The interaction of ISLAD with the TCR complex. (A) ISLAD only inhibits T-cell proliferative signals that result from TCR engagement through APCs. MOG35-55–specific line T cells were activated in the presence of ISLAD (1 µM) by the following agents: (1) MOG35-55 antigen and APCs, (2) CD3 and CD28 antibodies (2 µg/mL), and (3) PMA (50 ng/mL) and ionomycin (1 μM). The uninhibited T-cell proliferative responses were 707 ± 97 cpm and 14 822 ± 1541 cpm for CD3/CD28 antibodies and PMA/ionomycin, respectively. The background proliferation levels in the absence of CD3/CD28 antibodies and PMA/ionomycin were 66 ± 6 cpm and 105 ± 17 cpm, respectively. Results presented are the mean % inhibition ± SD of the proliferative response relative to the control (in the absence of HIV peptide) from a representative experiment (out of 2 experiments). (B) Biochemical analysis of ISLAD’s interaction with T-cell proteins is shown. Jurkat T cells were incubated with 1 μM fluorescently labeled rhodamine (Rho)-ISLAD or its mutant Rho-ISLAD (W/G), and then cross-linked and lysed. The T-cell proteins were resolved by SDS-PAGE, and proteins bound to Rho-peptides were detected by the fluorescence of rhodamine (the ladder of protein sizes is indicated in kDa). Subsequently, the gel was transferred to a membrane and probed for actin. (C) Jurkat T cells were incubated with Rho-ISLAD, lysed, and immunoprecipitated with antibodies to TCR-α or GFP. Bound proteins were separated by SDS-PAGE and analyzed for the presence of the fluorescently labeled peptides. Results are presented as the mean fold of fluorescence intensity relative to the control (GFP) ± SD (n = 2). (D-E) The images show the colocalization of ISLAD with cellular TCR-α by using confocal microscopy. Activated mMOG35-55 T cells were probed with antibodies against TCR-α, followed by staining with secondary FITC-labeled antibodies (green, left column) and with rhodamine fluorescent peptides (red, middle column). The right column is the merged image of the molecules. In (D), the molecules were stained with Rho-labeled ISLAD, and in (E) they were stained with the Rho-labeled control peptide, AMP. (F) The graphs shows the percentage of colocalization ± SD between TCR-α and the peptides (ISLAD or AMP). (G) ISLAD forms an α-helical structure in a lipid environment; the graphs shows the circular dichroism spectra of ISLAD and its mutant, ISLAD (W/G), in 1% lysophosphatidylcholine in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). (H) The graphs show the changes in the membrane-bound state of the NBD-labeled CP of the TCR-α TMD upon the addition of ISLAD. The fluorescent experiments were performed using the NBD-labeled CP from the TCR-α TMD. Fluorescence spectra excitation was set at 467 nm, and an emission scan was at 500-600 nm. NBD-CP was first added to a dispersion of PC/Chol (9:1) LUVs in PBS. This was followed by the addition of unlabeled ISLAD (left) and the control peptides ISLAD (E/G), (D/G) (middle), and ISLAD (W/G) (right) in several sequential doses. Fluorescence spectra were obtained in different NBD-labeled CP/HIV peptide ratios ranging from 40:1 to 5:1 (corresponding to the blue line up to the black line, respectively). Scale bars represent 2 µM. *P < .05; **P < .01.