Gp41 TMD co-localizes with CD3 within the TCR complex.

<p>Gp41 TMD-NBD and AMP-NBD were used to study peptide binding to the membranes of resting and activated T cells in combination with Sy5-labeled antibodies to CD3. The T cells had been activated by incubation with APC and the MOG 35–55 peptide. (<b>A</b>) Distribution of gp41 TMD-NBD, AMP-NBD and CD3 molecules in resting and activated T cells. (<b>B</b>) Co-localization of gp41 TMD-NBD with the CD3 molecules. (<b>C</b>) Co-localization of AMP-NBD with the CD3 molecules.</p

Gp41 TMD inhibits T-cell proliferation <i>in vitro</i>.

<p>T-cells were activated with the MOG 35–55 peptide and APC in the presence of gp41 TMD (black) or FP_5–13 AAAA as control peptide (grey) in three different concentrations (50, 25 and 5 µg/ml) and the proliferative responses were assayed. The data are presented as mean inhibitions+SD (<i>n</i> = 4 or more, ** <i>p</i><0.01). The uninhibited T-cell proliferative responses were 9382±891 cpm. The background proliferation in the absence of antigen was 232±36 cpm.</p

Fluorescence Energy Transfer (FRET) measurements reveal a specific interaction of the gp41 TMD and the TCRα CP.

<p>Fluorescence spectra were obtained at room temperature, with excitation set at 467 nm (5-nm slit) and emission scan at 500–600 nm (10-nm slits). NBD-labeled CP peptide was added first from a stock solution in DMSO (final concentration 0.1 µM and a maximum of 0.25% (v/v) DMSO) to a dispersion of PC LUV (100 µM) in PBS. This was followed by the addition of: <b>A</b>. Rho labeled gp41 TMD peptide <b>B</b>. Rho labeled TCRα CP <b>C</b>. Rho labeled TAR/PS TM control peptide. Fluorescence spectra were obtained in four different ratios of Rho-peptide∶NBD-peptide: 0∶4 (black line), 1∶4 (black triangle), 2∶4 (black circle) and 3∶4 (dashed line). The fluorescence values were corrected by subtracting the corresponding blank (buffer with the same vesicles concentration). Statistical analysis was performed at the pick measurements (∼537 nm, n = 5, * p<0.05).</p

Gp41 TMD inhibit T-cell activation induced by mitogenic antibodies to CD3.

<p>T-cells were activated with 1µg/ml anti-CD3 and APC in the presence of 50µg/ml gp41 TMD or TCRα CP and the proliferative responses were assayed. The uninhibited T-cell proliferative responses were 9103±208 cpm. The background proliferation in the absence of antigen was 257±41 cpm. The data are presented as normalized mean proliferation percentage (control, no peptide = 100%) + SD (<i>n</i> = 5 or more, ** <i>p</i><0.01). There was no significant statistical difference between the TCRα CP and the control.</p

HIV-1 gp41 extra-cellular domain.

<p>A scheme showing the functional regions within the extra-cellular portion of gp41 (aa 512–684).</p

Vaccination with HSP60 peptide Hu3 induces anti-ergotypic T cells.

<p>A. Anti-ergotypic proliferative response of LNC from rats vaccinated with PBS, Mt3 or Hu3 in IFA, taken 26 days after AA induction. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. * p<0.05 compared to the Mt3 group. B. Monoclonal antibodies to MHC-II/RT1.B, MHC-II/RT1.D or MHC-I were assayed for their ability to block the anti-ergotypic proliferative response. Results are presented as the percent of inhibition of proliferation±SEM of quadruplicate cultures. C. Anti-ergotypic cytokine response of LNC taken from rats vaccinated with PBS, Mt3 or Hu3 in IFA, 26 days after AA induction. IFNγ (IFNg), TGFβ1 (TGFb1), IL-10 and IL-4 were quantified in the culture supernatants after 72 hr of stimulation with 10⁵ activated or resting, irradiated, A2b cells per well. The results are presented as pg/ml±SEM of triplicate cultures. Three independent experiments produced similar results.</p

T-cell activation up-regulates cellular levels of HSP60.

<p>A. LNC were stimulated with Con A for 24, 48 or 72 hr, subjected to a 30 minutes 42°C heat shock (HS) or kept at 37°C (None). Cell lysates were prepared and HSP60 expression was analyzed by western blot with specific antibodies, and quantified (in arbitrary units). B. A2b T-cells were stimulated with various concentrations of the target peptide Mt176-90, a control peptide (Mt3) for 72 hr, or with medium alone (None). Cell lysates were prepared and HSP60 expression was analyzed by western blot with specific antibodies, and quantified (in arbitrary units). Two independent experiments produced similar results.</p