Kaluza v2

TILs and PBMCs were stained for 10-color flow cytometry (detailed antibody list, Supplementary Table 4). An extensive literature research focusing on druggable targets was performed to select immune-regulatory molecules. Representative literature and clinical studies underlying the classification in co-inhibitory or co-stimulatory molecules is included in Supplementary Table 1. Samples were acquired on a Gallios flow cytometer (Beckman Coulter, USA) and analyzed using Kaluza v2.1 (Kaluza, RRID:SCR_016182, Beckman Coulter, USA; gating strategy in Supplementary Fig. 12). FFPE sections containing tumor tissue and healthy tissue were selected for each patient. The tumor front was delineated digitally on scanned slides using Aperio ImageScope v12.4.0 (Leica, Germany). Whole section slides and tissue micro arrays (∅ 1.2 mm) of tumor specimens were stained on a Leica BOND-MAX or Roche Ventana platform according to the manufacturer’s instructions (details in Supplementary Table 4).

Cells were measured using a CytoFLEX cytometer (AS34240) and CytExpert v.2.4 software (both Beckman-Coulter, Brea, USA). Data were analyzed using Kaluza v.2.1 (Beckman Coulter). Gating strategies are shown in Supplementary Methods (Figures S1, S2, S3). Frequencies of cell populations determined by flow cytometry were multiplied with the absolute lymphocyte count per μL of whole blood (as determined by clinical CBCs) to estimate the abundance of T-cell and NK-cell subpopulations in peripheral blood. Background-corrected HCMV-specific T-cell frequencies were calculated by subtracting HIV-induced responses (considered unspecific background in HIV-negative individuals) from HCMV-induced responses.

Bone marrow samples were routinely examined by eight-color labelling method. During the examined period, the combination of antibodies was slightly changed in accordance with the new guideline (Supplementary Figure S1) [29 (link)]. Conventional FC analysis was performed by FACS Diva software v 8.0.3 (Becton Dickinson, San Jose, CA, USA). The raw data from the bi-dimensional FC examination was reanalyzed with novel protocols based on multidimensional dot-plots of Kaluza v 2.1 (Beckman Coulter, Brea, CA, USA) and Infinicyt v 2.0 (Cytognos, Salamanca, Spain) software.

For flow cytometry, phenotyping cells were grown in wells of 96 well plates. Conditioned medium from each well was removed, and cells were dissociated with TrypLE Select Enzyme (Gibco) and dissolved in conditioned medium supplemented with 5 μM Y27632. Intracellular staining of the cells was made by Alexa Fluor^® 594 anti-tyrosine hydroxylase antibodies (Cat. 818003 BioLegend, London, UK) with PerFix-nc-Kit (no centrifugation assay kit) (Beckman Coulter, Marseille, France, catalog # B10825). All procedures were performed according to the manufacturer’s recommendations.
Cell phenotyping was performed using a CytoFlex S (Beckman Coulter Biotechnology, Suzhou, China) flow cytometer equipped with 405 nm (violet), 488 nm (blue), and 638 nm (red and 561 nm (yellow-green) lasers. FSC (forward scattering) and SSC (side scattering) were used as triggering signals and for primary cell detection. Threshold was set using FSC to discriminate noise and debris.
Fluorescence was measured after excitation on a yellow-green laser with 590 nm light filter. The evaluation was carried out by counting at least 5000 cells.
The flow cytometry data were analyzed using CytExpert 2.4 (Beckman Coulter, Biotechnology, Suzhou, China) and Kaluza v2.1 (Beckman Coulter, Biotechnology, Suzhou, China) software.

WB samples were stimulated at 37 °C as described above for a total of 6 h for granulocyte and NK-cell stimulation and 24 h for T-cell stimulation. For NK-cell and T-cell analysis, brefeldin A (10 μg/mL, Sigma-Aldrich St. Louis, MO, USA), BD GolgiStop™ (0.67 µL/mL, BD Biosciences, San Jose, CA, USA), and CD107a-PE-Vio770 (10 µL/mL, Miltenyi, Bergisch Gladbach, Germany) were added to the stimulation tubes after 2 and 4 h, respectively. After incubation, cells were processed and stained as described before [16 (link)]. Fluorescent antibodies used in this study are listed in Supplementary Table S1. Cells were analyzed using a Cytoflex AS34240 flow cytometer (Beckman Coulter, Brea, CA, USA). Downstream data analysis was performed with Kaluza v2.1 (Beckman Coulter, Brea, CA, USA).

Flow cytometry data was analyzed with Kaluza v2.1 software (Beckman coulter, IN, USA) to obtain frequencies of positive cells and mean fluorescence intensities (MFI) of the cellular markers.
t-distributed Stochastic Neighbor Embedding (t-SNE) analyses were performed using FCS express version 6 (De Novo software, CA, USA). To this end, compensation was applied before subsequently gating APCs in all FCS files individually. APCs were gated as HLA-DR+CD19- single cells. Equal numbers of APCs were exported as separate files from each file and merged into a single FCS file including a file identifier. t-SNE was calculated based on expression of CD14, CD16, CD303, CD1c, CD141, PDL1, CD40, CD86, TLR2 and CD11c. Sampling options included an interval down sampling method, a Barnes- Hut approximation of 0.50, perplexity set to 60 and number of iterations to 2000.
Mann-Whitney U tests were performed to compare between GCA/PMR patients and HCs. Spearman rank correlations were calculated when indicated in the text. R (version 3.6.2) with the Seurat package (version 3.2.0) was used as described previously to analyze the scRNAseq data. All plots were created using GraphPad Prism version 9. P values <0.05 were considered statistically significant.