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

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

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

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.

<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