Figure 1.
Detection of regulatory T cells in secondary lymphoid organs and the brain of C57BL/6 and DEREG mice.
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 (A) and draining cervical lymph nodes (CLN) (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 (C), 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).
Figure 2.
Expansion of T lymphocytes with the superagonistic CD28 antibody D665.
(A) 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) Experimental setup used for the treatment of MV-infected mice with mAb D665. As a control an appropriate volume of PBS was injected. (C) 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). (D) 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).
Figure 3.
Expansion of T lymphocytes with the superagonistic CD28 antibody D665 induces virus replication and spread.
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 (A), and sections of infected control animals in the absence of mAb D665 at 28 dpi (B), and 14 dpi (C) are shown. The numbers of infected eGFP+ cells per brain (sections through the complete cerebrum of each animal were evaluated as described [33]) were determined microscopically in infected control C57BL/6 mice at 7, 14, 28 and 42 dpi (D, lanes 1–4) and in D665-treated mice at 28 and 42 dpi (D, lanes 5 and 6). The difference between control and D665-treated mice at 28 dpi was highly significant (P<0,0001).
Figure 4.
Depletion of Tregs leads to a reduction of the CNS infection.
(A) 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) 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. (C) 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 [33].
Figure 5.
Treg depletion causes an increase in virus-specific CD8+ T cells in the brain.
(A) The proportion of virus-specific DbMV-H22–30-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) and the number and proportion of DbMV-H22–30-pentamer+ CD8+ T cells (C) was determined in brains of 28 days infected control (DEREG−/−) and DEREG (DEREG−/+) mice, both treated with DT (Values ± SEM; n = 3).