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.

MethodsChemical 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.

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).

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).

ResultsT-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).

Figure 1

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).

Figure 2

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.