CTLA4-CD28 chimera delivers CD28 signals and enhances T-cell reactivity. (A) Conceptual design and mode of action of CTLA4-CD28 chimera (CTC28) and CTLA4-decoy (CTdc). + indicates stimulatory signal; −, inhibitory signal; and \\, abrogation of the signal. (B) Structure of retroviral constructs. EV is the empty retroviral vector (pMIG-w). CTC28 or CTdc cDNA was inserted in front of internal ribosomal entry site (IRES)–GFP cassette of pMIG-w. IRES-GFP cassette is used for tracing the virus-transduced cells. CTLA4 EC indicates extracellular domain of CTLA4 (amino acids 1-161); TM, transmembrane domain of CTLA4 (amino acids 162-189); CD28 CP, cytoplasmic domain of CD28 (amino acids 177-218); and LTR, long terminal repeat. (C-D) Jurkat T cells transiently transfected with the retroviral plasmids (10 μg), RE/AP luciferase plasmids (10 μg) and TK-Renilla luciferase plasmid (0.5 μg) were stimulated with plate-bound anti-CD3 plus soluble anti-CD28 or with plate-bound anti-CD3 plus anti-CTLA4 or control IgG for 6 hours. Firefly luciferase activity was measured from cell lysates. Transfection efficiency was normalized with Renilla luciferase activity. (E) Jurkat T cells transfected with retroviral plasmids were stimulated with anti-CD28 or anti-CTLA4 cross-linked by secondary antibodies for 30 minutes. Cell lysates were immunoblotted with anti–phospho-Akt or anti-Akt. (F-G) GFP-positive T cells were sorted after retroviral transduction of activated splenic T cells from normal mice and 3-day resting in the absence of stimulation. Then, the cells were stimulated with various concentrations of anti-CD3 in the presence of irradiated splenocytes for 48 hours. [³H]Thymidine (1 μCi) was added to the culture for additional 24 hours to measure cell proliferation (F), or the supernatant was harvested to measure IFN-γ production by ELISA (G). (H) Transduced splenic T cells activated with anti-CD3 (1 μg/mL) in the presence of irradiated splenocytes for 48 hours as described in panels F and G were stained with APC-labeled anti-CD4 and PE-Cy5–labeled anti-CD8 along with PE-labeled anti-CTLA4 (intracellular staining), or anti-CD28 (surface staining), or relevant isotype control antibodies. Then, GFP-positive populations were analyzed by flow cytometry. (I) Transduced splenic T cells were stimulated with plate-bound anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), or both in the presence or absence of anti-CTLA4 (10 μg/mL) cross-linked with plate-bound anti–hamster IgG (20 μg/mL). Seventy-two hours after stimulation, the supernatant was harvested to measure IFN-γ production by ELISA. Results are representative of 2 (E,H-I) or 3 independent experiments (C-D,F-G).