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To validate the most significant immune cells in children, the whole blood samples from 30 children with ASD and 30 age and sex-matched TD children were collected at the 3rd Affiliated Hospital of Zhengzhou University. All participants provided informed consent to participate in the study, which was approved by the Medical Ethics Committee of the 3rd Affiliated Hospital of Zhengzhou University (Ethical number 2020–56). All blood samples were processed within 8 h for flow cytometry with a mix of antibodies according to the manufacturer’s instructions. The antibodies purchased from BD Biosciences included CD45 (HI30, Cat# 564105) CD14 (M5E2, Cat# 561712), CD16 (3G8, Cat# 563692), and HLA-DR (G46-6, Cat# 560896). The data were analyzed using FlowJo software (TreeStar) and presented using the UMAP method.

All experiments were performed after the approval of the institutional Committee for Ethics in Research (Fundação Pró-Sangue, CEP#03, FMUSP). Healthy donors’ peripheral blood was obtained from leukoreduction chambers (41 (link)) after signed written informed consents. PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare, Uppsala, Sweden). For the generation of monocyte-derived DCs, PBMCs were either plated for 2 h for adherence of monocytes to the plastic and subsequent removal of non-adherent cells (42 (link)) or by positive magnetic selection (Milteny Biotec, Bergisch Gladbach, Germany) for the isolation of CD14⁺ monocytes. The monocytes were cultured at 37°C and 5% CO₂ in RPMI-1640 medium supplemented with 10% FBS, antibiotic-antimycotic (Thermo Fisher Scientific) and 50 ng/ml of IL-4 plus 50 ng/ml of GM-CSF (both from PeproTech, Rocky Hill, NJ, USA) for 5 days to obtain immature monocyte-derived dendritic cells (iDCs) (43 (link)). At day 5, iDCs cells were harvested, washed, counted, and activated for 24 h with previously transduced SK-MEL-147 cells at a 1:1 and 1:10 ratios. Cells were harvested and analyzed by flow cytometry using CD209 (DCN46, #551545, BD), HLA-DR (G46-6, #556643, BD), CD80 (L307.4, #340294, BD), CD83 (HB15e, #561132, BD), CD86 (2331, #561124, BD) or used for the priming of autologous T cells.

The following antibodies were used: CD80 (L307.4; BD Biosciences), CD86 (FUN-1; BD Biosciences), CD86 (IT2.2, BioLegend), CD25 (BC96; eBioscience), FoxP3 (236A/E7; eBioscience), CTLA-4 (BNI3; BD Biosciences), IFN-γ (B27; BD Biosciences), CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD45RA (HI100; eBioscience), CD38 (HIT2; BD Biosciences) and HLA-DR (G46-6; BD Biosciences). For surface marker staining, cells were washed and re-suspended in 50 µl of FACS buffer (PBS and 2% Goat serum) containing antibodies conjugated with fluorochromes. Reactions were incubated for 30 min on ice. Cycling CTLA-4 was stained for 30 min at 37°C. For intracellular staining of FoxP3, a FoxP3 staining kit (eBioscences) was used according to the manufacturer’s instructions. Flow cytometry data were analysed by FlowJo (TreeStar, Ashland, Oregon, USA).