Dendritic cell expression of the signaling molecule TRAF6 is critical for gut microbiota-dependent immune tolerance.

The intracellular signaling molecule TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). We now report that DC-specific deletion of TRAF6 (TRAF6ΔDC) resulted, unexpectedly, in loss of mucosal tolerance, characterized by spontaneous development of T helper 2 (Th2) cells in the lamina propria and eosinophilic enteritis and fibrosis in the small intestine. Loss of tolerance required the presence of gut commensal microbiota but was independent of DC-expressed MyD88. Further, TRAF6ΔDC mice exhibited decreased regulatory T (Treg) cell numbers in the small intestine and diminished induction of iTreg cells in response to model antigen. Evidence suggested that this defect was associated with diminished DC expression of interleukin-2 (IL-2). Finally, we demonstrate that aberrant Th2 cell-associated responses in TRAF6ΔDC mice could be mitigated via restoration of Treg cell activity. Collectively, our findings reveal a role for TRAF6 in directing DC maintenance of intestinal immune tolerance through balanced induction of Treg versus Th2 cell immunity.

IL-18 synergizes with IL-7 to drive slow proliferation of naive CD8 T cells by costimulating self-peptide-mediated TCR signals.

Naive T cell populations are maintained in the periphery at relatively constant levels via mechanisms that control expansion and contraction and are associated with competition for homeostatic cytokines. It has been shown that in a lymphopenic environment naive T cells undergo expansion due, at least in part, to additional availability of IL-7. We have previously found that T cell-intrinsic deletion of TNFR-associated factor (TRAF) 6 (TRAF6ΔT) in mice results in diminished peripheral CD8 T cell numbers. In this study, we report that whereas naive TRAF6ΔT CD8 T cells exhibit normal survival when transferred into a normal T cell pool, proliferation of naive TRAF6ΔT CD8 T cells under lymphopenic conditions is defective. We identified IL-18 as a TRAF6-activating factor capable of enhancing lymphopenia-induced proliferation (LIP) in vivo, and that IL-18 synergizes with high-dose IL-7 in a TRAF6-dependent manner to induce slow, LIP/homeostatic-like proliferation of naive CD8 T cells in vitro. IL-7 and IL-18 act synergistically to upregulate expression of IL-18R genes, thereby enhancing IL-18 activity. In this context, IL-18R signaling increases PI3K activation and was found to sensitize naive CD8 T cells to a model noncognate self-peptide ligand in a way that conventional costimulation via CD28 could not. We propose that synergistic sensitization by IL-7 and IL-18 to self-peptide ligand may represent a novel costimulatory pathway for LIP.

Enhancing CD8 T-cell memory by modulating fatty acid metabolism.

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (T(E)) populations, followed by the persistence of long-lived memory (T(M)) cells. Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to T(M) cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 T(M)-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 T(E)-cell responses, but have a profound defect in their ability to generate T(M) cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 T(M)-cell generation in the absence of TRAF6. This treatment also increased CD8 T(M) cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.

TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

Evaluation of a stabilized RSV pre-fusion F mRNA vaccine: Preclinical studies and Phase 1 clinical testing in healthy adults.

Human respiratory syncytial virus (RSV) causes a substantial proportion of respiratory tract infections worldwide. Although RSV reinfections occur throughout life, older adults, particularly those with underlying comorbidities, are at risk for severe complications from RSV. There is no RSV vaccine available to date, and treatment of RSV in adults is largely supportive. A correlate of protection for RSV has not yet been established, but antibodies targeting the pre-fusion conformation of the RSV F glycoprotein play an important role in RSV neutralization. We previously reported a Phase 1 study of an mRNA-based vaccine (V171) expressing a pre-fusion-stabilized RSV F protein (mDS-Cav1) in healthy adults. Here, we evaluated an mRNA-based vaccine (V172) expressing a further stabilized RSV pre-fusion F protein (mVRC1). mVRC1 is a single chain version of RSV F with interprotomer disulfides in addition to the stabilizing mutations present in the mDS-Cav1 antigen. The immunogenicity of the two mRNA-based vaccines encoding mVRC1 (V172) or a sequence-optimized version of mDS-Cav1 to improve transcriptional fidelity (V171.2) were compared in RSV-naïve and RSV-experienced African green monkeys (AGMs). V172 induced higher neutralizing antibody titers than V171.2 and demonstrated protection in the AGM challenge model. We conducted a Phase 1, randomized, placebo-controlled, clinical trial of 25 µg, 100 µg, 200 µg, or 300 µg of V172 in healthy older adults (60-79 years old; N = 112) and 100 µg, 200 µg, or 300 µg of V172 in healthy younger adults (18-49 years old; N = 48). The primary clinical objectives were to evaluate the safety and tolerability of V172, and the secondary objective was to evaluate RSV serum neutralization titers. The most commonly reported solicited adverse events were injection-site pain, injection-site swelling, headache, and tiredness. V172 was generally well tolerated in older and younger adults and increased serum neutralizing antibody titers, pre-fusion F-specific competing antibody titers, and RSV F-specific T-cell responses.

TRAF6 inhibits Th17 differentiation and TGF-beta-mediated suppression of IL-2.

Transforming growth factor-beta (TGF-beta) has an essential role in the generation of inducible regulatory T (iTreg) and T helper 17 (Th17) cells. However, little is known about the TGF-beta-triggered pathways that drive the early differentiation of these cell populations. Here, we report that CD4(+) T cells lacking the molecular adaptor tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) exhibit a specific increase in Th17 differentiation in vivo and in vitro. We show that TRAF6 deficiency renders T cells more sensitive to TGF-beta-induced Smad2/3 activation and proliferation arrest. Consistent with this, in TRAF6-deficient T cells, TGF-beta more effectively down-regulates interleukin-2 (IL-2), a known inhibitor of Th17 differentiation. Remarkably, TRAF6-deficient cells generate normal numbers of Foxp3-expressing cells in iTreg differentiation conditions where exogenous IL-2 is supplied. These findings show an unexpected role for the adaptor molecule TRAF6 in Smad-mediated TGF-beta signaling and Th17 differentiation. Importantly, the data also suggest that a main function of TGF-beta in early Th17 differentiation may be the inhibition of autocrine and paracrine IL-2-mediated suppression of Th17 cell generation.

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates.

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.

Upper and lower respiratory tract correlates of protection against respiratory syncytial virus following vaccination of nonhuman primates.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory illness in infants and the elderly. Although several vaccines have been developed, none have succeeded in part due to our incomplete understanding of the correlates of immune protection. While both T cells and antibodies play a role, emerging data suggest that antibody-mediated mechanisms alone may be sufficient to provide protection. Therefore, to map the humoral correlates of immunity against RSV, antibody responses across six different vaccines were profiled in a highly controlled nonhuman primate-challenge model. Viral loads were monitored in both the upper and lower respiratory tracts, and machine learning was used to determine the vaccine platform-agnostic antibody features associated with protection. Upper respiratory control was associated with virus-specific IgA levels, neutralization, and complement activity, whereas lower respiratory control was associated with Fc-mediated effector mechanisms. These findings provide critical compartment-specific insights toward the rational development of future vaccines.

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection.

The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation.

Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Regulation of RelB expression during the initiation of dendritic cell differentiation.

The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CbetaII (PKCbetaII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCbetaII also downregulated constitutive expression of NF-kappaB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCbetaII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCbetaII-mediated regulation of transcriptional initiation and elongation.

Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability.

Respiratory Syncytial Virus (RSV) infection is the leading cause of lower respiratory tract infection in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. The infectious RSV particle is decorated with a type I viral fusion (F) glycoprotein that structurally rearranges from a metastable prefusion form to a highly stable postfusion form. In people naturally infected with RSV, the neutralizing antibodies primarily recognize the prefusion conformation. Therefore, engineered RSV F protein stabilized in its prefusion conformation has been an attractive strategy for developing RSV F vaccine antigens. Long-term stability at 4 °C or higher is a desirable attribute for a RSV F subunit vaccine antigen. We have previously shown that a prefusion stabilized RSV F construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4 °C. Structure-based design was performed to improve the stability of the RSV F subunit vaccine. We identified additional mutations that further stabilize RSV F protein in its prefusion conformation by using binding to a previously described antigenic site I antibody 4D7 as the screening tool. In addition, we designed and identified variants with increased expression levels, which is another desirable attribute for a subunit vaccine. Our data suggested that an RSV F variant F111 is properly folded, and has improved heat stability as well as stability upon long-term storage at 4 °C. A mouse immunogenicity study demonstrated that no compromise in immunogenicity (both binding and neutralizing antibody levels) was observed with the introduction of these additional mutations.

Diminished expression of transcription factors nuclear factor kappaB and CCAAT/enhancer binding protein underlies a novel tumor evasion mechanism affecting macrophages of mammary tumor-bearing mice.

Interactions between malignant tumors and the host immune system shape the course of cancer progression. The molecular basis of such interactions is the subject of immense interest. Proinflammatory cytokines produced by macrophages are critical mediators of immune responses that contribute to the control of the advancement of neoplasia. We have shown that the expressions of interleukin 12 (IL-12) and inducible nitric oxide synthase (iNOS) are decreased in macrophages from mammary tumor-bearing mice. In this study, we investigated the causes of IL-12 dysregulation and found deficient nuclear factor kappaB (NFkappaB) and CCAAT/enhancer binding protein (C/EBP) expression and function in tumor bearers' peritoneal macrophages. The constitutive expressions of NFkappaB p50, c-rel, p65, and C/EBPalpha and beta, as well as the lipopolysaccharide-induced nuclear translocation and DNA binding of NFkappaB components and C/EBPalpha and beta, are profoundly impaired in macrophages from mice bearing D1-DMBA-3 tumors. Because similar findings occur with the iNOS gene, it seems that it represents a novel mechanism by which tumor-derived factors interfere with the host immune defenses.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

EGR-2 is not required for in vivo CD4 T cell mediated immune responses.

BACKGROUND: The zinc finger transcription factor EGR-2 has been shown to play an important role in the induction of T cell anergy and the regulation of peripheral T cell tolerance. In vitro, a prior study has show that T cells deficient in EGR-2 are hyperproliferative to IL-2 and produce elevated levels of the effector cytokine IFN-γ. EGR-2 deficient mice have increased levels of CD44(high) T cells in peripheral lymphoid organs, and with age, develop autoimmune-like features. PRINCIPAL FINDINGS: Here we show that despite increased numbers of cells bearing an activated CD44(high)CD62L(low) phenotype, T cells from young healthy EGR-2 deficient mice have normal proliferative and cytokine responses, and the mice themselves mount normal immune responses against minor histocompatibility antigens, and the pathogens Toxoplasma gondii and lymphocytic choriomeningitis virus. CONCLUSIONS: Our results indicate that EGR-2 is not required to mount normal acute in vivo immune responses against foreign antigens, and suggest instead that it may serve to regulate the response to chronic antigenic exposure, such as that which occurs to autoantigens.

Reactive oxygen species are not required for an arsenic trioxide-induced antioxidant response or apoptosis.

Arsenicals are both environmental carcinogens as well as therapeutic agents for the treatment of trypanosomiasis and more recently cancer. Arsenic trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has activity in multiple myeloma (MM). While signaling events associated with carcinogenesis have been well studied, it still remains to be determined which of these events are involved in anti-cancer signaling. To better define this response, gene expression profiling following ATO treatment of four MM cell lines was performed. The pattern was consistent with a strong antioxidative response, particularly of genes activated by Nrf2. While Nrf2 is expressed constitutively at the mRNA level, the protein is not detected in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized and present in the nucleus within 6 h of ATO treatment. Despite the activation of this antioxidative response, ROS may not be important in ATO-induced death. Inhibition of ATO-induced ROS with butylated hydroxyanisole (BHA) does not affect Nrf2 activation or cell death. Moreover, silencing Nrf2 had no effect on ATO-induced apoptosis. Together these data suggest that ROS is not important in the induction of the antioxidative response or cellular death by ATO.

Differentiation of acute and chronic myeloid leukemic blasts into the dendritic cell lineage: analysis of various differentiation-inducing signals.

PURPOSE: Ex vivo differentiation of myeloid leukemic blasts into dendritic cells (DCs) holds significant promise for use as cellular vaccines, as they may present a constellation of endogenously expressed known and unknown leukemia antigens to the immune system. Although variety of stimuli can drive leukemia --> DC differentiation in vitro, these blast-derived DCs typically have aberrant characteristics compared with DCs generated from normal progenitors by the same stimuli. It is not clear whether this is due to underlying leukemogenic mechanisms (e.g., specific oncogenes), genetic defects, stage of maturation arrest, defects in cytokine receptor expression or signal transduction pathways, or whether different stimuli themselves induce qualitatively dissimilar DC differentiation. METHODS: To assess what factors may contribute to aberrant leukemic blast --> DC differentiation, we have examined how the same leukemic blasts (AML and CML) respond to different DC differentiation signals--including extracellular (the cytokine combination GM-CSF + TNF-alpha + IL-4) and intracellular (the protein kinase C agonist PMA, the calcium ionophore A23187, and the combination of PMA plus A23187) stimuli. RESULTS: We have found that the same leukemic blasts will develop qualitatively different sets of DC characteristics in response to differing stimuli, although no stimuli consistently induced all of the characteristic DC features. There were no clear differences in the responses relative to specific oncogene expression or stage of maturation arrest (AML vs CML). Signal transduction agonists that bypassed membrane receptors/proximal signaling (in particular, the combination of PMA and A23187) consistently induced the greatest capability to activate T cells. Interestingly, this ability did not clearly correlate with expression of MHC/costimulatory ligands. CONCLUSIONS: Our findings suggest that signal transduction may play an important role in the aberrant DC differentiation of leukemic blasts, and demonstrate that direct activation of PKC together with intracellular calcium signaling may be an effective method for generating immunostimulatory leukemia-derived DCs.

Protein kinase C betaII plays an essential role in dendritic cell differentiation and autoregulates its own expression.

Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. The signal transduction pathways that regulate the ability of progenitors to undergo DC differentiation, as well as the specific characteristics of the resulting DC, are only beginning to be characterized. We have found previously that activation of protein kinase C (PKC) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC. We now report that PKC activation is also required for cytokine-driven DC differentiation from monocytes. Of the cPKC isoforms, only PKC-betaII was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. Transfection of PKC-betaII into the differentiation-resistant KG1a subline restored the ability to undergo DC differentiation in a signal strength-dependent fashion as follows: 1) by development of characteristic morphology; 2) the up-regulation of DC surface markers; 3) the induction of expression of the NFkappaB family member Rel B; and 4) the potent ability to stimulate allo-T cells. Most unexpectedly, the restoration of PKC-betaII signaling in KG1a was not directly due to overexpression of the transfected classical PKC (alpha, betaII, or gamma) but rather through induction of endogenous PKC-beta gene expression by the transfected classical PKC. The mechanism of this positive autoregulation involves up-regulation of PKC-beta promoter activity by constitutive PKC signaling. These findings indicate that the regulation of PKC-betaII expression and signaling play critical roles in mediating progenitor to DC differentiation.