HSP60-specific Anti-ergotypic T-cells control arthritogenic T-cells <i>in vitro</i>.

<p>LNC from Mt immunized rats (2.5×10⁵ per well) were activated with Mt176-90 for 72 hr in the presence of Anti-p277 or Anti-MBP T-cells (5×10⁴ per well). The secretion of IFNγ was determined by ELISA, the results are presented as pg/ml±SEM of triplicate cultures. The differences between the groups were significant (p<0.05) for antigen concentrations higher than 0.1 µg/ml. Three independent experiments produced similar results.</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

MHC class II-restricted recognition of activated T cells by HSP60-specific T-cells.

<p>A. Anti-ergotypic proliferative response of Anti-HSP60 or Anti-MBP T cell lines. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. B. Anti-ergotypic proliferative response of Anti-p277 or Anti-MBP T cell lines. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. C. 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 of the Anti-HSP60 and the Anti-p277 T cell lines. Results are presented as the percent of inhibition of proliferation±SEM of quadruplicate cultures. D. IFNγ (IFNg), TGFβ1 (TGFb1), IL-10 and IL-4 were quantified in the culture supernatants after 72 hr of stimulation of the Anti-MBP, Anti-p277 or Anti-HSP60 T cell lines with 10⁵ activated or resting, irradiated, A2b cells per well. The results are presented as pg/ml±SEM of triplicate cultures. Three to five independent experiments produced similar results.</p

Anti-ergotypic HSP60-specific T cells become anergic after interacting with activated T cells.

<p>Anti-ergotypic Anti-p277 T cells were stimulated for 3 days with irradiated, activated A2b cells (A2b) or with irradiated APC fed with the p277 peptide (APC). The Anti-p277 T cells were maintained for 4 additional days in culture, and stimulated with APC and p277 peptide, Con A or immobilized anti-TCR (αTCR) antibodies. T-cell proliferation (A) and IFNγ (B) release were measured after 3 days. The proliferative responses are presented as the ΔCPM (±SEM) (A), and the IFNγ as pg/ml±SEM (B) of triplicate cultures. Three to five independent experiments produced similar results.</p

HSP60-specific anti-ergotypic T-cells control arthritogenic T-cells <i>in vivo</i>.

<p>A and B. Anti-MBP or Anti-p277 T cells were injected ip into naïve Lewis rats and three days later AA was induced. Twenty-six days after AA induction, at the peak of AA, the AA clinical score (A) and the hind paw diameter (B) were determined. The bars represent the mean values ± SEM for each group of 8 rats. C. LNC were collected on day 26 after AA induction and the secretion of IFNγ upon stimulation with Mt176-90 was studied. The results are presented as pg/ml±SEM of triplicate cultures. Three independent experiments produced similar results. * p<0.05 and ** p<0.005 compared to the Anti-MBP group.</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

DNA vaccination with HSP60 induces anti-ergotypic T cells.

<p>A and B. Anti-ergotypic proliferative response of LNC from rats vaccinated with pcDNA3, pHSP65 or pHSP60 (A) or pcDNA3, pI or pII (B), taken 26 days after the induction of AA. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. * p<0.05 compared to the pHSP65 (A) or the pcDNA3 (B) groups. C. 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 quadriplicate cultures. D. Anti-ergotypic cytokine response of LNC taken from rats vaccinated with pcDNA3, pHSP65, pHSP60, pI or pII 26 days after the induction of AA. 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

The activation HSP60-specific anti-ergotypic T cells requires co-stimulation.

<p>Monoclonal antibodies to CD28, CD80 or CD86, or a control IgG (Control), were assayed for their ability to block the anti-ergotypic proliferative response of Anti-HSP60 T-cells (Line) or of LNC prepared from pHSP60-vaccinated rats (LNC). Results are presented as the percent of inhibition of proliferation±SEM of quadruplicate cultures. Three independent experiments produced similar results.</p