Flow cytometric analysis of lymphocyte subsets. (A) Peripheral blood and leukapheresis products were prepared for flow cytometry as detailed in “Flow cytometric analysis” and lymphocyte subpopulations were identified as follows: Total B cells, CD20⁺, CD3⁻; total T cells, CD3⁺, CD20⁻; NK cells, CD3⁻,CD20⁻, CD8⁺; mDCs, CD3⁻, CD20⁻, CD8⁻, HLA-DR⁺, CD11c⁺, CD123⁻; pDC: CD3⁻, CD20⁻, CD8⁻, HLA-DR⁺, CD11c⁻, CD123⁺; IgG⁺ B cells, CD3⁻, CD20⁺, IgG⁺,IgM⁻; IgM⁺ B cells, CD3⁻, CD20⁺, IgG⁻, IgM⁺; CD4⁺ T cells, CD20⁻, CD3⁺, CD4⁺,CD8⁻; CD8⁺ T cells, CD20⁻, CD3⁺, CD4⁻,CD8⁺; naive T cells, Tn, CD28⁺,CD95⁻ CD4 or CD8 T cells; central memory T cells, Tcm, CD28⁺,CD95⁺ CD4 or CD8 T cells; effector memory T cells, Tem, CD28⁻,CD95⁺ CD4 or CD8 T cells. Shown is a representative example of the following: First row: Lymphocyte gating based on forward- and side-scatter characteristics (left panel), subsequent CD3 versus CD20 gating (middle panel), subsequent CD4 versus CD8 gating (right panel). Second row: Subsequent IgG versus IgM gating (left panel) and subsequent CD95 versus CD28 gating (middle and right panels). Third row: mDC and pDC gating based on CD123 and CD11c. (B) Peripheral blood and leukapheresis products were prepared for flow cytometry. CD4⁺ Tregs were identified as CD3⁺,CD4⁺, CD25^high, CD127^low, FoxP3⁺ cells. Shown is a representative example of lymphocyte gating based on forward- and side-scatter characteristics (far left panel), subsequent identification of CD3⁺,CD4⁺ cells (second panel from the left), subsequent identification of CD25^high, CD127^low cells (third panel from the left), subsequent labeling with FoxP3 (red cells represent CD3⁺,CD4⁺, CD25^high, CD127^low cells labeled with FoxP3; and black cells, CD3⁺,CD4⁺, CD25^high, CD127^low cells labeled with the corresponding isotype control). The far right panel shows histogram analysis of FoxP3 fluorescence comparing FoxP3 labeling of putative Tregs (red), bulk CD4 cells (blue), or putative Treg cells labeled with the FoxP3 isotype control antibody (black). (C) Peripheral blood and leukapheresis products were prepared for flow cytometry as detailed in “Flow cytometric analysis,” and CXCR4 expression was determined for each subpopulation. Shown are representative histograms showing CXCR4 fluorescence for the individual cell populations (blue traces) compared with these cells labeled with the CXCR4 isotype control antibody (red traces).