Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01.

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

RORC2 is involved in T cell polarization through interaction with the FOXP3 promoter.

The process of Th cell differentiation toward polarized effector T cells tailors specific immunity against invading pathogens while allowing tolerance against commensal microorganisms, harmless allergens, or autologous Ags. Identification of the mechanisms underlying this polarization process is therefore central to understand how the immune system confers immunity and tolerance. The present study demonstrates that retinoic acid receptor-related orphan receptor C2 (RORC2), a key transcription factor in Th17 cell development, inhibits FOXP3 expression in human T cells. Although overexpression of RORC2 in naive T cells reduces levels of FOXP3, small interfering RNA-mediated knockdown of RORC2 enhances its expression. RORC2 mediates this inhibition at least partially by binding to two out of four ROR-responsive elements on the FOXP3 promoter. Knockdown of RORC2 promotes high FOXP3 levels and decreased expression of proinflammatory cytokines beta form of pro-IL-1, IL-6, IL-17A, IFN-gamma, and TNF-alpha in differentiating naive T cells, suggesting that the role of RORC2 in Th17 cell development involves not only induction of Th17-characteristic genes, but also suppression of regulatory T cell-specific programs. Together, this study identifies RORC2 as a polarizing factor in transcriptional cross-regulation and provides novel viewpoints on the control of immune tolerance versus effector immune responses.

Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in diseases.

Advancing our understanding of mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections could lead to effective and targeted therapies. Subsets of immune and inflammatory cells interact via ILs and IFNs; reciprocal regulation and counter balance among T(h) and regulatory T cells, as well as subsets of B cells, offer opportunities for immune interventions. Here, we review current knowledge about ILs 1 to 37 and IFN-γ. Our understanding of the effects of ILs has greatly increased since the discoveries of monocyte IL (called IL-1) and lymphocyte IL (called IL-2); more than 40 cytokines are now designated as ILs. Studies of transgenic or knockout mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided important information about IL and IFN functions. We discuss their signaling pathways, cellular sources, targets, roles in immune regulation and cellular networks, roles in allergy and asthma, and roles in defense against infections.

Unique phenotype of human tonsillar and in vitro-induced FOXP3+CD8+ T cells.

Forkhead box p3 (FOXP3) is known to program the acquisition of suppressive capacities in CD4(+) regulatory T cells (Treg), whereas its role in CD8(+) T cells is unknown. The current study investigates whether FOXP3 also acts as a Treg master switch in peripheral blood and tonsillar CD8(+) T cells. Single-cell analyses reveal the existence of a FOXP3(+)CD8(+) population in human tonsils, whereas FOXP3(+)CD8(+) T cells are rarely detected in peripheral blood. Tonsillar FOXP3(+)CD8(+) T cells exhibit a Treg phenotype with high CTLA-4 and CD45RO and low CD127 and CD69 expression. Interestingly, the tonsillar FOXP3(+)CD8(+) T cells are mostly CD25(negative) and some cells also express the proinflammatory cytokines TNF-alpha, IFN-gamma, or IL-17A. Particularly, IL-17A-expressing cells are present among FOXP3(+)CD8(+) T cells. Even though FOXP3 expression is at the detection limit in peripheral blood CD8(+) T cells ex vivo, it can be induced in vitro in naive CD8(+) T cells by polyclonal stimulation. The induced FOXP3(+)CD8(+) T cells are predominantly CD25(high) and CD28(high) and similar to tonsillar cells, they produce high levels of TNF-alpha, IFN-gamma, and granzyme B. However, IL-4 expression is mutually exclusive and IL-17A expression is not detectable. These FOXP3(+)CD8(+) T cells suppress the proliferation of CD4(+) T cells in cocultures, while showing no direct cytotoxic activity. In conclusion, the current study characterizes FOXP3-expressing CD8(+) T cells from human tonsils and shows that in vitro activation leads to FOXP3 expression in CD8(+) T cells and gain of suppressive activity.

Regulation of the foxp3 gene by the Th1 cytokines: the role of IL-27-induced STAT1.

Impaired functional activity of T regulatory cells has been reported in allergic patients and results in an increased susceptibility to autoimmune diseases. The master regulator of T regulatory cell differentiation, the transcription factor FOXP3, is required for both their development and function. Despite its key role, relatively little is known about the molecular mechanisms regulating foxp3 gene expression. In the present study, the effect of Th1 cytokines on human T regulatory cell differentiation was analyzed at epigenetic and gene expression levels and reveals a mechanism by which the STAT1-activating cytokines IL-27 and IFN-gamma amplify TGF-beta-induced FOXP3 expression. This study shows STAT1 binding elements within the proximal part of the human FOXP3 promoter, which we previously hypothesized to function as a key regulatory unit. Direct binding of STAT1 to the FOXP3 promoter following IL-27 stimulation increases its transactivation process and induces permissive histone modifications in this key region of the FOXP3 promoter, suggesting that FOXP3 expression is promoted by IL-27 by two mechanisms. Our data demonstrate a molecular mechanism regulating FOXP3 expression, which is of considerable interest for the development of new drug targets aiming to support anti-inflammatory mechanisms of the immune system.

GATA3-driven Th2 responses inhibit TGF-beta1-induced FOXP3 expression and the formation of regulatory T cells.

Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.

Differentiation and functional analysis of human T(H)17 cells.

BACKGROUND: T(H)17 cells are of pathologic relevance in autoimmune disorders and presumably also in allergy and asthma. Regulatory T (Treg) cells, in contrast, suppress inflammatory and allergen-driven responses. Despite these disparate functions, both T-cell subsets have been shown to be dependent on TGF-beta for their development. OBJECTIVE: The aim of the study was to analyze the differentiation and function of human T(H)17 cells in comparison with other T(H) cell subsets. METHODS: Naive human CD4(+) T cells were differentiated in vitro, and gene expression was analyzed by means of quantitative real-time PCR, ELISA, and immunofluorescence. The function of T(H) cell subsets was assessed by monitoring the response of primary bronchial epithelial cells in coculture experiments. RESULTS: In vitro differentiated T(H)17 cells differ from Treg and other T(H) cells in their potency to induce IL-6 and IL-1beta expression in primary bronchial epithelial cells. TGF-beta, IL-1beta, IL-6, and IL-23 are necessary during T(H)17 cell differentiation to acquire these functions, including IL-17 production. In contrast, TGF-beta alone is necessary and sufficient to induce the transcription factor RORC2. This transcription factor, previously thought to be specific for T(H)17 cells, is also expressed in Treg cells, CD25(+) cells, cytotoxic T cells, and natural killer T cells. CONCLUSION: This study demonstrates mechanisms of differentiation to human T(H)17 cells, a subset that effectively and uniquely modulates the function of primary bronchial epithelial cells.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

Molecular mechanisms underlying FOXP3 induction in human T cells.

FOXP3 is playing an essential role for T regulatory cells and is involved in the molecular mechanisms controlling immune tolerance. Although the biological relevance of this transcription factor is well documented, the pathways responsible for its induction are still unclear. The current study reveals structure and function of the human FOXP3 promoter, revealing essential molecular mechanisms of its induction. The FOXP3 promoter was defined by RACE, cloned, and functionally analyzed using reporter-gene constructs in primary human T cells. The analysis revealed the basal, T cell-specific promoter with a TATA and CAAT box 6000 bp upstream the translation start site. The basal promoter contains six NF-AT and AP-1 binding sites, which are positively regulating the trans activation of the FOXP3 promoter after triggering of the TCR. The chromatin region containing the FOXP3 promoter was bound by NF-ATc2 under these conditions. Furthermore, FOXP3 expression was observed following TCR engagement. Promoter activity, mRNA, and protein expression of T cells were suppressed by addition of cyclosporin A. Taken together, this study reveals the structure of the human FOXP3 promoter and provides new insights in mechanisms of addressing T regulatory cell-inducing signals useful for promoting immune tolerance. Furthermore, the study identifies essential, positive regulators of the FOXP3 gene and highlights cyclosporin A as an inhibitor of FOXP3 expression contrasting other immunosuppressants such as steroids or rapamycin.