B29-specific T cell proliferation in mice immunized with B29 after CD25⁺ T cell depletion.

<p>Mice were injected with anti-CD25 depleting antibody PC61 or with PBS as a control. 7 days after depletion of CD25^<b>+</b> cells, the mean percentage (± s.e.m.) of CD25^<b>+</b> cells (A) or FoxP3^<b>+</b> cells (B) was determined in total peripheral blood directly prior to immunization of n = 2–6 (A) or n = 1–3 (B) animals per group. Data of figure A are representative of 3 independent experiments. (C) 7 days after administration of anti-CD25 antibody (depicted as αCD25) or PBS, mice were immunized with Hsp70 peptide B29, or control peptide pOVA, and 10 days later splenocytes were restimulated with B29, mouse homologs mB29a or mB29b, or control peptide pOVA. Results are expressed as the mean relative cpm (cpm peptide / cpm medium only ± s.e.m.) obtained from of 3–4 animals per condition and are representative of 3 independent experiments. Background cpm values of the negative controls were as follows (all medium controls from left to right): medium control of B29-immunized mouse 1004 cpm; medium control of pOVA immunized mouse 738 cpm; medium control of B29-immuinized mouse + αCD25 prior to immunization 4088 cpm; medium control pOVA immunized mouse + αCD25 prior to immunization 2055. nd: not determined. P values are from an unpaired two-tailed Student t test in which the PBS group was compared to the anti-CD25 antibody treated group (A), or in which Hsp70 peptide (B29, mB29a, or mB29b) stimulation was compared to pOVA stimulation (B). *P < 0.05; **P <0.01; ***P < 0.001.</p

B29 induced Tregs are suppressive in vitro.

<p>Mice were either injected with anti-CD25 antibody to deplete CD25^<b>+</b> cells, or with PBS as a control. 7 days after injection, mice (n = 3 per treatment) were immunized with either B29 (upper graph) or pOVA (lower graph). 10 days later autologous CD4^<b>+</b>CD25^<b>-</b> responder cells (white bars) and CD4^<b>+</b>CD25^<b>+</b> cells were isolated and pooled for co-culture in various ratios in the presence of anti-CD3 antibody to activate the cells. As a control, also CD4^<b>+</b>CD25^<b>-</b> responder cells from naïve (N) donors (black bars) were used to test the suppressive capacity of B29-induced Tregs or pOVA-induced Tregs on the same population of responder T cells. ^<b>3</b>H-thymidine incorporation was determined and cpm data are shown as the mean of triplicate samples (± s.e.m.). % supp. is the proliferative response of responder T cells cultures alone, compared to responder T cells co-cultured with Tregs. Data shown are representative for two independent experiments. P values are from an unpaired two-tailed Student t-test in which cpm from CD4^<b>+</b>CD25^<b>-</b> cells were compared to cpm from CD4^<b>+</b>CD25^<b>+</b> cells. ** P < 0.01, *** P < 0.001.</p

Induction of CD4⁺CD25⁺ and CD4⁺FoxP3⁺ cells after peptide immunization in mice prior depleted from CD25⁺ cells.

<p>Mice were injected with anti-CD25 antibody or with PBS only. 7 days later, mice depleted from CD25^<b>+</b> cells were immunized with B29 (depicted as αCD25+B29). Mice that received PBS were immunized with B29 or pOVA (depicted as B29 or pOVA). 10 days after peptide immunization, mice were sacrificed and splenic CD4^<b>+</b> T cells were assessed for Treg markers and activation markers by flow cytometry. The results depicted are the mean percentages (± s.e.m.) of CD25^<b>+</b>, CD25^<b>-</b>, FoxP3^<b>+</b>, CD69^<b>+</b> and CD62L^<b>+</b> cells within the CD4+ T cell population of the spleen. Data are the mean of 8 animals per group. P values are from an unpaired two-tailed Student t-test in which the αCD25 + B29 group was compared to B29 group. * P < 0.05, ** P < 0.01.</p

Adoptive transfer of B29-induced Tregs reduces inflammation in a mouse model of rheumatoid arthritis.

<p>Mean arthritis scores of recipient mice after adoptive transfer of CD4^<b>+</b>CD25^<b>-</b> cells or CD4^<b>+</b>CD25^<b>+</b> cells from B29-immunized donors (injected with PBS 7 days prior to B29 immunization), or mice receiving CD4^<b>+</b>CD25^<b>+</b> cells from B29-immunized donors injected with anti-CD25 antibody 7 days prior to immunization (depicted as CD25+ αCD25). Recipient animals (n = 6–7 mice per group) received 3x10^<b>5</b> cells i.p. one day prior to the second PG immunization. Clinical scores were assessed over time and are depicted as the mean of the group (± s.e.m.). Data shown are representative for 2 experiments. P values are from a two-way ANOVA (all time points) followed by Bonferroni post hoc comparison. *P < 0.05.</p