Facsymphony a5

For measuring mitochondrial ROS, cells were stained with MitoSOX red mitochondrial superoxide dye (Thermo Scientific) at the concentration suggested by the manufacturer for 30 min at 37 °C. For calreticulin staining, cells were stained with calreticulin antibody (1 μg/ml) (Abcam, ab92516) for 30 min at RT, followed by staining with Alexa Fluoro 488 goat-anti-rabbit antibody (Invitrogen) for 30 min at RT. Stained cells were analyzed on BD FACSCanto II (BD Biosciences) with data analyzed by FlowJo (Tree Star). Residual tumors were resected from mice. Dissociation of tumor tissues into single cells was performed according to our previous protocol [20 (link)]. Dissociated single cells were stained with LIVE/DEAD Fixable Red Dead Cell Stain (Invitrogen, L34972) and antibodies of immune markers listed in Supplemental Table 2. Immunophenotyping was performed on Quanteon Flow Cytometer (Novocyte) and FACSymphony A5.2 (BD), and data analysis was performed using Flowjo (Tree Star). Gating strategies were included in Suppl. Fig. 19.

Flow cytometric analysis will be performed on a single centralised (Newcastle University) BD FACSymphony A5 flow cytometer (Becton Dickinson Biosciences, San Jose, California, USA, from here BDB). Daily internal quality control will be performed using Cytometry Setup and Tracking beads (BDB).
Leucocyte staining will be performed using antibody:fluorophore conjugates supplied by BDB. Samples will be processed according to a standard operating procedure.
At each time point, serum will be collected for measurement of PCT and CRP. These will be batched and processed by ELISA at the end of the recruitment period.

For H2-K^b Ova_257-264 (SIINFEKL)-specific CD8⁺ T-cell analysis, spleen cells were obtained 12 days after tumor inoculation and stained with AF647-coupled dextramer loaded with Ova_257-264 peptide (Dex^OT–I; Immudex, Copenhagen, Denmark), FITC-anti-CD11b, PerCP-anti-CD43, BV421-anti-CD8 (all from BioLegend, San Diego, CA, USA), Fixable Viability Dye eFluor 780, PE-Cy7-anti-CD62L (eBioscience), and BV510-anti-CD44 (Becton-Dickinson).
The frequency of CD8⁺ T cells specific for the H2-D^b-restricted Leader-Gag-derived epitope GagL_85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetI^GagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL_85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown in Supplementary Figure 2.

To generate single-cell suspensions for flow cytometry, a modified digestion protocol was used as previously described.^{39 (link)} Cell suspensions were stained with a previously established flow cytometry panel.³¹ Samples were acquired using a BD FACSymphony A5 (Becton Dickinson) and analyzed using FlowJo 10.2 (Treestar, Ashland, OR) software (www.flowjo.com).

U2OS cells with a stably integrated DR-GFP reporter^{71 (link)} were transfected with the indicated siRNAs 48 h prior to infection with adenovirus expressing I-SceI restriction enzyme at an MOI of 10. The percentage of GFP-positive cells was determined 48 h after infection by flow cytometry using a BD FACSymphony A5 (Becton Dickinson). Data were collected using BD FACS Diva software (v9, Becton Dickinson), and analysis was performed using FlowJo Software.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque™ Plus 206 (Amersham Pharmacia Biotech, Amersham, UK) density-gradient centrifugation and immediately frozen and stored in liquid nitrogen until use. The freezing medium contained 90% Fetal Bovine Serum (FBS) and 10% DMSO. Cells were stained with the appropriate combination of fluorochrome-conjugated antibodies to identify T-cell and B-cell subsets according to standard techniques. B-cell populations were identified as CD19+. Memory B cells (MBC) were gated as CD19+CD24+CD27+, and IgM and switched memory B cells were discriminated by the expression of IgM. T cell subsets were identified based on the expression of CD3, CD4, CD8, CD45RO and CCR7 markers by flow cytometry. An analysis of T cell subsets was performed in all 47 CVID patients and in 9 age-matched HD.
Immune cells were identified as follows: naïve CD4+ T cells (CD4+CD45RO−CCR7+), central memory CD4+ (CD4+CD45RO+CCR7−) T cells, naïve CD8+ T cells (CD8+CD45RO-) and central memory CD8+ T cells (CD8+CD45RO+CCR7−). The CD4+ naïve/memory ratio was calculated as a ratio of CD4+CD45RO−CCR7+ and CD4+CD45RO+CCR7−; the CD8+ naïve/memory ratio was calculated as a ratio of CD8+CD45RO−CCR7+ and CD8+CD45RO+CCR7−. Cells were acquired on a BD FACSymphony A5™. Data were analyzed with FlowJo ver. 10.