Figure 1: DNA/RNAs are incorporated into and induce costimulation of CD4⁺ T cells.

(a) Naive CD4⁺ T cells were cultured with indicated TLR ligands (Pam3: 5 μg ml⁻¹, MALP-2: 5 μg ml⁻¹, poly(I:C): 100 μg ml⁻¹, Flagellin: 1 μg ml⁻¹, Loxoribine: 100 μM, CpG-B: 5 μM) in the presence or absence of immobilized anti-CD3∈ Ab (10 μg ml⁻¹). After 48-hour incubation, IL-2 production and cell growth were assessed by ELISA and MTS assay, respectively. *P<0.05, Student’s t-test (compared with anti-CD3 alone). (b) Naive CD4⁺ T cells from WT or Myd88^−/−Trif^−/− mice were stimulated with the indicated TLR ligands or anti-CD28 (Clone: 37.51, 5 μg ml⁻¹) in the presence of immobilized anti-CD3∈ Ab. *P<0.05, Student’s t-test (compared with WT cells treated with Pam3). (c,d) Naive CD4⁺ T cells were stimulated with the indicated NAs (c) and ODNs (d) in the presence of immobilized anti-CD3∈ Ab. These T cells were incubated with the Cy5-labelled ODNs at 37 °C for 90 min and ODN uptake was determined by flow cytometry (d, upper). *P<0.05, Student’s t-test (compared with anti-CD3 alone). (e) Naive CD4⁺ T cells were incubated with 5 μM non-CpG-Cy5 for 90 min and dextran-Alexa Fluor 488 or LysoTracker for last 10 min for the subcellular staining of endosomes and lysosomes, respectively. Confocal microscopy data with differential interference contrast (DIC) images of representative cells are shown. Scale bars, 2.5 μm. Error bars indicate s.d. Data are representative of at least three independent experiments.