 2A). Ten clusters were identified by the FlowSOM analysis, while T cells in clusters 1 and 5 significantly increased in the co-culture with minocycline. The expression of CD69, a T-cell activation marker, was high in clusters 1 and 5 (figure 2B). Cluster 1 was identified as CD69 high CD8+ T cells and cluster 5 as CD69 high CD4+ T cells. The ViSNE analysis, a dimensionality reduction analysis using data from the flow cytometry analysis, similarly revealed that CD69 high T cells increased under co-culture conditions with minocycline (figure 2C). The CITRUS analysis, a method for the fully automated discovery of significant stratifying biological signatures using data from the flow cytometry analysis, also revealed that the population of several CD4+ or CD8+ T cell clusters with the high expression of CD69 increased under co-culture conditions with minocycline (figure 2D). The flow cytometry analysis consistently showed that the percentage of peripheral CD69+ CD4+ or CD8+ T cells collected from healthy donors significantly increased under co-culture conditions with minocycline, but not with nivolumab (figure 2E). CD69+ T cells were maintained over a 24-hour peak under the co-culture with minocycline, but decreased after the 24-hour peak under the co-culture with nivolumab or no compound (figure 2E). The expression of other T-cell markers, including CD25 and Foxp3, is shown in online supplemental figure S5 and S6. In these analyses, CD69-positive and CD25-positive cells increased not only in Foxp3-negative conventional T cells, but also in Foxp3-positive regulatory T (Treg) cells under the co-culture with minocycline. The CD69 mRNA expression of PBMCs co-cultured with minocycline was also upregulated (figure 2F). Additionally, CD69+ cells in isolated CD4+ or CD8+ T cells co-cultured in the BiTE-assay system were increased by minocycline (figure 2G).
Upregulation of CD69 by minocycline in human peripheral T cells. (A–D) A flow cytometry analysis of PBMCs from healthy donors after a 72-hour co-culture with and without 1 µM of minocycline or 1 µg/mL of nivolumab in the BiTE-assay system (N=4). (A) A FlowSOM clustering analysis was performed using the results of the flow cytometry analysis after gating CD3+ T cells. The result of one representative donor is shown. The percentage of clusters 1 and 5 to total CD3+ T cells is shown in bar graphs. (B) A heatmap showing selected marker expression in CD3+ T-cell clusters identified by FlowSOM. (C) A viSNE analysis was performed using the results of the flow cytometry analysis after gating CD3+ T cells. A representative viSNE plot of T cells colored by the expression levels of the selected markers is shown. (D) A CITRUS analysis was performed using the results of the flow cytometry analysis after gating CD3+ T cells. Clusters defined by the CITRUS analysis are depicted. Red-colored clusters showed cell subsets that had a significantly different abundance among the three groups (control, minocycline, and nivolumab). Clusters circled in yellow were abundant in the minocycline group. Histograms for selected markers in two representative clusters with significantly large differential expressions are shown. (E) The percentage of CD69+ CD4+ or CD8+ healthy-donor T cells cultured in the BiTE-assay system for 48 hours with and without 1 µM of minocycline or 1 µg/mL of nivolumab was measured by flow cytometry. The time course of changes in the percentage of CD69+ CD4+ or CD8+ T cells is also shown. (F) CD69 mRNA expression of PBMCs from healthy donors after a 6-hour co-culture with and without 1 µM of minocycline in the BiTE-assay system was assessed by qRT-PCR (N=3). Data are shown as a minocycline/control relative ratio modified by β-actin. (G) A flow cytometry analysis measured the percentage of CD69+ in isolated CD4+ or CD8+ T cells co-cultured with and without 1 µM of minocycline in the BiTE-assay system for 72 hours. Isolation was performed by negative selection using the CD4+ or CD8+ T Cell Isolation Kit (Miltenyi Biotec) (N=3). Data are shown as the mean±SEM. A one-way analysis of variance with Tukey’s post hoc test or a paired two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). BiTE, bispecific T cell engager; GzmB, granzyme B; MINO, minocycline; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; qRT-PCR, quantitative reverse transcription PCR.
We then examined T-cell subsets in the lung tumor tissues of patients with NSCLC co-cultured with minocycline in the BiTE-assay system. The baseline percentages of PD-1+ CD4+ or CD8+ T cells, Tim-3+ CD8+ T cells, and Foxp3+ CD4+ T cells in the lung tumor-infiltrating cells of patients with NSCLC were higher than those in healthy donor PBMCs before the co-culture in the BiTE-assay system (figure 3A). A CITRUS analysis was performed to identify the dominant T-cell subset under the co-culture with minocycline. The results obtained revealed that a cluster with CD69 high and GzmB high T cells in lung tumor tissues was dominant under co-culture conditions with minocycline (figure 3B). Consistent with the results of PBMCs, the flow cytometry analysis showed that the ratio of CD69+ cells in the CD4+ or CD8+ T cells of lung tumor tissues was significantly increased under co-culture conditions with minocycline (figure 3C, online supplemental figure S7A). The ratio of CD69+ GzmB+ cells in CD8+ T cells in lung tumor tissues also significantly increased in the presence of minocycline (figure 3D). In contrast to healthy donor PBMCs, the ratio of CD69+ cells in the CD8+ T cells of lung tumor tissues increased under co-culture conditions with nivolumab (online supplemental figure S7B). T-cell activation by nivolumab was estimated to be stronger in the lung tumor-infiltrating cells of patients with NSCLC than healthy donor PBMCs because of the high expression of PD-1 in lung tumor-infiltrating T cells (figure 3A). In summary, the expression of the T-cell activation marker CD69 was enhanced by the co-culture with minocycline not only in healthy donor peripheral T cells, but also in the lung-tumor infiltrating T cells of patients with NSCLC.
Upregulation of CD69 by minocycline in tumor-infiltrating T cells from surgically resected lung tumor tissues of patients with NSCLC. (A) Flow cytometry analysis of PBMCs from healthy donors or lung tumor-infiltrating cells from surgically resected lung tumor tissues before a co-culture in the BiTE-assay system (N=4). (B–D) Flow cytometry analysis of lung tumor-infiltrating cells collected from surgically resected lung cancer tissues of patients with NSCLC and co-cultured in the BiTE-assay system for 72 hours with and without 1 µM of minocycline (N=4). (B) A CITRUS analysis was performed using the results of the flow cytometry analysis on tumor-infiltrating CD3+ T cells. Clusters defined by the CITRUS analysis are depicted. Red-colored clusters show cell subsets that had a significantly different abundance among the control and minocycline groups. Clusters circled in yellow were abundant in the minocycline group. Histograms for selected markers in an identified cluster with significantly large differential expression are shown. (C) A flow cytometry analysis of the percentage of CD69+ CD4+ T cells or CD8+ T cells from the surgically resected lung tumor tissues of patients with NSCLC. (D) A flow cytometry analysis of the percentage of CD69+ GzmB+ CD8+ T cells from the surgically resected lung tumor tissues of patients with NSCLC. Data are shown as the mean±SEM. A paired two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). BiTE, bispecific T cell engager; GzmB, granzyme B; MINO, minocycline; NSCLC, non-small cell lung cancer; PBMCs, peripheral blood mononuclear cells; PD-1, programmed cell death-1; TIL, tumor-infiltrating lymphocyte.
TCR signal transduction was upregulated by minocycline
We investigated the signaling pathways in T cells that triggered the upregulation of CD69 expression by minocycline. Since CD69 expression is downstream of T-cell receptor (TCR) signal transduction, we focused on other factors related to TCR signal transduction. Nur77 has been identified as a protein whose expression does not increase other than downstream of TCR signal transduction (figure 4A).20 Accordingly, we investigated whether minocycline enhanced Nur77 expression using a flow cytometry analysis of PBMCs from healthy donors. Nur77+ CD4+ or CD8+ T cells increased under co-culture conditions with minocycline in the BiTE-assay system (figure 4B). The time course of changes in the expression of Nur77 in CD4+ or CD8+ T cells was similar to that of CD69 (figure 4B).
Upregulation of TCR signal transduction by minocycline. (A) A schematic representation of TCR signal transduction is depicted. (B) The percentage of Nur77+ CD4+ or CD8+ T cells cultured in the BiTE-assay system for 48 hours with and without 1 µM of minocycline was measured by flow cytometry (N=4). The time course of changes in the percentage of CD69+ CD4+ or CD8+ T cells is also shown. PBMCs used in the BiTE-assay system were collected from healthy donors. (C) The percentage of pZap70+ CD4+ or CD8+ T cells cultured in the BiTE-assay system for 72 hours with and without 1 µM of minocycline was measured by flow cytometry (N=5). PBMCs used in the BiTE-assay system were collected from healthy donors. (D) PBMC lysates were measured by ELISA for Zap70 phosphorylation in PBMCs cultured in the BiTE-assay system for 5 min with and without 1 µM of minocycline (N=4). PBMCs used in the BiTE-assay system were collected from healthy donors. (E) Upregulated or downregulated gene pathways of GO biological process in the presence of minocycline were analyzed using bulk RNA-seq data. CD8+ T cells isolated from healthy donor PBMCs after a 6-hour co-culture in the presence and absence of 1 µM of minocycline in the BiTE-assay system were used for RNA-seq (N=3). The top 40 gene pathways with significant differential expression between minocycline and the control group are shown by a hierarchical clustering tree. Data are shown as the mean±SEM. A one-way analysis of variance with Tukey’s post hoc test or a paired two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). BiTE, bispecific T cell engager; GO, Gene Ontology; MHC, major histocompatibility complex; MINO, minocycline; PBMCs, peripheral blood mononuclear cells; pZap70, phosphorylated Zap70; RNA-seq, RNA sequencing; TCR, T-cell receptor; TNF, tumor necrosis factor.
We then investigated Zap70 phosphorylation to validate the direct stimulation of TCR signaling by minocycline in healthy donor T cells co-cultured in the BiTE-assay system. The flow cytometry analysis of healthy donor peripheral CD4+ or CD8+ T cells showed that Zap70 phosphorylation was upregulated under 72-hour co-culture conditions with minocycline, but not nivolumab (figure 4C). Moreover, the analysis of PBMC lysates by ELISA revealed an increase in Zap70 phosphorylation under 5 min co-culture conditions with minocycline (figure 4D), indicating that the upregulation of Zap70 by minocycline occurred in the early phase. In contrast, the level of Zap70 phosphorylation was not significantly increased by the 5 min co-culture conditions with nivolumab (online supplemental figure S8). These results suggest that the enhancement observed in TCR signal transduction was a trigger augmenting T-cell cytotoxicity by minocycline.
To obtain a more detailed understanding of the immunological landscape enhanced by minocycline, we conducted bulk RNA-seq using peripheral CD8+ T cells isolated from healthy donor PBMCs after a 6-hour co-culture with minocycline in the BiTE-assay system. A pathway analysis of GO biological processes using GAGE revealed that minocycline upregulated “Positive regulation of lymphocyte activation”, “Regulation of lymphocyte proliferation”, “Lymphocyte mediated cytotoxicity”, “Positive regulation of cytokine production”, and “Response to tumor necrosis factor” (figure 4E). The results of the pathway analysis were in agreement with the results obtained on protein expression associated with T-cell activation and antitumor cytotoxicity enhanced by minocycline as described above. In summary, the T-cell activation markers associated with TCR signal transduction, Nur77 and Zap70 phosphorylation, were enhanced in the presence of minocycline. The GO pathways of T-cell activation and antitumor cytotoxicity were upregulated at the mRNA level. These results suggest that the upregulation of TCR signal transduction was a trigger for the enhancement in antitumor T-cell responses by minocycline.
Galectin-1 was a target molecule of minocycline in T-cell immunity
To identify target molecules of minocycline in T-cell immunity, we initially investigated direct target cells of minocycline. In the BiTE-assay system, tumor cells or PBMCs were pretreated with minocycline before a co-culture in the absence of minocycline. The results obtained showed that T-cell cytotoxicity was stronger against tumor cells pretreated with minocycline than against non-treated tumor cells (figure 5A,B). To identify tetracycline-binding molecules in tumor cells, we analyzed a data set from a previous study on affinity isolation in tandem with MS-based quantitative stable isotope labeling by amino acids in cell cultures for proteomics in order to detect doxycycline-binding molecules in tumor cells.21 We confirmed that doxycycline enhanced T-cell cytotoxicity against tumor cells (online supplemental figure S9). Among the 188 proteins binding to doxycycline, galectin-1 was associated with T-cell immunity (online supplemental table S3).21
Supplemental material
Inhibitory effects of minocycline on galectin-1 in T-cell immunity. (A) The cytotoxicity of T cells enhanced by tetracyclines was measured by the MTS assay after a 48-hour co-culture in the BiTE-assay system using PBMCs from healthy donors (N=3). A co-culture was conducted under three conditions; (1) PBMCs and U251 cells were both co-cultured±1 µM of minocycline in the BiTE-assay system. (2) After U251 cells only were cultured±1 µM of minocycline for 3 hours, PBMCs and U251 cells were co-cultured in the BiTE-assay system without minocycline. (3) After PBMCs only were cultured±1 µM of minocycline for 3 hours, PBMCs and U251 cells were co-cultured in the BiTE-assay system without minocycline. (B) The percentage of CD69+ CD4+ or CD8+ healthy-donor T cells cultured in the BiTE-assay system for 48 hours±1 µM of minocycline was measured by flow cytometry (N=3). A co-culture was conducted under the same three conditions as in (A). (C) SPRM was conducted to demonstrate the binding of minocycline to galectin-1 on control or galectin-1 KO U251 cells. The green site shows the presence of tumor cells on measurement chips and the red site shows the binding reaction of minocycline to tumor cells. (D) The percentage of CD69+ Jurkat cells after a 24-hour co-culture of BiTE and galectin-1 knockout or control U251 cells±1 µM of minocycline was measured by flow cytometry. Each experiment was performed in triplicate. (E) The percentage of CD69+ T cells, GzmB+ CD8+ T cells, or Nur77+ T cells cultured on 1 µg/mL of an anti-human CD3 antibody-coated plate±1 µM of minocycline±1 µM of recombinant galectin-1 protein was measured by flow cytometry (N=5). PBMCs used in the recombinant galectin-1 protein assay system were collected from healthy donors. Data are shown as the mean±SEM or the mean±SD. A one-way analysis of variance with Tukey’s post hoc test, a paired two-tailed Student’s t-test, or a two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). BiTE, bispecific T cell engager; GzmB, granzyme B; KO, knock-out; MINO, minocycline; MTS, 3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium; PBMCs, peripheral blood monocytes; SPRM, surface plasmon resonance microscopy; rGal-1, recombinant galectin-1 protein.
Galectin-1 is secreted from tumor cells and binds to their surface.22 Since galectin-1 on tumor cells was previously reported to inhibit T-cell activity,22 we examined the binding of minocycline to galectin-1 on tumor cells. SPRM cell-binding experiments showed more binding of minocycline to the surface of galectin-1-expressing control U251 cells than to that of galectin-1 knockout U251 cells (figure 5C and online supplemental figure S10). The BiTE-assay system revealed that minocycline enhanced CD69 expression in Jurkat cells co-cultured with galectin-1-expressing control U251 cells, but not galectin-1 knockout U251 cells (figure 5D). The recombinant galectin-1 protein suppressed CD69, Nur77, and granzyme B expression in human peripheral T cells stimulated with anti-CD3 antibodies (figure 5E). The effects of the recombinant galectin-1 protein on T cells were canceled by minocycline (figure 5E). Galectin-1 is reportedly a potent suppressor of not only antitumor T-cell activity, but also antitumor NK immune surveillance.22 23 We found that minocycline enhanced the cytotoxic activity of NK cells against tumor cells (online supplemental figure S11). These results indicate that galectin-1 is a target molecule of minocycline in T-cell immunity.
Antitumor effects of tetracyclines in murine models
To investigate the antitumor effects of tetracyclines in vivo, BALB/c mice subcutaneously engrafted with murine tumor cell lines were treated with tetracyclines (figure 6A). Since tetracyclines are orally administered in clinical practice, BALB/c mice received tetracyclines orally. To investigate the murine pharmacokinetics of tetracyclines, the plasma concentration of demeclocycline in mice orally administered demeclocycline at doses of 1 and 3 mg/kg was measured (online supplemental figure S12A). Since the enhancement in T-cell cytotoxicity by minocycline was sufficiently effective at 0.1 µM or higher in vitro (online supplemental figure S12B), we conducted a simulation of the plasma concentration of demeclocycline in mice receiving re-administration 6 hours after the oral administration of demeclocycline at a dose of 3 mg/kg (online supplemental figure S12C). The results of the simulation of pharmacokinetics suggested that administration twice a day was necessary to maintain effective plasma concentrations of tetracyclines (0.1 µM).
Antitumor effects of tetracyclines in murine models subcutaneously engrafted with murine tumor cell lines. (A) Between 1 and 30 mg/kg of tetracyclines was orally administered to BALB/c mice twice a day for 14 days from 5 days after the tumor inoculation. (B–D) Tumor growth curves from days 0 to 19 after the tumor inoculation (N=9–12). Demeclocycline was used in (B) and (D) and minocycline in (C). EMT6 was used in (B) and (C) and CT26 in (D). Data are shown as the mean±SEM. A two-tailed Student’s t-test was used to examine the significance of differences between the control group and each group (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). DMC, demeclocycline; MINO, minocycline.
When demeclocycline was administered orally at doses of 3, 10, and 30 mg/kg twice daily to BALB/c mice subcutaneously engrafted with EMT6 cells, tumor volumes were significantly lower in the demeclocycline-treated groups than in the control group (figure 6B). No significant difference was observed in the therapeutic efficacy of demeclocycline at doses of 3, 10, and 30 mg/kg. This was similar to the results showing that different concentrations of demeclocycline above the effective concentration (0.1 µM) resulted in similar enhancements in T-cell cytotoxicity in vitro (figure 1B).6 Minocycline also exerted the same antitumor effects as demeclocycline (figure 6C). We then examined another murine tumor cell line, CT26 and found that the tumor volumes of CT26 tumor-bearing BALB/c mice were significantly lower in the demeclocycline-treated groups than in the control group (figure 6D). Therefore, the oral administration of tetracyclines exhibited antitumor therapeutic efficacy in murine models subcutaneously engrafted with murine tumor cell lines.
In vivo antitumor T-cell immunity enhanced by demeclocycline
To investigate whether the antitumor efficacy of tetracyclines was mediated by T-cell cytotoxicity, an anti-mouse CD8a inhibitor was administered prior to 3 mg/kg of demeclocycline twice daily in BALB/c mice subcutaneously engrafted with EMT6 cells (figure 7A). Since the anti-mouse CD8a inhibitor completely canceled the antitumor effects of demeclocycline (figure 7A), these effects of demeclocycline were confirmed to be mediated by T-cell immunity. In contrast, anti-mouse CD4 inhibitors exerted antitumor effects (online supplemental figure S12D), which was consistent with previous findings.24 25
In vivo antitumor T-cell immunity enhanced by demeclocycline. (A) A demeclocycline dose of 3 mg/kg was orally administered to BALB/c mice twice a day for 14 days from 5 days after the EMT6 inoculation with 400 µg of an anti-CD8 inhibitor or IgG2a isotype control antibody via an intraperitoneal injection 4 days after the tumor inoculation. Tumor growth curves from days 0 to 19 after the tumor inoculation are shown (N=9 or 10 per group). (B) (left) Antigen-specific gp70-tetramer+ peripheral CD3+ T cells in CT26-engrafted BALB/c mice treated with and without 3 mg/kg of demeclocycline twice daily for 14 days from 5 days after the tumor inoculation were analyzed by flow cytometry on day 19 after the tumor inoculation (N=6). (right) Antigen-specific gp70-tetramer+ tumor-infiltrating CD3+ T cells collected from CT26-engrafted BALB/c mice treated with and without 3 mg/kg of demeclocycline twice daily for 7 days from 5 days after the tumor inoculation were analyzed by flow cytometry on day 12 after the tumor inoculation (N=8). (C–D) Tumor-infiltrating cells collected from CT26-engrafted BALB/c mice treated with and without 3 mg/kg of demeclocycline twice daily for 7 days from 5 days after the tumor inoculation were analyzed by flow cytometry after a 4-hour PMA and ionomycin stimulation on day 12 after the tumor inoculation (N=8). (C) Tumor-infiltrating IFN-γ+ CD8+ T cells from CT26-engrafted BALB/c mice treated with and without 3 mg/kg of demeclocycline twice daily were analyzed by flow cytometry. (D) A CITRUS analysis was performed using the results of a flow cytometry analysis of tumor-infiltrating CD8+ T cells. Clusters defined by the CITRUS analysis are depicted. A red-colored cluster circled in yellow shows a cell subset that had a significantly high abundance in the demeclocycline group. Histograms for selected markers in the identified cluster with significantly large differential expressions are shown. (E) Tumor-infiltrating cells collected from CT26-engrafted BALB/c mice treated with and without 3 mg/kg of demeclocycline twice daily for 7 days from 5 days after the tumor inoculation were analyzed by flow cytometry without the PMA and ionomycin stimulation on day 12 after the tumor inoculation (N=8). A CITRUS analysis was performed using the results of the flow cytometry analysis of tumor-infiltrating CD8+ T cells. Clusters defined by the CITRUS analysis are depicted. Red-colored clusters show cell subsets that had a significantly different abundance among the control and demeclocycline groups. Clusters circled in yellow were abundant in the demeclocycline group. Histograms for selected markers in the identified clusters with significantly large differential expressions are shown. Data are shown as the mean±SEM. A two-tailed Student’s t-test was used to examine the significance of differences between samples (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). DMC, demeclocycline; IFN-γ, interferon-gamma; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; PMA, phorbol 12-myristate 13-acetate; TIL, tumor-infiltrating lymphocyte; GzmB, granzyme B.
We also investigated T-cell immunological changes induced by tetracyclines in vivo. We examined tumor antigen-specific T cells in CT26-bearing BALB/c mice orally administered 3 mg/kg of demeclocycline twice daily. A flow cytometry analysis of PBMCs in CT26-bearing BALB/c mice showed that peripheral tumor antigen-specific gp70-tetramer+ T cells were significantly increased by the administration of demeclocycline (figure 7B). Similarly, demeclocycline slightly increased tumor-infiltrating gp70-tetramer+ T cells in CT26-bearing BALB/c mice (figure 7B). To investigate whether tumor-infiltrating T cells producing IFN-γ were increased by the oral administration of demeclocycline, a flow cytometry analysis of tumor-infiltrating lymphocytes from CT26-bearing BALB/c mice after a co-culture with PMA and an ionomycin stimulation for 4 hours was conducted. Tumor-infiltrating IFN-γ+ CD8+ T cells significantly increased in CT26-engrafted BALB/c mice treated with demeclocycline (figure 7C). A CITRUS analysis using data from the flow cytometry analysis of tumor-infiltrating CD8+ T cells also revealed that a CD8+ T cell subset consisting of IFN-γ+ CD69+ gp70-tetramer+ T cells was abundant in CT26-engrafted mice treated with demeclocycline (figure 7D). Additionally, a CITRUS analysis using data from the flow cytometry analysis of tumor-infiltrating lymphocytes without the PMA and ionomycin stimulation showed that a subset consisting of Ki-67+ gp70-tetramer+ T cells was also abundant in CT26-engrafted BALB/c mice treated with demeclocycline (figure 7E). Therefore, the in vivo antitumor effects of tetracyclines were mediated by T-cell immunity including the activation and proliferation of tumor antigen-specific T cells.
In summary, the present results demonstrated that tetracyclines enhanced antitumor T-cell cytotoxicity in vitro and in vivo. Furthermore, the upregulation of TCR signal transduction was a trigger that augmented T-cell cytotoxicity by minocycline. RNA-seq revealed that GO pathways associated with T-cell activation and antitumor cytotoxicity were upregulated by minocycline. The enhancement in antitumor T-cell immunity by tetracyclines was observed not only in peripheral T cells, but also in lung tumor-infiltrating T cells in mice and patients with NSCLC.
Discussion
In the present study, we found that tetracyclines enhanced antitumor T-cell responses in human peripheral T cells, murine models, and the lung tumor tissues of patients with NSCLC. Tetracyclines enhanced Zap70 signaling, which was not affected by the anti-PD-1 antibody, nivolumab under the same conditions.
In our analysis, the phosphorylation of Zap70 was enhanced by minocycline 5 min after T-cell activation. On the other hand, nivolumab did not enhance Zap70 signaling during that period. Previous findings on the effects of PD-1 on Zap70 phosphorylation are controversial. PD-1/PD-L1 complex formation has been shown to result in the phosphorylation of ITSM (immunoreceptor tyrosine-based switch motif) and ITIM (immunoreceptor tyrosine-based inhibitory motif) in the PD-1 cytoplasmic tail, which recruits Src homology region 2 domain-containing phosphatase 1/2, leading to the dephosphorylation of Zap70.26 A quantitative analysis of the dephosphorylation of Zap70 indicated that Zap70 phosphorylation did not decrease within 10 min of PD-1/PD-L1 complex formation.27 However, anti-PD-1 blockade increased Zap70 phosphorylation in murine CD8+ T cells 48 hours after the stimulation with anti-CD3/CD28 antibodies and PD-L1-immunoglobulin.28 Based on these findings and the present results, tetracyclines enhance Zap70 signaling in T cells during the early phase after T-cell activation, whereas nivolumab appears to inhibit the dephosphorylation of Zap70 at a different phase after the blockade of the PD-1/PD-L1 complex. In the present study, we analyzed Zap70 phosphorylation in peripheral T cells expressing PD-1 at lower levels than lung tumor-infiltrating T cells. Therefore, difficulties were associated with detecting the enhancement in Zap70 phosphorylation by nivolumab in peripheral T cells. The direct target molecule of tetracycline has been suggested to directly affect Zap70 signaling independently of the PD-1/PD-L1 pathway. Due to the different points of action of tetracyclines and nivolumab, we estimated that additive antitumor effects were observed by the combination of these agents. Regarding clinical studies, a retrospective analysis of 690 patients treated with ICIs for advanced cancer showed that tetracycline was positively associated with OS in patients with melanoma, despite the effects of antibiotics having a negative impact on survival.29
In the present study, tetracyclines enhanced CD4+ and CD8+ T-cell responses. Moreover, activation markers of Treg cells were increased by tetracyclines. We did not confirm whether tetracyclines enhanced the suppressive activity of Treg cells. The anti-PD-1 antibody has been shown to bind to PD-1 on Treg cells and enhance the suppressive activity of these cells.28 30 31 The anti-PD-1 antibody is considered to exhibit antitumor efficacy under the condition of predominant effector T-cell activation over Treg cell activation.32 Although tetracyclines may enhance the immunosuppressive activity of Treg cells, the present results indicated that antitumor T-cell responses were enhanced by tetracyclines based on the balance between effector and Treg cells.
Tetracyclines reportedly exhibit anti-inflammatory activity through multiple mechanisms.33 34 They generally exert antibiotic effects by binding to the 30S ribosome and inhibiting protein synthesis by bacteria.35 The anti-inflammatory properties of tetracyclines are considered to be off-target actions. Tetracyclines exhibit anti-inflammatory activity by inhibiting matrix metalloproteinases,36 inducible nitric oxide synthase,37 cyclooxygenase-2,38 and phospholipase A2.39 In the present study, we showed that tetracyclines enhanced Zap70 signaling, which did not directly correlate with previously reported anti-inflammatory activities. Tetracyclines have been shown to inhibit the activation, proliferation, and cytokine production of T cells.40–43 The concentration of minocycline in these in vitro studies ranged between 20 and 25 µg/mL. However, the concentrations of tetracyclines in these studies were higher than those in the blood of humans analyzed by LC-MS/MS after the administration of tetracyclines with antibiotics (135 mg of minocycline).44 The maximum plasma concentration in healthy subjects after the administration of 135 mg of minocycline was approximately 1 µg/mL (equal to 2.2 µM).44 In our analysis, tetracyclines enhanced antitumor T-cell responses at concentrations between 0.1 and 1 µM in vitro, which were also applied to our in vivo experiments. The maximum plasma concentration in mice after the administration of 3 mg/kg of demeclocycline was approximately 0.3 µM in our experiment. The difference observed in the effects of tetracyclines between previous studies and our analysis was attributed to the concentrations of tetracyclines used. In contrast to the 30S ribosome for the target of tetracyclines as antibiotics, we identified galectin-1 as a target for tetracyclines as reagents to enhance antitumor T-cell immunity. We estimated that the optimal concentrations of tetracyclines were different for the interaction with 30S ribosome or galectin-1.
Previous studies reported that tetracyclines caused autoimmune adverse events, indicating T-cell activation by tetracyclines. Autoimmune adverse events induced by tetracyclines include autoimmune hepatitis,45 systemic lupus erythematosus,46 and vasculitis.47 48 Tetracyclines are one of the most frequent drugs to cause autoimmune adverse events, and although the underlying mechanisms remain unclear, activated T cells are reportedly involved. Regarding autoimmune hepatitis, T cells dominantly infiltrate and are activated in liver tissues.49 50 These findings indicate that tetracyclines modulate T-cell activities, leading to autoimmune adverse events.
There are a number of limitations that need to be addressed. Although we demonstrated that tetracyclines enhanced antitumor T-cell responses via the Zap70 signaling pathway, the direct target molecule of tetracycline in this machinery remains unclear. In the present study, we analyzed T cells in the lung tumor tissues of patients with NSCLC. However, the effects of tetracyclines on T cells in the tumor tissues of patients with other cancers have yet to be examined. Based on our murine experiment including murine colorectal and breast cancer cells, tetracyclines appeared to enhance antitumor T-cell responses in the tumor tissues of patients with various cancers.
In conclusion, we showed that tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. Further clarification of the underlying mechanism of action will contribute to the development of novel cancer immunotherapy.
Supplemental material
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
The study protocol was approved by the Institutional Ethics Committee of Osaka University Hospital (IRB number 13266). The retrospective clinical study was approved by the Institutional Ethics Committee of Osaka University Hospital (IRB number 22097). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We acknowledge the NGS core facility at the Research Institute for Microbial Diseases of Osaka University for sequencing and data analyses, the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine for the use of the SPRm 200AP system, Kiko Tech Co., Ltd. for their technical support with the SPRm 200AP system, and Tomomi Isono for the management of clinical specimens. Illustrations were created with https://BioRender.com.
References
Supplementary materials
Supplementary Data
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Footnotes
Contributors MT: Conceptualization, methodology, validation, formal analysis, investigation, data curation, visualization, writing—original draft. KI: Conceptualization, methodology, validation, formal analysis, investigation, data curation, visualization, writing—original draft, writing—review and editing. MH: Methodology, investigation, writing—review and editing. AU: Methodology, investigation, writing—review and editing. AT: Methodology, investigation, writing—review and editing. J-IH: Methodology, investigation, writing—review and editing. YT: Supervision, writing—review and editing. YS: Investigation, writing—review and editing. AK: Supervision, writing—review and editing. HW: Supervision, writing—review and editing. KI is responsible for the overall content as guarantor.
Funding This work was supported by JST SPRING, Grant Number JPMJSP2138, by Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP23ama121054, and by JSPS KAKENHI Grant Number 21K08153.
Competing interests There are no competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.