Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette-Guérin

Abstract

Both CD4(+) and CD8(+) T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) on the CD8(+) T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8(+) T cells specific to the mycobacterial Mtb32a(309-318) epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8(+) T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D(b-)Mtb32a(209-318) peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8(+) T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8(+) T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8(+) T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8(+) T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-gamma in response to Mtb32a(209-318) peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8(+) T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.

Figure 1

Induction of a T cytotoxic 1 (Tc1)-type T-cell response after immunization with major histocompatibility complex (MHC) H-2^b class I-restricted mycobacterial epitopes. B6 mice were inoculated with mycobacterial Ag85B_291–299(striped bars), PstS-3_285–293 (open bars), Mtb32a_309–318 (hatched bars), or control OVA_257–264 (closed bars) peptide, emulsified with incomplete Freund’s adjuvant (IFA), into the footpads on day 0. On day 10 after immunization, CD8⁺ T cells were purified from the inguinal and popliteal lymph node (LN) cells, and cultured with the JAWSII dendritic cell (DC) line in the presence (+) or absence (−) of the relevant peptide. The concentration of interferon-γ (IFN-γ) in the culture supernatants was analyzed using enzyme-linked immunosorbent assay (ELISA.) Data were expressed as the mean ± standard deviation. The data are representative of two independent experiments.

Figure 2

Kinetics of the appearance of mycobacterial antigen-specific CD8⁺ T cells in the lung and mediastinal lymph node (LN) cells following infection with Mycobacterium tuberculosis. B6 mice were infected intratracheally (i.t.) with 1 × 10⁴ colony-forming units (CFU) of M. tuberculosis, and the lung lymphocytes and mediastinal LN cells were prepared on the indicated days after the infection. The cells were surface-stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 monoclonal antibody (mAb), phycoerythrin (PE)-conjugated anti-CD8α mAb and allophycocyanin (APC)-conjugated D^b-Mtb32a pentamer, the analysis gate was set on the CD19⁻ CD8⁺ fraction, and the percentage of D^b-Mtb32a pentamer⁺ CD8⁺ T cells was determined using flow cytometry (FCM). The representative FCM profiles (a, b) and the absolute number of D^b-Mtb32a pentamer⁺ CD8⁺ T cells (c, d) in the lung (a, c) and mediastinal LNs (b, d) were demonstrated. The data in panels c and d are the mean and standard deviation for a group of five mice at each time-point. The bars with the same letter differ significantly (P< 0·05) in the post-hoc test. The data shown are representative of three independent experiments. Med LN, mediastinal LN; Mtb, Mycobacterium tuberculosis.

Figure 3

Kinetics of Mtb32a-specific CD8⁺ T cells in the inguinal and popliteal lymph nodes (LNs) after subcutaneous (s.c.) vaccination with 2·5 × 10⁶ colony-forming units (CFU) of Mycobacterium bovis bacille Calmette–Guérin (BCG). At the designated time-points after the infection, the cells were collected and surface-stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 monoclonal antibody (mAb), phycoerythrin (PE)-conjugated anti-CD8α mAb and allophycocyanin (APC)-conjugated D^b-Mtb32a pentamer. The analysis gate was set on CD19⁻ CD8α⁺ T cells, the percentage of D^b-Mtb32a pentamer⁺ cells was determined and the absolute number of the D^b-Mtb32a pentamer⁺ CD8⁺ T cells was calculated. The data are the mean and standard deviation for a group of five mice at each time-point. Bars with the same letter differed significantly (P< 0·05) in post-hoc testing. The data are representative of three independent experiments.

Figure 4

Kinetics of Mtb32a-specific CD8⁺ T-cell numbers in the lung and mediastinal lymph node (LN) cells after Mycobacterium tuberculosis infection of mice with or without Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccination. B6 mice were vaccinated subcutaneously with 2·5 × 10⁶ colony-forming units (CFU) of M. bovis BCG into the footpads (open bars). Another group of mice were unimmunized (closed bars). Four weeks later, the mice were challenged intratracheally (i.t.) with 1 × 10⁴ CFU of M. tuberculosis. At the time-points indicated after infection with M. tuberculosis, the lung lymphocytes (a) and mediastinal LN cells (b) were prepared. The cells were then surface-stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 monoclonal antibody (mAb), phycoerythrin (PE)-conjugated anti-CD8 mAb and allophycocyanin (APC)-conjugated D^b-Mtb32a pentamer. The cells were analyzed by flow cytometry (FCM) to determine the percentage of D^b-Mtb32a pentamer⁺ CD8⁺ T cells, and the absolute number of the D^b-Mtb32a pentamer⁺ CD8⁺ T cells was calculated. The data of each time-point represent the mean and standard deviation for a group of five mice. *P< 0·05 in the Student’s t-test compared with the absolute number of Mtb32a-specific CD8⁺ T cells in unimmunized mice. The data are representative of three independent experiments. Med LN, mediastinal LN; Mtb, Mycobacterium tuberculosis.

Figure 5

Mtb32a peptide-induced interferon-γ (IFN-γ) production by CD8⁺ T cells. B6 mice were vaccinated subcutaneously with 2·5 × 10⁶ colony-forming units (CFU) of Mycobacterium bovis bacille Calmette–Guérin (BCG) into the footpads (+) or were unvaccinated (−). Four weeks later, the mice were challenged intratracheally (i.t.) with 1 × 10⁴ CFU of Mycobacterium tuberculosis. On days 7 and 14 after the M. tuberculosis infection, the lung lymphocytes were collected, and CD8⁺ T cells were enriched and cultured for 24 hr with the JAWSII dendritic cell (DC) line in the presence of Mtb32a_309–318 peptide. The concentrations of IFN-γ in the culture supernatants were determined using enzyme-linked immunosorbent assays (ELISAs) and the mean and standard deviation for a group of five mice are shown. *P< 0·05 in the Student’s t-test compared with unvaccinated mice. The data are representative of two independent experiments.