CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2: Characterizing CD4+ T cells in Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Blockade of N-Glycosylation Promotes Antitumor Immune Response of T Cells.

Adoptive cellular therapy and its derivative, chimeric AgR T cell therapy, have achieved significant progress against cancer. Major barriers persist, however, including insufficient induction of cytotoxic T cells and exhaustion of tumor-infiltrating lymphocytes. In this study, we discovered a new role for 2-deoxy-d-glucose (2DG) in enhancing the antitumor activity of human T cells against NKG2D ligand-expressing tumor cells. Human T cells treated with 2DG upregulated the NK-specific transcription factors TOX2 and EOMES, thereby acquiring NK cell properties, including high levels of perforin/granzyme and increased sensitivity to IL-2. Notably, rather than inhibiting glycolysis, 2DG modified N-glycosylation, which augmented antitumor activity and cell surface retention of IL-2R of T cells. Moreover, 2DG treatment prevented T cells from binding to galectin-3, a potent tumor Ag associated with T cell anergy. Our results, therefore, suggest that modifying N-glycosylation of T cells with 2DG could improve the efficacy of T cell-based immunotherapies against cancer.

Biomarkers of vascular disease in diabetes: the adipose-immune system cross talk.

Experimental and clinical studies aimed at investigating the mechanism(s) underlying vascular complications of diabetes indicate that a great number of molecules are involved in the pathogenesis of these complications. Most of these molecules are inflammatory mediators or markers generated by immune or adipose tissue. Some of them, i.e. resistin and sortilin, have been shown to be involved in the cross talk between adipocytes and inflammatory cells. This interaction is an attractive area of research, particularly in type 2 diabetes and obesity. Other proteins, such as adiponectin and visfatin, appear to be more promising as possible vascular markers. In addition, some molecules involved in calcium/phosphorus metabolism, such as klotho and FGF23, have an involvement in the pathogenesis of diabetic vasculopathy, which appears to be dependent on the degree of vascular impairment. Inflammatory markers are a promising tool for treatment decisions while measuring plasma levels of adipokines, sortilin, Klotho and FGF23 in adequately sized longitudinal studies is expected to allow a more precise characterization of diabetic vascular disease and the optimal use of personalized treatment strategies.

Endothelial Sox17 promotes allergic airway inflammation.

BACKGROUND: IL-33, levels of which are known to be increased in patients with eosinophilic asthma and which is suggested as a therapeutic target for it, activates endothelial cells in which Sry-related high-mobility-group box (Sox) 17, an endothelium-specific transcription factor, was upregulated. OBJECTIVE: We investigated the relationship between Sox17 and IL-33 and the possible role of Sox17 in the pathogenesis of asthma using a mouse model of airway inflammation. METHODS: We used ovalbumin (OVA) to induce airway inflammation in endothelium-specific Sox17 null mutant mice and used IL-33 neutralizing antibody to evaluate the interplay between IL-33 and Sox17. We evaluated airway inflammation and measured levels of various cytokines, chemokines, and adhesion molecules. We also carried out loss- or gain-of-function experiments for Sox17 in human endothelial cells. RESULTS: Levels of IL-33 and Sox17 were significantly increased in the lungs of OVA-challenged mice. Anti-IL-33 neutralizing antibody treatment attenuated not only OVA-induced airway inflammation but also Sox17 expression in pulmonary endothelial cells. Importantly, endothelium-specific deletion of Sox17 resulted in significant alleviation of various clinical features of asthma, including airway inflammation, immune cell infiltration, cytokine/chemokine production, and airway hyperresponsiveness. Sox17 deletion also resulted in decreased densities of Ly6chigh monocytes and inflammatory dendritic cells in the lungs. In IL-33-stimulated human endothelial cells, Sox17 showed positive correlation with CCL2 and intercellular adhesion molecule 1 levels. Lastly, Sox17 promoted monocyte adhesion to endothelial cells and upregulated the extracellular signal-regulated kinase-signal transducer and activator of transcription 3 pathway. CONCLUSION: Sox17 was regulated by IL-33, and its genetic ablation in endothelial cells resulted in alleviation of asthma-related pathophysiologic features. Sox17 might be a potential target for asthma management.

Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells.

BACKGROUND: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. PRINCIPAL FINDINGS: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1ß and IFN-γ gene expression. Also, TGF-ß and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. CONCLUSIONS: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.

The expression of HMGB1 on microparticles released during cell activation and cell death in vitro and in vivo.

High mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that is a prototypic alarmin that can stimulate innate immunity and drive the pathogenesis of a wide range of inflammatory diseases. While HMGB1 can be released from both activated and dying cells, its biochemical and immunological properties differ depending on the release mechanism, resulting from redox changes and posttranslational modifications including acetylation. In addition to release of HMGB1, cell death is associated with the release of microparticles. Microparticles are small membrane-bound vesicles that contain cytoplasmic, nuclear and membrane components. Like HMGB1, microparticles display immunological activity and levels are elevated in diseases characterized by inflammation and vasculopathy. While studies have addressed the immunological effects of HMGB1 and microparticles independently, HMGB1, like other nuclear molecules, is a component of microparticles. Evidence for the physical association of HMGB1 comes from Western blot analysis of microparticles derived from RAW 264.7 macrophage cells stimulated by lipopolysaccharide (LPS) or induced to undergo apoptosis by treatment with etoposide or staurosporine in vitro. Analysis of microparticles in the blood of healthy volunteers receiving LPS shows the presence of HMGB1 as assessed by flow cytometry. Together, these findings indicate that HMGB1 can be a component of microparticles and may contribute to their activities. Furthermore, particle HMGB1 may represent a useful biomarker for in vivo events that may not be reflected by measurement of the total amount of HMGB1 in the blood.

HMGB in mollusk Crassostrea ariakensis Gould: structure, pro-inflammatory cytokine function characterization and anti-infection role of its antibody.

BACKGROUND: Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB's antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB's antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4-12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4-12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates. CONCLUSIONS/SIGNIFICANCE: Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.

High-mobility group box family of proteins: ligand and sensor for innate immunity.

Recent discoveries in signal-transducing innate receptors have illustrated the close link between innate and adaptive immunity. These advances revisit a fundamental issue of immunology, the recognition of self and non-self molecules by the immune system. Indeed, mounting evidence has been provided that the sensing of self-derived molecules by the immune system is important for health and disease. The high-mobility group box (HMGB) proteins, particularly HMGB1, are self-derived immune activators that have multiple functions in the regulation of immunity and inflammation. In this review, we summarize current knowledge of the function of HMGB proteins, as a ligand that can evoke inflammatory responses, and as a sensor for nucleic-acid-mediated immune responses.

Suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (HMGBs).

The activation of innate immune responses by nucleic acids is central to the generation of host responses against pathogens; however, nucleic acids can also trigger the development and/or exacerbation of pathogenic responses such as autoimmunity. We previously demonstrated that the selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by high-mobility group box proteins (HMGBs). From this, we reasoned that nonimmunogenic nucleotides with high-affinity HMGB binding may function as suppressing agents for HMGB-mediated diseases, particularly those initiated and/or exacerbated by nucleic acids. Here we characterize an array of HMGB-binding, nonimmunogenic oligodeoxynucleotides (ni-ODNs). Interestingly, we find that binding affinity is rather independent of nucleotide sequence, but is instead dependent on length and structure of the deoxyribose backbone. We further show that these ni-ODNs can strongly suppress the activation of innate immune responses induced by both classes of nucleic acid-sensing receptors. We also provide evidence for the suppressive effect of an ni-ODN, termed ISM ODN, on the induction of adaptive immune responses and in mouse models of sepsis and autoimmunity. We discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of these ni-ODNs in therapeutic interventions.

The dual role of the neuroinflammatory response after ischemic stroke: modulatory effects of hypothermia.

Neuroinflammation is a key element in the ischemic cascade after cerebral ischemia that results in cell damage and death in the subacute phase. However, anti-inflammatory drugs do not improve outcome in clinical settings suggesting that the neuroinflammatory response after an ischemic stroke is not entirely detrimental. This review describes the different key players in neuroinflammation and their possible detrimental and protective effects in stroke. Because of its inhibitory influence on several pathways of the ischemic cascade, hypothermia has been introduced as a promising neuroprotective strategy. This review also discusses the influence of hypothermia on the neuroinflammatory response. We conclude that hypothermia exerts both stimulating and inhibiting effects on different aspects of neuroinflammation and hypothesize that these effects are key to neuroprotection.

Enhancement of antibody response by high mobility group box protein-1-based DNA immunization.

High mobility group box protein 1 (HMGB1), a major non-histone protein, released from the cells induces dendritic cell (DC) maturation and Th1 polarization. While DNA immunization has become an attractive method for eliciting the production of antibodies (Abs) in animals injected with DNA encoding an antigen, the Ab responses induced by DNA immunization remain relatively weak. In this study, we investigated the release of an HMGB1-conjugated ovalbumin (HMGB1 OVA conjugate, HMGB1-OVA) from necrotic cells and the Ab responses to HMGB1-OVA following DNA immunization. HMGB1-OVA was released from 293T cells after induction of necrosis in vitro and from the muscle into the serum after DNA immunization followed by electroporation. DCs pulsed with the supernatant of necrotic 293T cells containing HMGB1-OVA induced DO11.10 CD4+ T cell proliferation and interferon-γ secretion more potently than DCs pulsed with the cell supernatant containing OVA. DNA immunization with an expression plasmid for HMGB1-OVA by intramuscular injection elicited enhanced Th1-type Ab responses to OVA. Moreover, DNA immunization with a plasmid vector for release-type HMGB1 mutant-conjugated OVA elicited an even stronger response than DNA immunization with wild-type HMGB1-OVA. HMGB1-based DNA immunization described here has the potential to enhance the immunogenicity of antigens and elicit stronger Th1-type Ab response.

Inefficient clearance of dying cells in patients with SLE: anti-dsDNA autoantibodies, MFG-E8, HMGB-1 and other players.

Systemic lupus erythematosus (SLE) is a complex disease resulting from inflammatory responses of the immune system against several autoantigens. Inflammation is conditioned by the continuous presence of autoantibodies and leaked autoantigens, e.g. from not properly cleared dying and dead cells. Various soluble molecules and biophysical properties of the surface of apoptotic cells play significant roles in the appropriate recognition and further processing of dying and dead cells. We exemplarily discuss how Milk fat globule epidermal growth factor 8 (MFG-E8), biophysical membrane alterations, High mobility group box 1 (HMGB1), C-reactive protein (CRP), and anti-nuclear autoantibodies may contribute to the etiopathogenesis of the disease. Up to date knowledge about these key elements may provide new insights that lead to the development of new treatment strategies of the disease.

HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

Innate immune mechanisms in ischemia/reperfusion.

Ischemia/reperfusion (I/R) injury remains a major problem in solid organ transplantation, as it adversely impacts both short and long term outcomes. It has been well established that the innate immune system plays a significant role in the pathogenesis of I/R injury. In contrast, the proximal molecular signaling events that initiate activation of the innate immune system are less clear. Recent findings have demonstrated that Toll-like receptors (TLR) play a role in I/R injury. Specifically, TLR4 is central to early activation of the innate immune response in the setting of I/R. Furthermore, recent evidence has shown that endogenous molecules, such as high mobility group box-1 (HMGB1) and others that are released from ischemic, damaged, or dying cells and tissues in the setting I/R, can serve as triggers for activation of the innate immune system and exacerbate tissue injury. This evolving body of literature, which has provided insight into the early molecular events that activate the innate system after I/R, is reviewed here.

Frequent and specific immunity to the embryonal stem cell-associated antigen SOX2 in patients with monoclonal gammopathy.

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A(*)0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

Frequency of SOX Group B (SOX1, 2, 3) and ZIC2 antibodies in Turkish patients with small cell lung carcinoma and their correlation with clinical parameters.

BACKGROUND: Expression of neuroectodermal markers is a key feature of small cell lung carcinoma (SCLC). Although immune responses against a number of these proteins have been associated with paraneoplastic neuronal disease (PND), most patients with SCLC have anti-neuroectodermal antibodies in the absence of PND. Whether these immune responses affect the clinical outcome in SCLC is critical in understanding the potential value of these proteins as cancer vaccine targets as well as in the pathogenesis of PND. METHODS: The authors investigated the frequency of immunoglobulin G autoantibodies against Sry-like high-mobility group box (SOX)1, 2, 3 and Zinc-finger gene of the cerebellum (ZIC)2 proteins in stored serum samples from 90 patients utilizing the lambda-phage plaque assay. Data obtained from patient records were utilized to measure clinical correlates of seroreactivity. RESULTS: Antibodies to SOX1 were present in 28% of patients and another 28% had anti-ZIC2 antibodies, classifying these as some of the most frequent antibody responses observed in SCLC. None had autoimmune paraneoplastic disease. Antibody titers were frequently as high as > or = 1:10(6) and were stable for < or = 6 months after diagnosis. Seroreactivity against either SOX1 or ZIC2 correlated with younger age, lower lactate dehydrogenase levels, and better response to initial therapy. CONCLUSIONS: The frequent and stable presence of SOX Group B and/or ZIC2 antibodies in SCLC, but not in healthy individuals examined, indicates they are serological markers of SCLC. However, the correlation between known clinical parameters of less aggressive disease and seroreactivity suggests that these antibodies are indicators of better prognosis in SCLC and warrants further studies to clarify the nature of the underlying immune responses.