Figure 7.

BPI suppresses Treg differentiation. (A) In vitro Treg differentiation of CD4⁺ splenic T cells from 5-week-old or 12-week-old Lck-BPI Tg and WT mice. Foxp3-positive CD4⁺ T cells were analyzed by flow cytometry. (B) Suppression of CFSE-labeled CD3⁺ T cells by Treg cells from 5-week-old and 12-week-old Lck-BPI Tg or wild-type mice, presented as CFSE dilution in responding T effector cells cultured with Treg cells at a ratio of 4:1. T cells were stimulated with plate-bound anti-CD3 antibody for 96 h. (C) The structure of the gene-trap BPI gene. LacZ, β-galactosidase; Neo, neomycin phosphotransferase genes; SA, splicing acceptor; the box with numbers, the exon of BPI; blue triangle, Flp recombination target (FRT) site; brown triangle, loxp site; dotted arrow, the primers for genotyping PCR. (D) PCR analyses of BPI wild-type and mutant allele in the genomic DNA from mouse tails. The PCR products of the higher band (754 bp) indicate wild-type (WT) allele, and the lower band (717 bp) indicates BPI mutant allele. KO, BPI-deficient. (E) Immunoblotting analysis of BPI and tubulin protein levels in peripheral blood T cells of wild-type and BPI-deficient mice. Relative fold changes (means of three independent experiments) were normalized to tubulin levels and are shown at the bottom of the BPI panel. WT, wild-type; KO, BPI-deficient. (F) In vitro Treg differentiation of CD4⁺ T cells from 12-week-old and 22-week-old wild-type (WT) and BPI-deficient (BPI KO) mice. Foxp3-positive CD4⁺ T cells were analyzed by flow cytometry. (G) Suppression of CFSE-labeled CD3⁺ T cells by Treg cells isolated from 12-week-old and 22-week-old BPI-deficient (KO) or wild-type mice, presented as CFSE dilution in responding T effector cells cultured with Treg cells at a ratio of 4:1. T cells were stimulated with plate-bound anti-CD3 antibody for 96 h. Data shown (A, B, D, E, F, and G) are representative of at least three independent experiments.