CA and ADJ (2 cm away from tumor) were collected by experienced operators from 11 breast cancer patients who underwent surgical treatment in the General Surgery Department of Beijing Friendship Hospital, Capital Medical University. The sample volume is about 1 cm in diameter and weighs 100-200 mg. The specimen was stored in pre-cooled RRMI-1640 (Corning) medium and transferred to the laboratory for prompt processing immediately. EDTA tubes were applied to collect 4 ml paired peripheral blood of the patients (PB) within 4 hours before surgery, and 5 peripheral blood samples were obtained from healthy volunteers (HB). This study was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University, and all participants signed the written informed consent.
Tissues were processed into 1 mm3 fragments by surgical scissors, mixed with HBSS (Solarbio Science &Technology Co. Ltd., Beijing, China) containing 0.03% type IV collagenase (Sigma), 0.01% DNase I (Sigma), 10% fetal bovine serum (FBS) (Gibco), and digested by Gentle-MACS dissociator with B-01 mode at 37 ° C for 45 minutes. Then 10ml Dulbecco’s phosphate buffered saline (DPBS) containing 2%FBS was added to neutralize digestive enzymes. Tissues were filtered through 50-mesh and 70-mesh strainers, washed with DPBS containing 2%FBS, centrifuged at 300g for 4 minutes to collect precipitates. Peripheral blood mononuclear cells (PBMC) were prepared by Ficoll (DAKEWE) gradient centrifugation.
Single cells for CyTOF were stained with 0.5uM cisplatin (Fluidigm), washed twice, fixed with 1.6% paraformaldehyde (Sigma) for 10 minutes at room temperature, then resuspended with cryopreserved solution containing 10% Dimethyl sulfoxide (Sigma) and cryopreserved.
Conjugated antibodies and multi-metal labeling kits were purchased from Fluidigm. Pure-antibodies were purchased from Biolegend and conjugated with Maxpar multi-metal labeling kits in our laboratory according to the manufacturer’s instructions. The CyTOF antibody list is shown in Table S1. The cryopreserved cells were thawed and rinsed twice with cell staining buffer (CSB, Biolegend), and stained with antibodies targeting cell surface receptors at room temperature for 30 minutes. After incubation, the samples were processed with nuclear antigen staining perm (Fluidigm) for washing and membrane breaking and then incubated with antibodies targeting intracellular molecules at room temperature for 30 minutes and washed twice by DPBS. Then cells were resuspended using Ir Intercalator (Fluidigm) and stored at 4℃ overnight, washed twice with CSB and Distillation-Distillation H2O the next day, suspended with 10% EQTM Four Element Calibration Beads (Fluidigm) for loading.
CyTOF data acquisitions and analysis
CyTOF data were obtained by a HeliosTM mass cytometer (Fluidigm) from the Beijing Institute of Hepatology and analyzed visually using viSNE (Amir el, Davis et al. 2013). viSNE is a dimensional-reduction method implemented by Barnes-Hut acceleration of the T-SNE algorithm (Kobak and Berens 2019). Cytobank (http://www.cytobank.org/), FlowJo Software V10 (Treestar, Ashland, OR, USA), and R3.6.1(http://www.R-project.org) were applied for data analysis.
Humanized TNBC cell line MDA-MB-231 and HER2+ cell line SK-BR-3 were purchased from China Infrastructure of Cell Line Resource. The cells were cultured with RPMI-1640 medium containing 10%FBS in a constant temperature incubator at 37℃ and 5%CO2. The cells were stored in a -80℃ refrigerator or in liquid nitrogen using serum-free cryopreserved solution (Cellregen).
Human DNT expansion
15 ml peripheral blood from healthy volunteer was collected for DNT expansion. This study was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (2021-P2-064). All participants has signed the written informed consent. PBMC was collected via Ficoll gradient centrifugation. CD4 and CD8 cells were removed with negative sorting kit (STEMCELLTM Technology) to enrich DNT cells from PBMC (Merims, Li et al. 2011). X-VIVO (Lonza) medium was used to purify and expand DNT cells. DNT cells were cultured in 24-well plates coated with purified anti-CD3 (5ug / mL) (Biolegend) for 5 days, adding human recombinant IL-2 (25ng / mL) (peprotech) and purified anti-CD28 (3ug / mL) (Biolegend) at D0, D3, and D5 to the medium. Fresh X-VIVO medium containing IL-2 (25ng / mL) and anti-CD3 (100ng / mL) was added on D7, D10 and D13. DNT cells cultured for 7 to 14 days were used for subsequent experiments.
All fluorochrome-conjugated anti-human antibodies against CD45 (Cat:304062), CD3 (Cat:300306), CD4 (Cat: 317444; Cat: 301051), CD8 (Cat:300539), LAG3 (Cat:369206), PD1 (Cat: 329918), NKG2D (Cat:320808), HLA-DR (Cat307604:), CD274 (Cat: 329708), MICA / MICB (Cat: 320906), Annexin V (AV) (Cat: 422201), Human TruStain FcX™ (Cat: 422302) were purchased from Biolegend company. Data were collected using the Aria II Flow Cytometer (BD Biosciences), Attune NxT (Thermo), or Cytoflex (Beckman) and analyzed by FlowJo Software V10.
DNT cells and MDA-MB-231 or SK-BR-3 cells were co-cultured in 96-well plates for 24h with the effector to target (E:T) ratios of 0:1, 1:1, 2.5:1, 5:1, 10:1. RPMI-1640 complete medium was used in this experiment. In some assays, DNT was pre-stimulated with LAG3Ab (10ug / mL) (offered by Innovent Company) for 1 hour prior to co-culture. The specific killing of DNTs against breast cancer cells was calculated by:. In co-culture systems, the apoptotic breast cancer cells were defined as CD45-Annexin V+.
Quantitative real-time PCR
DNT cells were cultured in X-VIVO medium with or without LAG3Ab (10ug / ml) for 48 hours, and washed twice with DPBS. Total RNA was extracted using Trizol (Sigma) reagent and cDNA was synthesized by Prime scriptTM RT Reagent Kit (Perfect Real Time) (TAKARA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed by ABI 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). mRNA relative expression was calculated by . The genes and primer sequences are shown in Table 1.
TCGA data analysis
TCGA data were calculated by some interactive web resources. Lag3 expression in subgroups of breast cancer were analyzed by UALCAN (http://ualcan.path.uab.edu/) (Chandrashekar, Bashel et al. 2017). The clinical outcome relevance between lag3 expression and cancer subtypes was determined by “outcome module” of the tumor immune estimation resource (TIMER 2.0, http://timer.cistrome.org/) (Li, Fu et al. 2020). Cox regression was used in this section, and the hazard ratio, p value for Cox model, and the log-rank p value for KM curve are shown on the KM curve plot. We also applied TIMER 2.0 to explore the correlation of LAG3 (lag3), PD1 (pdcd1), NKG2D (klrk1) expression with immune infiltration level in breast cancer. The partial Spearman’s correlation was conducted to perform this association analysis. “Gene_Corr module” of TIMER 2.0 was used to explore the correlation between interested genes in TNBC. Gene expression profiling interactive analysis (GEPIA, http://gepia.cancer-pku.cn/) analyzed the correlation of the pair-wise gene expression in breast cancer. Pearson was utilized in this calculation (Tang, Li et al. 2017). Relations between abundance of tumor-infiltrating lymphocytes and lag3 expression in diverse cancer types were evaluated by TISIDB (http://cis.hku.hk/TISIDB/) (Ru, Wong et al. 2019).
Experimental data was calculated by Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Comparisons were made by the student’s t test and one-way ANOVA analysis. *p < 0.05; **p < 0.01; ***p < 0.001. Data represent SEM.