Fig. 5.
Fig. 5. CD28i functions as an integral factor to increase the CD28 costimulation signal. / (A) Tyrosine phosphorylation of CD28i induced by crosslinking of CD28. CD28i-HA–transfected Jurkat T cells were serum starved overnight. Cells were incubated with anti-CD28 Ab or anti-CD43 Ab for 30 minutes on ice. Anti–mouse IgG Ab was added to start activation at 37°C for 2 minutes and 5 minutes and then cell lysates were prepared with 1% Nonidet P40. Immunoprecipitation was performed with anti-HA Ab, and immunoprecipitates analyzed by immunoblotting. The top panel shows the data blotted with anti–P-Tyr (indicated as p-Tyr). The same membrane was blotted with anti-HA Ab and is shown in the bottom (indicated as CD28iHA). Lane 1: not stimulated. Lane 2: CD28 stimulated for 2 minutes. Lane 3: CD28 stimulated for 5 minutes. Lane 4: CD43 stimulated for 2 minutes. (B) Association of CD28i with PI3-kinase p85α induced by crosslinking of CD28. CD28i-HA–transfected Jurkat T cells were serum starved overnight. Cells were incubated with CHO cells or mouse B7-1–transfected CHO cells at 37°C for 5 minutes and then cell extracts were prepared with 1% Nonidet P40. Immunoprecipitation was performed with anti-HA Ab and immunoprecipitates analyzed by immunoblotting. The top panel shows the data blotted with anti–PI3-kinase p85α. The same membrane was blotted with anti-HA Ab and is shown in the bottom panel (indicated as CD28i-HA). Lane 1: not stimulated. Lane 2: CHO-B7-1–stimulated. Lane 3: CHO-stimulated. Lane 4: whole-cell lysate of Jurkat T cells. The arrow indicates the position of PI3-kinase p85α. H indicates the position of the Ig heavy chain. (C) HA-tagged CD28i is functional in inducing costimulation. HA-tagged CD28i and an IL-2 reporter plasmid were transfected into Jurkat T cells. Sixteen hours later, cells were stimulated for 6 hours with PHA and PMA in the presence of anti-HA Ab or anti-CD28 Ab as indicated on the left. Cell extracts were examined for IL-2 promoter activity by luciferase assays. The data represent 3 different experiments with similar results. (D) CD28i enhances CD28 signals in Jurkat T cells. The CD28i-HA or the control plasmid (pcDNA3), and the IL-2 reporter plasmid, were transfected into Jurkat T cells. Sixteen hours later, cells were stimulated for 6 hours with PHA and PMA in the presence of anti-CD28 Ab, anti-CD2 Ab, or anti-CD7 Ab as indicated on the left. Cell extracts were studied for IL-2 promoter activity by luciferase assays. The data represent 5 different experiments with similar results. (E) CD28i enhances the human CD28 signaling in a mouse T-cell hybridoma, DC28. The human CD28 transfectant of mouse T-cell hybridoma DC28 was transfected with CD28i-HA or pcDNA3 and the IL-2 reporter plasmid. Sixteen hours later, cells were stimulated for 6 hours with CHO or CHO-B7-1 in the presence of antimouse CD3 Ab as indicated on the left. The data represent 3 different experiments with similar results.

CD28i functions as an integral factor to increase the CD28 costimulation signal.

(A) Tyrosine phosphorylation of CD28i induced by crosslinking of CD28. CD28i-HA–transfected Jurkat T cells were serum starved overnight. Cells were incubated with anti-CD28 Ab or anti-CD43 Ab for 30 minutes on ice. Anti–mouse IgG Ab was added to start activation at 37°C for 2 minutes and 5 minutes and then cell lysates were prepared with 1% Nonidet P40. Immunoprecipitation was performed with anti-HA Ab, and immunoprecipitates analyzed by immunoblotting. The top panel shows the data blotted with anti–P-Tyr (indicated as p-Tyr). The same membrane was blotted with anti-HA Ab and is shown in the bottom (indicated as CD28iHA). Lane 1: not stimulated. Lane 2: CD28 stimulated for 2 minutes. Lane 3: CD28 stimulated for 5 minutes. Lane 4: CD43 stimulated for 2 minutes. (B) Association of CD28i with PI3-kinase p85α induced by crosslinking of CD28. CD28i-HA–transfected Jurkat T cells were serum starved overnight. Cells were incubated with CHO cells or mouse B7-1–transfected CHO cells at 37°C for 5 minutes and then cell extracts were prepared with 1% Nonidet P40. Immunoprecipitation was performed with anti-HA Ab and immunoprecipitates analyzed by immunoblotting. The top panel shows the data blotted with anti–PI3-kinase p85α. The same membrane was blotted with anti-HA Ab and is shown in the bottom panel (indicated as CD28i-HA). Lane 1: not stimulated. Lane 2: CHO-B7-1–stimulated. Lane 3: CHO-stimulated. Lane 4: whole-cell lysate of Jurkat T cells. The arrow indicates the position of PI3-kinase p85α. H indicates the position of the Ig heavy chain. (C) HA-tagged CD28i is functional in inducing costimulation. HA-tagged CD28i and an IL-2 reporter plasmid were transfected into Jurkat T cells. Sixteen hours later, cells were stimulated for 6 hours with PHA and PMA in the presence of anti-HA Ab or anti-CD28 Ab as indicated on the left. Cell extracts were examined for IL-2 promoter activity by luciferase assays. The data represent 3 different experiments with similar results. (D) CD28i enhances CD28 signals in Jurkat T cells. The CD28i-HA or the control plasmid (pcDNA3), and the IL-2 reporter plasmid, were transfected into Jurkat T cells. Sixteen hours later, cells were stimulated for 6 hours with PHA and PMA in the presence of anti-CD28 Ab, anti-CD2 Ab, or anti-CD7 Ab as indicated on the left. Cell extracts were studied for IL-2 promoter activity by luciferase assays. The data represent 5 different experiments with similar results. (E) CD28i enhances the human CD28 signaling in a mouse T-cell hybridoma, DC28. The human CD28 transfectant of mouse T-cell hybridoma DC28 was transfected with CD28i-HA or pcDNA3 and the IL-2 reporter plasmid. Sixteen hours later, cells were stimulated for 6 hours with CHO or CHO-B7-1 in the presence of antimouse CD3 Ab as indicated on the left. The data represent 3 different experiments with similar results.

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