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

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

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

Bystander activation of irrelevant CD4⁺ T cells following antigen-specific vaccination occurs in the presence and absence of adjuvant

<div><p>Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4⁺ T cells. Intramuscular injection with the highly immunogenic Complete Freund’s Adjuvant (CFA) led to local <i>in vivo</i> proliferation and activation of intravenously transferred CD4⁺ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund’s Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.</p></div

Antigen-specific boost leads to local proliferation of transferred hPG-specific T cells.

<p>Animals received a prime at d₀ (PBS, OVA+PBS or OVA+CFA) followed by an i.v. transfer of labeled CD90.1⁺CD4⁺ T cells at d₂₀ and a boost at d₂₁ (PBS, OVA+PBS or OVA+IFA) before analysis at d₂₄. (A,B) Percentage of (A) proliferation of and (B) CD62L expression in CFSE-labeled CD90.1⁺CD4⁺ cells in spleen and LN. (C,D) IFN-γ-ELISpot assay with 48h <i>ex vivo</i> hPG-peptide restimulation of (D) splenocytes and (C) LNs. The ELISpot was performed in duplo (LN) and triplo (spleen). Data shown are the means+SEM of two independent experiments (each 2–5 animals per group). Differences between two groups were determined with an unpaired two-tailed student’s t-test. Differences between the three groups were determined with a one-way ANOVA (two-tailed) with Dunnett’s multiple comparison test. P < 0.05 was considered significant. CSC: cytokine secreting cell, ndLN: non-draining LN.</p

Set up of <i>in vivo</i> transfer studies.

<p>CD4⁺ T cells were isolated from TCR Tg mice which have CD4⁺ T cells specific for hPG peptide. The (CD90.1⁺)CD4⁺ T cells were CFSE-labeled and i.v. transferred to donor mice at d_-1 (approach 1) or d₂₀ (approach 2). At d₀ acceptor mice received an i.m. injection (50 μl) in the left quadriceps with CFA or PBS, sometimes supplemented with hPG-peptide or OVA. Animals were sacrificed at d₃ or d₂₄. Animals sacrificed on d₂₄ received a booster shot (right quadriceps, d₂₁) with IFA or PBS, sometimes supplemented with OVA.</p

Onset of disease and maximum arthritis scores.

<p>Hsp70-mB29a peptide loaded PLGA, PLGA-TMC nanoparticles or PBS control (10 µg) were given i.n. on day −7, −5 and −3 and arthritis was induced by PG/DDA immunization on day 0 and 21. Arthritis symptoms were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026684#s4" target="_blank">materials and methods</a>. Day of onset and maximum arthritis scores were depicted as mean ± SEM. of n = 3 mice per group of one experiment.</p

Enhanced OVA-specific CD4⁺ T-cell proliferation, after nanoparticle administration.

<p><b>A and D.</b> OVA-specific CFSE labeled CD4⁺ T-cells were transferred to BALB/c recipient mice one day prior to vaccination. Mice received a single i.n. application of 30 µg of sOVA or OVA encapsulated into PLGA, PLGA-TMC or TMC-TPP nanoparticles. For induction of a non-mucosal response, mice received a single i.m. immunization in the hind limbs. At 72 h post OVA administration, <i>in vivo</i> T-cell division was addressed in spleen, nose-draining NALT and CLN as well as the thigh-draining ILN. Data are representative for at least 3 i.n. and 2 i.m. independent transfer studies. <b>B, C and E.</b> Total mRNA was purified from single cell suspensions from NALT, CLN, and ILN. Relative mRNA expression to HPRT of Foxp3 was determined 72 h post OVA application. Cells isolated from NALT were pooled per group. LN data are representative for at least 3 to 5 mice per group; mean ± SEM. Statistically significant: *, <i>P</i><0.05.</p

Injection with hPG peptide results in proliferation and activation of transferred hPG-T cells at d₃.

<p>Animals received an i.v. transfer of CFSE-labeled hPG specific CD4⁺ T cells at d_-1 and an i.m. injection with PBS or hPG peptide in PBS at d₀ before analysis at d₃. (A) Representative FACS-histogram and -gating strategy of CFSE-labeled hPG-specific donor CD4⁺ T cells in the iliac LN after hPG peptide injection. (B,C) Percentage of (B) proliferation of and (C) CD69 expression on CFSE-labeled CD4⁺ T cells in spleen and LN. (D,E) IFN-γ-ELISpot assay with 48h <i>ex vivo</i> hPG peptide restimulation of (D) splenocytes and (E) LNs. The ELISpot was performed in duplo (LN) and triplo (spleen). 1 animal per group was used. This experiment was performed twice. ND: not determined, hPG: hPG peptide, CSC: cytokine secreting cell, med: medium, ndLN: non-draining LN.</p