Anti h 2kb

The following antibodies were used for flow cytometry analysis: anti-IFNγ (XMG1.2, 1:50), anti-CD45.1 (A20, 1:100), anti-CD45.2 (104, 1:100), anti-IL1β-APC (NJTEN3, 1:50), anti-CD44 (IM7, 1:100), anti-CD62l (MEL-14, 1:100), anti-F4/80 (BM8, 1:100), anti-CCR7 (4B12, 1:100), anti-CD4 (RM4-5, 1:200), anti-CD8α (53-6.7, 1:200), anti-H-2K^b (AF6-88.5.5.3, 1:200), anti-Ly6G (RB6-8C5, 1:100), anti-MHC class II (I-A/I-E) (M5/114.15.2, 1:100) and anti-CD11c (N418, 1:100) were from eBioscience. Anti-CD103 (2E7, 1:100) was from Biolegend. Annexin V apoptosis Detection kit (556547) was from BD Pharmingen. For intracellular cytokine staining, Cytofix/Cytoperm plus kit (BD, cat. 55508) was used. For Foxp3 staining, Foxp3 staining buffer set (eBioscience, cat. 00-5523-00) was used. Multiple-colour flow cytometric analysis was performed using FACSAria (BD Biosciences; Franklin Lakes, NJ, USA). For FACS sorting, cells stained with FACS
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.

Cells were stained with antibodies from Biolegend (anti-CD4, RM4-5; anti-CD8, 53-5.8; anti-CD11b, M1/70; anti-CD11c, N418; anti-Ly6G, 1A8; anti-Ly6C, HK1.4; anti-Gr1, RB6-8C5; anti-MHC-II, M5/114.15.2; anti-CD40, 3/23; anti-CD80, 16-10A1; anti-CD86, GL-1; anti-CX3CR1, SA011F11; anti-PDL1, MIH7; anti-IL4, 11B11; anti-CD44, IM7; anti-CD62L, MEL-14; anti-Ki67, 11F6; anti-CD25, PC61; anti-H2Kb, AF6-88.5; anti-H2Kd, SF1-1.1), eBioscience (anti-FoxP3, FJK-16S). For intracellular staining, FoxP3/Transcription Factor Staining Buffer Set was used (eBioscience, 00-5523-00). Flow cytometry was determined by Canto-II instrument (BD) and analyzed by FlowJo v10.
For cell sorting, CD11b⁺ cells were pre-enriched with anti-CD11b microbeads (Miltenyi Biotec, 130-097-142). Enriched CD11b⁺ cells were stained with anti-CD11b and anti-Gr-1 for FACS by using an MoFlo Astrios system (Beckman Coulter).
Detailed reagents information is listed in Table S2.

To investigate the cytotoxic effect of Rv3841 on the DCs, DCs (1 × 10⁶ cells/mL) were incubated with 10 μg/mL Rv3841 for 24 h. After 24 h of treatment, the cells were stained with FITC-conjugated annexin V and PI from R&D Systems (Minneapolis, MN, USA). Cell toxicity was detected according to the manufacturer's instructions. Then, samples were detected on the FACSCanto II with FACSDiva and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA). To investigate the surface molecules, after 24 h of Rv3841 protein treatment, the cells were stained with PE-conjugated anti-CD80, anti-CD86, anti-H-2Kb (MHC class I), and anti-I-Ab (MHC class II) with FITC-conjugated anti-CD11c antibodies from eBioscience (San Diego, CA, USA) for 30 min at 4°C. The fluorescence was measured by flow cytometry.