Generation and characterization of CD137 KO mice. (A) The targeting map of the CD137 genomic locus. The signal peptide with the ATG starting code and first 6 exons encoding extracellular and transmembrane regions of murine CD137 were replaced with the Neo cassette. A short open bar labeled “Probe” indicates the position of the 3′ end probe for screening of ES cells, and PCR indicates the position of PCR products in screening of CD137-deficient mice using primers. Shaded boxes represent exons within the murine CD137 open reading frame. (B) Southern blotting of heterozygous and homozygous CD137 mutants in the gDNA from targeted ES cells. The upper band (6691 bp) shows the targeted fragment, and the lower one (6147 bp) represents the one from the normal genome. (C) Splenocytes from WT B6 or CD137 KO mice were activated by Con A for 24 hours, and live cells were stained for CD137 or PD-1 gated on CD3⁺ cells by specific mAbs and subsequently analyzed by flow cytometry. Shaded histograms indicate isotype control; open histograms, anti-CD137 or anti-PD1. (D) Whole splenocytes or purified T cells from CD137KO or WT control mice and were activated by Con A or plate-bound CD3 mAb at the indicated concentrations. [³H]TdR was included in the cultures 16 hours before harvesting. The results are from one representative of 2 independent experiments with similar results. (E) Splenocytes from untreated WT or CD137 KO mice were stained for CD44 and CD62L, gated on CD8⁺ or CD4⁺ cells, respectively. The results are from one representative of 2 independent experiments with similar results.