Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Th17 Cells Provide Mucosal Protection against Gastric Trypanosoma cruzi Infection.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and gamma interferon (IFN-γ) are important players in host defense. More recently, Th17 cells and interleukin 17A (IL-17A) have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect in the mucosa, the initial site of parasite invasion in many human cases. We found that IL-17RA knockout (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reduced with IL-17A neutralization or reversed by loss of macrophage NADPH oxidase activity. Consistently with this, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection and identify a novel protective mechanism involving the induction of NADPH oxidase activity by IL-17A. These studies provide important insights for Chagas vaccine development and, more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Automated generation of immature dendritic cells in a single-use system.

Dendritic cells (DCs) are an indispensable part of studying human responses that are important for protective immunity against cancer and infectious diseases as well as prevention of autoimmunity and transplant rejection. These cells are also key elements of personalized vaccines for cancer and infectious diseases. Despite the vital role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remains a comparatively under-developed and inefficient process. DCs are present in very low concentrations (<1%) in blood, thus they must be generated from monocytes and the current methodology in DC generation involves a laborious process of static culture and stimulation with cytokines contained in culture medium. Herein, we describe an automated fluidic system, MicroDEN, that allows for differentiation of monocytes into immature-DCs (iDCs) utilizing continuous perfusion of differentiation media. Manual steps associated with current ex vivo monocyte differentiation are vastly reduced and an aseptic environment is ensured by the use of an enclosed cartridge and tubing network. Benchmark phenotyping was performed on the generated iDCs along with allogeneic T-cell proliferation and syngeneic antigen-specific functional assays. MicroDEN generated iDCs were phenotypically and functionally similar to well plate generated iDCs, thereby demonstrating the feasibility of utilizing MicroDEN in the broad range of applications requiring DCs.

New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells.

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5(flox/flox) (control) and ATG5(flox/flox) Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.

Dipterinyl calcium pentahydrate inhibits intracellular mycobacterial growth in human monocytes via the C-C chemokine MIP-1ß and nitric oxide.

Tuberculosis remains one of the top three leading causes of morbidity and mortality worldwide, complicated by the emergence of drug-resistant Mycobacterium tuberculosis strains and high rates of HIV coinfection. It is important to develop new antimycobacterial drugs and immunomodulatory therapeutics and compounds that enhance antituberculous immunity. Dipterinyl calcium pentahydrate (DCP), a calcium-complexed pterin compound, has previously been shown to inhibit human breast cancer cells and hepatitis B virus (HBV). DCP inhibitory effects were attributed to induction of apoptosis and/or increased production of interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we tested the ability of DCP to mediate inhibition of intracellular mycobacteria within human monocytes. DCP treatment of infected monocytes resulted in a significant reduction in viability of intracellular but not extracellular Mycobacterium bovis BCG. The antimicrobial activity of DCP was comparable to that of pyrazinamide (PZA), one of the first-line antituberculosis drugs currently used. DCP potentiated monocyte antimycobacterial activity by induction of the cysteine-cysteine (C-C) chemokine macrophage inflammatory protein 1ß (MIP-1ß) and inducible nitric oxide synthase 2. Addition of human anti-MIP-1ß neutralizing antibody or a specific inhibitor of the l-arginase-nitric oxide pathway (N(G)-monomethyl l-arginine [l-NMMA] monoacetate) reversed the inhibitory effects of DCP on intracellular mycobacterial growth. These findings indicate that DCP induced mycobacterial killing via MIP-1ß- and nitric oxide-dependent effects. Hence, DCP acts as an immunoregulatory compound enhancing the antimycobacterial activity of human monocytes.

Granzyme A produced by γ(9)δ(2) T cells induces human macrophages to inhibit growth of an intracellular pathogen.

Human γ(9)δ(2) T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ(9)δ(2) T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ(9)δ(2) T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ(9)δ(2) T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ(9)δ(2) T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ(9)δ(2) T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ(9)δ(2) T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

Tumor-derived γδ regulatory T cells suppress innate and adaptive immunity through the induction of immunosenescence.

Fundamentally understanding the suppressive mechanisms used by different subsets of tumor-infiltrating regulatory T (Treg) cells is critical for the development of effective strategies for antitumor immunotherapy. γδ Treg cells have recently been identified in human diseases including cancer. However, the suppressive mechanisms and functional regulations of this new subset of unconventional Treg cells are largely unknown. In the current studies, we explored the suppressive mechanism(s) used by breast tumor-derived γδ Treg cells on innate and adaptive immunity. We found that γδ Treg cells induced immunosenescence in the targeted naive and effector T cells, as well as dendritic cells (DCs). Furthermore, senescent T cells and DCs induced by γδ Treg cells had altered phenotypes and impaired functions and developed potent suppressive activities, further amplifying the immunosuppression mediated by γδ Treg cells. In addition, we demonstrated that manipulation of TLR8 signaling in γδ Treg cells can block γδ Treg-induced conversion of T cells and DCs into senescent cells in vitro and in vivo. Our studies identify the novel suppressive mechanism mediated by tumor-derived γδ Treg cells on innate and adaptive immunity, which should be critical for the development of strong and innovative approaches to reverse the tumor-suppressive microenvironment and improve effects of immunotherapy.

Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection.

Mucosa-associated invariant T (MAIT) cells are a unique population of αß T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vß T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection.

Transcriptional suppression of IL-27 production by Mycobacterium tuberculosis-activated p38 MAPK via inhibition of AP-1 binding.

Mycobacterium tuberculosis remains a major global challenge to human health care, and the mechanisms of how M. tuberculosis evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that bacillus Calmette-Guérin (BCG) and viable M. tuberculosis as well as M. tuberculosis lysates could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and M. tuberculosis lysate stimulation of macrophages induced activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. M. tuberculosis lysate-induced p28 transcription was dependent on MyD88 signaling pathway. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after M. tuberculosis lysate stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to M. tuberculosis lysate stimulation. Furthermore, blockade of p38 by SB203580 enhanced M. tuberculosis-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhanced the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective tuberculosis immunity.

Co-administration of a plasmid DNA encoding IL-15 improves long-term protection of a genetic vaccine against Trypanosoma cruzi.

BACKGROUND: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone. METHODOLOGY: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8(+) T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation. CONCLUSION: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.

Immune responses to gp82 provide protection against mucosal Trypanosoma cruzi infection

The potential use of the Trypanosoma cruzi metacyclic trypomastigote (MT) stage-specific molecule glycoprotein-82 (gp82) as a vaccine target has not been fully explored. We show that the opsonization of T. cruzi MT with gp82-specific antibody prior to mucosal challenge significantly reduces parasite infectivity. In addition, we investigated the immune responses as well as the systemic and mucosal protective immunity induced by intranasal CpG-adjuvanted gp82 vaccination. Spleen cells from mice immunized with CpG-gp82 proliferated and secreted IFN-γ in a dose-dependent manner in response to in vitro stimulation with gp82 and parasite lysate. More importantly, these CpG-gp82-immunized mice were significantly protected from a biologically relevant oral parasite challenge.

Induction of B- and T-cell responses to cruzipain in the murine model of Trypanosoma cruzi infection.

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. The parasite is transmitted mucosally, with both intra- and extracellular life stages in the human host. Cruzipain, the major cysteinyl proteinase of T. cruzi, has been shown to be antigenic in both humans and mice during infection with the parasite. We extend these observations, showing here that multiple murine immune subsets of potential importance for vaccine-induced protection can be induced by cruzipain. Cruzipain-specific serum IgG responses were induced during chronic infection with T. cruzi. In addition, T. cruzi mucosal infection stimulated the development of cruzipain-specific secretory IgA detectable in fecal extracts from infected mice. Cruzipain-specific type 1 cytokine responses characterized by the production of IFN-gamma but not IL-4 were also detectable during murine infection. Furthermore, immunization of mice with a DNA vaccine encoding cruzipain was shown to stimulate cytotoxic T lymphocyte (CTL) responses capable of recognizing and lysing T. cruzi-infected cells. The induction of serum antibody, mucosal IgA, Th1 cytokine and CTL responses by cruzipain in mice supports the use of this parasite protein for further efforts in T. cruzi vaccine development.