Migration of BM-DC to the lymph nodes.

<p>BM-DC were incubated with Pb labeling with CFSE and injected into the lung. After 12 and 24 hrs the lymph nodes cells were analyzed by citometry. A-Figure representative of 3 different experiments, where the gate represent positive cells, determined by FMO, as described in the M&M. The numbers represent the percentage of positive cells. B- At 12 and 24 hrs after Pb infection, the absolute numbers of CFSE+/CD11c+ were determined in the lymph nodes. Error bars represent the SEM. *p<0.05 when compared with only DC.</p

DC transport yeast form of <i>Pb</i> to draining lymph nodes.

<p>Yeast form of Pb were labeling with FITC as described in the M&M. After labeling, 1×10⁶<i>Pb</i> were injected intratracheally. After 12 h, the lymph node cells were removed and analyzed by cytometry. Animals injected with FITC only were used as controls. The Figure is representative of 3 different experiments. The number represent the percentage of positive cells.</p

Migration and phenotype of DC in lung during Pb infection.

<p>A- At several time points after Pb infection, the absolute numbers of DC (MHC-II+/CD11c+) were determined in the lung. Error bars represent the SEM. B-Phenotype of lung DC after Pb infection, the gate represent positive cells, determined by FMO, as described in the M&M. This figure is representative of 3 different experiments. The numbers represent the percentage of positive cells. The experiment was performed at least 3 times. *p<0.05 when compared with PBS treatment.</p

Migration of lung DC to the lympho nodes.

<p>CFSE and Pb were injected into the lung and after 12 and 24 hrs the reginal lympho nodes cells were analyzed to the presence of CFSE+/CD11c+ by citometry. A-Figure representative of 3 different experiments where the gate represent positive cells, determined by FMO, as described in the M&M. The numbers represent the percentage of positive cells. B- At 12 and 24 hrs after CFSE and Pb infection, the absolute numbers of CFSE+/CD11c+ were determined in the lympho nodes. Error bars represent the SEM. *p<0.05 when compared with only CFSE.</p

Cytokines.

<p>BALB/c mice (seven/group) were infected with 1x10⁶ of <i>P</i>. <i>brasiliensis</i> yeast. On 7 and 14 days, the animals were treateds with pMAC/PS-scFv. As control, the BALB/c mice was treateds with PBS (50μL) or empty vector. After seven days of last burst, the lung (A) and lynph nodes (B) cells were cultivated in vitro for 24 hours and the IFN-γ, IL-12 and IL-4 were measured by ELISA. *p<0.05 e **p<0,001. Results are representative of three independent experiments.</p

Humoral response.

<p>BALB/c mice (seven/group) were infected previously with <i>P</i>. <i>Brasiliensis</i> yeast (1x10⁶) by intratracheal pathway. The animals were treateds twice with pMAC/PS-scFv and total IgG was measured (A). The ratio between IgG2a/IgG1 was analyse (B) *p<0.05. Results are representative of three independent experiments.</p

Phenotype of DCs and T cells.

<p>BALB/c mice (seven/group) were immunized via intramuscular injection of PBS, pMAC/PS or pMAC/PS-scFv. After 7 days the lymph nodes were obtained and the DCs CD11c⁺/CD8⁺, CD11c⁺/CD40 and CD11c⁺/DEC205 (A) and CD4⁺ (B) and CD8⁺ (C) T cells were analyzed by flow cytometer. *p<0.05. A. Flow cytometry graph: one representative experiment is shown. B and C: Data are expressed as the number of cells obtained with specific Ab. Results are representative of three independent experiments.</p

DCs molecule expression.

<p>DCs were transfected with pMAC/PS-scFv. After 48 hours the total DCs were stained with anti-MHCII/CD11c, anti-CD86, anti-CD40 and anti-CD80. The data were analyzed by flow cytometer. Results are representative of three independent experiments.</p

Phenotype of lung cells.

<p>BALB/c mice (seven/group) were infected with <i>P</i>. <i>brasiliensis yeast</i> (1x10⁶). After that, the animals received twice treatments with PBS (50μL), DC (1x10⁶), DC-pMAC/PS (1x10⁶ cells plus 20ng/mL DNA) and DC-pMAC/PS-scFv (1x10⁶ cells plus 20ng/mL DNA). After seven days from the last treatment, the lung cells were stained with anti-CD3e/CD4 (A), anti-CD3e/CD8 (B) and anti-CD40/CCR7 (C). As control of the infection, BALB/c mice received only PBS (50μL) by intratracheal pathway. The data was analyzed by flow cytometer. Flow cytometry graphs: one representative experiment is shown.</p

CD4⁺ and CD8⁺ T cells activation.

<p>Lymph node cells from seven BALB/c mice per group were obtained after intramuscular immunization with PBS, pMAC/PS or pMAC/PS-scFv. 3x10⁵ cells were cultivated with 20 μg/mL of gp43 antigen. After that, the cells were stained with CFSE and after 72 hours, lymphoproliferation of CD4⁺ (A) and CD8⁺ (B) was analyzed by flow cytometry. As a positive control, we used concanavalin mitogen (ConA). *p<0.05. Results are representative of three independent experiments.</p