Figure 2
Figure 2. Inhibition of MOG35-55–specific T cells by ISLAD. MOG35-55–specific line T cells were cultured in microtiter plates with irradiated syngeneic splenocytes as APCs and MOG35-55 in the presence or absence of several HIV peptides. Their proliferative response was measured in an H3-thymidine proliferation assay. (A) The graph shows the inhibition of proliferation by HIV peptides 1-4 with increasing concentrations of 2.5 µM, 5 µM, and 10 µM (gray, black, and white, respectively). Results presented are the mean % inhibition ± SE of the proliferative response to MOG35-55 peptide relative to the control (in the absence of HIV peptides) from a representative experiment (out of 4 experiments). Uninhibited T-cell proliferative responses were 7866 ± 563 cpm. The background proliferation in the absence of antigen was 132 ± 35 cpm. (B) The graph shows dose-dependent inhibition of MOG35-55–specific T-cell proliferation of peptide 3 (ISLAD) starting from the nanomolar concentration range. (C) ISLAD inhibits IFN-γ secretion by MOG35-55–stimulated T cells. T cells were cultured with APCs and MOG35-55 in the presence of 1 µM of ISLAD. After 48 hours, the media were collected and levels of IFN-γ were detected by ELISA. Results are mean ± SE, n = 3. (D) ISLAD does not inhibit TNF-α secretion by LPS-stimulated macrophages. RAW264.7 macrophages were incubated for 2 hours in the presence of 1 µM of ISLAD, and then stimulated with LPS (10 ng/mL) for 5 hours. The media were collected, and the levels of TNF-α were detected by ELISA. Results are mean ± SE, n = 3. (E) ISLAD is not toxic to T cells. Jurkat T cells and MOG35-55–specific T cells were incubated with 20 µM of ISLAD for 4 hours. Then the viability of the cells was analyzed by an XTT cytotoxicity assay. Results are the mean % viability ± SD from the control (cells with no peptide added), n = 3. (F) ISLAD is not hemolytic. Red blood cells were incubated with 100 µM of ISLAD for 1 hour. Hemolytic activity was measured by the release of hemoglobin into the media (OD 540 nm). Triton (1% vol/vol) served as a control for a hemolytic agent. Results are mean ± SD, n = 8. (G) The figure shows the generation of motif-related mutations in ISLAD. The conserved Trp residues and the conserved acidic residues (Glu and Asp) were mutated to Gly. (H) The graph shows the inhibition of MOG35-55–specific T-cell proliferation by the mutant peptides ISLAD (W/G) and ISLAD (D/G), (E/G) with increasing concentrations of 5 µM and 10 µM (gray and black, respectively). *P < .05; **P < .01. RBC, red blood cells.

Inhibition of MOG35-55–specific T cells by ISLAD. MOG35-55–specific line T cells were cultured in microtiter plates with irradiated syngeneic splenocytes as APCs and MOG35-55 in the presence or absence of several HIV peptides. Their proliferative response was measured in an H3-thymidine proliferation assay. (A) The graph shows the inhibition of proliferation by HIV peptides 1-4 with increasing concentrations of 2.5 µM, 5 µM, and 10 µM (gray, black, and white, respectively). Results presented are the mean % inhibition ± SE of the proliferative response to MOG35-55 peptide relative to the control (in the absence of HIV peptides) from a representative experiment (out of 4 experiments). Uninhibited T-cell proliferative responses were 7866 ± 563 cpm. The background proliferation in the absence of antigen was 132 ± 35 cpm. (B) The graph shows dose-dependent inhibition of MOG35-55–specific T-cell proliferation of peptide 3 (ISLAD) starting from the nanomolar concentration range. (C) ISLAD inhibits IFN-γ secretion by MOG35-55–stimulated T cells. T cells were cultured with APCs and MOG35-55 in the presence of 1 µM of ISLAD. After 48 hours, the media were collected and levels of IFN-γ were detected by ELISA. Results are mean ± SE, n = 3. (D) ISLAD does not inhibit TNF-α secretion by LPS-stimulated macrophages. RAW264.7 macrophages were incubated for 2 hours in the presence of 1 µM of ISLAD, and then stimulated with LPS (10 ng/mL) for 5 hours. The media were collected, and the levels of TNF-α were detected by ELISA. Results are mean ± SE, n = 3. (E) ISLAD is not toxic to T cells. Jurkat T cells and MOG35-55–specific T cells were incubated with 20 µM of ISLAD for 4 hours. Then the viability of the cells was analyzed by an XTT cytotoxicity assay. Results are the mean % viability ± SD from the control (cells with no peptide added), n = 3. (F) ISLAD is not hemolytic. Red blood cells were incubated with 100 µM of ISLAD for 1 hour. Hemolytic activity was measured by the release of hemoglobin into the media (OD 540 nm). Triton (1% vol/vol) served as a control for a hemolytic agent. Results are mean ± SD, n = 8. (G) The figure shows the generation of motif-related mutations in ISLAD. The conserved Trp residues and the conserved acidic residues (Glu and Asp) were mutated to Gly. (H) The graph shows the inhibition of MOG35-55–specific T-cell proliferation by the mutant peptides ISLAD (W/G) and ISLAD (D/G), (E/G) with increasing concentrations of 5 µM and 10 µM (gray and black, respectively). *P < .05; **P < .01. RBC, red blood cells.

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