Overreactive HVEM-deficient T cells in vitro. (A) HVEM –/ – T cells are more sensitive to stimulation by suboptimal anti-CD3 mAb as shown by ³H-thymidine incorporation assay. Data represent ³H-thymidine incorporation in cpm ± SD. One of 4 independent experiments is shown. (B) Splenocytes (2 × 10⁵ per well) from WT and HVEM –/ – mice were cultured in 96-well plates with 0, 0.3, 0.6, and 1.2 μg/ml ConA in vitro. HVEM –/ – mice showed significantly increased response to ConA at all concentrations (P < 0.01). Data represent 1 of 6 independent experiments. (C) Cytokine levels in culture supernatants from B were determined by cytokine bead assay. The cells from HVEM –/ – mice also produced significantly higher levels of cytokines (P < 0.001). Representative data are shown from 1 of 3 independent experiments. (D) HVEM deficiency in both T cells and APCs contributes to the higher T cell responses to ConA. T cells from HVEM –/ – mice (T-KO) showed higher proliferation than T cells from WT mice in response to ConA when APCs from WT mice were provided (P < 0.01), and APCs from HVEM –/ – mice (APC-KO) also provided better stimulation than APCs from WT mice (P < 0.01) when T cells from WT mice were used as responders. Representative data are shown from 3 independent experiments. (E) Increased HVEM –/ – T cell response but not LIGHT –/ – T cell response to ConA in vitro. Splenocytes from WT HVEM –/ – and LIGHT ^{–/ –} mice were collected and cultured with ConA. The proliferation was determined by ³H-thymidine incorporation assay (P < 0.01), and cytokine levels were determined by cytokine bead assay (P < 0.05).