Generation of stable cells lines expressing CD8/28 chimeric receptors. A, schematic representation of the CD8/28 chimeric receptor showing the extracellular domain and transmembrane domain of CD8α fused to the cytoplasmic domain of CD28 as well as a list of all the CD8/28 chimeric mutants used and mentioned in this study. B, expression of CD8/28 chimeric receptor (black line) and the endogenous CD28 (filled gray histogram) was determined by flow cytometry in either Jurkat T cells or Jurkat T cells stably expressing all the chimeric receptors used in this study. Cells were stained with phycoerythrin-conjugated CD8α antibody, and fluorescein isothiocyanate-conjugated CD28 antibody.

TCR signals negatively regulate CyclinT1-HEXIM1 interaction. A, Jurkat cells stably expressing the indicated CD8/28 chimeric receptors were serum-starved for 12–16 h prior to activating with 0.1 μg/ml anti-CD3 and 1.0 μg/ml anti-CD8, whereas wild-type Jurkat T cells were activated with 0.1 μg/ml anti-CD3 and 1.0 μg/ml anti-CD28 for 30 min before adding 5 μg/ml goat-anti-mouse for 4 h. Nuclear extracts were prepared, and CyclinT1 immunoprecipitation was carried out. The samples were run on a SDS-PAGE gel, Western blot analysis assessed the amount to CyclinT1, and an anti-HEXIM1 Western blot was done to determine the amount of HEXIM1 associated with CyclinT1. Blots were quantified in the panel on the right. B, CD4⁺ T cells were isolated from human peripheral blood and were left unstimulated or activated with 0.1 μg/ml anti-CD3, 0.1 μg/ml anti-CD3, or 1.0 μg/ml anti-CD28 alone. Receptors were cross-linked by adding 5 μg/ml goat-anti-mouse for 4 h. Nuclear extracts were prepared, and an anti-HEXIM1 immunoprecipitation was carried out. Western blot analysis determined the amount of HEXIM1 immunoprecipitated and the amount of CyclinT1 associated with HEXIM1. Blots were quantified in the lower panel. C, nuclear extracts were prepared from Jurkat cells activated through the CD3 and CD28 receptors as described above, and an anti-HEXIM1 immunoprecipitation was carried out. Western blot analysis determined the amount of HEXIM1 immobilized as well as CyclinT1 associated with HEXIM1 (quantified in the panel below). D, CD4⁺ T cells were treated with 50 μm LY294002 for 4 h prior to preparing nuclear extracts. HEXIM1 was immunoprecipitated, and Western blot analysis determined the levels of HEXIM1 and CyclinT1 (quantified in the panel below). For all panels, nuclear extracts (NE) were probed with the indicated antibodies as an input control. Data shown are from a single experiment, and quantification was performed from at least two independent experiments.

NF-κB activation is indispensable for CD28-mediated HIV-1 transcription. A, Jurkat cells stably expressing the CD8/28 chimeric receptors FYFF, FFYF, FFFY, and YFFY were activated as described above. Nuclear extracts were prepared and incubated with radiolabeled NF-κB probe for 20 min, and samples were run on a 6% polyacrylamide gel. Reactions were also performed in the presence of a 100-fold excess of specific competitor or nonspecific competitor. B, electrophoretic mobility shift assays were performed as in A in the presence of anti-p50 and anti-p65 antibodies. Gel-shifted complexes are denoted by arrowheads; supershifted complexes are indicated by bolded arrowheads. Data shown are from a single experiment that is representative of three independent experiments. C, Jurkat cells were electroporated with wild-type LTR-luciferase and mKB-LTR luciferase constructs. Cells were either left unstimulated, or stimulated with 0.1 μg/ml anti-CD3, or 0.1 μg/ml anti-CD3 and 1.0 μg/ml anti-CD28, followed by 5 μg/ml secondary antibody (goat anti-mouse) to cross-link the receptors. Eight hours post-activation the cells were lysed, and luciferase activity was measured. The luciferase activity is shown as -fold induction wherein luciferase units corresponding to the unstimulated samples were set to 1.

Model for CD28-mediated control of HIV-1 transcription. CD28 enhances HIV transcription by inducing NF-κB activation. Tyr-188 modulates negative signals from Tyr-173 and positive signals emanating from Tyr-191 and Tyr-200. Recruitment of PI3K to Tyr-173 increases P-TEFb association with the HEXIM/7SK RNP to negatively regulate HIV-1 Tat-dependent transcription.

Specific tyrosine residues in the cytoplasmic domain regulate HIV-1 transcription. Jurkat cells expressing the indicated CD8/28 chimeric receptors were infected with NL4-3 luciferase virus for 16 h. Post infection cells were either left unstimulated, or stimulated with 0.1 μg/ml anti-CD3 plus 1.0 μg/ml anti-CD8α, or with 1.0 μg/ml anti-CD28, followed by 5 μg/ml secondary antibody (goat anti-mouse) to cross-link the receptors. Eight hours post-activation the cells were lysed, and luciferase activity was measured. The luciferase activity is shown as a percentage of endogenous response, wherein the CD3 plus CD28 response of each cell line was set to a 100%. The dotted line represents the luciferase activity corresponding to activation through CD3 alone. The data shown were done in triplicate and represent one experiment of a total of three independent infections. A two-tailed t test was performed on paired samples comparing CD3 responses to CD3 plus CD8 responses. *, p < 0.01; **, p < 0.005.

Vav activation is not necessary for induction of HIV transcription. A, Jurkat T cells stably expressing the CD8/28 chimeric receptors FYFF, FFYF, and FFFY were activated as indicated with antibodies against CD3, CD28, and CD8, for 5 min after cross-linking the receptors. Vav was immunoprecipitated from cell lysates, and Vav phosphorylation was determined by anti-phosphotyrosine Western blot. Vav phosphorylation downstream the chimeric receptors (CD8 activation) were compared directly to the endogenous CD28 receptor. B, Jurkat T cells were infected with NL4-3 luc virus and activated with anti-CD3 and CD28 antibodies in the presence of CsA. Six hours post activation luciferase assay was performed. The unstimulated response was set as 1, and -fold activation has been plotted. Data shown are from a single experiment that is representative of three independent experiments.

CD28-mediated signals induce recruitment of NF-κB to proviral LTR. A, Jurkat cells expressing the indicated CD8/28 chimeric receptors were infected with pHXB-PLAP virus. Infected cells were then activated for 6 h, and chromatin was isolated. Chromatin immunoprecipitations were performed using p65 antibody. FLAG antibody was used as a nonspecific antibody control. Real-time PCR was carried out for the −150 to +76 region of the HIV LTR, and the data are plotted as a percentage of input immunoprecipitated in each reaction. B, Jurkat cells expressing the indicated CD8/28 chimeric receptors were infected with pHXB-PLAP virus. Infected cells were then activated for 6 h, and chromatin was isolated. Chromatin immunoprecipitations were performed using RNA pol II antibody. FLAG antibody was used as a nonspecific antibody control. Real-time PCR was carried out for −150 to +76 region of the HIV LTR, and the data are plotted as a percentage of input immunoprecipitated in each reaction. C, Jurkat cells expressing the indicated CD8/28 chimeric receptors were infected with pHXB-PLAP virus. Infected cells were then activated for 6 h, and chromatin was isolated. Chromatin immunoprecipitations were performed using RNA pol II antibody. FLAG antibody was used as a nonspecific antibody control. Real-time PCR was carried out for +5396 to +5531 region of the HIV, and the data are plotted as a percentage of input immunoprecipitated in each reaction.