Expression and functional characterization of CD158a and CD158b on P1 malignant CD4⁺ T cells and an in vitro–derived T-cell clone. (A) PBMCs and the P1 cell line were subjected to a double immunostaining using a PE-conjugated anti–TCR-Vβ8 mAb, and an anti-CD158k mAb plus FITC-conjugated goat anti–mouse IgM antibodies (top panel). Alternatively, cells were stained with a PE-conjugated anti-CD158a (middle panel) or anti-CD158b (bottom panel) mAb. (B) The CDR3 size analysis of the TCR-Vβ8 transcript from P1 PBMCs (top panel) or derived cell line (bottom panel) was performed. Following total RNA extraction and reverse transcription, cDNA were amplified by PCR with Vβ8- and Cβ-specific primers. The unlabeled amplification products were elongated using a nested fluorescent Cβ and Jβ2.5 primers. The samples were subjected to electrophoresis and analyzed on an automated sequencer. (C) Expanded normal CD4⁺CD158b⁺ lymphocytes, the P1-derived cell line, or PBMCs were activated by incubation with an anti-CD3 mAb together with an isotype-matched anti-CD16 (white histogram), anti-CD158a (gray) or anti-CD158b (black) mAb. When necessary, the anti-CD16 mAb was used instead of the anti-CD3 mAb (concentration “0”) alone, or in combination with an anti-KIR mAb. Concentrations of the anti-CD3 mAb used are indicated. Results are expressed as the mean of triplicates ± SD. (D) Sorted and expanded CD4⁺CD158b⁺ T cells from a healthy donor, or the P1-derived cell line, were surface biotinylated and left untreated (−) or incubated in the presence of vanadate (+). An aliquot of each NP40 cell lysate, corresponding to 5 × 10⁵ cell equivalent, was collected, and immunoprecipitations were performed on the remaining samples using an anti-CD16 (C), anti-CD158a, or anti-CD158b mAb. The immunoprecipitates were separated by SDS–8% PAGE and transferred onto a nitrocellulose membrane. The immunoprecipitated receptors were first revealed by incubation of the blot with streptavidin-peroxidase, and an ECL detection system (top panel). The position of the short (KIR-S) and long (KIR-L) isoforms is indicated. After a dehybridization step, the membrane was reprobed with the antiphosphotyrosine mAb 4G10 (middle panel), stripped, and incubated with the purified anti–SHP-1 polyclonal antibodies (bottom panel). Total cell lysates from control or activated cells were similarly probed with the anti–SHP-1 antibodies to ensure protein expression. (E) Cells were incubated in the presence of control murine IgG, anti-CD3 and/or anti-CD158a or anti-CD158b mAb. Following activation, cell lysates were prepared and subjected to gel electrophoresis. Immunoblotting was then performed sequencially using the indicated antiphosphoprotein antibodies. Equal protein loading was verified by detection of total Erk1/2. The concentrations of the antibodies used for cell activation are given in micrograms per milliliter.