Scientific Papers

FLT3L-induced virtual memory CD8 T cells engage the immune system against tumors | Journal of Biomedical Science

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Reagents and antibodies

For the generation of Alb-FLT3LL protein constructs, albumin was first amplified with PCR using cDNA template of albumin (NM_000477) purchased from transOMIC technologies (Huntsville, AL) and a set of primers, 5′-AAATCTAGAGCCACCATGAAGTGGGTAACCTTT-3′ and 5′-TTTGAATTCGGCTAAGGCGTCTTTGCATC-3′. The amplified product was then cloned into the XbaI/EcoRI sites of pcDNA3 vector (Invitrogen Corp., Carlsbad, California, USA).

Next, for the generation of pcDNA3-alb-FLT3L, FLT3 ligand was first PCR amplified using cDNA template of human FLT3 ligand (AAA90950.1) gene synthesized from Genscript (Piscataway, NJ) and a set of primers, 5′-TTTGAATTCACCCAGGACTGCTCCTTCCAA-3′ and 5′-AAAGGATCCTCACGAGGTCAGGAGATCGAG-3′. The amplified product was then cloned into the EcoRI/BamHI sites of pcDNA3-alb.

All plasmid constructs were confirmed by DNA sequencing. Albumin-FLT3L proteins were expressed using Expi293F expression system kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. Expi293F cells were transfected with pcDNA3-alb-FLT3L. Proteins were purified by HiTrap Albumin column (GE Healthcare Life Sciences, Marlborough, MA). Recombinant human FLT3L proteins were purchased from GeneScript (Piscataway, NJ).

The antibodies and reagents used in flow cytometry analysis are as follows:

Antibodies

Catalog Number

Company

PE/Dazzle™ 594 anti-mouse/human CD44

103056

BioLegend

APC/Cyanine7 anti-mouse CD49d Antibody

103635

BioLegend

PE/Cyanine7 anti-mouse CCL5 (RANTES) Antibody

149105

BioLegend

PE anti-mouse CD5 Antibody

100607

Biolegend

PerCP/Cyanine5.5 anti-mouse Ly-6C Antibody

128011

BioLegend

FITC anti-mouse CD314 (NKG2D) Antibody

115711

BioLegend

Alexa Fluor® 647 anti-mouse EOMES Antibody

157703

Biolegend

PE/Cyanine5 anti-mouse CD69 Antibody

104510

Biolegend

APC anti-mouse IFN-γ Antibody

505810

Biolegend

Brilliant Violet 421™ anti-mouse Ki-67 Antibody

652411

Biolegend

APC/Fire™ 750 anti-mouse CD8a Antibody

100766

BioLegend

Zombie AquaTM Fixable Viability Kit

423102

Biolegend

Brilliant Violet 650™ anti-mouse CD8a Antibody

100742

Biolegend

Brilliant Violet 785™ anti-mouse CD3 Antibody

100231

Biolegend

APC/Fire™ 750 anti-mouse CD3 Antibody

100247

Biolegend

Brilliant Violet 421™ anti-mouse CD11c Antibody

117330

Biolegend

PE/Cyanine7 anti-mouse I-A/I-E Antibody

107630

Biolegend

PE/Dazzle™ 594 anti-mouse/human CD44 Antibody

103056

Biolegend

CD44 Monoclonal Antibody (IM7), PE

12-0441-82

eBioscience™

FITC Anti-CD8 alpha antibody [KT15]

Ab22504

abcam

T-Select H-2Kb OVA Tetramer-SIINFEKL-PE

TB-5001-M

MBL

Ethics approval

All animals were housed and handled in the animal facility of the Johns Hopkins Medical Institution under specific-pathogen-free conditions. All procedures were performed according to approved protocols by the Johns Hopkins Medical Institutions Animal Care and Use Committee and the National Institutes of Health.

Mice

6-week-old female C57BL/6 mice were purchased from Taconic Biosciences (Germantown, NY). OT-1, and STAT1−/− mice of the C57BL/6 background were purchased from Jackson Laboratories (Farmington, CT). All mice were maintained at the Johns Hopkins University School of Medicine (Baltimore, MD) animal facility under specific pathogen-free conditions.

Cell culture

Mouse melanoma B16-OVA cells were acquired as a generous gift from Dr. Charles Drake and maintained in complete DMEM media supplemented with 10% FBS, 1% l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, 2 mM sodium pyruvate, and 2 mM non-essential amino acid.

In vitro CD44high T cell activation and proliferation

Lymphocytes were taken from the spleen and lymph nodes of C57BL/6, OT-1, and STAT1−/− mice with or without FLT3L treatment. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5% BSA/PBS (FACS buffer). Cells were stained with PE-conjugated CD44 antibodies in the staining buffer, washed, and resuspended in the staining buffer. To assess the functions of CD44high CD8 T cells, CD44-positive cells were isolated using the EasySep™ PE Positive Selection Kit II (Stem Cell Technologies). The isolation efficiency was confirmed by flow cytometry analysis. For the T cell proliferation assay, lymphocytes (1 × 106 cells/ml) were suspended in phosphate-buffered saline (PBS) and labeled with the CellTrace Violet™ (Thermo Fisher Scientific, Waltham, MA) at a concentration of 10 μM at 37 °C for 20 min. After labeling, an excess of 10% RPMI/FBS was added to the samples to quench the reaction. Following centrifugation and extensive washing, cells were resuspended in 200 ml of 10% RPMI/FBS with T cell stimulant: either α-CD3 (1.25 ug/ml) and α-CD28 (0.25 ug/ml) antibodies for lymphocytes from C57BL/6 mice, or OVA257-264 (SIINFEKL, 10 or 50 ng/ml) peptides for lymphocytes from OT-1 mice for 24 to 48 h. Cells were then harvested for analysis by flow cytometry.

Flow cytometry and cell sorting

Blood samples were obtained from the mouse facial vein by lancet and collected into EDTA-coated tubes. Lymphocytes harvested from the lymph nodes or spleen were prepared in single-cell suspensions. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5%BSA/PBS (FACS buffer). Single staining controls of ultracomp ebeads (Thermo Fisher Scientific, Waltham, MA) were used to set a compensation matrix for each experiment. FMO and isotype staining controls were used to set gating and determine non-specific binding. Before antibody staining, Zombie Aqua live/dead (BioLegend, San Diego, CA) was used for dead cell discrimination according to manufacturer instructions. Fc Block was used prior to antibody staining. Antibody and tetramer dilutions were determined through titration. All antibody mixes were stained for at least 30 min at 4 degrees Celsius. Data collection was done on a 13-color Beckman Coulter CytoFLEX S, and analysis was done with FlowJo 10.4 software (FlowJo LLC). BD FACSAria™ Fusion sorter was used for cell sorting at the Johns Hopkins School of Medicine CRB HP Flow Core facility.

Intracellular staining

For intranuclear staining to measure cytokine production, mouse lymphocytes were treated with protein transport inhibitor Brefeldin A (BioLegend, San Diego, CA) for 16 h. Cells were then collected, and prepared as described in the flow cytometry section. At the end of extracellular staining, cells were permeabilized using eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA) and stained for intracellular cytokines.

Adoptive cell transfer

Lymphocytes were collected from the spleen and lymph nodes of C57BL/6 mice with or without administration of FLT3L treatment, and single-cell suspensions were prepared. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5% BSA/PBS (FACS buffer). Cells were stained with fluorescence-conjugated CD3, CD8, and CD44 antibodies in the staining buffer, washed, and then resuspended in the staining buffer. The CD44high CD8 T cells were sorted by flow cytometry for adoptive transfer via retro-orbital injection.

Bulk RNA-sequencing analysis

For bulk RNA-sequencing, CD44high CD8 T cells preparation followed the same procedure as described for adoptive cell transfer. Total RNA was extracted with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer instruction. The preparation of the RNA library and transcriptome sequencing was conducted by Novogene Co., LTD (Beijing, China). Genes with adjusted p-value < 0.05 and |log2(Fold Change) |> 0 were considered as differentially expressed.

Tumor experiments

For the tumor experiments, 2 × 105 B16-ova cells were inoculated subcutaneously in the abdomen of each C57BL/6 mouse. Tumor growth was monitored by digital caliper reading and the size was measured and calculated using the formula length*length*width*0.5. The endpoints of the experiment were determined once the diameter of tumors surpassed 2 cm or when any loss in body weight was observed as per our approved animal protocols. Tumors and draining lymph nodes were harvested for flow cytometry analysis. For tumor infiltration leukocyte analysis, tissue was collected from mice and placed in FACS buffer with magnesium and calcium. After mincing with scissors into ~ 2 mm pieces, tissue digestion enzymes including Collagenase I, Collagenase IV, and DNase I were added to samples and incubated for 20 min at 37 degrees Celsius. Excess 10% RPMI/FBS was then added to the samples to quench the reaction. Following centrifugation, samples were filtered using a 70-mm cell strainer. Then, tumor samples were purified by Ficoll gradient. Cells were resuspended in 10% RPMI/FBS and loaded onto Ficoll-Paque Plus (GE Healthcare Life Sciences, Marlborough, MA) under a centrifugation condition of 400×g at 25 °C for 30 min. The lymphocyte layer was harvested and washed twice with phosphate-buffered saline solution (PBS), followed by centrifugation at 400×g at 25 °C for 10 min. Samples were then counted, plated at equal cell numbers, and prepared for flow cytometry. The draining lymph nodes were dissociated using a syringe plunger and 70 uM filter and washed using FACS buffer. Cell suspensions were then RBC lysed, washed, and resuspended in FACS buffer for downstream analysis.

In vitro dendritic cell-T cell coculture assay

For plasmacytoid (pDC) enrichment, splenocytes were harvested from FLT3L-treated C57BL/6 mice and pDC were isolated according to the manufacturer’s instructions using the EasySep™ Mouse Plasmacytoid DC Isolation Kit (STEMCELL, Vancouver, Canada). After isolation, pDC cells were resuspended in complete RPMI media supplemented with 10% FBS, 1% l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, 2 mM sodium pyruvate, 2 mM non-essential amino acid, and 50 µM 2β-mercaptoethanol. For FLT3L-preconditioned BMDCs preparation, bone marrow was collected from the femurs of C57BL/6 mice after euthanasia. Bone marrow cells were isolated by flushing femurs with complete RPMI media. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in complete RPMI media. Cells were then resuspended in complete RPMI and seeded at 1 × 10cells/ml in a 6-well plate with 20 ng/ml recombinant GM-CSF (Peprotech, Waltham, MA) or 200 ng/ml recombinant human FLT3L (GeneScript, Piscataway, NJ) for 7 days. On day 3, cells received an additional 2 ml of fresh complete RPMI with recombinant GM-CSF or FLT3L proteins. On Day 7, culture media was removed and centrifuged at 300×g for 5 min to recover non-adherent cells. After centrifugation, the supernatant was aspirated, and the BMDCs were resuspended in complete RPMI media.

For naïve CD8 T cell isolation, lymphocytes were harvested from the lymph nodes and spleen, and processed into single-cell suspensions. RBCs were lysed as previously described. Naïve CD8 T cells were enriched according to the manufacturer’s instructions using the EasySep™ Mouse Naïve CD8+ T Cell Isolation Kit (STEMCELL, Vancouver, Canada). After isolation, T cells were resuspended in complete RPMI media. For coculture experiments, different ratios of pDCs or FLT3L-preconditioned BMDCs were incubated with 2 × 105 naïve CD8 T cells in 96-well round-bottom tissue culture plates. After 3 days of coculture, cells were collected and stained with zombie Aqua live/dead (BioLegend, San Diego, CA), CD3, CD8α, and CD44 for flow cytometry analysis.

Statistical analysis

All data are expressed as means ± standard error of the mean (S.E.M). The statistical significance was determined by one-way ANOVA with Tukey–Kramer multiple comparison or Student’s t-test using Prism 9 software (GraphPad, CA, USA). In all circumstances, p-values ≤ 0.05 were considered significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

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