CD8/γ-induced degranulation, cytokine production, and survival in FcRγ-deficient BMMCs. (A) Establishment of FcRγ-deficient BMMCs expressing CD8/γ. Bone marrow cells were infected with CD8/γ and cultured as described in “Materials and methods.” (i) BMMCs were stained with FITC-labeled anti-CD8 and analyzed by FACSCalibur (gray shaded histogram). The open histogram indicates uninfected control cells. (ii) Total cell lysates were blotted with polyclonal anti-CD8 (top) and anti-FcRγ (bottom). (B) Degranulation and prostaglandin synthesis upon CD8/γ cross-linking. FcRγ-deficient BMMCs expressing the CD8/γ chimera were stimulated with indicated amount of soluble anti-CD8 for 30 minutes, and β-hex release (left) and PGD₂ production (right) were measured. (C) Cytokine production upon CD8/γ cross-linking. Cells were stimulated as described for panel B for 4 hours (□) and 24 hours (▪). Production of IL-6 (left) and TNF (right) was determined by ELISA. (D) Survival induction upon CD8/γ cross-linking. Cells were stimulated with the indicated amount of anti-CD8 in the absence of IL-3. Viability was assessed by propidium iodide (PI) staining. (E) Comparison of divalent versus monovalent anti-CD8 on survival induction. CD8/γ-expressing cells were left untreated or treated with 3 μg/mL whole anti-CD8 or anti-CD8 Fab in the absence of IL-3. After 4 days of treatment, viability was determined as described. (F) Degranulation and survival in Lyn-deficient BMMCs expressing CD8/γ. WT BMMCs or Lyn-deficient BMMCs expressing CD8/γ were established as described in “Materials and Methods.” β-hex release (left) and survival after IL-3 depletion for 4 days (right) were determined. Data were means ± SDs of triplicate assays.