Background: Microarray experiments capture details of cellular responses upon perturbations, including Mycobacterium tuberculosis (Mtb) infection.Results: Novel bioinformatics approach “Express Path Analysis” identifies Src at the core of the network regulating divergent responses to virulent and avirulent Mtb infection.Conclusion: Src acts through regulating key antimycobacterial properties, acidification of phagolysosome and autophagy induction.Significance: Results may help evolve novel host targeted anti-tuberculosis therapy strategy.

Mycobacterium tuberculosis cell wall glycolipid, Lipoarabinomannan, can inhibit CD4+ T cell activation by down-regulating phosphorylation of key proximal TCR signaling molecules Lck, CD3ζ, ZAP70 and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an antigen encounter, yet remain viable and hyporesponsive. We tested whether LAM-induced inhibition of CD4+ T cell activation resulted in CD4+ T cell anergy. The presence of LAM during primary stimulation of P25TCR-Tg murine CD4+ T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25TCR-Tg CD4+ T cells primed in the presence of LAM also exhibited decreased response upon re-stimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hypo-responsiveness to re-stimulation was not due to apoptosis, generation of FoxP3-positive regulatory T cells or inhibitory cytokines. Acquisition of the anergic phenotype correlated with up-regulation of GRAIL (gene related to anergy in lymphocytes) protein in CD4+ T cells. Inhibition of human CD4+ T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM pre-treatment abrogated LAM induced hypo-responsiveness. In addition, exogenous IL-2 reversed defective proliferation by down-regulating GRAIL expression. These results demonstrate that LAM up-regulates GRAIL to induce anergy in Ag-reactive CD4+ T cells. Induction of CD4+ T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.

SUMMARY We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4+ T cells and up-regulate TCR triggered- IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4+ T cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4+ T cells express TLR2 and respond to TLR2 co-stimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T cell TLR2 in priming of CD4+ T cells in vivo, naïve MTB Ag85B specific TCR transgenic CD4+ T cells (P25 TCR-Tg) were adoptively transferred into Tlr2-/- recipient mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3Cys-SKKKK (P3CSK4). TLR2 engagement during priming resulted in increased numbers of IFN-γ secreting P25 TCR-Tg T cells one week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4+ T cells increases MTB-Ag specific responses and may contribute to protection against MTB infection.