CD4+T cells were incubated for 72 h in 96-well flat-bottom plates (Becton Dickinson)
CD4+T cells were incubated for 72 h in 96-well flat-bottom plates (Becton Dickinson). cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (51) on CD4+T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtbcan directly engage receptors on CD4+T cells and regulate their function. Rv2468c-induced costimulation of CD4+T cells could have implications for TB immune pathogenesis andMtbadjuvant effect. == Introduction == Mtb, the causative agent of TB, is one of the leading causes of death among adults worldwide. Closing significant gaps in understanding the complex host-pathogen interaction inMtbinfection and disease is necessary to develop more effective drug treatments and vaccines to prevent TB. Cell-mediated immunity is required for control Rabbit polyclonal to RAB1A ofMtbinfection Sivelestat sodium salt and preventing progression to disease. TCR+CD4+T cells are the primary mediators of protective immunity againstMtbinfection [16]. Sivelestat sodium salt Involvement of cytotoxic CD8+T cells, TCR (V2V2+) T cells, CD1-restricted T cells, as well as NK T cells has also been demonstrated [7,8]. Although necessary to control infection, T cell activation may also mediate tissue damage. Excessive T cell activation likely contributes to the tissue destruction of cavitary TB [9]. Thus, regulation of protective immune responses toMtbrequires a careful balance between activation and inhibition of T cells. Regulation of T cell function by intracellular pathogens, such asMtb, is traditionally considered the indirect result of altered APC function. These indirect mechanisms include inhibition of MHC class II and class I antigen processing, disruption of DC maturation, and induction of inhibitory cytokines (IL-10, TGF-) [1014].Mtbcan also regulate T cell function directly through mycobacterial molecules released by infected macrophages. Small vesicles or exosomes containing mycobacterial glycolipids, as well as lipoproteins and protein antigens, are found in the supernatants of infected macrophages [15,16]. We previously demonstrated that PIM binds directly to VLA-5 on CD4+T cells, resulting in T cell adhesion to FN [17].Mtblipoproteins also can directly costimulate CD4+T cells via TLR2 expressed on activated T cells [18]. Direct effects on T cells by mycobacterial molecules released by infected cells provide an alternative mechanism for regulation of cell-mediated immune responses inMtbinfection and disease. Stimulatory effects may protect the host through amplification of protective T cell immunity or alternatively contribute to T cell-mediated tissue damage. Inhibitory effects on T cells may contribute toMtbsurvival and immune evasion. Identification of mycobacterial molecules and mechanisms involved in direct regulation of Sivelestat sodium salt T cell function will contribute to better understanding the hostpathogen interaction in infections withMtband other intracellular bacteria. In screening for mycobacterial molecules with direct effects on lymphocytes, we identified fractions of mycobacterial lysate with costimulatory effects on human CD4+T cells. This resulted in identifying Rv2468c/MT2543, an uncharacterized mycobacterial protein with a FN-like RGD motif. Rv2468c triggers signals through integrin VLA-5, which when combined with TCR signaling, leads to T cell proliferation and cytokine secretion. We report the identification of Rv2468c inMtbsubcellular fractions and the mechanism involved in the ability of Rv2468c to affect CD4+T cell function. Our findings suggest thatMtbhas a wide variety of strategies to directly regulate lymphocyte functions, including expression of molecules that share motifs with host proteins. == MATERIALS AND METHODS == == Mycobacterial strains and subcellular fractions == Mtbstrain H37Ra lysate was extracted with Triton X-114, followed by phenol, and the hydrophobic extract was subsequently fractionated by preparative electroelution, as described [18,19]. Thirty fractions were obtained and analyzed in 13% SDS polyacrylamide gels, followed by Western blotting with antilipoprotein mAb and anti-BCG. Subcellular fractions from lab strain H37Rv, clinical isolates CDC1551 and HN878, and the Rv2468c-deficient transposon mutant strain (JHU-2468c) were obtained through the Tuberculosis Vaccine Testing and Research Materials Contract (NIAID HHSN266200400091C) at Colorado State University (Fort Collins, CO, USA). == LC-MS analysis == Electroelution fractions were analyzed in 13% SDS polyacrylamide gels stained with Coomassie blue. A 15- to 17-kDa band was excised from the gel and subjected to ingel digestion with trypsin or chemotrypsin [20]. Digests were resolved by LC-MS using.