Immune Cell Profiling by Flow Cytometry

Immune cells were phenotyped by using flow cytometry at baseline and on Days 1, 2, 7, 14, 28, and 56 as previously described [23 (link)] using an LSRII cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo Software (TreeStar, Ashland, OR, USA). All samples were gated to exclude dead cells, debris, and doublets. Dendritic cells (CD3-CD14-CD16-CD19-CD20-CD56-HLA-DR+) were defined as myeloid (mDCs, CD11c+) or plasmacytoid (pDCs, CD123+) and further classified by BDCA1, CCR7, CD86, and CD11b expression. Monocytes (CD19-CD3-HLA-DR+) were subdivided by CD14/CD16 expression and further with CD86 and CCR5. Lymphocytes (CD14−) were divided to T cells (CD3+), B cells (CD19+), and natural killer cells (CD3-CD19-CD56dimCD16+) and further categorized based on CD4 (CD4+ and CD4− for the Leukocyte Panel) and CD8 (for T cell phenotyping), CCR7, CD86, CD11b, HLA-DR, CCR5, CD38, and CD69. Activated T cells were defined by CD38 and HLA-DR co-expression or intracellular co-expression of Ki67 and Bcl-2. CD4 responders were defined at Days 4, 14, or 28 by percentage of CD3+CD4+ CD38+HLA-DR+ effector T cells at least 2 standard deviations (SD) above the mean of all baseline samples. CD8+ T cell responders were defined at Days 7, 14, or 28 by percentage of CD3+CD8+ CD38+HLA-DR+ effector T cells at least 3 SD above the mean of all baseline samples.