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Metabolic reprogramming of cancer cells creates a unique tumor microenvironment (TME) characterized by the limited availability of nutrients, which subsequently affects the metabolism, differentiation, and function of tumor-infiltrating T lymphocytes (TILs). TILs can also be inhibited by tumor-derived metabolic waste products and low oxygen. Therefore, a thorough understanding of how such unique metabolites influence mammalian T cell differentiation and function can inform novel anti-cancer therapeutic approaches. Here, we highlight the importance of these metabolites in modulating various T cell subsets within the TME, dissecting how these changes might alter clinical outcomes. We explore potential TME metabolic determinants that might constitute candidate targets for cancer immunotherapies, ideally leading to future strategies for reprogramming tumor metabolism to potentiate anti-cancer T cell functions.

IntroductionTuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a serious threat to human health. Vaccination with BCG prevents the development of the most severe forms of TB disease in infants and was recently shown to prevent Mtb infection in previously uninfected adolescents. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. However, our understanding of the effects of BCG vaccination on γδ T cell responses is incomplete.MethodsIn this study we performed γδ T cell receptor (TCR) repertoire sequencing of samples provided pre- and post-BCG vaccination from 10 individuals to identify specific receptors and TCR clones that are induced by BCG.ResultsOverall, there was no change in the diversity of γTCR or δTCR clonotypes in post- vs pre-BCG samples. Furthermore, the frequencies of TCR variable and joining region genes were minimally modulated by BCG vaccination at either the γTCR or δTCR loci. However, the γTCR and δTCR repertoires of individuals were highly dynamic; a median of ~1% of γTCR and ~6% of δTCR in the repertoire were found to significantly expand or contract in post- vs pre-BCG comparisons (FDR-q < 0.05). While many of the clonotypes whose frequency changed after BCG vaccination were not shared among multiple individuals in the cohort, several shared (i.e., “public”) clonotypes were identified with a consistent increase or decrease in frequency across more than one individual; the degree of sharing of these clonotypes was significantly greater than the minimal sharing that would be expected among γTCR and δTCR repertoires. An in vitro analysis of Mtb antigen-reactive γδ T cells identified clonotypes that were similar or identical to the single-chain γTCRs and δTCRs that changed consistently after BCG vaccination; pairings of γTCRs and δTCRs that increased after BCG vaccination were significantly over-represented among the Mtb-reactive γδ T cells (p = 1.2e-6).DiscussionThese findings generate hypotheses about specific γδTCR clonotypes that may expand in response to BCG vaccination and may recognize Mtb antigens. Future studies are required to validate and characterize these clonotypes, with an aim to better understand the role of γδ T cells in Mtb immunity.

The scientific response to the SARS-CoV-2 virus outbreak has been nothing short of breathtaking. Within 7 months of virus discovery, over 75,000 virus isolates around the world were sequenced and w...

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.

M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naïve and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects.

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 γ9δ2T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion ofGZMAfrom γ9δ2T 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.

Failure of the most recent tuberculosis (TB) vaccine trial to boost bacillus Calmette–Guérin–mediated anti-TB immunity despite the induction of Th1-specific central memory cell and effector memory cell responses highlights the importance of identifying optimal T cell targets for protective vaccines. In this study, we describe a novel, Mycobacterium tuberculosis–specific IFN-γ+CD4+ T cell population expressing surface markers characteristic of naive-like memory T cells (TNLM), which were induced in both human (CD45RA+CCR7+CD27+CD95−) and murine (CD62L+CD44−Sca-1+CD122−) systems in response to mycobacteria. In bacillus Calmette–Guérin–vaccinated subjects and those with latent TB infection, TNLM were marked by the production of IFN-γ but not TNF-α and identified by the absence of CD95 expression and increased surface expression CCR7, CD27, the activation markers T-bet, CD69, and the survival marker CD74. Increased tetramer-positive TNLM frequencies were noted in the lung and spleen of ESAT-61–20–specific TCR transgenic mice at 2 wk postinfection with M. tuberculosis and progressively decreased at later time points, a pattern not seen with TNF-α+CD4+ T cells expressing naive cell surface markers. Importantly, adoptive transfer of highly purified TNLM alone, from vaccinated ESAT-61–20–specific TCR transgenic mice, conferred equivalent protection against M. tuberculosis infection in the lungs of Rag−/− mice when compared with total memory populations (central and effector memory cells). Thus, TNLM may represent a memory T cell population that, if optimally targeted, may significantly improve future TB vaccine responses.

Tuberculosis (TB) remains a tremendous global health problem, with 1/4 of the world's population infected and causing 1 million deaths annually. Intradermal Bacillus Calmette-Guérin (BCG) vaccine given during infancy protects against severe forms of acute disease but does not prevent chronic infection or development of pulmonary TB. TB vaccine mucosal targeting potentially could induce mucosal resident immune cells with increased protective capacity against pulmonary infection and disease. Sublingual (SL) administration of vaccines may be an optimal mucosal delivery platform based on the high absorptive capacity of this mucosal surface, the extensive lymphoid tissue, and published preclinical studies demonstrating efficacy of SL vaccination against other pathogens. To this end, we performed preliminary testing of sublingual TB vaccines. Vaccination of mice with SL BCG elicited potent mycobacteria-specific T cell responses which persisted 16 weeks post-immunization. The magnitudes of the T cell responses were similarly induced after sublingual, intranasal, and subcutaneous BCG vaccination. Interestingly, serum mycobacteria-specific antibody responses and systemic recovery of BCG post-vaccination were lower after SL BCG compared with systemic BCG immunization. However, more importantly, SL BCG vaccinated mice were significantly protected against an aerosolized virulent M. tuberculosis challenge (P 0.0001 compared to unvaccinated mice). Furthermore, this protection was long-lived, persisting for 16 weeks with1 log CFU reduction compared with naïve challenged mice in both lungs and spleens (P 0.0001 and P 0.0028, respectively). These exciting results provide strong support for further studies exploring the mechanisms of protective immunity induced by sublingual BCG vaccination.

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.

SummaryAntigen classes other than proteins can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. We sought to define the roles of donor unrestricted T (DURT) cells, including MR1-reactive MAIT cells, CD1b-reactive glucose monomycolate (GMM)-specific T cells, CD1d-reactive NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis. We characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in infants or BCG-revaccination in adults. BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CD1b-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CD1d- restricted NKT cells. By contrast, BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of IFN-γ-expressing CD4+ T cells with a CD26+CD161+TRAV1-2− phenotype in infants. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies.

Background Herpes zoster (HZ) infection increases dementia risk, but it is not known if herpes zoster vaccination is associated with lower risk for dementia. We determined if HZ vaccination, compared to no HZ vaccination, is associated with lower risk for incident dementia. Methods and findings Data was obtained from Veterans Health Affairs (VHA) medical records (10/1/2008–9/30/2019) with replication in MarketScan® commercial and Medicare claims (1/1/2009-12/31/2018). Eligible patients were ≥65 years of age and free of dementia for two years prior to baseline (VHA n = 136,016; MarketScan n = 172,790). Two index periods (either start of 2011 or 2012) were defined, where patients either had or did not have a HZ vaccination. Confounding was controlled with propensity scores and inverse probability of treatment weighting. Competing risk (VHA) and Cox proportional hazard (MarketScan) models estimated the association between HZ vaccination and incident dementia in all patients and in age (65–69, 70–74, ≥75) and race (White, Black, Other) sub-groups. Sensitivity analysis measured the association between HZ vaccination and incident Alzheimer’s dementia (AD). HZ vaccination at index versus no HZ vaccination throughout follow-up. VHA patients mean age was 75.7 (SD±7.4) years, 4.0% were female, 91.2% white and 20.2% had HZ vaccination. MarketScan patients mean age was 69.9 (SD±5.7) years, 65.0% were female and 14.2% had HZ vaccination. In both cohorts, HZ vaccination compared with no vaccination, was significantly associated with lower dementia risk (VHA HR = 0.69; 95%CI: 0.67–0.72; MarketScan HR = 0.65; 95%CI:0.57–0.74). HZ vaccination was not related to dementia risk in MarketScan patients aged 65–69 years. No difference in HZ vaccination to dementia effects were found by race. HZ vaccination was associated with lower risk for AD. Conclusions HZ vaccination is associated with reduced risk of dementia. Vaccination may provide nonspecific neuroprotection by training the immune system to limit damaging inflammation, or specific neuroprotection that prevents viral cytopathic effects.

Natural killer (NK) cells are an important cell subset of the innate immune system. Accumulating evidence indicates that NK cells are dysfunctional in the tumor microenvironment during cancer development. However, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here, we used both the mouse E0771 breast cancer and B16F0 melanoma tumor models to mimic different clinical stages of human cancers and characterized the dynamic distributions and qualities of NK cells in different organs and tumors associated with tumor progression. We found that NK cells are dynamically involved in the immune responses to cancer with different distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, NK cells exhibit effector properties. In the later cancer stages, NK cells have impaired cytotoxic capacities and dysfunctional states. Importantly, we found that melanoma and breast cancer cells promote NK cells to become senescent NK cells in vitro and in vivo in tumor microenvironments. These studies provide a better understanding of the dynamic and functional role of NK cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK cells for cancer treatment. the Melanoma Research Alliance, American Cancer Society, and the National Institutes of Health.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response., Author summary The mechanisms of SARS-CoV-2 induced excessive inflammatory response associated with disease severity needs to be critically studied for therapeutic intervention. Cytokine storm is typical of macrophage activation; which leads to tissue damage, lung injury, and acute respiratory distress syndrome. Lung epithelial cells; a primary host for SARS coronavirus infection, can generate a cytokine response leading to an expanded pathology. Our present study was designed to understand SARS-CoV-2 and human epithelial cell interactions, by delineating their contribution to inflammatory cytokine systems with special emphasis on ectopic expression of the viral spike protein. We observed that SARS-CoV-2 infection or spike protein expression in human epithelial cells inhibits ACE2 expression leading to the induction of AT1 signaling, and promotion of IL-6/soluble IL-6R release. IL-6 is one of the major mediators of the hyper-inflammatory response, and can exert signaling through two main pathways referred to as classical or trans- signaling to coordinate prominent pro-inflammatory properties. Our experimental observations suggest the potential for IL-6 trans-signaling during SARS-CoV-2 infection of epithelial cells. These observations will aid in the development of new therapeutic modalities for combating COVID-19 associated disease severity. Aggressive tissue damage via increased IL-6 secretion and other cytokines leading to a hyper-inflammatory response are in line with COVID-19 disease severity. We propose that SARS-CoV-2 infection in pulmonary epithelial cells induces IL-6 trans-signaling for secretion of chemokines; like MCP-1, from pulmonary vascular endothelial cells to attract monocytes/macrophages and create a hyper-inflammatory state leading to enhanced disease severity and acute respiratory distress syndrome.

TH17 cells have been extensively investigated in inflammation, autoimmune diseases, and cancer. The precise molecular mechanisms for TH17 cell regulation, however, remain elusive, especially regulation at the post-transcriptional level. Tristetraprolin (TTP) is an RNA-binding protein important for degradation of the mRNAs encoding several proinflammatory cytokines. With newly generated T cell-specific TTP conditional knockout mice (CD4CreTTPf/f), we found that aging CD4CreTTPf/f mice displayed an increase of IL-17A in serum and spontaneously developed chronic skin inflammation along with increased effector TH17 cells in the affected skin. TTP inhibited TH17 cell development and function by promoting IL-17A mRNA degradation. In a DSS-induced colitis model, CD4CreTTPf/f mice displayed severe colitis and had more TH17 cells and serum IL-17A compared with wild-type mice. Furthermore, neutralization of IL-17A reduced the severity of colitis. Our results reveal a new mechanism for regulating TH17 function and TH17-mediated inflammation post-transcriptionally by TTP, suggests that TTP might be a novel therapeutic target for the treatment of TH17-mediated diseases.

Francisella tularensis (F. tularensis) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPAR&gamma, nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC&ndash, MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon &alpha, /&beta, signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-&kappa, B, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1&alpha, Il-1&beta, and TNF typically activated following infection were suppressed. The NF-&kappa, B pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host&ndash, pathogen interactions.

TH17 cells have been extensively investigated in inflammation, autoimmune diseases, and cancer. The precise molecular mechanisms for TH17 cell regulation, however, remain elusive, especially regulation at the post-transcriptional level. Tristetraprolin (TTP) is an RNA-binding protein important for degradation of the mRNAs encoding several proinflammatory cytokines. With newly generated T cell-specific TTP conditional knockout mice (CD4CreTTPf/f), we found that aging CD4CreTTPf/f mice displayed an increase of IL-17A in serum and spontaneously developed chronic skin inflammation along with increased effector TH17 cells in the affected skin. TTP inhibited TH17 cell development and function by promoting IL-17A mRNA degradation. In a DSS-induced colitis model, CD4CreTTPf/f mice displayed severe colitis and had more TH17 cells and serum IL-17A compared with wild-type mice. Furthermore, neutralization of IL-17A reduced the severity of colitis. Our results reveal a new mechanism for regulating TH17 function and TH17-mediated inflammation post-transcriptionally by TTP, suggests that TTP might be a novel therapeutic target for the treatment of TH17-mediated diseases.

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 (

A comprehensive strategy to control hepatitis C virus (HCV) infection needs a vaccine. Our phase I study with recombinant HCV E1/E2 envelope glycoprotein (EnvGPs) as a candidate vaccine did not induce a strong immune response in volunteers. We analyzed the interactions of HCV EnvGPs with human monocyte-derived macrophages as antigen-presenting cells. HCV E2 induced immune regulatory cytokine interleukin (IL)-10 and soluble CD163 (sCD163) protein expression in macrophages from 7 of 9 blood donors tested. Furthermore, HCV E2 enhanced Stat3 and suppressed Stat1 activation, reflecting macrophage polarization toward M2 phenotype. E2-associated macrophage polarization appeared to be dependent of its interaction with CD81 leading endothelial growth factor receptor (EGFR) activation. Additionally, E2 suppressed the expression of C3 complement, similar to HCV-exposed dendritic cells (DCs), implying potential impairment of immune cell priming. Conclusion: Our results suggest that E2 EnvGP may not be an ideal candidate for HCV vaccine development, and discrete domains within E2 may prove to be more capable of elliciting a protective immune response. (Hepatology 2018).

Protective efficacy of Bacillus Calmette-Guerin (BCG) may be affected by the methods and routes of vaccine administration. We have studied the safety and immunogenicity of oral (PO) and/or intradermal (ID) administration of BCG in healthy human subjects. No major safety concerns were detected in the 68 healthy adults vaccinated with PO and/or ID BCG. Although both PO and ID BCG could induce systemic Th1 responses capable of IFN-γ production, ID BCG more strongly induced systemic Th1 responses. In contrast, stronger mucosal responses (TB-specific secretory IgA and bronchoalveolar lavage T cells) were induced by PO BCG vaccination. To generate preliminary data comparing the early gene signatures induced by mucosal and systemic BCG vaccination, CD4+ memory T cells were isolated from subsets of BCG vaccinated subjects pre- (Day 0) and post-vaccination (Days 7 and 56), rested or stimulated with BCG infected dendritic cells, and then studied by Illumina BeadArray transcriptomal analysis. Notably, distinct gene expression profiles were identified both on Day 7 and Day 56 comparing the PO and ID BCG vaccinated groups by GSEA analysis. Future correlation analyses between specific gene expression patterns and distinct mucosal and systemic immune responses induced will be highly informative for TB vaccine development.

Summary Background Bacille Calmette–Guerin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. Methods The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. Results IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor β (IL2Rβ), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFβR1) and, in agreement with the peptide microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination. Conclusions BCG vaccination modifies the host's immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals.

Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were analyzed using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was produced to study the enzymatic role in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments looked at GzmA internalization. We have evidence that Granzyme A’s intact active site is not necessary to inhibit the intracellular replication of mycobacteria as the S195A variant inhibits mycobacterial infection in MGIA as Wild Type (WT) GzmA. The proteomic approach identified two pathways: ER-stress response and purinergic channel receptor activation are important for GzmA-mediated intracellular mycobacterial inhibition. These results were validated in separate volunteers by western blot. Confocal microscopy experiments show that GzmA is internalized within the infected cell and clears the infection. Modulation of key proteins in the discovered pathways are being targeted to confirm our proteomic results and to test potential novel therapies that emulate GzmA. It will also be investigated if GzmA co-localizes in these cellular compartments. These studies will lead to a better understanding of GzmA biology during infection and to the development of novel host-directed therapeutics for control of Mtb infection.

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis. We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette–Guérin (BCG) in healthy, tuberculin skin test–positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ–expressing CD8 T, γδ T, CD3+CD56+ NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis– or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ–expressing BCG-reactive CD3+CD56+ NKT-like cells and CD3−CD56dim and CD3−CD56hi NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

Tuberculosis (TB) is still a major global health problem. A third of the world's population is infected with Mycobacterium tuberculosis. Only ~10% of infected individuals develop TB but there are 9 million TB cases with 1.5 million deaths annually. The standard prophylactic treatment regimens for latent TB infection take 3-9 months, and new cases of TB require at least 6 months of treatment with multiple drugs. The management of latent TB infection and TB has become more challenging because of the spread of multidrug-resistant and extremely drug-resistant TB. Intensified efforts to find new TB drugs and immunotherapies are needed. Immunotherapies could modulate the immune system in patients with latent TB infection or active disease, enabling better control of M. tuberculosis replication. This review describes several types of potential immunotherapies with a focus on those which have been tested in humans.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids, several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Failure of the most recent tuberculosis (TB) vaccine trial to boost BCG mediated anti-TB immunity despite highly durable Th1-specific central (TCM) and effector (TEM) memory cell responses, highlights the importance of identifying optimal T cell targets for protective vaccines. Here we describe a novel, Mycobacterium tuberculosis (Mtb)-specific IFN-γ+CD4+ T cell population expressing surface markers characteristic of naïve T cells (TNLM), that were induced in both human (CD45RA+CCR7+CD27+CD95-) and murine (CD62L+CD44-Sca-1+CD122-) systems in response to mycobacteria. In BCG vaccinated subjects and those with latent TB infection, TNLM cells, compared to bonafide naïve CD4+ T cells were identified by absence of CD95 expression and had increased expression CCR7 and CD27, the activation markers T-bet, CD69 and PD-1 and the survival marker CD74. Increased TNLM frequencies were noted in the lung and spleen of wild type C57BL6 mice at 2 weeks after infection with Mtb, and progressively decreased at later time points, a pattern not seen in TNF-α+CD4+ T cells expressing naïve cell surface markers. Importantly, adoptive transfer of highly purified TNLM from vaccinated ESAT-61-20-specific TCR transgenic mice conferred superior protection against Mtb infection in Rag-/- mice when compared with total meory populations (central and effector memory cells). Thus, TNLM cells may represent a memory T cell population that if optimally targeted may significantly improve future TB vaccine responses.

Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

Chagas disease is a major neglected tropical disease caused by persistent chronic infection with the protozoan parasite Trypanosoma cruzi. An estimated 8 million people are infected with T. cruzi, however only 2 drugs are approved for treatment and no vaccines are available. Thus there is an urgent need to develop vaccines and new drugs to prevent and treat Chagas disease. In this work, we identify T cell targets relevant for human infection with T. cruzi. The trans-sialidase (TS) gene family is a large family of homologous genes within the T. cruzi genome encoding over 1,400 members. There are 12 highly conserved TS gene family members which encode enzymatically active TS (functional TS; F-TS), while the remaining TS family genes are less conserved, enzymatically inactive and have been hypothesized to be involved in immune evasion (non-functional TS; NF-TS). We utilized immunoinformatic tools to identify HLA-A2-restricted CD8(+) T cell epitopes conserved within F-TS family members and NF-TS gene family members. We also utilized a whole-genome approach to identify T cell epitopes present within genes which have previously been shown to be expressed in life stages relevant for human infection (Non-TS genes). Thirty immunogenic HLA-A2-restricted CD8(+) T cell epitopes were identified using IFN-γ ELISPOT assays after vaccination of humanized HLA-A2 transgenic mice with mature dendritic cells pulsed with F-TS, NF-TS, and Non-TS peptide pools. The immunogenic HLA-A2-restricted T cell epitopes identified in this work may serve as potential components of an epitope-based T cell targeted vaccine for Chagas disease.

Vaccines against mucosally invasive, intracellular pathogens must induce a myriad of immune responses to provide optimal mucosal and systemic protection, including CD4+ T cells, CD8+ T cells, and Ab-producing B cells. In general, CD4+ T cells are known to provide important helper functions for both CD8+ T cell and B cell responses. However, the relative importance of CD4+ T cells, CD8+ T cells, and B cells for mucosal protection is less clearly defined. We have studied these questions in detail using the murine model of Trypanosoma cruzi infection. Despite our initial hypothesis that mucosal Abs would be important, we show that B cells are critical for systemic, but not mucosal, T. cruzi protective immunity. B cell–deficient mice developed normal levels of CD8+ effector T cell responses early after mucosal T. cruzi infection and T. cruzi trans-sialidase vaccination. However, after highly virulent systemic challenge, T. cruzi immune mice lacking T. cruzi–specific B cells failed to control parasitemia or prevent death. Mechanistically, T. cruzi–specific CD8+ T cells generated in the absence of B cells expressed increased PD-1 and Lag-3 and became functionally exhausted after high-level T. cruzi systemic challenge. T. cruzi immune serum prevented CD8+ T cell functional exhaustion and reduced mortality in mice lacking B cells. Overall, these results demonstrate that T. cruzi–specific B cells are necessary during systemic, but not mucosal, parasite challenge.

Both live attenuated influenza vaccines (LAIV) and inactivated influenza vaccines (IIV) induce protective immunity against influenza. There is evidence that LAIV induces superior protection in children, whereas IIV may induce superior protection in adults. The immune mechanisms responsible for these differences have not been identified. We previously compared LAIV and IIV in young children of 6 to 36 months of age, and we demonstrated that while both induced similar hemagglutination inhibition (HAI) antibody responses, only LAIV induced significant increases in T cell responses. In the present study, 37 healthy adult subjects of 18 to 49 years of age were randomized to receive seasonal influenza vaccination with LAIV or IIV. Influenza virus-specific HAI, T cell, and secretory IgA (sIgA) responses were studied pre- and postvaccination. In contrast to the responses seen in young children, LAIV induced only minimal increases in serum HAI responses in adults, which were significantly lower than the responses induced by IIV. Both LAIV and IIV similarly induced only transient T cell responses to replication-competent whole virus in adults. In contrast, influenza virus-specific sIgA responses were induced more strongly by LAIV than by IIV. Our previous studies suggest that LAIV may be more protective than IIV in young children not previously exposed to influenza virus or influenza vaccines due to increased vaccine-induced T cell and/or sIgA responses. Our current work suggests that in adults with extensive and partially cross-reactive preexisting influenza immunity, LAIV boosting of sIgA responses to hemagglutinin (HA) and non-HA antigenic targets expressed by circulating influenza virus strains may be an important additional mechanism of vaccine-induced immunity.

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 naive/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 naive 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.

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. The effects of this highly purified GzmA on mycobacterial replication inside human macrophages are being investigated using systems biology. A 2D-DIGE proteomic approach of PBMC-adherent cells has been performed to identify putative pathways involved in the control of mycobacterial replication. Our preliminary analysis of two PPD+ volunteers indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Verification of these results is being performed to confirm the overall mechanism, and to exclude unrelated signals. Elucidation of the detailed pathways involved in pathogen clearance could lead to the development of novel chemotherapeutic and/or immunotherapeutic approaches for control of Mtb infection.

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle to successful tumor immunotherapy. Dissecting the regulations between energy metabolism and functionality in human Treg cells will provide insights for developing novel immunotherapies against cancer. We discovered that human naturally occurring Treg (nTreg) and tumor-associated Treg cells exhibit distinct metabolic profiles with predominant dependence on glucose metabolism compared with effector T cells. Activated nTreg and tumor-associated Treg cells depend on accelerated glucose consumption for their cellular senescence induction and suppressive activity in responder T cells during their cross-talk. We further identified that TLR8 signaling selectively inhibits glucose uptake and suppresses metabolic processes of glycolysis in human Treg cells, resulting in reversal of Treg suppressive function and cellular senescence induction. In addition, TLR8 signaling activation downregulates mTORC1-HIF1α signaling in Treg cells that control molecular processes of Treg glucose metabolism and suppressive functions. Our in vivo studies further demonstrated that TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity and immunotherapy efficacy in a melanoma adoptive transfer T cell therapy model. These studies identify novel mechanistic links between innate signaling and metabolic regulation of human Treg suppression, and provide a new strategy and concept for tumor immunotherapy.

Understanding the role of different subtypes of tumor-infiltrating lymphocytes (TIL) in the immunosuppressive tumor microenvironment is essential for improving cancer treatment. Enriched γδ1 T-cell populations in TILs suppress T-cell responses and dendritic cell maturation in breast cancer, where their presence is correlated negatively with clinical outcomes. However, mechanism(s) that explain the increase in this class of regulatory T cells (γδ Treg) in patients with breast cancer have yet to be elucidated. In this study, we show that IP-10 secreted by breast cancer cells attracted γδ Tregs. Using neutralizing antibodies against chemokines secreted by breast cancer cells, we found that IP-10 was the only functional chemokine that causes γδ Tregs to migrate toward breast cancer cells. In a humanized NOD-scid IL-2Rγnull (NSG) mouse model, human breast cancer cells attracted γδ Tregs as revealed by a live cell imaging system. IP-10 neutralization in vivo inhibited migration and trafficking of γδ Tregs into breast tumor sites, enhancing tumor immunity mediated by tumor-specific T cells. Together, our studies show how γδ Tregs accumulate in breast tumors, providing a rationale for their immunologic targeting to relieve immunosuppression in the tumor microenvironment. Cancer Res; 73(20); 6137–48. ©2013 AACR.

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., Author Summary Chronic infection with the intracellular parasite Trypanosoma cruzi results in Chagas disease, an illness endemic in more than 20 countries that leads to life-threatening cardiac and gastrointestinal dysfunction. Although CD4+ Th1 cells are known to be protective against T. cruzi infection, less is known about the role of other CD4+ T cell subsets. We demonstrate that CD4+ Th17 cells are also highly protective against T. cruzi infection, even outperforming Th1 cells in protection from T. cruzi-related death, despite the standard conception that Th17 cells are only important in the defense against extracellular pathogens. The novel discovery that Th17 cells are significantly more protective than Th1 cells against T. cruzi infection has increased our understanding of the advantageous immune responses for this major human pathogen, and may guide future efforts toward vaccine development.

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., Author Summary Chagas disease, driven by persistent infection with the intracellular protozoan parasite Trypanosoma cruzi, continues to be a major cause of morbidity and mortality in the Americas. Infections with complex pathogens induce T cell responses to a variety of proteins and epitopes in a specific and sometimes predictable pattern of immune dominance. Extensive research has been conducted with the goal of broadening vaccine-induced T cell responses, for example to better protect against pathogen immune escape due to epitope mutation. We show that T. cruzi trans-sialidase (TS) induces complex immunodominant and subdominant T cell responses after both T. cruzi infection and TS vaccination. In order to study the importance of immunodominant and subdominant TS epitopes, we generated TS vaccines designed to abolish T cell responses to the immunodominant TS CD8+ T cell epitope. These vaccines indeed abolished CD8+ T cell responses to the immunodominant epitope, but interestingly also eliminated T cell responses to many subdominant epitopes. Furthermore, vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. Thus, increasing the breadth of T cell epitope recognition may not necessarily enhance protective immunity, and in fact, may be detrimental to the desired goals.

IFN-γ-producing cytotoxic T lymphocytes are essential for host defense against viral infection and cancer. Here we show that the RNA-binding tristetraprolin, encoded by Zfp36, is needed for CD8+ T-cell production of IFN-γ in vivo. When activated in vitro, however, IFN-γ production by naive wild type and tristetraprolin-deficient CD8+ T-cells is comparable. IL-27 is overproduced by tristetraprolin-deficient macrophages and increased systemically in tristetraprolin-deficient mice. Tristetraprolin suppresses IL-27 production by promoting p28 mRNA degradation. Importantly, deletion of IL-27 receptor WSX-1 in tristetraprolin-deficient mice (WSX-1/tristetraprolin double knockout) leads to a reduction in cytotoxic T lymphocyte numbers. Moreover, tumor growth is accelerated, not only in tristetraprolin-deficient mice after cytotoxic T lymphocyte depletion, but also in WSX-1/tristetraprolin double knockout mice, with substantial reduction in the number of tumor cytotoxic T lymphocytes. This study describes a regulatory pathway for IL-27 expression and cytotoxic T lymphocyte function mediated by tristetraprolin, contributing to regulation of antitumour immunity., IL-27 is one of a number of cytokines that can induce antitumour CD8+ T cell responses. Here the authors show that TTP, encoded by Zfp36, degrades p28 to inhibit IL-27 production by macrophages and is thereby a negative regulator of the antitumour response.

Understanding and dissecting the role of different subsets of regulatory tumor-infiltrating lymphocytes (TILs) in the immunopathogenesis of individual cancer is a challenge for anti-tumor immunotherapy. High levels of γδ regulatory T cells have been discovered in breast TILs. However, the clinical relevance of these intratumoral γδ T cells is unknown. In this study, γδ T cell populations were analyzed by performing immunohistochemical staining in primary breast cancer tissues from patients with different stages of cancer progression. Retrospective multivariate analyses of the correlations between γδ T cell levels and other prognostic factors and clinical outcomes were completed. We found that γδ T cell infiltration and accumulation in breast tumor sites was a general feature in breast cancer patients. Intratumoral γδ T cell numbers were positively correlated with advanced tumor stages, HER2 expression status, and high lymph node metastasis but inversely correlated with relapse-free survival and overall survival of breast cancer patients. Multivariate and univariate analyses of tumor-infiltrating γδ T cells and other prognostic factors further suggested that intratumoral γδ T cells represented the most significant independent prognostic factor for assessing severity of breast cancer compared with the other known factors. Intratumoral γδ T cells were positively correlated with FOXP3+ cells and CD4+ T cells but negatively correlated with CD8+ T cells in breast cancer tissues. These findings suggest that intratumoral γδ T cells may serve as a valuable and independent prognostic biomarker, as well as a potential therapeutic target for human breast cancer.

Regulatory T (Treg) cells have broad suppressive activity on host immunity, but the fate and function of suppressed responder T cells remains largely unknown. In the present study, we report that human Treg cells can induce senescence in responder naive and effector T cells in vitro and in vivo. Senescent responder T cells induced by human Treg cells changed their phenotypes and cytokine profiles and had potent suppressive function. Furthermore, Treg-mediated molecular control of senescence in responder T cells was associated with selective modulation of p38 and ERK1/2 signaling and cell-cycle–regulatory molecules p16, p21, and p53. We further revealed that human Treg-induced senescence and suppressor function could be blocked by TLR8 signaling and/or by specific ERK1/2 and p38 inhibition in vitro and in vivo in animal models. The results of the present study identify a novel mechanism of human Treg cell suppression that induces targeted responder T-cell senescence and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression.

Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ 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.

Background Despite the availability of Bacille Calmette Guerin (BCG) vaccines, Mycobacterium tuberculosis currently infects billions of people and millions die annually from tuberculosis (TB) disease. New TB vaccines are urgently needed. Methods We studied the ability of AERAS-402, a recombinant, replication-deficient adenovirus type 35 expressing the protective M. tuberculosis antigens Ag85A, Ag85B, and TB10.4, to boost BCG immunity in an area of low TB endemicity. Results In volunteers primed with BCG 3 or 6 months prior to AERAS-402 boosting, significant CD4+ and CD8+ T cell responses were induced. Ag85-specific responses were more strongly boosted than TB10.4-specific responses. Frequencies of TB-specific CD8+ T cells reached > 50 fold higher than pre-AERAS boosting levels, remarkably higher than reported in any previous human TB vaccine trial. Multiparameter flow cytometric assays demonstrated that AERAS-402-boosted CD4+ and CD8+ T cells were multifunctional, producing multiple cytokines and other immune effector molecules. Furthermore, boosted T cells displayed lymphoproliferative capacity, and tetramer analyses confirmed that antigen-specific CD8+ T cells were induced. BCG and AERAS-402 vaccinations given 3 and 6 months apart appeared equivalent. Conclusions Our results indicate that AERAS-402 is a promising TB vaccine candidate that can significantly enhance both CD4+ and CD8+ TB-specific T cell responses after BCG priming. ClinicalTrials.gov Identifier: NCT01378312.

Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic T. cruzi protection. We evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival T. cruzi challenges. T. cruzi-immune CCR5−/− and wild-type C57BL/6 mice were generated by repeated infectious challenges with T. cruzi. CCR5−/− and wild-type mice developed equivalent levels of cellular, humoral, and protective mucosal responses. However, CCR5−/−-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5−/−-immune mice resulted in decreased mucosal inflammatory responses, reduced T. cruzi-specific Ab-secreting cells, and significantly less mucosal T. cruzi protection, confirming an important role for CCL5 in optimal immune control of T. cruzi replication at the point of initial mucosal invasion. To investigate further the mechanism responsible for mucosal protection mediated by CCL5–CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell coactivator.

Optimizing the development of modern molecular vaccines requires a complex series of interdisciplinary efforts involving basic scientists, immunologists, molecular biologists, clinical vaccinologists, bioinformaticians and epidemiologists. This review summarizes some of the major issues that must be carefully considered. The intent of the authors is to briefly describe key components of the development process to give the reader an overview of the challenges faced from vaccine concept to vaccine delivery. Every vaccine requires unique features based on the biology of the pathogen, the nature of the disease and the target population for vaccination. This review presents general concepts relevant for the design and development of ideal vaccines protective against diverse pathogens.

Accumulating evidence suggests that Th17 cells and Tregs may exhibit development plasticity and that CD4(+) Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs). We showed that Th17 clones generated from TILs can differentiate into IFN-γ-producing and FOXP3(+) cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells. We further demonstrated that T-cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage-specific transcriptional factor and cytokine genes. In addition to expressing IFN-γ and FOXP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Tregs. We further demonstrated that the Th17-derived Tregs, unlike naturally occurring CD4(+) CD25(+) Tregs, did not reconvert back into Th17 cells even under Th17-biasing cytokine conditions. These results provide the critical evidence that human tumor-infiltrating Th17 cells can differentiate into Tregs and indicate a substantial developmental plasticity of Th17 cells.

Chagas' disease, induced by Trypanosoma cruzi , is a common cause of infectious myocarditis. Recent clinical treatment trials and vaccine studies indicate that chagasic immunopathology is directed against the parasite and not self-antigens. Therefore, vaccines may have the potential to protect against disease progression. Certain combinations of mouse and parasite strains produce significant histopathology and can be used for safety analyses of new vaccination strategies. The goals of this study were to determine (1) whether the development of chagasic cardiomyopathy in the murine model could be identified by electrocardiogram (ECG); and (2) whether these potential chagasic ECG changes would correlate with histopathologic findings. Groups of BALB/c, C57BL/6, and C3H mice were infected with different parasite strains (Tulahuén, Brazil, or Sylvio-X10/4) and evaluated weekly by ECG. Selected tissues from subsets of mice were harvested periodically for blinded histologic evaluation. Significantly increased proportions of BALB/c mice infected with Brazil and Tulahuén strain parasites displayed prolonged QT intervals. Prolonged mean QT intervals detected in infected BALB/c mice significantly correlated with chagasic histopathologic changes. These results indicate that ECG can be used as a non-invasive method to screen for immune-mediated damage resulting in chagasic cardiomyopathy in the murine model.

Bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, responses to BCG are heterogeneous and limited options exist when BCG therapy fails. Preclinical data in animal models of bladder cancer (BC) suggests that priming with percutaneous BCG vaccine prior to intravesical BCG instillation can enhance BCG-specific immunity and improve outcome. To study the safety, immunogenicity and preliminary efficacy of this approach, we administered percutaneous BCG 21 days prior to intravesical BCG instillation in “prime patients” with NMIBC (n=13) in a prospective single-arm clinical trial. Immune responses and clinical outcomes were monitored and compared to a contemporaneous cohort of “control patients” (n=9) receiving intravesical BCG without prior percutaneous BCG. Priming was well tolerated and no grade ≥3 adverse events were observed. Compared to control, prime patients had improvement in both local and systemic measures of BCG-specific immunity, scored by increased post-BCG urinary IL-8 and IL-17A and increased circulating CD4, CD8, and γδ T cell proliferation and effector function in response to BCG. Furthermore, ex vivo cytotoxicity of circulating NK and γδ T cells against RT4 BC was significantly increased in prime patient after BCG treatment and was mediated in part by NKG2D, suggesting an important role of innate effector immune mechanisms underlying BCG’s antitumor activity. Remarkably, no prime patients progressed whereas 3 out of 9 control patients progressed to muscle-invasive disease and underwent cystectomy. Thus, BCG priming safely enhances BCG-specific immunity and could improve outcome for patients with NMIBC.