To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model was used to follow Vγ2Vδ2 + T cell responses to mycobacterial infections. These phosphoantigen-specific γδ T cells displayed major expansion during Mycobacterium bovis Bacille Calmette-Guérin (BCG) infection and a clear memory-type response after BCG reinfection. Primary and recall expansions of Vγ2Vδ2 + T cells were also seen during Mycobacterium tuberculosis infection of naı̈ve and BCG-vaccinated macaques, respectively. This capacity to rapidly expand coincided with a clearance of BCG bacteremia and immunity to fatal tuberculosis in BCG-vaccinated macaques. Thus, Vγ2Vδ2 + T cells may contribute to adaptive immunity to mycobacterial infections.
Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8(+) T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244(+)CD8(+) T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8(+) T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244-depressed CD8(+) T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244-expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection.
Abstract Ursolic acid (UA) has proved to have broad-spectrum anti-tumor effects, but its poor water solubility and incompetent targeting property largely limit its clinical application and efficiency. Here, we synthesized a nanoparticle-based drug carrier composed of chitosan, UA and folate (FA-CS-UA-NPs) and demonstrated that FA-CS-UA-NPs could effectively diminish off-target effects and increase local drug concentrations of UA. Using MCF-7 cells as in vitro model for anti-cancer mechanistic studies, we found that FA-CS-UA-NPs could be easily internalized by cancer cells through a folate receptor-mediated endocytic pathway. FA-CS-UA-NPs entered into lysosome, destructed the permeability of lysosomal membrane and then got released from lysosomes. Subsequently, FA-CS-UA-NPs localized into mitochondria but not nuclei. The prolonged retention of FA-CS-UA-NPs in mitochondria induced overproduction of ROS and destruction of mitochondrial membrane potential and resulted in the irreversible apoptosis in cancer cells. In vivo experiments showed that FA-CS-UA-NPs could significantly reduce breast cancer burden in MCF-7 xenograft mouse model. These results suggested that FA-CS-UA-NPs could further be explored as an anti-cancer drug candidate and that our approach might provide a platform to develop novel anti-cancer drug delivery system.
Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.
The possibility that CD4(+) T cells can act as "innate-like" cells to contain very early Mycobacterium tuberculosis dissemination and function as master helpers to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes during development of adaptive immunity against primary tuberculosis (TB) has not been demonstrated. We showed that pulmonary M. tuberculosis infection of CD4-depleted macaques surprisingly led to very early extrapulmonary M. tuberculosis dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during M. tuberculosis infection revealed the ability of CD8(+) T cells to compensate and rapidly differentiate to Th17-like/Th1-like and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. Whereas CD3(-) non-T lymphocytes in the presence of CD4(+) T cells developed predominant Th22-like and NK-like (perforin production) responses to M. tuberculosis infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3(-) lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNF-α, IL-17, IL-22, and perforin at the endpoint of more severe TB, but they presented pulmonary IL-4(+) T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22(+) and perforin(+) cells despite the presence of IL-17(+) and IL-4(+) cells. These results implicate a previously unknown innate-like ability of CD4(+) T cells to contain extrapulmonary M. tuberculosis dissemination at very early stage. Data also suggest that CD4(+) T cells are required to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes and to prevent rapid TB progression during M. tuberculosis infection of nonhuman primates.
The possibility that mycobacterial infections induce variant cytokine mRNA encoding a functionally distinct protein for immune regulation has not been addressed. In this study, we reported that Mycobacterium tuberculosis and bacillus Calmette-Guérin infections of macaques induced expression of variant IL-4 (VIL-4) mRNA encoding a protein comprised of N-terminal 97 aa identical with IL-4, and unique C-terminal 96 aa including a signaling-related proline-rich motif. While VIL-4 could be stably produced as intact protein, the purified VIL-4 induced apparent expansion of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)-specific Vgamma2Vdelta2 T cells in dose- and time-dependent manners. The unique C-terminal 96 aa bearing the proline-rich motif (PPPCPP) of VIL-4 appeared to confer the ability to expand Vgamma2Vdelta2 T cells, since simultaneously produced IL-4 had only a subtle effect on these gammadelta T cells. Moreover, VIL-4 seemed to use IL-4R alpha for signaling and activation, as the VIL-4-induced expansion of Vgamma2Vdelta2 T cells was blocked by anti-IL-4R alpha mAb but not anti-IL-4 mAb. Surprisingly, VIL-4-expanded Vgamma2Vdelta2 T cells after HMBPP stimulation appeared to be heterologous effector cells capable of producing IL-4, IFN-gamma, and TNF-alpha. Thus, mycobacterial infections of macaques induced variant mRNA encoding VIL-4 that functions as growth factor promoting expansion of HMBPP-specific Vgamma2Vdelta2 T effector cells.
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