Immune cell numbers
Peripheral blood samples from children were obtained at the age of 10
years.16 Absolute counts of CD3+ T
cells and CD19+ B cells per μLblood were determined
with diagnostic lyse-no-wash protocol and detailed immunophenotyping was
performed with 11-color flow cytometry (LSRII Fortessa, BD Biosciences).
We determined naive (CD45RO-CCR7+),
effector memory RO-positive T cells (TemRO;
CD45RO+CCR7-) and effector memory
RA-positive T cells (TemRA;
CD45RO-CCR7-) within
CD4+ and CD8+lineages.16,21,22 Within Treg cells, the
differentiation in naive (CD45RA+) and memory
(CD45RA-) was determined.16 Finally,
the following T helper (Th) cell subsets
(CD4+CD45RA-) were determined after
exclusion of Treg cells: Th1
(CCR6−CXCR3+CCR4−),
Th2
(CCR6−CXCR3−CCR4+),
Th17
(CCR6+CXCR3−CCR4+CCR10-),
Th17.1(CCR6+CXCR3+CCR4–)
and Th22
(CCR6+CXCR3–CCR4+CCR10+).s
In addition CD27+ and CD27-IgG+, IgA+, IgE+CD19+CD38dimIgD-memory B cell subsets were defined.16 Gating
strategies for immune cell determination were published
previously.16