Anti cd4 fitc rm4 5

α-galactosylceramide (α-GalCer) was purchased from Funakoshi (KRN7000).
The antibodies and CD1d tetramer used for cell-surface staining were as follows: α-GalCer-loaded APC-conjugated CD1d tetramer (cat#E001-4B; ProImmune), anti-NK1.1-PE (PK136; BD Biosciences), anti- CD4-FITC (RM4-5; BD Biosciences), anti-CD8-PE (53–6.7; BD Biosciences), anti-CD24-PE (M1/69; BioLegend), antip-CD24-APC (M1/69; BioLegend), anti-CD44-APC (IM7; BioLegend), anti-CD3εantibody-PE (145-2C11; eBioscience), anti-CD3εantibody-violetFluor 450 (17A2; TONBO Bioscience), anti-B220 antibody-PerCP/Cy5.5 (RA3-6B2; BioLegend), anti-IL17Rb-PE (MUNC33; eBioscience), and anti-CD19-PE (eBio1D2; eBioscience). All antibodies were diluted and used according to the manufacturer’s protocols.
A flow cytometric analysis (FACS) was performed using a Gallios flow cytometer (Beckman Coulter) or FACSCalibur cytometer (BD Biosciences), and the results were analyzed using the FlowJo software program (Tree Star).

IFN-γ secretion following twice-stimulation with antigens in vivo and in vitro sequentially was performed [28 (link)]. Firstly, mice were stimulated with BCG (Danish 1331, 1 × 10⁶ CFU/dose) by intraperitoneal injection (i.p.) at 20 weeks after the final immunization. Nine days later, lymphocytes were isolated and stimulated with mixed antigens of ESAT-6, Ag85B, Rv2626c and HspX (2 μg/mL of each protein) for 4 h in vitro. Then, cells were incubated for 5–6 h with BD GolgiPlug™ (including brefeldin A, BD, USA) at 37 °C. Subsequently, cells were stained with anti-CD4-FITC (RM4-5, BD, USA) and anti-CD8-PerCP-Cy5.5 (53-6.7, BD, USA) at 4 °C for 30 min. Later, cells were permeabilized (Cytofix/Cytoperm kit, BD, USA) and intracellular cytokine (ICC) staining of anti-IFN-γ (XMG1.2, BD, USA) was performed at 4 °C for 30 min as previous reported [29 (link)]. All samples were run on ACEA NoveCyte.