Profiling HPV-16-specific T cell responses reveals broad antigen reactivities in oropharyngeal cancer patients.

Cellular immunotherapeutics targeting the human papillomavirus (HPV)-16 E6 and E7 proteins have achieved limited success in HPV-positive oropharyngeal cancer (OPC). Here we have conducted proteome-wide profiling of HPV-16-specific T cell responses in a cohort of 66 patients with HPV-associated OPC and 22 healthy individuals. Unexpectedly, HPV-specific T cell responses from OPC patients were not constrained to the E6 and E7 antigens; they also recognized E1, E2, E4, E5, and L1 proteins as dominant targets for virus-specific CD8+ and CD4+ T cells. Multivariate analysis incorporating tumor staging, treatment status, and smoking history revealed that treatment status had the most significant impact on HPV-specific CD8+ and CD4+ T cell immunity. Specifically, the breadth and overall strength of HPV-specific T cell responses were significantly higher before the commencement of curative therapy than after therapy. These data provide the first glimpse of the overall human T cell response to HPV in a clinical setting and offer groundbreaking insight into future development of cellular immunotherapies for HPV-associated OPC patients.

Designing an effective vaccine to prevent Epstein-Barr virus-associated diseases: challenges and opportunities.

INTRODUCTION: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a number of clinical manifestations. Primary EBV infection in young adolescents often manifests as acute infectious mononucleosis and latent infection is associated with multiple lymphoid and epithelial cancers and autoimmune disorders, particularly multiple sclerosis. Areas covered: Over the last decade, our understanding of pathogenesis and immune regulation of EBV-associated diseases has provided an important platform for the development of novel vaccine formulations. In this review, we discuss developmental strategies for prophylactic and therapeutic EBV vaccines which have been assessed in preclinical and clinical settings. Expert commentary: Major roadblocks in EBV vaccine development include no precise understanding of the clinical correlates of protection, uncertainty about adjuvant selection and the unavailability of appropriate animal models. Recent development of new EBV vaccine formulations provides exciting opportunities for the formal clinical assessment of novel formulations.

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance.

BACKGROUND: Application of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a 'niche' so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained. METHODS: BM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells. RESULTS: Immune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM. CONCLUSIONS: Rapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.

Ultraviolet B induces high mobility group box 1 release from mouse peritoneal macrophages in vitro via caspase-1 mediated secretion pathway.

High mobility group box 1 (HMGB1) protein is a unique non histone nuclear protein that acts extracellularly as a mediator of delayed inflammation. Sub lethal dose of UVB triggers the release of cytokines from macrophages (MΦs). Adding to the panoply of UVB induced cytokines; it is reported that UVB induces HMGB1 release from mouse peritoneal MΦs in time and partially dose dependent manner, independent of TNF-α. UVB also enhanced the transcription of HMGB1 gene and expression of cellular protein, which influences its subsequent release. HMGB1 is secreted by an unconventional secretion pathway of unknown mechanism. Caspase-1 has been shown to function as a general regulator of stress induced unconventional secretion for a number of cytokines. In the present study, we have observed that pharmacological inhibitors specific for caspase-1 (ZVAD and YVAD) abrogated UVB induced HMGB1 release from MΦs. This effect was most likely mediated via physical interaction between HMGB1 and active caspase-1 (p10 and p20) as demonstrated by immunoprecipitation. In addition, it was found that HMGB1 and active caspase-1 p20 release depends on UVB mediated enhancement of intracellular Ca(2+). Thus our data suggests that optimal dose of UVB (50 mJ/cm(2)) induces HMGB1 upregulation and active release from mouse peritoneal MΦs which is mediated by caspase-1 in a Ca(2+) dependent manner.

High mobility group box 1 protein synergizes with lipopolysaccharide and peptidoglycan for nitric oxide production in mouse peritoneal macrophages in vitro.

Extracellular high mobility group box 1 (HMGB1) protein and nitric oxide (NO) has been credited with multiple inflammatory functions using in vivo and in vitro systems. Therefore, delineating their regulation may be an important therapeutic strategy for the treatment of sepsis. In the present study, it is demonstrated that recombinant HMGB1 (rHMGB1) synergizes with sub threshold concentration of TLR2 agonist (PGN; 1 µg/ml) as well as with TLR4 agonist (LPS; 1 ng/ml) to induce NO release in mouse peritoneal macrophages. The enhanced iNOS expression was also observed at the transcription and translational level. Co-incubation of macrophages with rHMGB1 with either PGN or LPS showed enhanced expression of TLR2, TLR4 and RAGE. TLR2, TLR4 or RAGE knockdown macrophages effectively inhibited the rHMGB1+PGN or LPS induced NO synergy. It was further observed that the JNK MAPK inhibitor SP600125 attenuated the PGN+rHMGB1 induced iNOS/NO synergy whereas p38 MAPK inhibitor SB908912 inhibited iNOS/NO synergy induced by LPS+rHMGB1. It was also observed that the activation of NF-κB is essential for the synergy as the pharmacological inhibition or siRNA knockdown of NF-κB (cRel) significantly reduced the rHMGB1+PGN or rHMGB1+LPS induced enhanced iNOS/NO expression. Altogether, the data suggests that the co-incubation of macrophages with rHMGB1 with either LPS or PGN induces the synergistic effect on iNOS expression and NO release by the upregulation of surface receptors (TLR2, TLR4 and RAGE) which in turn amplifies the MAPKs (p38 and JNK) and NF-κB activation and results in enhanced iNOS expression and NO production.

Peptidoglycan induced expression of peroxisome proliferator-activated receptor γ in mouse peritoneal macrophages: role of ERK and JNK MAP kinases.

The peroxisome proliferator-activated receptor (PPAR) γ plays an important role in macrophage inflammatory homeostasis. Here we investigate the cross talk between PPARγ and TLR2 signaling pathway in mouse peritoneal macrophages. Real time RT-PCR and immunoblot analysis revealed that peptidoglycan (PGN) treatment of macrophages leads to biphasic effect on PPARγ expression i.e. an early upregulation and a late suppression. Inhibition of ERK MAP kinase by PD98059 abolished the early and rapid induction of PPARγ, while the inhibition of JNK MAP kinase by SP600125 nullifies the late inhibitory effect on the PPARγ expression in a dose-dependent manner. Furthermore, PPARγ knockdown macrophages showed enhanced NF-κB activity after PGN treatment. PGN treatment also enhances PPARγ interaction with p65 as observed by immunoprecipitation. This interaction may inhibit NF-κB (p65) activity as increased nuclear localization of p65 was observed in PPARγ knockdown macrophages after PGN treatment. PPARγ knockdown also increased the PGN-induced inflammatory cytokines (TNF-α, IL-1ß, IL-12p40) production. Thus, our observations suggest that PGN induces PPARγ expression which is regulated by MAPKs activation and this enhanced PPARγ in turn attenuate NF-κB activity probably via enhancing p65 nuclear export. These results provide insight into how these pathways could be modulated in inflammatory diseases.

Role of mitogen-activated protein kinases in peptidoglycan-induced expression of inducible nitric oxide synthase and nitric oxide in mouse peritoneal macrophages: extracellular signal-related kinase, a negative regulator.

The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38(mapk) in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages; however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity.

Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro.

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages.

Role of prostaglandin E2 in peptidoglycan mediated iNOS expression in mouse peritoneal macrophages in vitro.

Many extracellular stimuli, e.g. microbial products, cytokines etc., result in the expression of inducible nitric oxide synthase (iNOS) in macrophages. However, it is not known whether expression of the iNOS gene in response to microbial products is a primary response of macrophages, or is the result of paracrine/autocrine signalling induced by endogenous biomolecules that are synthesised as a result of host cell-microbe interaction. In this paper we demonstrate that iNOS expression in mouse peritoneal macrophages in response to bacterial peptidoglycan (PGN) is a secondary effect requiring autocrine signalling of endogenously produced prostaglandin E2, and that PGN stimulation is mandatory, but not sufficient in itself, for induction of iNOS expression.

Protein kinase Cdelta and protein tyrosine kinase regulate peptidoglycan-induced nuclear factor-kappaB activation and inducible nitric oxide synthase expression in mouse peritoneal macrophages in vitro.

Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. Nitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MPhi) against microorganisms. This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide release caused by peptidoglycan from Staphylococcus aureus in mouse peritoneal macrophages. Protein tyrosine kinase inhibitor, genestein and PKCdelta inhibitor, rottlerin attenuated the PGN-induced expression of iNOS and NO. H-7, a PKC inhibitor did not significantly affected the PGN-induced iNOS expression and NO release. NF-kappaB inhibitor, curcumin also inhibited PGN-induced NO release. Treatment of MPhi with PGN caused an increase in protein tyrosine kinase activity, expression and activation of PKCdelta, IkappaB phosphorylation and p65 (NF-kappaB) nuclear translocation. The PGN-induced IkappaB phosphorylation and p65 nuclear translocation was inhibited in macrophages pretreated with rottlerin and genestein. No paracrine or autocrine effect of TNF-alpha on PGN-induced iNOS expression and NO release was observed. These observations suggest that PGN induces enhanced expression of iNOS and NO production through activation of protein tyrosine kinases and PKCdelta, which in turn initiates NF-kappaB activation and translocation to nucleus.