I a 1 e pe

The mouse anti-CHIKV-nsP2 monoclonal antibody, used in this study was developed by us [58 (link)]. The anti-CHIKV-E2 monoclonal antibody was a kind gift from Dr. Manmohan Parida, DRDE, Gwalior, India. HRP linked secondary antibodies, H-2k^d PE, I-A/I-E PE, isotype PE, isotype APC and HSP90 antibodies were purchased from BD Biosciences (San Jose, CA, USA). CD86 APC and CD80 APC were purchased from eBiosciences (San Diego, CA, USA). The monoclocal antibodies for cleaved caspase-3 (Asp175), cleaved caspase-8 (Asp387) and caspase-9 (C9) were purchased from cell signaling technology (Danvers, MA, USA). The anti-mouse Alexa Fluor 488 was purchased from Invitrogen (Carlsbad, CA, USA). Mouse IgG1 isotype control, GAPDH and β-actin were purchased from Abgenex India Pvt. Ltd. (Bhubaneswar, India). Saponin, Anisomycin and Bovine serum albumin fraction V were purchased from Sigma-Aldrich. 17-Allylaminogeldanamycin (17-AAG) and Z-VAD-FMK were purchased from Merck Millipore (Billerica, MA, USA).

Tumor pieces were fixed overnight with Periodate-Lysine-Paraformaldehyde [48 (link)] at 4°C and Immunofluorescence on tumor slices was performed as previously described [17 (link), 49 (link)]. Immunostaining was performed by first blocking Fc receptors with anti-FCR (BD Pharmingen), then staining for 1 h RT or at 4°C overnight was performed with primary antibodies specific for CD8-PerCP, CD45-APC, IA-IE-PE, CD11b-FITC, Ly6C-APC, CD31-biotin (all from BD Pharmingen), fibronectin, gp38/podoplanin, F4/80-biotin (all from Biolegend) or F4/80-PE (AbD Serotec). Immunodetection was performed using anti-Rat or anti-rabbit antibodies coupled to 488, 568 or 647 (BD Pharmingen) and streptavidin- Alexa Fluor 488 or 647 (Invitrogen). Slices were then counter-stained with Hoechst for 10 min at room temperature. Antibodies were diluted in PBS, 0.5% BSA, 2% human serum. Images were obtained with a spinning disk microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x and a 63x objective. All images were acquired with MetaMorph 7 imaging software (Molecular Devices) and analysed with ImageJ. For staining of dying cells in vivo, vaccinated TC1-GFP bearing mice received an intra-tumoral injection of propidium iodide (1 mg/ml) and were sacrificed 30 min later.

Cells were washed with PBS (Life Technologies, Darmstadt, Germany) and dead cells were excluded by viability dye staining (eBioscience^TM Fixable Viability Dye eFluor^TM 506) for 20 min on ice. Prior to the surface staining of CD8⁺ T cells or BMDCs, cells were washed twice with staining buffer (1% BSA, 0,02% NaN₃ in PBS). To analyze activated CD8⁺ T cells, cells were stained for CD8 using anti-CD8-PB (eBioscience) for 30 min on ice. Thereafter, intracellular staining was performed, as described below. Alternatively, BMDCs were analyzed for their SIINFEKL/K^b-loading ability using anti-CD11c-PE, -I-A/I-E-PB, -Kb-PE/Cy7, and -SIINFEKL/H2-K^b-PE/Cy7 (all from eBioscience) or for their maturation phenotype using anti-CD11c-APC/Cy7, -CD86-APC, I-A/I-E-PE antibodies (all BD Pharmingen), or anti-CD40-PB antibody (Biolegend). Antibodies were added after blocking of Fc-receptors using CD16/32 antibodies (Fc-Block^TM, BD Biosciences). After incubation on ice for 30 min, cells were washed and fixed with 1% paraformaldehyde and stored at 8°C until flow cytometry was performed using BD FACS Canto II (BD Biosciences, Heidelberg, Germany).