Figure 3.

Humoral and cellular immune responses of rBCG-DisA-immunized mice. (A) The immunization and Mtb challenging scheme. Female BALB/c mice were subcutaneously (s.c.) immunized with 1 × 10⁶ CFU BCG and rBCG-DisA, respectively, and PBS was used as negative control. At the fourth week after immunization, a group of three mice were sacrificed to determine immunological characteristics. At the same time, three mice were challenged intravenously (i.v) with 5 × 10⁴ CFU virulent Mtb H37Rv strain, and mice injected s.c. with PBS (Naïve) were used as control groups. Eight weeks post-challenge, mice were sacrificed to detect immunological characteristics. (B) Four weeks after immunization, sera (1:200) from different groups were collected and total IgG titers of anti-BCG proteins were detected using ELISA. (C) Splenic lymphocyte proliferation was assayed using MTS agent after stimulating with BCG and DisA protein, respectively, for 72 h. (D) Splenic lymphocyte proliferation was determined using CFSE agent after stimulating with BCG proteins (25 μg/mL) for 7 days, and detected by flow cytometry. (E) Percentages of CD4⁺ and CD8⁺ T cells of splenic lymphocytes from each group, and the ratios of CD4/CD8 were calculated as shown. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.