Cd196 bv605

Blood samples were collected prior to the first administration of the treatment (baseline) and processed by a centralized laboratory within the first 24 h after their collection. Samples of whole blood were analyzed by flow cytometry to determine the percentage and absolute number of T lymphocyte subpopulations using the following combination of monoclonal antibodies per panel: CD3-V450, CD4 PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Biosciences), CD183 AF488, CD196 BV605, and CD45 AF700 (BioLegend, San Diego, CA, USA). The absolute cell number quantification was performed as previously reported [16 (link)]. Samples were acquired on a LSR II Fortessa flow cytometer (BD Biosciences, San José, CA, USA).
The following T cell subpopulations were analyzed: CD4⁺ naïve, CD4⁺ T_CM, Th1_CM, Th1Th17_CM, Th2_CM, Th17_CM, CD4⁺ T_EMRA, CD4⁺ T_EM, Th1_EM, Th1Th17_EM, Th2_EM, Th17_EM, CD8⁺ naïve, CD8⁺ T_CM, CD8⁺ T_EMRA, CD8⁺ T_EM, double positive (CD4⁺CD8⁺), and double negative (CD4⁻CD8⁻) T cells. Analysis was performed using the FACSDiva software (BD Biosciences). The gating strategy for the subpopulations analyzed in whole blood is shown in Figure 1 and previously described by Quirant-Sánchez et al. [22 (link)].

After 5 days of culture, PBMC co-cultured with mature DC (mDC), VitD3-tolDC (100% VitD3-tolDC) and 50% VitD3-tolDC (50% VitD3-tolDC + 50% mDC) in the presence and absence of IFN-beta were harvested and stained using CD3-V450, CD4-PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Bioscience), CD183 AF488, CD196 BV605 and CD45 AF700 (Biolegend, San Diego, CA, USA) for T cell analysis; and CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, CD3 V450, HLA-DR V500 (BD Biosciences) and CD45 AF700 (Biolegend) for Treg analysis. Monoclonal antibodies were incubated for 20 min at room temperature and protected from the light. Samples were washed and a total of 50,000 CD3+ events were acquired on an LSR Fortessa flow cytometer (BD Biosciences). Both panels were analyzed using FACSDiva software (BD Biosciences). The gating strategy used to analyze the desired T cell subpopulations was previously reported [28 (link)].