Fitc anti mouse cd4 antibody

To measure expressions of surface molecules, cells were stained with FITC anti-mouse major histocompatibility complex (MHC)-II antibody (1:200; Biolegend, 107606), BV510 anti-mouse CD3 antibody (1:200; Biolegend, 100234), FITC anti-mouse CD4 antibody (1:200; Biolegend, 100405) or APC anti-mouse CD25 antibody (1:200; Biolegend, 101909) in FACS buffer for 40 min. For intracellular staining, cells were permeabilized, followed by fixation using the Foxp3 staining buffer kit prior to staining with PE anti-mouse Foxp3 antibody (1:100; Biolegend, 320007), BV421 anti-mouse Foxp3 antibody (1:100; Biolegend, 126419), or PE-conjugated S100A9 antibody (1:100; Cell Signaling Technology, 93941). Flow cytometry analyses were performed on a LSR II instrument and raw data were retrieved. Results were analyzed by use of FlowJo software version 10.0.

Antigen-specific T cell immune responses were assayed on a multicolor flow cytometer (BD Biosciences). After collecting the splenocytes from immunized or unimmunized mice, 2 × 10⁶ cells (100 μL) per sample were stimulated with the SARS-CoV-2 S-protein peptides for 4 h at 37 °C. Brefeldin A (Thermo Scientific) was then added into splenocytes and incubated for 6 h. Stimulated cells were washed in PBS/0.5% BSA and stained with APC/Fire 750 anti-mouse CD3 antibody (BioLegend, San Diego, CA, USA), FITC anti-mouse CD4 antibody (BioLegend), and Brilliant Violet 510 anti-mouse CD8a antibody (BioLegend) surface markers. The cells were then fixed using a Fixation/Permeabilization Solution Kit (BD Biosciences) and stained with PE anti-mouse IFN-γ antibody (BioLegend), PE anti-mouse IL-2 antibody (BioLegend), and PE anti-mouse IL-4 antibody (BioLegend). Data were analyzed with FlowJo software.

CSPCM was provided by HeBei TaiFeng Biotechnology Co., Ltd. (Handan, China). CTX CAS#6055-19-2(C₇H₁₅Cl₂N₂O₂P·H₂O) was purchased from Source Leaf Biotechnology Co., LTD(Shanghai, China). Eastep® Super Total RNA Extraction kit was purchased from Promega (Beijing) Biotechnology Co., LTD (Beijing, China). PrimeScript™ RT reagent Kit with gDNA Easer, TB Green® Premix Ex Taq™ II(Tli RNaseH Plus) were purchased from Takara Biomedical Technology (Beijing) Co., LTD (Beijing, China). BCA Protein Assay Kit was purchased from Beijing ComWin Biotechnology Co., LTD. (Beijing, China). SDS-PAGE Gel preparation kit was purchased from Biomed Genentech Inc. (Beijing, China). Rabbit Anti-Foxp3 Polyclonal Antibody, Rabbit Anti-RORγt Polyclonal Antibody and Mice Anti-GAPDH Polyclonal Antibody were purchased from Beijing Bioss Biotechnology Co. LTD (Beijing, China). FITC anti-mouse CD3 Antibody, APC anti-mouse CD4 Antibody, PE anti-mouse IL-17A Antibody, FITC anti-mouse CD4 Antibody, APC anti-mouse CD25 Antibody, PE anti-mouse FOXP3 Antibody, True-Nuclear™ Transcription Factor Buffer Set, True Nuclear TM 4X Fix Concentrate, True Nuclear TM 10X Per and True Nuclear TM Fix Diluent were purchased from BioLegend, Inc (California, United States).

Blood was collected from mice (50 μL in each staining tube). Each tube received 1 × Lysis Buffer (Biolegend, San Diego, CA, USA) followed by incubation at 37 °C for 10 to 15 min. The cells were centrifuged at 300 × g for 5 min. The supernatant was removed and the cells were stained with peridinin chlorophyll protein complex (PerCP) anti-mouse CD45 antibody, APC anti-mouse CD3 antibody, PE anti-mouse CD8b antibody, FITC anti-mouse CD4 Antibody, True-Nuclear™ One Step Staining Mouse Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD25 PE/CD4 PerCP), and FITC anti-mouse IgG1 antibody, PE anti-mouse IgG1 antibody, and APC anti-mouse IgG1 antibody (all from Biolegend, San Diego, CA, USA) as an isotype control. Samples were run on a BD Canto II flow cytometer. The percentage of proliferating T cells was analyzed using FlowJo version 10.

A total of 60 µL blood was collected and stained with 10 µL E7-specific or p15E-specific tetramer (both provided by NIH tetramer core facility) to a final 500 times dilution for 1 hour on ice with constant shaking. Aqua fluorescent reactive dye for live and dead staining (Invitrogen, catalog no. L34957) in a final 500 times dilution, FITC anti-mouse CD4 Antibody (BioLegend, catalog no. 100406) and APC anti-mouse CD8a Antibody (BioLegend, catalog no. 100712) in a final 200 times dilution was added to the testing samples to a total volume of 100 µL and incubated for 30 minutes on ice with constant shaking. After one wash with cold PBS and centrifugation at 1,350 rpm for 3 minutes, the cells were resuspended in 200 µL FACS buffer and ready for examination by using BD LSRFortessa X-20 cytometer. Data were processed by Flowjo (version 10).

The mice were divided into five groups, the PBS group (NS), the OVA (20 µg per mouse) mixed with free CpG (20 µg OVA plus 8 µg CpG per mouse, OVA/CpG) group, the OVA mixed with alum adjuvant (20 µg OVA plus 100 µg Alum adjuvant per mouse, OVA/Alum) group, the OVA (0.2 mg/mL) admixed with the Ncom Gel (20 µg OVA plus 100 µL Ncom Gel per mouse, OVA/Ncom Gel) group and the Ncom Gel only (100 µL Ncom Gel per mouse) group. Female C57BL/6 mice were immunized on the 1^st, 15^th and 22^nd days. Next, spleen single-cell suspensions were obtained from immunized mice seven days after the last immunization, seeded in 24-well plates, restimulated with OVA (50 µg/mL) and incubated for 72 h. After incubation, spleen cells were centrifuged and washed twice, then the cultures were collected for IFN-γ detection. The obtained cells were stained with FITC anti-mouse CD8a antibody and FITC anti-mouse CD4 antibody (Biolegend, San Diego, CA, USA), respectively. Then, the labeled cells were fixed with paraformaldehyde for 30 min and washed twice. Then, the fixed cells were permeabilized with Triton X-100 (Sigma-Aldrich, Saint Louis MO, USA) and stained with PE anti-mouse IFN-γ antibody (Biolegend, San Diego, CA, USA). Finally, the cells were resuspended and analyzed by flow cytometry (ACEA NovoCyte, San Diego, California, USA).

C57BL/6 mice were purchased from Clea (Clea Japan, Inc., Tokyo, Japan). Total CD4⁺ T cells isolated from mouse spleen were prepared using a FITC anti-mouse CD4 antibody (Cat#100510, RM4–5, BioLegend) and anti-FITC-microbeads (Cat#130-097-050, Miltenyi Biotec). Naive CD4⁺ T cells (CD44^low, CD62L^high) were prepared using a MojoSort Mouse CD4 T cell Isolation Kit (Cat#480033), BioLegend). Biotinylated anti-CD44 (Cat#103004, IM7) and anti-CD25 antibodies (Cat#102004, PC61) were purchased from BioLegend. CD4⁺ T cells (7.5 × 10⁵) were stimulated using immobilized anti-TCR-β mAb (3 µg/mL, H57–597, BioLegend) plus an anti-CD28 mAb (1.0 µg/mL, cat#40–0281, TONBO Biosciences) for 2 days, and then, the cells were further expanded under similar conditions for an additional 3 days. IL-2 conditions: IL-2 (10 ng/mL, Pepro Tech); Th2 conditions: IL-2 (10 ng/mL, Pepro Tech), IL-4 (1 ng/mL, Pepro Tech), and anti-IFN-γ mAb (2.5 µg/mL, cat#40–731, TONBO Biosciences). Th1 conditions: IL-2 (10 ng/mL, Pepro Tech), IL-12 (1 ng/mL, Pepro Tech), and anti-IL-4 mAb (2.5 µg/mL, cat#40–731, TONBO Biosciences). The cultured cells were subjected to intracellular staining, ELISA assay, and RT-PCR. All animal experiments received approval from the Ehime University Administrative Panel for Animal Care. All animal care was conducted in accordance with the guidelines of Ehime University.