Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+) /CD4(+) Foxp3(+) cells

Abstract

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette-Guérin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+) ) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Figure 1

Total number and frequency of spleen CD4⁺ and CD4⁺ Foxp3⁺ cells from immunized mice. BALB/c mice were immunized with the DNA-heat-shock protein 65 (hsp 65), BCG/DNA-hsp 65 and BCG/CFP-CpG vaccines. Mice were immunized with BCG or injected with pVAX plasmid or saline as experimental controls. Fifteen days after the completion of the immunization schedule, the total number and frequency of effector (CD4⁺) and regulatory (CD4⁺Foxp3⁺) T cells were evaluated in the spleen (a). Spleen cells were first gated by size (forward scatter) and granularity (side scatter) and then by CD4 and Foxp3 expression. The results are expressed as the means ± SEM of individual animals analysed in each different group. Total number of spleen CD4⁺ cells and total number of spleen CD4⁺ Foxp3⁺ cells (b). Ratio of total CD4⁺/CD4⁺ Foxp3⁺ cells and ratio of the frequency of CD4⁺/CD4⁺ Foxp3⁺ cells (c). The total number of cells is representative of two independent experiments with the following experimental n: DNA-hsp 65, 6; BCG/DNA-hsp 65, 8; BCG/CFP-CpG, 6; pVAX, 7; BCG, 4. The cell frequency data are representative of three independent experiments (DNA-hsp 65, 8; BCG/DNA-hsp 65, 8; BCG/CFP-CpG, 6; pVAX, 10; BCG: 12). *P < 0·05 compared with non-immunized mice (black dotted line). &P < 0·05 compared with non-immunized mice (grey dotted line). Horizontal bars represent the significant difference between groups.

Figure 2

Number of colony-forming units (CFU) in the lung and spleen and correlations of CFU, CD4⁺/CD4⁺ Foxp3⁺ ratio and CD4⁺ Foxp3⁺ cells from immunized mice. Different groups of mice were challenged with 1 × 10⁵ bacilli of Mycobacterium tuberculosis 30 days after immunization (a). Lung and spleen CFU counts were evaluated on day 30 post challenge (b). The total number of lung CD4⁺ (c) and total number of lung CD4⁺ Foxp3⁺ cells (d) are shown. The ratio of CD4⁺/CD4⁺ Foxp3⁺ cells (e), the ratio of lung CFU counts and CD4⁺/CD4⁺ Foxp3⁺ cells (f), the ratio of lung CFU counts and total number of CD4⁺Foxp3⁺ cells (g) are shown. The cell number is expressed as the mean ± SEM for individual animals. The CFU counts are expressed as Log₁₀ of the mean ± SEM for individual animals. The data are representative of two independent experiments (DNA-heat-schock protein (hsp) 65, 7; BCG/DNA-hsp 65, 7; BCG/CFP-CpG, 8; pVAX, 4; BCG, 7). *P < 0·05 compared with non-immunized and infected mice (black dotted line); &P < 0·05 compared with non-immunized and infected mice (grey dotted line); #P < 0·05 compared with non-immunized, uninfected mice (solid line). Horizontal bars represent the significant difference between groups.

Figure 3

Histological analysis of lungs. Mice were vaccinated with DNA-heat-shock protein (hsp) 65, BCG/DNA-hsp 65 or BCG/CFP-CpG and challenged with Mycobacteirum tuberculosis as described in Fig. 2. Pathology was evaluated on the upper right lobe of the lung on day 30 post-challenge (original magnification, 100 ×). Asterisks show the areas represented in 400 × magnification (upper right quadrant). Arrow shows a foamy macrophage. Head arrow shows neutrophils.

Figure 4

Evaluation of the regulatory T cells in vivo after immunization with the DNA-heat-shock protein (hsp) 65 or BCG/DNA-hsp 65 vaccine. BALB/c mice were infected intratracheally with 1 × 10⁵ bacilli. Thirty days after infection, 50 μg Mycobacterium tuberculosis antigen (Mtb Ag) in the presence or absence of CD4⁺ CD25⁺ cells, which were obtained from infected or uninfected mice, were administered in the right footpad. The left footpad received saline (a). Oedema measurement was performed 48 hr after the administration of antigen (b). BALB/c mice were infected, and 30 days after infection, they received 50 μg M. tuberculosis antigens in the right footpad in the presence or absence of CD4⁺ CD25⁺ cells that were obtained from immunized or non-immunized mice (c). Oedema measurements were performed at 24 and 48 hr (d) after antigen administration. BALB/c mice were immunized with BCG/DNA-hsp 65 and challenged 30 days after prime boost vaccination. Three days before immunization and challenge, the mice were treated with the monoclonal antibody (mAb) PC61 (e). Representative flow cytometry data demonstrating the percentages of CD4⁺ CD25⁺ lymphocytes in the blood of mice treated with 500 μg mAb PC61 during immunization (f). Colony-forming unit (CFU) counts were evaluated 30 days after challenge (g). The results are expressed as the mean ± SEM. The data are representative of two experiments. For the delayed type hypersensitivity (DTH) assay, with CD4⁺ CD25⁺ cells purified from uninfected or infected mice, seven mice were used. For the DTH assay, with CD4⁺ CD25⁺ cells purified from immunized or non-immunized mice, 8–14 mice were used. For the treatment with anti-CD25, eight or nine mice were used. Horizontal bars represent the significant difference between the groups. *P < 0·05 compared with non-immunized, infected and treated with immunoglobulin control mice; #P < 0·05 compared with mice non-immunized, infected and treated with mAb PC61.