Expression levels of Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specific T cells during stationary level lung infection in mice

Abstract

Mycobacterium tuberculosis lung infection in mice was controlled at an approximately stationary level after 20 days of log linear growth. Onset of stationary level infection was associated with the generation by the host of T helper type 1 (Th1) immunity, as evidenced by the accumulation of CD4 Th1 cells specific for the early secretory antigen (ESAT-6) of M. tuberculosis encoded by esat6, and for a mycolyl transferase (Ag85B) encoded by fbpB. CD4 T cells specific for these antigens were maintained at relatively high numbers throughout the course of infection. The number of CD4 T cells generated against ESAT-6 was larger than the number generated against Ag85B, and this was associated with a higher transcription level of esat6. The total number of transcripts of esat6 increased during the first 15 days of infection, after which it decreased and then approximately stabilized at 10(6.5) per lung. The total number of fbpB transcripts increased for 20 days of infection before decreasing and then approximately stabilizing at 10(4.8) per lung. The number of transcripts of esat6 per colony-forming unit of M. tuberculosis fell from 8.6 to 0.8 after day 15, and of fbpB from 0.3 to less than 0.02 after day 10, suggesting that at any given time during stationary level infection the latter gene was expressed by a very small percentage of bacilli. Expressed at an even lower level was an M. tuberculosis replication gene involved in septum formation (ftsZ), indicating that there was no significant turnover of the M. tuberculosis population during stationary level infection.

Figure 1

Course of M. tuberculosis infection (a) in the lungs of mice infected with approximately 10² CFU of M. tuberculosis via the airborne route. The M. tuberculosis grew progressively for approximately 20 days before further growth was inhibited and the level of infection was stabilized by acquired immunity. The means ± SD of five mice are shown per time-point. Kinetics of development of acquired immunity (b) in terms of the accumulation in the lungs of T cells capable of making IFN-γ in response to ESAT-6 (1–20) peptide, or Ag85B (240–260) peptide in an 18-hr Elispot assay. The means ± SD of the number of spots in triplicate wells using pooled cells of four mice are shown per time-point. Identical results were obtained in two separate experiments.

Figure 2

Evidence that cells that made IFN-γ in response to ESAT-6 (1–20) or Ag85B (240–260) peptide as measured with the Elispot assay were CD4 T cells. Flow cytometric analysis (a) showing that magnetic depletion of lung cells treated with biotin-conjugated, anti-CD4 monoclonal antibody and then with streptavidin-coupled magnetic nanoparticles was approximately 95% successful at removing CD4 cells. Depletion of CD4 T cells (b) resulted in loss (open bars) of most cells capable of making IFN-γ (closed bars) in response to ESAT-6 and Ag85B peptide.

Figure 3

Changes against time of infection (a) in the log₁₀ copy number per lung of mRNA for esat6, fbpB and ftsZ, as quantified by real-time RT-PCR. Changes in the log10 CFU of M. tuberculosis are also shown. The number of esat6 transcripts increased for the first 15 days of infection, and of fbpB and ftsZ for the first 20 days, after which transcript numbers declined briefly and then approximately stabilized. When transcripts of esat6, fbpB and ftsZ were expressed as copies per CFU of M. tuberculosis (b) the number of esat6 transcripts declined from 8·6 to 0·8 per CFU, of fbpB from 0·3 to 0·02 per CFU, and of ftsZ from 0·14 to 0·007. Therefore, most M. tuberculosis were not transcribing the latter two genes at any one time of infection, particularly after infection became stationary. Means ± SD for M. tuberculosis RNA from lungs of four or five mice per time-point.