IL10 does not mediate the observed suppression of active IL-1β release.

<p>a) memory CD4+CD45RO+ T-cells cultured overnight in the presence of monocytes with or without αCD3 and/or IFNβ were restimulated with PMA/Ionomycin in the presence of Brefeldin A for 4 h and stained for IL-10 and IFNγ (representative of 3 independent experiments); b) upregulation of <i>IL10</i> mRNA expression in resorted memory T-cells was confirmed by qPCR (n = 3; n.d. = not detected); c) inhibition of IL-10 (10 ug/ml) and/or IFNγ (10 ug/ml) by specific blocking antibodies did not affect the suppressive effect on IL-1β release by activated T-cells in the presence of IFNβ (n = 4) (% suppression is calculated by subtracting from 1 the ratio of IL-1β release from activated T-cells in the presence of IFNβ and non-activated T-cells in the absence of IFNβ and multiplying by 100; **p<0.01 employing repeated measures ANOVA with post-hoc Bonferroni adjustment for multiple comparisons to avoid random correlations).</p

Inhibition of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is ATP-specific.

<p>IL-1β release after incubation with a) Alum (400 ug/ml) (n = 3), b) MSU crystals (300 ug/ml) (n = 3) and c) MDP (10 ug/ml) (n = 3) was not inhibited by co-incubation of monocytes with IFNβ-primed CD4+CD45RO+ memory T-cells (Data are shown as means ± SD of duplicate cultures; ***p<0.01 employing repeated measures ANOVA with post-hoc Bonferroni adjustment for multiple comparisons to avoid random correlations).</p

Suppression of IL-1β release is cell contact independent and mediated by a soluble factor of 23–38 kDa MW.

<p>a) Blocking OX40L and CD40L pathways by the use of inhibitory antibodies did not affect the suppressive effect of activated T-cells on monocytes in the presence of IFNβ (n = 3; % suppression calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039576#pone-0039576-g004" target="_blank">Fig 4</a>); b) <i>CD39</i> mRNA expression by memory T-cells resorted after co-culture with monocytes was measured by qPCR (n = 3) (Data are shown as means ± SD of duplicate cultures; *p<0.01 employing repeated measures ANOVA with post-hoc Bonferroni adjustment for multiple comparisons to avoid random correlations); c) transfer of supernatant conditioned by a co-culture of monocytes and activated memory CD4+ T-cells in the presence of IFNβ onto fresh monocytes reproduced the suppressive effect on IL-1β release (n = 4); d) Kinetics of the inhibitory factor release and determination of molecular weight; supernatants of monocytes co-cultured with activated human memory T-cells in the presence of IFNβ were removed at the indicated time-points and fresh monocytes were cultured in the conditioned supernatants for 16 h before stimulation with LPS and activation with ATP (representative of 2 independent experiments); e) supernatants were filtered through molecular cut-off filters or f) SEC fractionated and molecular weight fractions in a 1∶1 ratio with fresh medium were used for the culture of fresh monocytes. IL-1β secretion was measured and inhibition of release was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039576#pone-0039576-g004" target="_blank">Fig 4</a> (representative of 2 independent experiments).</p

IFNβ inhibits IL-1β release in the presence of activated human CD4+CD45RO+ memory T-cells.

<p>Monocytes were incubated overnight in the absence or presence of unstimulated or soluble αCD3 (0.5 ug/ml) activated CD4+CD45RO+CD45RA- memory T-cells with or without 1000 IU/ml IFNβ. The next day 100 ng/ml LPS was added to the co-culture for 4 h, followed by addition of 500 uM ATP for 45 min; a) IL-1β (n = 8) and b) TNFα levels (n = 5) were measured in the supernatant by ELISA. There was a significant reduction of IL-1β release in the presence of activated T-cells and IFNβ; TNFα secretion was not affected; c) memory T-cells were incubated with IFNβ either in the presence or absence of 2 ug/ml αCD3 and αCD28 for 18 h, washed twice with PBS and co-incubated with monocytes for 14 h. The release of IL-1β by monocytes after stimulation with LPS and ATP was suppressed by co-incubation with activated IFNβ-primed memory T-cells (n = 3) (Data are shown as means ± SD of duplicate cultures; *p<0.05; ***p<0.001 employing repeated measures ANOVA with post-hoc Bonferroni adjustment for multiple comparisons to avoid random correlations); d) immunoblot of pro-IL-1β cleavage and pro-caspase-1 levels in the cell lysate of monocytes.</p

Activated CD4+ CD45RO+ memory T-cells decrease secretion of soluble caspase-1 and P2X7R mRNA expression in monocytes leading to a reduced response upon ATP binding.

<p>a) sCaspase-1 levels in the supernatant of monocytes was measured by ELISA (n = 8). A significant decrease of soluble Caspase-1 in the supernatant of cells co-cultured with activated memory T-cells was observed both in the presence and absence of IFNβ; b) intracellular levels of pro-IL-1β measured with an ELISA specific for the immature precursor of IL-1β showed a significant increase in pro-IL-1β levels in the presence of human activated CD4+CD45RO+ memory T-cells (n = 3), cell lysates from adherent monocytes were obtained after removal of non-adherent T-cells after addition of LPS and prior to incubation with ATP c) the ratio of intracellular pro-IL-1β to released soluble Caspase-1 is significantly increased in the presence of human activated CD4+CD45RO+ memory T-cells (n = 3). d) <i>P2X7R</i> mRNA expression in monocytes cultured overnight with activated CD4+CD45RO+ memory T-cells is decreased with and without addition of exogenous IFNβ (n = 3), mRNA was isolated from adherent monocytes after removal of non-adherent T-cells after addition of LPS and prior to incubation with ATP (Data are shown as means ± SD of duplicate cultures; *p<0.05; **p<0.01 employing repeated measures ANOVA with post-hoc Bonferroni adjustment for multiple comparisons to avoid random correlations); e) Ca²⁺-influx measurement in monocytes cultured with activated human CD4+CD45RO+ memory T-cells in the presence of IFNβ demonstrates the absence of slow sustained Ca²⁺-influx mediated by ATP binding to the purinergic P2X7-receptor (representative of 3 independent experiments); f) Annexin V staining of monocytes coincubated with T-cells and stimulated with LPS was measured by flow cytometry (representative flow cytometry histogram of n = 5).</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

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

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