Treg depletion causes an increase in virus-specific CD8+ T cells in the brain.

<p>(<b>A</b>) The proportion of virus-specific D^bMV-H_22–30-pentamer⁺ CD8⁺ T cells of all CD8⁺ T cells was determined in spleen, lymph nodes, and brains of MV-infected C57BL/6 mice at days 3, 7, 10, 14, and 28 post infection (n = 3). MV-specific cells were gated as CD19-negative lymphocytes to exclude pentamer⁺ CD19⁺ cells. The total number of CD8⁺ T cells (<b>B</b>) and the number and proportion of D^bMV-H_22–30-pentamer⁺ CD8⁺ T cells (<b>C</b>) was determined in brains of 28 days infected control (DEREG^−/−) and DEREG (DEREG^−/+) mice, both treated with DT (Values ± SEM; n = 3).</p

Detection of regulatory T cells in secondary lymphoid organs and the brain of C57BL/6 and DEREG mice.

<p>Mice were i.c. infected and analyzed at 3, 7, 10, 14, and 28 dpi as indicated. The percentages of regulatory CD4⁺ Foxp3⁺ T cells all lymphocytes in spleen (<b>A</b>) and draining cervical lymph nodes (CLN) (<b>B</b>) of rMV-green-infected and PBS-injected (ctrl) animals were determined using C57BL/6 mice. Foxp3⁺ T cells were quantified by flow cytometry after staining with antibodies to CD25, CD4, and Foxp3, and gating on positive cells. In brains (<b>C</b>), total cell numbers of Foxp3-GFP⁺ Tregs were determined using of rMV-infected and PBS-injected (ctrl) DEREG mice. Mean values ± SEM are presented (n = 3).</p

Expansion of T lymphocytes with the superagonistic CD28 antibody D665.

<p>(<b>A</b>) Two week old uninfected C57BL/6 mice were i.p. injected with 100 µg mAb D665 or PBS (control) and analyzed 3 days later. Lymphocytes were isolated from the spleen and lymph nodes (12 per mouse; 6 cervical, 4 axillary and 2 inguinal lymph nodes). FACS dot plot examples for CD4⁺ Foxp3⁺ T cells in the lymph nodes are shown (left panels: ctrl and +mAb D665 with percentages of all gated lymphocytes). Right panel: quantitative evaluation of the proportion of Foxp3⁺ T cells (percent of all CD4⁺ T cells) in spleen and lymph nodes of D665-treated and control animals (mean values ± SEM, n = 4, P<0.01). (<b>B</b>) Experimental setup used for the treatment of MV-infected mice with mAb D665. As a control an appropriate volume of PBS was injected. (<b>C</b>) The total number of lymphocytes in the spleen and draining lymph nodes (LN) (n = 3; P<0.01), and total number of percoll-isolated cells in brains of D665-treated and control animals (n = 3). (<b>D</b>) Quantitative evaluation of CD4⁺ Foxp3⁺ Tregs in the spleen and LN of D665-treated and control animals (percent CD4⁺Foxp3⁺ cells of all CD4⁺ T cells; n = 3, P<0.002 and P<0.02, respectively).</p

Depletion of Tregs leads to a reduction of the CNS infection.

<p>(<b>A</b>) Adult DEREG (DEREG^−/+) mice were i.p. injected with 1 µg diphtheria toxin (DT) or with an appropriate volume of PBS (ctrl) at 6 consecutive days and analyzed the next day. Lymphocytes were isolated from the spleen and LN (6 cervical, 4 axillary, and 2 inguinal). FACS dot plot examples for regulatory CD4⁺ Foxp3-GFP⁺ T cells in the lymph nodes (left panels), and a quantitative evaluation of Foxp3-GFP⁺ T cells (percentage of all lymphocytes, right panel) from spleen and LN (mean values ± SEM, n = 4, P<0.01) are shown. (<b>B</b>) Experimental setup for the treatment of young MV-infected DEREG mice with DT at day 17, 18, and 20 post infection and analysis at 28 dpi. (<b>C</b>) Quantitative evaluation of the number of infected eGFP⁺ cells at 28 dpi in DEREG (DEREG^−/+) and control (DEREG^−/−) mice both infected i.c. with rMV-green and treated with DT. The reduction of mean values from 50 to 8 was significant, with P = 0,0098. The number of infected eGFP⁺ cells per brain was determined by microscopic evaluation of 100 µm sections through the complete cerebrum as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033989#pone.0033989-Schubert1" target="_blank">[33]</a>.</p

Expansion of T lymphocytes with the superagonistic CD28 antibody D665 induces virus replication and spread.

<p>Consecutive coronal brain sections (100 µm sections) were prepared from complete rMV-green-infected mouse cerebra and analyzed using the UV microscope. Overviews and details of a typical section of an infected brain of a mouse treated with mAb D665 and analyzed at 28 dpi (<b>A</b>), and sections of infected control animals in the absence of mAb D665 at 28 dpi (<b>B</b>), and 14 dpi (<b>C</b>) are shown. The numbers of infected eGFP⁺ cells per brain (sections through the complete cerebrum of each animal were evaluated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033989#pone.0033989-Schubert1" target="_blank">[33]</a>) were determined microscopically in infected control C57BL/6 mice at 7, 14, 28 and 42 dpi (<b>D</b>, lanes 1–4) and in D665-treated mice at 28 and 42 dpi (<b>D</b>, lanes 5 and 6). The difference between control and D665-treated mice at 28 dpi was highly significant (P<0,0001).</p

BAC-Foxp3^Cre-GFP-driven GFP expression faithfully reflects Foxp3 protein expression during thymic Treg cell lineage commitment.

<p>Direct assessment of Foxp3 protein expression using mAbs to Foxp3 after flow cytometric isolation of GFP⁺ cells from BAC-Foxp3^Cre-GFP mice. (A) Presort analysis of CD25 and GFP expression among gated DP and CD4SP cells from BAC-Foxp3^Cre-GFP mice after magnetic bead enrichment of CD25⁺ cells, as well as postsort analysis are depicted, as indicated. CD25^?GFP^? DP cells were included for comparison (left). (B) Foxp3 expression of sorted cells, as revealed by intracellular staining (ICS) with mAbs to Foxp3. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate.</p

Genetic lineage tracing of Foxp3⁺ Treg cells in BAC-Foxp3^Cre-GFP × R26Y mice.

<p>Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP⁻YFP⁻ and GFP⁻/GFP⁺YFP⁺ populations among (<b>A</b>) CD25⁺ CD4SP thymocytes and (<b>B</b>) CD4⁺CD25⁺ T cells from LNs of BAC-Foxp3^Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4⁺CD25⁺ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP⁻YFP⁻ (left) and GFP⁻/GFP⁺YFP⁺ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (<b>C</b>) Percentages of GFP⁺ cells among <i>ex vivo</i> populations of YFP⁻CD25⁺ CD4SP thymocytes (left) and peripheral YFP⁻CD4⁺CD25⁺ T cells (right). (<b>D</b>) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP⁻YFP⁻ and GFP⁻/GFP⁺YFP⁺ cells presented in (<b>A and B</b>).</p

Infidelity of BAC-Foxp3^Cre-GFP-dependent GFP expression.

<p>(<b>A-C</b>) Tracking Foxp3⁺ cells that lack GFP expression during thymic Treg cell lineage commitment. (<b>A</b>) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3^Cre-GFP mice before and after magnetic bead enrichment of CD25⁺ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (<b>B</b>) Percentages and (<b>C</b>) numbers of Foxp3⁺ cells (ICS) among GFP⁻ and GFP⁺ cells that had been sorted from DP and CD4SP thymocyte compartments. (<b>D-F</b>) Tracking Foxp3⁺ Treg cells that lack GFP expression in peripheral lymphoid tissues. (<b>D</b>) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4⁺ T cells from LNs before and after magnetic bead enrichment of CD25⁺ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (<b>E</b>) Percentages and (<b>F</b>) numbers of Foxp3-expressing (ICS) CD4⁺CD25⁺ T cells among GFP⁻ and GFP⁺ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3^Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.</p

Comparative quantification of GFP⁺ DP cells.

<p>Flow cytometric isolation of GFP⁺ DP thymocytes from (<b>A</b>) Foxp3^GFP, BAC-Foxp3^Cre-GFP and (<b>B</b>) Foxp3^IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25⁺GFP⁺ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (<b>C</b>) Quantification of GFP⁺ thymocytes. Percentages (left) and numbers (right) of GFP⁺ DP cells (top) and GFP⁺ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (<b>A,B</b>). All mice were six weeks old. (<b>D</b>) Numbers of GFP⁺ DP thymocytes from eleven-week-old Foxp3^GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3^Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).</p

Culture conditions demonstrating eGFP and Foxp3 expression dichotomy amongst in vitro generated iTregs from DEREG mice.

<p>(A) Dot plots display eGFP and Foxp3 expression among the live CD4⁺ gated DEREG cells. Shown are <i>ex vivo</i> stained T cells (left), <i>in vitro</i> differentiated iTregs using soluble anti-CD3, TGF-β, RA and GM-CS- or Flt3L-derived BMDC (second and third panels respectively) and iTregs using plate-bound anti-CD3, anti-CD28, TGF-β and RA (right). (B) Differential expression of various surface antigens on eGFP⁺Foxp3⁻ (dotted line, upper panel) and eGFP⁺Foxp3⁺ (solid line, upper panel) iTregs and eGFP⁻Foxp3⁻ (dotted line, lower panel) and eGFP⁻Foxp3⁺ (solid line, lower panel) cells differentiated in the presence of TGF-β, RA, soluble anti-CD3 and DC. Gray histograms represent isotype controls. Graphs shown are representative of four individual experiments.</p