Anti cd4 antibody

Fresh cell suspension from the spleen was firstly stained with Fixable Viability Stain 700 (BD), anti-CD4 antibody (Biolegend) and anti-CD8 antibody (eBioscience); then PE Mouse Anti-Ki-67 Set (BD Pharmingen™, cat# 556027) was used to detect Ki-67 level in T cells.

Freshly isolated cells were incubated with Fc Block for 25 min at 4 °C and then stained with anti-CD4 antibody (#GK1.5, BioLegend), anti-CD25 antibody (#PC61, Biolegend) and anti-CD3e antibody (#145-2C11, Biolegend). Intracellular staining of Foxp3 was conducted by using the Foxp3/transcription factor staining buffer set, according to the instructions provided by the manufacturer (eBioscience). For Foxp3 staining, Foxp3-antibody (#fjk-16s, eBioscience) was added, followed by additional double washing. The washed cells were analyzed with FACSVerse (BD Biosciences). Tregs were identified by gating on the CD4⁺ Foxp3⁺ cell population.

TC-1-GSDMD-NT cells on the slides were collected and treated with 4% paraformaldehyde containing 0.1% Triton X-100 for 10 min, followed by three washes with PBS. Then, the cells were blocked with 2% BSA in PBS for 1 h and incubated with rabbit polyclonal anti-GSDMD antibody (Affinity, AF4012; 1:100 in 2% BSA) for 2 h at RT, followed by three washes with PBS. Next, the samples were incubated with FITC-conjugated secondary antibody (1:2,000 in 1% BSA, Proteintech) for 2 h at RT. After washing five times with PBS, the nuclei were stained with DAPI (ab104139, Abcam) for 10 min. All of the samples were examined by confocal microscopy. Tumor tissues were collected and fixed in formalin, embedded in paraffin, and sectioned. After deparaffinization and hydration, the slides were immersed in EDTA antigen retrieval buffer. Then, the slides were incubated with anti-CD4 antibody (Biolegend) and anti-Foxp3 antibody (Biolegend) or rabbit polyclonal anti-GSDMD antibody (Affinity, AF4012, 1:100) overnight at 4 °C. Next, Cy3-conjugated secondary antibody (Servicebio, GB21303; 1:2,000 in 2% BSA) was added, followed by incubation at room temperature for 1 h. Then, the slides were incubated with DAPI (ab104139, Abcam) at RT for 10 min. All of the sections were examined by fluorescence microscopy.

To deplete T cells, on days 9 and 13 after final immunization, mice were intraperitoneally injected with 200 μg of anti-CD4 antibody (clone: GK1.5), isotype control antibody for anti-CD4 antibody (clone: RTK4530; BioLegend), 200 μg of anti-CD8 antibody (clone: 53.6.7), isotype control antibody for anti-CD8 antibody (clone: 2A3; BioXcell, West Lebanon, NH). To deplete alveolar macrophages on day 1 after final immunization, mice were intranasally injected with 30 μL of clodronate-loaded liposomes (Clophosome A, FormuMax Scientific Inc., Sunnyvale, CA, USA) or control liposome (FormuMax Scientific Inc.) under anesthesia. On 10 days after the final immunization, mice were intranasally challenged with 1.2 × 10³ TCID₅₀ of PR8 in 30 μL PBS under anesthesia. Body weights and survival of challenged mice were monitored.

Cucurbit[n]urils (n = 6, 7 and 8) were synthesized, purified and kindly provided by the Nikolaev Institute of Inorganic Chemistry (Novosibirsk, Russia). Before the experiments, CB[n] was diluted in the culture medium RPMI-1640. CB[6] and CB[7] were prepared at a concentration 0.3, 0.5 and 1 mM; CB[8] at 0.01 mM and in a PBS solution; CB[6] and CB[7] at 0.2, 0.5, 1 and 2 mM; CB[8] at 0.01 mM. RPMI-1640 (Biowest LLC, Riverside, MO, USA), gentamicin (Dalfarma, Khabarovsk, Russia), tienam (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA), FCS (Hyclone, Chicago, IL, USA), tabs for preparing phosphate-buffered saline (PBS) (AppliChem GmbH, Darmstadt, Germany), Na2EDTA (Helicon, Moscow, Russia), Ficoll (BioClot, Aidenbach, Germany), urografin 76% (Schering, Berlin, Germany), human albumin solution (Microgen, Moscow, Russia), Triton^™ X-100 (Dow Corning, Midland, MI, USA, WST-1 assay kit (Takara Bio, Kusatsu, Japan), LDH-Cytox™ Assay Kit (BioLegend, San Diego, CA, USA), PE Annexin V Apoptosis Detection Kit with 7ADD (BioLegend, San Diego, CA, USA), anti-CD3 antibody (BioLegend, San Diego, CA, USA) and anti-CD4 antibody (BioLegend, San Diego, CA, USA).
The Ethical Committee of RIFCI, Russia, approved the study design and the recruitment of subjects. Subjects provided written informed consent. The relevant guidelines and regulations were followed when performing the experiments.

Freshly isolated PBMCs were seeded in 48-well plates at a final concentration of 2 × 10⁶ cells/ml in complete RPMI1640 medium supplemented with 2 mM L-glutamine, 100 IU/100 mg/ml penicillin/streptomycin (Sigma), and 10% fetal bovine serum (Gibco) and were stimulated by the cell stimulation cocktail (500X) containing phorbol 12-myristate 13-acetate and ionomycin (eBioscience, ThermoFisher) for 4 h at 37°C. After washing with SB, the cells were surface-stained with anti-CD4 antibody (clone: RPA-T4, BioLegend) for 20 min on ice. Then, the cells were permeabilized and fixed with a fixation/permeabilization solution (BD Bioscience) according to the manufacturer's instructions. The cells were stained with IL-21 PE (clone: 3A3-N2), IL-4 Alexa Fluor 488 (clone: 8D4-8), IL-17A PE (clone: eBio64), IFN-γ Alexa Fluor 488 (clone: 4S.B3), or IL-10 Alexa Fluor 488 (clone: JES3-9D7, eBiosciences Thermofisher) for 30 min on ice. The results were acquired by flow cytometry as discussed above.

Density-gradient centrifugation was used to isolate PBMCs from healthy donor samples or patients with MM by Ficoll-Paque (General Electric). T cells were isolated from PBMCs with anti-CD3 microbeads (Miltenyi Biotec, 130-050-101) and were cultured in AIM V Medium (Thermo Fisher Scientific) with 5% human AB serum (MilliporeSigma) and 400 IU IL-2 (R&D Systems). Dynabeads of human T-activator CD3/CD28 (Thermo Fisher Scientific, 11161D) were added for T cell expansion and activation (26 (link)). T cells were activated for 2–3 days prior to transduction.
Lentivirus particles were used to transduce T cells. T cells and concentrated lentiviruses were added into RetroNectin-precoated plates (Takara Bio) (26 (link)). Cells were cultured in AIM V Media for 24 hours, and the transduction step was repeated. After 24 hours, cells were washed with PBS and cultured in fresh media for 7 days. CAR-T cells were detected by BD FACSVerse flow cytometry with anti-CD34 antibodies (BioLegend, clone: 561, no. 343606), anti-CD3 antibodies (BioLegend, clone: HIT3a, no. 300318), and CD34 microbeads (Miltenyi Biotec, 130-046-703) for isolation. CD4 or CD8 phenotypes of CAR-T cells were detected by flow cytometry using anti-CD4 antibodies (BioLegend, clone: RPA-T4, no. 300508) and anti-CD8 antibodies (BioLegend, clone: SK1, no. 344722).