Role of cell-cell interaction and HLA recognition in acquisition of TGN1412 reactivity during high-density culture. (A) Cytokine release from cells precultured at high (107 cells/mL or 2 × 106 cells/cm2) or at low (106 cells/mL or 2 × 105 cells/cm2) density for 2 days, or at low density with a high-density transwell insert (106/107), and restimulated with 1 μg/mL TGN1412 for 24 hours. (B) Colocalization of CD3 with tyrosine-phosphorylated proteins in situ. Human lymph node sections were costained with anti–CD3 mAb (red) and anti–pTyr (green). Anti–CD45 mAb was included as a negative control. Appropriate isotype controls were used. (C) Fresh and high-density PBMCs were stained with anti–CD3 mAb (red) and anti–pTyr (green). An isotype control for pTyr was included. For statistical analysis of colocalization, see supplemental Figure 3. (D) High-density preculture of PBMCs was performed as described in Figure 1B. Fab fragments of mAb to HLA class I (W6/32) or HLA class II (Tü39) were added at 10 μg/mL. Secondary cultures were performed as in Figure 1. (E) The presence of Lck inhibitor PP1 throughout preculture affects TGN1412 response in secondary culture but is without effect when added as a short pulse before stimulation (washout control), and it fully blocks the response when added at the onset of the 24-hour stimulation. Two-way ANOVA: ***P ≤ .0001. Data are mean ± SD of triplicate samples.
