Abstract
γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.
Highlights
␥9␦2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination
We found that ␥9␦2 T cells capable of inhibiting intracellular M. tuberculosis growth represent only a subset of the phosphoantigen (IPP and hydroxy-3-methyl-but2-enyl pyrophosphate (HMBPP))-responsive ␥9␦2 T cells
We first verified that expansion of protective ␥9␦2 T cells did not require infection of host cells with viable mycobacteria by comparing the ability of M. tuberculosis whole-cell lysates (MtbWL)-expanded ␥9␦2 T cells and live BCG-expanded ␥9␦2 T cells to inhibit intracellular mycobacterial growth
Summary
␥9␦2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded ␥9␦2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive ␥9␦2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection Based on this premise, we have been searching for M. tuberculosis antigens capable of inducing a unique subset of mycobacterium-protective ␥9␦2 T cells. Specific to M. tuberculosis, earlier biochemical analysis identified 4 molecules from M. tuberculosis lysates, termed TUBag to -4, that stimulated the proliferation of a human ␥9␦2 T cell clone (G115) [26] These TUBag compounds have been shown to be active in the nanomolar range (i.e., with bioactivities up to 30,000fold higher than that of isopentenyl pyrophosphate [IPP]), suggesting that these molecules could account for most of the ␥9␦2. Synthetic bromohydrin pyrophosphate (BrHPP) is a strong activator of ␥9␦2 T cells and has been tested for tumor immunotherapy and as a vaccine component [21, 32,33,34]
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