**2. Materials and methods**

#### **2.1 In vivo effects of different mycobacterial lysates over the subsequent activation of DCs**

Separate groups of 3-5 BALB/c female mice were intratracheally primed with each lysate (40g/mice in 40L) from the three strains used in our experiments (lyCan: M. canettii lysate; lyH37: Mtb H37Rv lysate; lyBei: M. Beijing lysate). Five hours after the mycobacterial lysate, mice were given an intranasal (i.n.) challenge with LPS to assess in DCs the potential effects of prior mycobacterial lysate treatment. Control groups included a) mice primedboosted both with LPS, or b) mice treated with endotoxin-free saline solution before the LPS challenge, c) mice treated only with endotoxin-free saline solution without further LPS.

mycobacterial replication rate is diminished (B. J. Rogerson et al., 2006) while diverse immune evasion mechanisms avoid bacterial killing by T cell-activated macrophages (J. A. Armstrong & P. D. Hart, 1971) and cytotoxic CD8+ T cells (E. M. Weerdenburg et al., 2010). The analysis of these evasion mechanisms used by MTC strains, however, have barely been

To evaluate the in vivo differences in host-pathogen interaction across the wide range of virulence among MTC strains we used three mycobacterial strains as representative of low (*Mycobacterium canettii*), intermediate (*Mycobacterium tuberculosis* H37Rv), and high (*Mycobacterium beijing*) virulence degrees. The recently defined *Mycobacterium tuberculosis* Beijing (M. Beijing) strains are associated with high virulence and multidrug resistance (I. Parwati et al., 2010), and cause in mice a quick increase of cellular infiltrate with high numbers of colony forming units in the lungs (J. Dormans et al., 2004). Conversely, smoothtype *Mycobacterium tuberculosis* Canettii (M. canettii) strains rarely cause TB in humans and in the experimental mouse model show low cellular infiltrate with limited chronic infection (M. Fabre et al., 2010). Interestingly, our previous results assessing the mechanisms causing the difference in virulence showed an inverse correlation between strain virulence and in vivo cytotoxic responses, as well as higher bacterial burden in the lungs of M. Beijing

We decided to assess a profile of dendritic cell maturation and T cell exhaustion in vivo during pulmonary infection with these three mycobacterial strains. Since MTC strains are intracellular pathogens, T cells have an important role in mediating cytolysis of infected cells and to induce activation of other immune cells (Y. He et al., 2001; S. Inoue et al., 2005; E. A. Murphy et al., 2001; S. C. Oliveira et al., 2002). For intracellular pathogens like MTb, one way to subvert T cell responses could well be by altering activation/maturation of DCs. Importantly, DCs play an important role both in inducing effector cytotoxic T cells in vivo as well as in Ag-surveillance of mucosal surfaces and in the uptake and transport of mycobacterial bacilli to the lung draining lymphoid tissue, the mediastinal lymph nodes (MedLN)(G. S. Garcia-Romo et al., 2004; A. Pedroza-Gonzalez et al., 2004; A. J. Wolf et al.,

We aimed to describe our recent findings regarding the differential stimulation of DCs and T cells by MTC strains with different virulence. We consider that increasing the research on the differences among MTC strains pathogen-host interactions in vivo might help to better

**2.1 In vivo effects of different mycobacterial lysates over the subsequent activation** 

Separate groups of 3-5 BALB/c female mice were intratracheally primed with each lysate (40g/mice in 40L) from the three strains used in our experiments (lyCan: M. canettii lysate; lyH37: Mtb H37Rv lysate; lyBei: M. Beijing lysate). Five hours after the mycobacterial lysate, mice were given an intranasal (i.n.) challenge with LPS to assess in DCs the potential effects of prior mycobacterial lysate treatment. Control groups included a) mice primedboosted both with LPS, or b) mice treated with endotoxin-free saline solution before the LPS challenge, c) mice treated only with endotoxin-free saline solution without further LPS.

understand, among other things, the underlying limitations of anti-TB vaccines.

comparatively assessed (L. Quintero-Macias et al., 2011).

infected mice (L. Quintero-Macias et al., 2011).

2007).

**of DCs** 

**2. Materials and methods** 

Then, 10 hours after LPS challenge, a time which is around the peak of lung DC activation induced by LPS alone, we obtained cell suspensions from lung, BAL (Bronchio-Alveolar Lavage) and MedLN to assess CD86 expression in (Gr1-, MHC-II hi, CD11c+) DCs. Mycobacterial lysates were prepared by one of us (I. Estrada-Garcia, ENCB-IPN).
