Search

Abstract Accumulating evidence suggests the immunosuppressive microenvironments created by malignant tumors represent a major obstacle for effective anti‐tumor immunity. A better understanding of the suppressive mechanisms mediated by tumor microenvironments and the development of strategies to reverse the immune suppression are major challenges for the success of tumor immunotherapy. Here, we report that human tumor cells can induce senescence in naïve/effector T cells, exhibiting potent suppressive function in vitro and in vivo. We further show that tumor‐derived endogenous cyclic adenosine monophosphate (cAMP) is responsible for the induction of T‐cell senescence. Importantly, activation of TLR8 signaling in tumor cells can block the induction and reverse the suppression of senescent naïve and tumor‐specific T cells in vitro and in vivo, resulting in enhanced anti‐tumor immunity. These studies identify a novel mechanism of human tumor‐mediated immune suppression and provide a new strategy to reverse tumor immunosuppressive effects for tumor immunotherapy.

Th17 cells are a subset of CD4+ T cells known to play a central role in the pathogenesis of many autoimmune diseases, as well as in the defense against some extracellular bacteria and fungi. However, Th17 cells are not believed to have a significant function against intracellular infections. In contrast to this paradigm, we have discovered that Th17 cells provide robust protection against Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas disease. Th17 cells confer significantly stronger protection against T. cruzi-related mortality than even Th1 cells, traditionally thought to be the CD4+ T cell subset most important for immunity to T. cruzi and other intracellular microorganisms. Mechanistically, Th17 cells can directly protect infected cells through the IL-17A-dependent induction of NADPH oxidase, involved in the phagocyte respiratory burst response, and provide indirect help through IL-21-dependent activation of CD8+ T cells. The discovery of these novel Th17 cell-mediated direct protective and indirect helper effects important for intracellular immunity highlights the diversity of Th17 cell roles, and increases understanding of protective T. cruzi immunity, aiding the development of therapeutics and vaccines for Chagas disease.

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.

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.

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.

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.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 (P6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation.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.

BackgroundSerum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M. tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations.MethodWe constructed a high-content peptide microarray with 61 M. tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/-.FindingsThree distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB-, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB- does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope 'hotspots' at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background.ConclusionsA uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize biologically relevant targets useful for development of novel TB diagnostics and vaccines.

Objectives: To understand where transmission of Zika virus has the highest likelihood to occur in the contiguous United States with regard to its transmission both sexually and via Aedes aegypti mosquito bites., Methods: We evaluated the 2 routes of transmission risk with predictors of sexually transmitted infections (percentage women of childbearing age, birthrate, gonorrhea and chlamydia rates, concentrated disadvantage) as a surrogate for unprotected sexual activity and the demographic distribution of the A. aegypti mosquito across 3108 counties in the contiguous United States., Results: We found that 507 counties had the highest risk of virus exposure via mosquito vector or unprotected sexual activity; these were concentrated in southern states extending northward along the Atlantic coast and southern California, with the highest predicted risk in Mississippi counties., Conclusions: Identifying areas with higher transmission risk can inform prevention strategies and vector control, and assist in planning for diagnosis and treatment.