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 (CCR6CXCR3+CCR4), Th2 (CCR6CXCR3CCR4+), Th17 (CCR6+CXCR3CCR4+CCR10-), Th17.1(CCR6+CXCR3+CCR4) and Th22 (CCR6+CXCR3CCR4+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