Amit A. Lugade

Higher Frequencies of GARP+CTLA-4+Foxp3+ T Regulatory Cells and Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Are Associated with Impaired T-Cell Functionality

The extent to which T-cell-mediated immune surveillance is impaired in human cancer remains a question of major importance, given its potential impact on the development of generalized treatments of advanced disease where the highest degree of heterogeneity exists. Here, we report the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). Using multi-parameter fluorescence-activated cell sorting analysis, we quantified the cumulative frequency of regulatory T cells (Treg), exhausted CD4(+) helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concurrent views on the overall level of immune dysfunction in these inoperable patients. We documented augmented numbers of Tregs, MDSC, PD-1(+)-exhausted T cells, and increased levels of immunosuppressive cytokines in patients with HCC, compared with normal controls, revealing a network of potential mechanisms of immune dysregulation in patients with HCC. In dampening T-cell-mediated antitumor immunity, we hypothesized that these processes may facilitate HCC progression and thwart the efficacy of immunotherapeutic interventions. In testing this hypothesis, we showed that combined regimens to deplete Tregs, MDSC, and PD-1(+) T cells in patients with advanced HCC restored production of granzyme B by CD8(+) T cells, reaching levels observed in normal controls and also modestly increased the number of IFN-γ producing CD4(+) T cells. These clinical findings encourage efforts to restore T-cell function in patients with advanced stage disease by highlighting combined approaches to deplete endogenous suppressor cell populations that can also expand effector T-cell populations.

Cigarette Smoke Exposure Exacerbates Lung Inflammation and Compromises Immunity to Bacterial Infection

The detrimental impact of tobacco on human health is clearly recognized, and despite aggressive efforts to prevent smoking, close to one billion individuals worldwide continue to smoke. People with chronic obstructive pulmonary disease are susceptible to recurrent respiratory infections with pathogens, including nontypeable Haemophilus influenzae (NTHI), yet the reasons for this increased susceptibility are poorly understood. Because mortality rapidly increases with multiple exacerbations, development of protective immunity is critical to improving patient survival. Acute NTHI infection has been studied in the context of cigarette smoke exposure, but this is the first study, to our knowledge, to investigate chronic infection and the generation of adaptive immune responses to NTHI after chronic smoke exposure. After chronic NTHI infection, mice that had previously been exposed to cigarette smoke developed increased lung inflammation and compromised adaptive immunity relative to air-exposed controls. Importantly, NTHI-specific T cells from mice exposed to cigarette smoke produced lower levels of IFN-γ and IL-4, and B cells produced reduced levels of Abs against outer-membrane lipoprotein P6, with impaired IgG1, IgG2a, and IgA class switching. However, production of IL-17, which is associated with neutrophilic inflammation, was enhanced. Interestingly, cigarette smoke–exposed mice exhibited a similar defect in the generation of adaptive immunity after immunization with P6. Our study has conclusively demonstrated that cigarette smoke exposure has a profound suppressive effect on the generation of adaptive immune responses to NTHI and suggests the mechanism by which prior cigarette smoke exposure predisposes chronic obstructive pulmonary disease patients to recurrent infections, leading to exacerbations and contributing to mortality.

Anti-inflammatory IgG Production Requires Functional P1 Promoter in β-Galactoside α2,6-Sialyltransferase 1 (ST6Gal-1) Gene

Background: β-Galactoside α2,6-sialyltransferase 1 (ST6Gal-1) action is essential for the anti-inflammatory activity in intravenous immunoglobulin (IVIG) therapy. Results: Fc sialylation changes in accordance to the severity of inflammation. Inactivation of the P1 promoter abrogated IgG Fc sialylation. Conclusion: Fc sialylation depends on ST6Gal-1 in the circulation. Defective Fc sialylation is a mechanism for the generally proinflammatory tendencies of the P1-ablated mutant mouse (Siat1ΔP1). Significance: Anti-inflammatory bioactivity of IVIG requires sialylated Fc. The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.

Altered eosinophil profile in mice with ST6Gal-1 deficiency: an additional role for ST6Gal-1 generated by the P1 promoter in regulating allergic inflammation

Cumulative evidence indicates that the sialyltransferase ST6Gal‐1 and the sialyl‐glycans, which it constructs, are functionally pleiotropic. Expression of the ST6Gal‐1 gene is mediated by six distinct promoter/regulatory regions, and we hypothesized that these promoters may be used differentially to produce ST6Gal‐1 for different biologic purposes. To examine this hypothesis, we compared a mouse with a complete deficiency in ST6Gal‐1 (Siat1 null) with another mouse that we have created previously with a disruption only in the P1 promoter (Siat1ΔP1). We noted previously greater neutrophilic inflammation associated with ST6Gal‐1 deficiency. Here, we report that ST6Gal‐1‐deficient mice also have significantly elevated eosinophilic responses. Upon i.p. thioglycollate elicitation, eosinophils accounted for over 20% of the total peritoneal inflammatory cell pool in ST6Gal‐1‐deficient animals, which was threefold greater than in corresponding wild‐type animals. A principal feature of allergic respiratory inflammation is pulmonary eosinophilia, we evaluated the role of ST6Gal‐1 in allergic lung inflammation. Using OVA and ABPA experimental models of allergic airways, we showed that ST6Gal‐1 deficiency led to greater airway inflammation characterized by excessive airway eosinophilia. The severity of airway inflammation was similar between Siat1ΔP1 and Siat1 null mice, indicating a role for P1‐generated ST6Gal‐1 in regulating eosinophilic inflammation. Colony‐forming assays suggested greater IL‐5‐dependent eosinophil progenitor numbers in the marrow of ST6Gal‐1‐deficient animals. Moreover, allergen provocation of wild‐type mice led to a significant reduction in P1‐mediated ST6Gal‐1 mRNA and accompanied decline in circulatory ST6Gal‐1 levels. Taken together, the data implicate ST6Gal‐1 as a participant in regulating not only Th1 but also Th2 responses, and ST6Gal‐1 deficiency can lead to the development of more severe allergic inflammation with excessive eosinophil production.

Transgenic Plant-Based Oral Vaccines

Oral vaccines offer significant advantages over needle-based vaccines for achieving universal childhood vaccination goals. The expression of vaccine antigens in transgenic plants has the potential to provide a convenient, safe approach for oral vaccination and thus a feasible alternative to traditional parenteral vaccines. Many developments in the field have ushered in improvements such as enhanced protein antigen expression for the use of plants as factories for vaccine production, and facilitated studies pertaining to immunogenicity of candidate vaccines. Oral delivery of plant-based vaccines offers the benefit of antigen protection within the harsh intestinal environment. Within the gut, mucosal immune cells are poised to respond to pathogens, but can also be exploited to elicit protective immune responses to oral vaccines. Inclusion of mucosal adjuvants during immunization with the vaccine antigen has been an important step towards the success of plant-based vaccines. This review discusses the mechanisms that control mucosal immune responses and highlights some of the studies and the results achieved following immunization with transgenic plants.

Nrf2 Regulates Chronic Lung Inflammation and B-Cell Responses to Nontypeable Haemophilus influenzae

Nrf2 is a leucine zipper transcription factor that protects against oxidant-induced injury. Nontypeable Haemophilus influenzae is responsible for frequent disease exacerbations in patients with chronic obstructive pulmonary disease and is responsible for causing otitis media in young children. We hypothesized that Nrf2 would limit inflammatory responses to nontypeable H. influenzae. The objective of this study was to assess the role of Nrf2 in chronic lung inflammation and regulation of immune responses to nontypeable H. influenzae in mice. Wild-type (C57BL/6) mice and Nrf2(-/-) mice were instilled by oropharyngeal aspiration of 1 × 10(6) colony-forming units of live, nontypeable H. influenzae (NTHI) twice a week for 4 to 16 consecutive weeks to generate a chronic inflammatory milieu within the lungs that models chronic bronchitis. Nrf2(-/-) mice had increased lymphocytic airway inflammation compared with WT mice after NTHI challenge. Although the extent of NTHI-induced peribronchovascular inflammation did not significantly differ between the genotypes, plasma cell infiltration was significantly more abundant in Nrf2(-/-) mice. Most strikingly, Nrf2(-/-) mice generated significantly enhanced and persistent levels of serum antibodies against P6, a key outer membrane protein of NTHI. Lung dendritic cells from Nrf2(-/-) mice challenged with NTHI had increased activation markers compared with dendritic cells from similarly treated WT mice. Nrf2 regulates NTHI-induced airway inflammation characterized by lymphocytic and plasma cell infiltration and the activation of lung dendritic cells and B-cell responses in mice. Nrf2 may be a potential therapeutic target in limiting the bacterial infection-induced airway inflammation that drives exacerbations of chronic obstructive pulmonary disease.

Oral transgenic plant-based vaccine for hepatitis B

In addition to improving vaccine formulations in order to elicit robust and long-lasting immune responses, there is also an increasing need for improving the manner in which these vaccines are delivered. As most current vaccines are administered by injection by a health care giver, there is the ever-present danger of needlestick injuries. Therefore, needle-free vaccinations are a viable option toward limiting needle-associated injuries and additionally increasing compliance with vaccination schedules, as both children and adults have an aversion to injections. Noninvasive methods of vaccination will also facilitate speed of vaccine delivery and likely also reduce cost, both important factors for health care in developing countries. One alternative to current injectable immunizations is orally delivered vaccines. A specific approach that we and others are evaluating is the use of transgenic plant tissue that expresses vaccine antigens for oral immunization. Herein, we review the development of an oral HBV vaccine expressed in transgenic potato tubers and the immune responses generated in human subjects given this novel vaccine.

Single low-dose un-adjuvanted HBsAg nanoparticle vaccine elicits robust, durable immunity

UNLABELLED Chitosan nanoparticles were evaluated as a vaccine delivery system for hepatitis B surface antigen (HBsAg) in the absence of adjuvant. Nano-encapsulated HBsAg (HBsAg chitosan-NP) was endocytosed more rapidly and efficiently by dendritic cells compared to soluble HBsAg. FRET analysis demonstrated that intact nanoparticles were taken up by DCs. To determine the immunogenicity of adjuvant-free nano-encapsulated HBsAg, mice were immunized with a single dose of non-encapsulated HBsAg, HBsAg chitosan-NP, or HBsAg alum. Mice immunized with adjuvant-free nanoparticle elicited anti-HBs antibodies at significantly higher titers compared to mice immunized with HBsAg alum. Elevated numbers of BAFF-R(+) B cells and CD138+ plasma cells account for the heightened anti-HBs response in nanoparticle immunized mice. Increases in Tfh cells provide a mechanism for the accumulation of anti-HBs secreting cells. Thus, chitosan nanoparticle vaccines represent a promising un-adjuvanted platform to generate robust and durable immunity to HBsAg and other subunit antigens following a single low-dose administration. FROM THE CLINICAL EDITOR In this study, chitosan nanoparticle vaccines are demonstrated as a promising un-adjuvanted platform to generate robust and durable immunity to HBsAg and other subunit antigens following a single low-dose administration in a murine model. The authors also demonstrated superior antibody response induction compared with non-encapsulated HBs antigen and HBsAg aluminum.

Murine Model of Chronic Respiratory Inflammation

The respiratory mucosa is exposed to the external environment each time we breathe and therefore requires a robust and sophisticated immune defense system. As with other mucosal sites, the respiratory mucosal immune system must balance its response to pathogens while also regulating inflammatory immune cell-mediated tissue damage. In the airways, a failure to tightly control immune responses to a pathogen can result in chronic inflammation and tissue destruction with an overzealous response being deleterious for the host. Chronic obstructive pulmonary disease (COPD) is the fourth most common cause of death in the US and both the prevalence of and mortality rate of this disease is increasing annually. COPD is characterized by intermittent disease exacerbation. The causal contribution of bacterial infections to exacerbations of COPD is now widely accepted, accounting for at least 50% of all exacerbations. Non-typeable Haemophilus influenzae and Moraxella catarrhalis (both gram-negative bacteria) along with Streptococcus pneumoniae (a gram-positive bacterium) are the three most common bacterial pathogens that cause respiratory tract infections in COPD patients. The colonization of bacteria in the lower airways is similar to a low-grade smoldering infection that induces chronic airway inflammation. Chronic low-grade infection can induce a persistent inflammatory response in the airways and parenchyma. Inefficient removal of bacteria from the lower respiratory tract is characteristic of chronic bronchitis. Inflammation is believed to be central to the pathogenesis of exacerbations, but a clear understanding of the inflammatory changes during an exacerbation of COPD has yet to emerge. As bacterial colonization of the lung in COPD patients is a chronic inflammatory condition highlighted by frequent bouts of exacerbation and clearance, we sought to reproduce this chronic pathogen-mediated inflammation in a murine model by repeatedly delivering the intact, whole, live bacteria intra-tracheally to the lungs.

Lipid Motif of a Bacterial Antigen Mediates Immune Responses via TLR2 Signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2−/− DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively; our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.