Analysis of the mechanisms involved in NKG2D down-modulation. (A) Acidic treatment does not alter NKG2D expression on NKG2D^low cells. Total PBMCs were stimulated with SEB for 5 days and FACS analysis was performed on CFSE⁺CD3⁺CD8⁺ cells. The C-type lectin receptor CD94 was used as control. ■ indicate normal staining; , acidic elution; and □, control of the elution. The latter staining was performed by adding the anti-NKG2D or anti-CD94 mAb and, before adding the secondary GAM-PE mAb, cells were briefly washed with the acidic buffer. The lower staining demonstrates that the acidic treatment is able to break the antigen-Ab interaction. (B) Large amounts of intracellular NKG2D are found in NKG2D^low cells. NKG2D^high and NKG2D^low cells were harvested, fixed, and permeabilized to analyze the total amount of NKG2D (TOT = extracellular + intracellular) on CD3⁺CD8⁺ cells. Staining of extracellular (EC) NKG2D was performed as usual. In the right panel, results are presented as the mean plus or minus SD of the percentage of EC/TOT NKG2D obtained from 4 donors. (C) Clathrin-dependent endocytosis is involved in NKG2D down-modulation. Total or CD4-depleted PBMCs were stimulated with SEB for 4 days, sucrose (0.1 M) was added and left for an additional 18 hours. NKG2D and LFA-1 (used as control) expression was evaluated on CFSE⁺CD3⁺CD8⁺ cells. Treatment with sucrose did not reduce viability and CD3⁺CD8⁺ T-cell proliferation (data not shown). (D) NKG2D and DAP10 transcripts, quantitated by real-time PCR, in SEB-activated CD8⁺ T cells. Total or CD4-depleted PBMCs were stimulated with SEB and cells were harvested at different times as indicated. CD8⁺ T lymphocytes were further purified by immunomagnetic positive selection, total RNA was isolated, and real-time PCR was performed. Data were normalized by the amount of β-actin mRNA. Data are shown as the mean cycles of normalized NKG2D or DAP10 expression plus or minus SD (triplicates). All panels derive from 1 representative donor of 3 (D) or 4 (A-C) tested.