Increased Virulence in M. massiliense Infection Figure 6. Increased CD4+ Foxp3+IL-10+ regulatory T cell expression in the lungs and spleens of IFNc-KO mice infected with M. massiliense CRM-0019. Lung cells obtained from mice infected with M. massiliense CIP 108297 (closed square) and CRM-0019 (open circle) were assayed by multi-parametric flow cytometry on days 30 and 60. Panel A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD4+ T cells expression of Foxp3+. Panel B shows that mice infected with M. massiliense CRM-0019 had a significant increase in CD4+Foxp3+ and CD4+Foxp3+ IL-10+ regulatory T cells in the lungs and spleens at 30 and 60 days of infection compared to M. massiliense CIP 108297-infected mice. Results are expressed as the mean number of CD4+Foxp3+ and CD4+Foxp3+ IL-10+ (6 SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p,0.050. doi:10.1371/journal.pone.0024726.g006 development of immunosuppressive regulatory T cells. Earlier reports have shown that clinical isolates of M. tuberculosis vary in their ability to induce regulatory T cells with possible consequenc- es on virulence and host protective immunity [23,37]. Moreover, there is evidence that patients with active tuberculosis have raised levels of circulating regulatory T cells [38,39]. Thus, regulatory T cell induction may be inherent in mycobacterial host immunity and not just responding to the acute lung damage caused by highly virulent M. tuberculosis. Our study suggests that IL-10, a product of regulatory T cells as well as macrophages, may play a role in maintaining the stability of persistent NTM disease as has also been suggested in the case of Bordetella pertussis infection [40]. A likely correlate of our important findings is that the ability of some M. massiliense isolates and, perhaps, closely related Mycobac- terium species, to persist in vivo through increased resistance to the bactericidal mechanisms of macrophages and the modulation of T PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24726