KIR3DL2 inhibitory function on SS patients’ malignant T-cell clone. (A) Sorted CD4⁺ T cells from SS patients were left untreated (−) or incubated with anti-KIR3DL2 monoclonal antibody (mAb) AZ158 plus goat anti-mouse IgG antibodies or with the phosphatase inhibitor vanadate. Cell lysates were prepared and subjected to immunoprecipitation using the anti-KIR3DL2 mAb AZ158. The immunoprecipitates were separated by SDS-8% PAGE, transferred onto a nitrocellulose membrane, and then probed in series with anti-phosphotyrosine (Tyr) and anti-SHP-1 antibodies. Arrows indicate the position of phosphorylated KIR3DL2 and SHP-1. Results shown (patient 2) are representative of all patients tested (n = 3). (B) Sorted CD4⁺ T-cells from SS patients were incubated with anti-CD3ε or/and AZ158 mAb, as indicated. An isotype-matched control mAb was used to equalize the amount of antibodies used in each condition. Cross-linking was induced by the addition of goat anti-mouse Igs except for resting condition (NT). After lysis, the antibody-targeted molecules were collected and the resulting immunoprecipitates subjected to electrophoresis and western blotting procedures. The immunoblot was revealed with an anti-phospho-CD3ζ mAb (upper panel) and was reprobed after dehybridization using an anti-CD3ζ mAb (middle panel). CD3ε immunoblotting was performed to assess efficient cell targeting and immunoprecipitation (lower panel). Arrow indicates the position of phospho-CD3ζ. Results shown (patient 3) are representative of all patients tested (n = 3). (C) Peripheral blood mononuclear cells from SS patients were preloaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) and left untreated (NT) or stimulated with platebound anti-CD3ε and AZ158 mAb alone or in combination, as indicated. After 4 days of culture, cells were collected and immunolabelings performed using anti-TCRVβ-PE, anti-CD3-PE-Cy5, and anti-CD4-PE-Cy7 mAb. The percentages of dividing cells among the malignant (left) and the nonmalignant (right) CD4⁺ T-cell populations are presented. Results shown corresponded to patient 3, whose tumoral clone was identified as TCR-Vβ14⁺, and are representative of experiments performed on 3 SS patients. (D) Peripheral blood mononuclear cells of SS patients were activated as described for panel C for 6 days. After immunostaining with anti-CD3-FITC, -TCRVβ-PE, and -CD4-PE-Cy7 mAbs, and 7-AAD, cells were analyzed by flow cytometry. The percentage of 7-AAD⁺ apoptotic cells within the malignant (left) and the nonmalignant (right) CD4⁺ T cells is indicated for each condition of incubation. Results shown (patient 5, with a TCR-Vβ9⁺ malignant clone) are representative of experiments performed on 3 SS patients. Ig (H) and (L), Ig heavy and light chain; NT, nontreated.