Hla dr ecd

CSF and EDTA blood samples were obtained from all patients and processed as described previously (19 (link)). Briefly, cells from CSF or 100 μl EDTA blood were incubated in VersaLyse buffer (Beckman Coulter; Brea, CA) for 10 min and subsequently washed three times with PBS supplemented with 2% heat-inactivated FCS and 2 mM EDTA. Following incubation with fluorochrome-conjugated antibodies (CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange, all Beckman Coulter), cells were acquired on a Navios flow cytometer (Beckman Coulter). Analysis was conducted with Kaluza V1.2. Lymphocytes, monocytes, and granulocytes were selected based on forwards scatter channel, sideward scatter channel, CD14, and CD45 expression characteristics. Lymphocytes subsets were selected as CD3⁺CD4⁺ (T helper cells), CD3⁺CD8⁺ (cytotoxic T cells), CD3⁺HLA-DR⁺ (activated T cells), CD3⁻CD56⁺ (NK cells), CD3⁻CD19⁺ (B cells), CD3⁻CD19⁺CD138⁺ (plasma cells), whereas monocyte subsets were selected as CD14⁺CD16⁻ (classical monocytes), CD14⁺CD16⁺ (non-classical monocytes) cells.

Basophil activation tests were performed as reported in Santos et al. (2014) (link) at weeks 0, 4, 12, 16, 52, and 104. First, we performed dose-finding experiments in 10 HDM-allergic patients and 5 non-atopic controls. Heparinized whole blood (100 μl) was stimulated for 30 min at 37°C with Der p extract (ALK-Abello A/S, Horsholm, Denmark) diluted in phosphate-buffered saline (PBS, Sigma Diagnostics, St. Louis) at serial sixfold dilutions (150, 15, 1.5, 1.5 × 10^–1, 1.5 × 10^–2, 1.5 × 10^–3 μg/ml). The two allergen concentrations that evoked maximal (i.e., 15 μg/ml) and submaximal (i.e., 0.15 μg/ml) cell stimulation were chosen for the study to investigate the time course of BAT during the SCIT. Before erythrocyte lysis, cells were stained with CD123-PE-Cy5 (BD), CD203c-PE, HLA-DR-ECD, and CD63-FITC (Beckman Coulter). Basophils gated as SSC^low/CD203c⁺/CD123⁺/HLA-DR^– were detected by flow cytometry (Beckman Coulter Epics XL-MCL, United States) and analyzed using FCS Express software (version 4).

Whole blood collected immediately before the first VSV gag administration and then 1 and 7 days after was stained in TruCOUNT tubes as previously described (Elizaga et al., 2018 (link); Hensley et al., 2012 ; Hensley-McBain et al., 2014 (link)) using the following antibody staining panel: (antibodies from BD Biosciences, unless otherwise indicated): CD14–V450, CD19–V450, CD45–AmCyan, CD4–FITC, CD8–PerCP-Cy5.5, CD123–PE, HLA-DR–ECD (Beckman Coulter), CD86–PE-Cy5, CD56–PE-Cy7, CD11c–APC, CD3–Alexa700 and CD16–APC-Cy7. We used these measurements in Figures 6G and 6H to validate changes in cell type abundance that were detected by scRNA-seq.