Figure 4.

Activation-induced surface expression of MICA molecules on MICAgen splenocytes. (A) MICA is barely detectable on total splenocytes of MICAgen mice in contrast to splenocytes of H2-K^b-MICA mice. Freshly isolated splenocytes were stained for surface MICA and assessed flow cytometry. (B) Differential low MICA expression by subsets of MICAgen splenocytes. Freshly isolated splenocytes were stained for surface MICA in addition to various immune markers, and gated for B cells (CD19⁺CD3⁻), T cells (CD19⁻CD3⁺), NK cells (CD11b⁺Gr1⁻NKp46⁺), and myeloid cells (CD11b⁺Gr1⁺), respectively. (A,B) MICA stainings (biotinylated BAMO1 plus SA-BV421) of (subgated) splenocytes from MICAgen mice (red line) are overlayed with those of H2-K^b-MICA mice (blue line) and nontgLM (black line), and negative control stainings (biotinylated irrelevant mouse IgG1 plus SA-BV421) of MICAgen mice (red filled). (C,D) Strong induction of surface MICA expression by activated MICAgen splenocytes. Freshly isolated splenocytes of MICAgen mice were treated (C) with phorbol myristate acetate (PMA) plus ionomycin (PMA/I) or (D) with lipopolysaccharide (LPS) for various times and subsequently MICA cell surface expression of lymphocyte subsets monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. (C,D) MICA stainings of subgated splenocytes treated for 0 (red line), 8 (green line), and 24 h (blue line) are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment is also overlayed (filled light blue). (E) Activation-induced surface MICA expression on MICAgen splenocytes is transient. Freshly isolated splenocytes of MICAgen mice were cultured in presence of PMA/I for either 0.5 (left) or 2 h (right), extensively washed, and subsequently cultured for up to 96 h. MICA surface expression on B cells monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. MICA stainings of B cells before stimulation with PMA/I (red line), and 24 (blue), 48 (black), 72 (green), or 96 h (gray) after begin of stimulation are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment are also overlayed (filled blue). (F) Activation of MICAgen splenocytes results in de novo induced MICA glycoprotein expression. Freshly isolated splenocytes of nontgLM, MICAgen, and H2-K^b-MICA mice were treated for 24 h with LPS or PMA/I or left untreated (NT), and subsequently, PNGaseF-treated cell lysates were analyzed by immunoblotting with biotinylated BAMO1. Detection of actin as loading control.