CD1d-lipid antigen recognition by the γδ TCR.

The T cell repertoire comprises αß and γδ T cell lineages. Although it is established how αß T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human Vδ1(+) γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α-galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A' pocket of CD1d, in which the Vδ1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.

Adenovirus vector produced Zika virus-like particles induce a long-lived neutralising antibody response in mice.

Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.

The efficacy and safety of weekly vinorelbine in relapsed malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rapidly progressive invariably lethal tumor. Treatment options remain limited and the outcome in relapsed disease is poor warranting new therapeutic options. Following our previous experience in the first-line setting, we conducted a phase 2 open-label non-comparative study to assess the safety and efficacy of weekly vinorelbine chemotherapy, each cycle consisting of 30 mg/m(2) for 6 weeks, in patients with previous exposure to chemotherapy. In 63 individuals with relapsed MPM who had not received previous vinorelbine, we observed an objective response rate of 16% and an overall survival of 9.6 months (95% confidence interval 7.3-11.8 months). The main grade III/IV toxicity observed was neutropenia and toxicity was similar to weekly vinorelbine when used in the first-line setting. Weekly vinorelbine appeared to have a reasonable response rate with an acceptable toxicity profile in the second-line treatment of MPM. Its use should be prospectively evaluated in a randomised trial in the first or second-line therapy of MPM.

Attenuation of a dengue virus replicon by codon deoptimization of nonstructural genes.

The overwhelming increase of dengue virus (DENV) infections in recent years shows that current strategies to combat dengue do not work. The lack of a highly effective dengue vaccine and the limited effectivity of vector controls exacerbate this situation. To point the way to a novel method of creating DENV vaccine candidates, here we disrupted the codon usage in a DENV-2 reporter replicon to generate variants with different replication characteristics. Six different mutated constructs containing stretches of altered codon usage in the non-structural genes were generated. The mutated sequences were deoptimized to the least favorable codons for human cells. We studied the replication efficiency of these constructs by measuring luciferase reporter activity, relative RNA fold change, and NS1 secretion. Our findings showed that the level of virus attenuation is closely associated with the amount of codon deoptimization. Indeed, replication was completely abolished in extensively-deoptimized constructs D2Rep-6 and D2Rep-5, intermediate with constructs D2Rep-4 (771 bp silent mutations) and D2Rep-3 (756 bp silent mutations) and restored almost to wildtype levels with constructs D2Rep-2 (394 silent mutations) and D2Rep-1 (48 silent mutations). We also determined that the position of codon deoptimization within the genome is crucial to the degree of attenuation observed. Based on our analysis, we propose that the design for an ideal DENV vaccine candidate could include 700-1500 silent mutations within the NS2A and NS3 genes. Our results suggest that codon deoptimization is an ideal strategy that can readily be used to develop a DENV vaccine candidate with "fine-tuned" attenuation.

Recent advances in dengue pathogenesis and clinical management.

This review describes and commentates on recent advances in the understanding of dengue pathogenesis and immunity, plus clinical research on vaccines and therapeutics. We expand specifically on the role of the dermis in dengue virus infection, the contribution of cellular and humoral immune responses to pathogenesis and immunity, NS1 and mechanisms of virus immune evasion. Additionally we review a series of therapeutic intervention trials for dengue, as well as recent clinical research aimed at improving clinical diagnosis, risk prediction and disease classification.

The molecular bases of δ/αß T cell-mediated antigen recognition.

αß and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αß and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αß T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-ß chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αß T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αßTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αßTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αßTCR binding. Our findings highlight how components from αß and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.

Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells.

The ability of CD4(+)Foxp3(+) regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7(+)CCR5(-) lymph node-seeking to a CCR7(-)CCR5(+) inflammation-seeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression.

Proliferative signals mediated by CD28 superagonists require the exchange factor Vav1 but not phosphoinositide 3-kinase in primary peripheral T cells.

Almost all responses of naive T cells require co-stimulation, i.e. engagement of the clonotypic TCR with relevant antigen/MHC and the co-stimulatory molecule CD28. How CD28 contributes to T-cell proliferation remains poorly understood, with widely conflicting reports existing which may reflect different methods of co-ligating receptors. Some CD28 mAb, however, can stimulate T-cell proliferation without the need for TCR co-ligation, and thus provide unique tools to dissect proliferative signals mediated through CD28 alone. Using primary peripheral T cells from CD28-transgenic mice, we show that both the YMNM and Lck-binding motifs, but not the Itk-binding motif, in CD28 are required for proliferation. Given that the YMNM motif recruits both phosphoinositide 3-kinase (PI3K) and the exchange factor Vav1, we investigated the role of these two molecules in CD28-mediated proliferation. In p110delta(D910A/D910A) transgenic T cells, which are defective in PI3K activation following CD28 ligation, proliferation was comparable to that in wild-type cells. By contrast, T-cell proliferation was abolished in Vav1(-/-) cells. Although we did not address the role of Grb2 in CD28 signalling, these results indicate that CD28 can mediate Lck- and Vav1-dependent proliferative signals independently of PI3K.

Enhanced glucocorticoid receptor signaling in T cells impacts thymocyte apoptosis and adaptive immune responses.

To study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses.

Glucocorticoids engage different signal transduction pathways to induce apoptosis in thymocytes and mature T cells.

Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well as caspase-3, - 8, and -9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B into the cytosol, nuclear condensation, and processing of caspase-8 and -3. According to our model, activation of caspase-3 by caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the apoptotic program induced by GCs comprises cell type-specific as well as common features.

Unexpected features of acute T lymphoblastic lymphomas in Notch1IC transgenic rats.

Dysregulated Notch signaling accounts for the majority of acute T lymphoblastic leukemia/lymphoma (T-ALL) cases in humans. Here, we characterize lymphomas from Notch1IC transgenic rats, which develop T-ALL shortly after weaning, and show that they display a number of previously undocumented features. Starting from monoclonal thymic tumors, the CD4(+)CD8alphaalpha(+) lymphoma cells infiltrate the bone marrow and then spread to secondary lymphoid and non-lymphoid organs. However, major hallmarks of T-ALL cells in other murine models and human patients, such as constitutive NF-kappaB activity and increased levels of anti-apoptotic proteins, are remarkably absent in Notch1IC lymphomas. In contrast, CD30, a classic marker of Hodgkin lymphomas, is overexpressed in these tumors. Intriguingly, enforced Notch1 signaling up-regulates expression of Notch3, which has also been implicated in the pathogenesis of T-ALL. By blocking endogenous Notch signaling, we could demonstrate that Notch1IC is sufficient to induce sustained preTCR expression in transgenic thymocytes but not for their progression to the double-positive stage. This suggests that other Notch activities may also contribute to the phenotype of the transgenic rats. In summary, we anticipate this new animal model will help to further elucidate the role of Notch1 in the pathogenesis of T-ALL.

Molecular mechanisms of glucocorticoids in the control of inflammation and lymphocyte apoptosis.

The immune system must be tightly controlled not only to guarantee efficient protection from invading pathogens and oncogenic cells but also to avoid exaggerated immune responses and autoimmunity. This is achieved through interactions amongst leukocytes themselves, by signals from stromal cells and also by various hormones, including glucocorticoids. The glucocorticoids are a class of steroid hormones that exert a wide range of anti-inflammatory and immunosuppressive activities after binding to the glucocorticoid receptor. The power of these hormones was acknowledged many decades ago, and today synthetic derivatives are widely used in the treatment of inflammatory disorders, autoimmunity and cancer. In this review, we summarize our present knowledge of the molecular mechanisms of glucocorticoid action, their influence on specific leukocytes and the induction of thymocyte apoptosis, with an emphasis on how molecular genetics has contributed to our growing, although still incomplete, understanding of these processes.

Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection.

Notch1 is involved in directing cell fate decisions in a variety of developmental scenarios. Extending previous experiments in mice, we generated transgenic rats expressing the intracellular domain of Notch1 in the thymus. Importantly, this leads to sustained expression of the pre-TCR throughout thymocyte development, accompanied by a reduction of alphabetaTCR complexes. In addition, re-expression of RAG-1 and RAG-2 in TCRbeta(+) cells is impaired, and the Valpha repertoire is altered. Consequently, thymocytes in transgenic rats do not undergo positive selection and largely fail to progress to the single positive stage. According to our model, the previously reported effects of Notch1 on the CD4/CD8 cell fate decision may be explained by a differential sensitivity of the two lineages toward altered TCR signaling.

cFLIPL inhibits tumor necrosis factor-related apoptosis-inducing ligand-mediated NF-kappaB activation at the death-inducing signaling complex in human keratinocytes.

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-kappaB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIP(L) interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIP(L) in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIP(L). Functional analysis revealed that relative cFLIP(L) levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIP(L) specifically blocked TRAIL-induced NF-kappaB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIP(L)-overexpressing keratinocytes, although cFLIP(L) did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIP(L)-overexpressing cells. Taken together, our data demonstrate that cFLIP(L) is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-kappaB activation and subsequent proinflammatory target gene expression. Hence, cFLIP(L) modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.

CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells.

CpG oligodeoxynucleotides (CpG ODNs) stimulate immune cells via the Toll-like receptor 9 (TLR9). In this study, we have investigated the effects of CpG ODNs on latent human immunodeficiency virus (HIV) infection in human T cells. Treatment of the latently infected T cell line ACH-2 with CpG ODNs 2006 or 2040 stimulated HIV replication, whereas no effects were evident when ODNs without the CpG motif were used. CpG-induced virus reactivation was blocked by chloroquine, indicating the involvement of TLR9. In contrast to the responsiveness of ACH-2 cells, CpG ODNs failed to activate HIV provirus in the latently infected Jurkat clone J1.1. We also studied the effects of CpG ODNs on productive HIV infection and found enhancement of viral replication in A3.01 T cells, whereas again no stimulating effects were observed in Jurkat T cells. CpG ODN treatment activated NF-kappaB in ACH-2 cells, which was similarly triggered in uninfected A3.01 T cells following exposure to CpG ODNs, indicating that TLR9-induced signal transduction was not dependent on proviral infection. Our study demonstrates that CpG ODNs directly trigger the activation of NF-kappaB and reactivation of latent HIV in human T cells. Our results point to a novel role for CpG ODNs as stimulators of HIV replication and open new avenues to eradicate the latent viral reservoirs in HIV-infected patients treated with antiretroviral therapy.