Redundant role of PAD2 and PAD4 in the development of cardiovascular lesions in a mouse model of Kawasaki Disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. While circulating neutrophils are increased and activated during acute KD, it is unclear whether neutrophils and neutrophil extracellular traps (NETs) contribute to the pathogenesis of KD. Peptidylarginine deiminase 4 (PAD4), an enzyme involved in protein citrullination and essential for NETs formation, is implicated in the pathogenesis of various diseases. Here, we used the Lactobacillus casei cell wall extract (LCWE)-induced mouse model of KD vasculitis to determine the contribution of PAD4 in KD vasculitis. We found that the pan-PADs inhibitor, Cl-amidine, significantly reduced LCWE-induced cardiovascular lesions, but neutrophil-specific Padi4 KO mice did not impact development of KD vasculitis. While in vitro treatment of macrophages, which highly express Padi4, with Cl-amidine inhibited IL-1ßsecretion, macrophage-specific Padi4 KO mice did not reduce the lesions. Padi4-/- mice also developed KD vasculitis, AFM30a, a PAD2 inhibitor, significantly reduced KD vasculitis in Padi4-/- mice, indicating a compensatory role of PAD2 in PAD4 deficiency. We also identified several citrullinated proteins in macrophages with constitutively active NLRP3 inflammasome that were inhibited by Cl-amidine treatment, suggesting that protein citrullination participates in NLRP3 inflammasome activation. These data indicate a dispensable role for PAD4-dependent NETs formation, and a redundant role of PAD2 and PAD4 in this murine KD vasculitis. The cardioprotective effects of Cl-amidine to reduce the severity of murine KD vasculitis is not limited to PAD4 inhibition and may include decreased citrullination in the inflammasome pathway.

PD-L1 and VEGF dual blockade enhances anti-tumor effect on brain metastasis in hematogenous metastasis model.

Immunotherapy improves survival outcomes in cancer patients, but there is still an unmet clinical need in the treatment of brain metastases. Here, we used a mouse model to investigate the antitumor effect of programmed death-ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) dual blockade on metastatic brain tumors and evaluated immune responses during treatment. After establishing hematogenous brain metastasis by transplanting murine bladder carcinoma MBT2 cells stably expressing secNLuc reporter via the internal carotid artery of C3H/HeNCrl mice, we observed the formation of metastases not only in the brain parenchyma but also in the ventricles. The observed pathological areas showed that metastases in the ventricle were histologically larger than that in the brain parenchyma. Regarding the total tumor burden in the whole brain as revealed by Nluc activities, the combination of anti-PD-L1 antibody and anti-VEGF antibody showed a stronger anti-tumor effect than each single agent. Anti-PD-L1 antibody alone enhanced CD8+ T cell priming in regional lymph nodes, increased the proportion of activated CD8+ T cells in whole brain, and increased the density of CD8+ cells in the brain parenchyma. Furthermore, anti-VEGF antibody alone decreased microvessel density (MVD) in ventricular metastases, and the combination treatment increased intratumoral CD8+ cell density in the brain parenchyma and ventricular metastases. These results suggest that PD-L1 blockade enhanced cancer immunity not only in brain metastases lesions but also in the regional lymph nodes of the metastases, and that the addition of VEGF blockade increased the antitumor effect by increasing the infiltration of activated CD8+ T cell and decreasing MVD.

The intestinal microbiota contributes to the development of immune-mediated cardiovascular inflammation and vasculitis in mice.

Alterations in the intestinal microbiota contribute to the pathogenesis of various cardiovascular disorders, but how they affect the development of Kawasaki disease (KD), an acute pediatric vasculitis, remains unclear. We report that depleting the gut microbiota reduces the development of cardiovascular inflammation in a murine model mimicking KD vasculitis. The development of cardiovascular lesions was associated with alterations in the intestinal microbiota composition and, notably, a decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Oral supplementation with either of these live or pasteurized individual bacteria, or with short-chain fatty acids (SCFAs) produced by them, attenuated cardiovascular inflammation. Treatment with Amuc_1100, the TLR-2 signaling outer membrane protein from A. muciniphila , also decreased the severity of vascular inflammation. This study reveals an underappreciated gut microbiota-cardiovascular inflammation axis in KD vasculitis pathogenesis and identifies specific intestinal commensals that regulate vasculitis in mice by producing metabolites or via extracellular proteins acting on gut barrier function. IN BRIEF: It remains unclear whether changes in the intestinal microbiota composition are involved in the development of cardiovascular lesions associated with Kawasaki disease (KD), an immune-mediated vasculitis. Jena et al. observe alterations in the intestinal microbiota composition of mice developing vasculitis, characterized by reduced A. muciniphila and F. prausnitzii . Oral supplementation with either of these bacteria, live or pasteurized, or with bacteria-produced short-chain fatty acids (SCFAs) or Amuc_1100, the TLR-2 signaling outer membrane protein of A. muciniphila , was sufficient to alleviate the development of cardiovascular lesions in mice by promoting intestinal barrier function. HIGHLIGHTS: Absence or depletion of the microbiota decreases the severity of vasculitis in a murine model mimicking KD vasculitis. Supplementation of B. wadsworthia and B. fragilis promotes murine KD vasculitis. Decreased abundances of F. prausnitzii and A. muciniphila are associated with the development of cardiovascular lesions in mice. Supplementation with either live or pasteurized A. muciniphila and F. prausnitzii, or the TLR-2 signaling Amuc_1100, reduces the severity of vasculitis by promoting gut barrier function.

Dysfunction of sinus macrophages in tumor-bearing host induces resistance to immunotherapy.

Sinus macrophages in draining lymph nodes (DLNs) are involved in anti-tumor immune reactions. CD169 (Sialoadhesin, Siglec-1) is expressed on sinus macrophages and is considered a surrogate marker for the immunostimulatory phenotype of macrophages. In this study, the significance of sinus macrophages in immunotherapy was evaluated using mouse models. Treatment with anti-programmed death-ligand 1 (PD-L1) antibody suppressed the subcutaneous tumor growth of MC38 and E0771 cells but was not effective against MB49 and LLC tumors. Decreased cytotoxic T-lymphocyte (CTL) infiltration in tumor tissues and CD169 expression in sinus macrophages were observed in MB49 and LLC cells compared to corresponding parameters in MC38 and E0771 cells. The anti-tumor effects of the anti-PD-L1 antibody on MC38 and E0771 cells were abolished when sinus macrophages in DLNs were depleted, suggesting that sinus macrophages are involved in the therapeutic effect of the anti-PD-L1 antibody. Naringin activated sinus macrophages. Naringin inhibited tumor growth in MB49- and LLC-bearing mice but did not affect that in MC38- and E0771-bearing mice. The infiltration of CTLs in tumor tissues and their activation were increased by naringin, and this effect was impaired when sinus macrophages were depleted. Combination therapy with naringin and anti-PD-L1 antibody suppressed MB49 tumor growth. In conclusion, CD169-positive sinus macrophages in DLNs are critical for anti-tumor immune responses, and naringin suppresses tumor growth by activating CD169-positive sinus macrophages and anti-tumor CTL responses. The activation status of sinus macrophages has been suggested to differ among tumor models, and this should be investigated in future studies.

Effect of Bevacizumab on a Human Breast Cancer Model that Exhibited Palbociclib-resistance by RB Knockout.

BACKGROUND/AIM: Although CDK4/6 inhibitors have been increasingly used in combination with hormonal agents to treat hormone-receptor positive and human epithelial growth factor receptor 2-negative breast cancer, the mechanism of CDK4/6 inhibitor resistance and its impact on established therapy for post-resistance, especially bevacizumab combined with chemotherapy, are unclear. MATERIALS AND METHODS: Sensitivity of RB knockout MCF7 clones to CDK4/6 inhibitors was evaluated in vitro. One RB knockout clone was subcutaneously implanted in nude mice and the effects of bevacizumab on volume and microvessel density (MVD) of tumors were investigated. RESULTS: Palbociclib did not exhibit antitumor efficacy against the RB knockout tumor, in contrast to the parental MCF7 xenograft model. Bevacizumab significantly exhibited antitumor efficacy and suppressed the MVD both in RB knockout and parental MCF7 xenograft models. CONCLUSION: Bevacizumab inhibited tumor growth by suppressing MVD in the CDK4/6 inhibitor-resistant tumor acquired due to RB loss, suggesting its efficacy also in patients after treatment with CDK4/6 inhibitors.

PD-L1 blockade exhibits anti-tumor effect on brain metastasis by activating CD8+ T cells in hematogenous metastasis model with lymphocyte infusion.

Brain metastases are common complication in cancer patients. Immune checkpoint inhibitors show therapeutic benefits also in patients with central nervous system (CNS) metastases. However, their antitumor effects on metastatic tumors and their underlying mechanisms are still poorly understood. In this study we investigated the antitumor effect of anti-programmed death-ligand 1 (PD-L1) antibody on metastatic brain tumors and evaluated immune responses during treatment. We employed a hematogenous brain metastasis xenograft model using immunodeficient mice with murine lymphocyte infusions. A human non-small-cell lung cancer (NSCLC) cell line stably expressing NanoLuc® reporter (Nluc-H1915) was inoculated from the internal carotid artery of SCID mice. After metastases were established (24 days after inoculation), splenocytes prepared from H1915-immunized BALB/c mice were injected intravenously and mouse IgG or anti-PD-L1 antibody treatment was started (day 1). Evaluated by Nluc activity, tumor volume in the brain on day 14 was significantly lower in anti-PD-L1-treated mice than in mouse IgG-treated mice. Furthermore CD8+ cells were primarily infiltrated intratumorally and peritumorally and anti-PD-L1 treatment induced a significantly higher proportion of Granzyme B (GzmB)+ cells among CD8+ T cells. The antitumor effect of anti-PD-L1 antibody on brain metastasis is thought to be achieved by the enhanced activation of infiltrated CD8+ T cells into metastatic brain tumor. These results suggest that anti-PD-L1 antibody-containing regimens may be promising treatment options for cancer patients with brain metastases.

Loss of testosterone impairs anti-tumor neutrophil function.

In men, the incidence of melanoma rises rapidly after age 50, and nearly two thirds of melanoma deaths are male. The immune system is known to play a key role in controlling the growth and spread of malignancies, but whether age- and sex-dependent changes in immune cell function account for this effect remains unknown. Here, we show that in castrated male mice, neutrophil maturation and function are impaired, leading to elevated metastatic burden in two models of melanoma. Replacement of testosterone effectively normalized the tumor burden in castrated male mice. Further, the aberrant neutrophil phenotype was also observed in prostate cancer patients receiving androgen deprivation therapy, highlighting the evolutionary conservation and clinical relevance of the phenotype. Taken together, these results provide a better understanding of the role of androgen signaling in neutrophil function and the impact of this biology on immune control of malignancies.

Bevacizumab suppresses the growth of established non-small-cell lung cancer brain metastases in a hematogenous brain metastasis model.

Brain metastases are common in patients with non-small-cell lung cancer (NSCLC). The efficacy of bevacizumab, an anti-vascular endothelial growth factor (VEGF) humanized antibody, has been demonstrated in patients with nonsquamous NSCLC. We established a transplantable NSCLC cell line (Nluc-H1915) that stably expresses NanoLuc® reporter and confirmed the correlation between total Nluc activity in tumor and tumor volume in vivo. SCID mice inoculated with these cells through the internal carotid artery formed reproducible brain metastases, in which human VEGF was detected. Next, after metastases were established in the model mice (15-17 days), they were intraperitoneally administered weekly doses of human immunoglobulin G (HuIgG) or bevacizumab. Nluc activity in the brain was significantly lower in bevacizumab-treated mice than in HuIgG-treated mice. Additionally, bevacizumab concentration in the brain was higher in mice with brain metastasis than in normal mice, and bevacizumab was primarily observed in brain metastasis lesions. The microvessel density in brain metastasis was lower in bevacizumab-treated mice than in HuIgG-treated mice. We believe bevacizumab's anti-proliferative effect on brain metastasis is due to anti-angiogenic activity achieved by its penetration into brain metastases; this suggests that a bevacizumab-containing regimen may be a promising treatment option for patients with NSCLC brain metastasis.

Intestinal Permeability and IgA Provoke Immune Vasculitis Linked to Cardiovascular Inflammation.

Recent experimental data and clinical, genetic, and transcriptome evidence from patients converge to suggest a key role of interleukin-1ß (IL-1ß) in the pathogenesis of Kawasaki disease (KD). However, the molecular mechanisms involved in the development of cardiovascular lesions during KD vasculitis are still unknown. Here, we investigated intestinal barrier function in KD vasculitis and observed evidence of intestinal permeability and elevated circulating secretory immunoglobulin A (sIgA) in KD patients, as well as elevated sIgA and IgA deposition in vascular tissues in a mouse model of KD vasculitis. Targeting intestinal permeability corrected gut permeability, prevented IgA deposition and ameliorated cardiovascular pathology in the mouse model. Using genetic and pharmacologic inhibition of IL-1ß signaling, we demonstrate that IL-1ß lies upstream of disrupted intestinal barrier function, subsequent IgA vasculitis development, and cardiac inflammation. Targeting mucosal barrier dysfunction and the IL-1ß pathway may also be applicable to other IgA-related diseases, including IgA vasculitis and IgA nephropathy.

Cisplatin Augments Antitumor T-Cell Responses Leading to a Potent Therapeutic Effect in Combination With PD-L1 Blockade.

BACKGROUND: Immune checkpoint inhibitors have marked antitumor effect. However, monotherapy benefits only a limited population of patients, and further improvement of their effects is required. Here the therapeutic effect and immune response during anti-PD-L1 plus cisplatin combination therapy were investigated in a mouse model. MATERIALS AND METHODS: E.G7-OVA, expressing ovalbumin (OVA) gene as a model tumor antigen, was subcutaneously inoculated into syngeneic mice and treated with anti-PD-L1 with/without cisplatin. The tumor growth and activation status of immune cells were evaluated. RESULTS: The anti-PD-L1 plus cisplatin combination resulted in a potent antitumor effect leading to tumor shrinkage compared to anti-PD-L1 or cisplatin alone, even though each alone, significantly inhibited tumor growth compared to the control group. During treatment, all groups, including that treated with cisplatin alone, had increased CD8+ T-cell infiltration into tumor tissues compared with the control group, and the therapeutic effect was diminished by CD8+ cell depletion. Aside from its direct cytotoxic effect, cisplatin alone increased chemokine levels and expression of immune checkpoint molecules on CD8+ T-cells in the tumor site. The combination effectively activated OVA-specific CD8+ T-cells. Furthermore, anti-PD-L1 alone and in combination with cisplatin, but not cisplatin alone, induced interferon-gamma-producing CD4+ T-cells. CONCLUSION: These findings provide a rationale for anti-PD-L1 plus cisplatin becoming a promising combination therapy for patients with cancer.

T-Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation.

Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.

Topoisomerase I inhibitor, irinotecan, depletes regulatory T cells and up-regulates MHC class I and PD-L1 expression, resulting in a supra-additive antitumor effect when combined with anti-PD-L1 antibodies.

Anti-PD-L1 antibodies inhibit interactions between PD-L1 and PD-1 and interactions between PD-L1 and B7-1, thereby reinvigorating anticancer immunity. Although there are numerous ongoing clinical studies evaluating combinations of standard chemotherapies and anti-PD-L1 antibodies, irinotecan has not yet been investigated in this context so there is little information about its compatibility with anti-PD-L1 antibodies. Here we investigated the efficacy of anti-PD-L1 antibody in combination with irinotecan and the role of irinotecan in the tumor-immunity cycle in an FM3A murine tumor model. Despite a transient decrease in lymphocytes in the peripheral blood after irinotecan treatment, the antitumor activity of anti-PD-L1 antibody plus irinotecan was significantly greater than each agent alone. Irinotecan in combination with anti-PD-L1 antibody enhanced proliferation of CD8+ cells in both tumors and lymph nodes, and the number of tumor-infiltrating CD8+ cells was higher than either irinotecan or anti-PD-L1 antibody monotherapy. Irinotecan was found to decrease the number of Tregs in lymph nodes and tumors, and specific depletion of Tregs by anti-folate receptor 4 antibodies was found to enhance the proliferation of CD8+ cells in this model. In addition, irinotecan augmented MHC class I expression on tumor cells and concurrently increased PD-L1 expression on tumor cells and tumor-infiltrating immune cells. These results indicate that irinotecan may enhance the effect of T cell activation caused by anti-PD-L1 treatment by reducing Tregs and augmenting MHC class I-mediated tumor antigen presentation, and concurrent upregulation of PD-L1 expression can be blocked by the anti-PD-L1 antibody. These interactions may contribute to the superior combination effect.

Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis.

Interleukin-1ß (IL-1ß) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1ß requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1ß secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC's effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1ß in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

OBJECTIVE: Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. METHODS: We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. RESULTS: LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. CONCLUSION: The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans.

Role of Interleukin-1 Signaling in a Mouse Model of Kawasaki Disease-Associated Abdominal Aortic Aneurysm.

OBJECTIVE: Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model. METHODS AND RESULTS: We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1ß mAb blocked LCWE-induced AAA formation. CONCLUSIONS: Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1ß play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse Model: Role of Both IL-1α and IL-1ß.

OBJECTIVE: Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1ß signaling are required for the Lactobacillus casei cell wall extract-induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c(+) dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract-induced KD mouse model of vasculitis. APPROACH AND RESULTS: Using mouse knockout models and antibody depletion, we found that both IL-1α and IL-1ß were required for Lactobacillus casei cell wall extract-induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. CONCLUSIONS: Our results suggest that IL-1α and IL-1ß, as well as CD11c(+) dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1α and IL-1ß and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): preparation and immunological analysis of vaccine efficacy.

To elucidate the immunologic mechanisms of artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which indicated a great vaccine efficacy in human cancers, we prepared ovalbumin (OVA)-H/K-HELP by conjugating killer and helper epitopes of OVA-model tumor antigen via a glycine-linker. Vaccination of C57BL/6 mice with OVA-H/K-HELP (30 amino acids) but not with short peptides mixture of class I-binding peptide (8 amino-acids) and class II-binding peptide (17 amino-acids) combined with adjuvant CpG-ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotides), induced higher numbers of OVA-tetramer-positive CTL with concomitant activation of IFN-γ-producing CD4(+) Th1 cells. However, replacement of glycine-linker of OVA-H/K-HELP with other peptide-linker caused a significant decrease of vaccine efficacy of OVA-H/K-HELP. In combination with adjuvant CpG-ODN, OVA-H/KHELP exhibited greater vaccine efficacy compared with short peptides vaccine, in both preventive and therapeutic vaccine models against OVA-expressing EG-7 tumor. The elevated vaccine efficacy of OVAH/K-HELP might be derived from the following mechanisms: (i) selective presentation by only professional dendritic cells (DC) in vaccinated draining lymph node (dLN); (ii) a long-term sustained antigen presentation exerted by DC to stimulate both CTL and Th1 cells; (iii) formation of three cells interaction among DC, Th and CTL. In comparative study, H/K-HELP indicated stronger therapeutic vaccine efficacy compared with that of extended class I synthetic long peptide, indicating that both the length of peptide and the presence of Th epitope peptide were crucial aspects for preparing artificially synthesized H/K-HELP vaccine.

The key role of IL-6-arginase cascade for inducing dendritic cell-dependent CD4(+) T cell dysfunction in tumor-bearing mice.

Evaluation of immune dysfunction during the tumor-bearing state is a critical issue in combating cancer. In this study, we initially found that IL-6, one of the cachectic factors, suppressed CD4(+) T cell-mediated immunity through downregulation of MHC class II by enhanced arginase activity of dendritic cells (DC) in tumor-bearing mice. We demonstrated that administration of Ab against IL-6R (anti-IL-6R mAb) greatly enhanced T cell responses and inhibited the growth of tumor in vivo. We also found that IL-6 upregulated the expression of arginase-1 and arginase activity of DC in vitro. Tumor-infiltrating CD11c(+) DC exhibited upregulated mRNA expression of arginase-1 but reduced expression of MHC class II in parallel with the increase in serum IL-6 levels at the late stage in tumor-bearing hosts. However, the administration of anti-IL-6R mAb into tumor-bearing mice inhibited both the downmodulation of MHC class II and the upregulation of arginase-1 mRNA levels in DC. Furthermore, we noted that N(ω)-hydroxy-L-arginine or L-arginine, an arginase-1 inhibitor, blocked the reduction in MHC class II levels on CD11c(+) DC during the tumor-bearing state. Finally, we demonstrated that the administration of N(ω)-hydroxy-L-arginine at the peritumor site significantly enhanced CD4(+) T cell responses and inhibited tumor growth. Thus, IL-6-mediated arginase activation and the subsequent reduction in MHC class II expression on DC appeared to be critical mechanisms for inducing dysfunction of the immune system in the tumor-bearing state. Blockade of the IL-6-arginase cascade is a promising tool to overcome the dysfunction of antitumor immunity in tumor-bearing hosts.

The development of IL-17/IFN-γ-double producing CTLs from Tc17 cells is driven by epigenetic suppression of Socs3 gene promoter.

The plasticity of T lymphocytes induced by epigenetic modifications of gene promoters may play a pivotal role in controlling their effector functions, which are sometimes causally associated with immune disorders. IL -17-producing T cells, which induce type 17 immune responses, are newly identified pathogenic effector cells. The type 1 signature cytokine IFN-γ strongly inhibits their differentiation, indicating a mutually exclusive relationship between type 17- and type 1-immune responses. However, many reports indicate the presence of a unique IL-17/IFN-γ-double producing T-cell subset in various inflammatory settings, although the mechanisms responsible for their development and their precise functions remain unclear. Here, we demonstrate that IL-12 permits the conversion of mouse IL-17-producing CD8(+) T (Tc17) cells to IL-17/IFN-γ-double producing CD8(+) T (Tc17/IFN-γ) cells, and that this conversion is due to repressive epigenetic modifications of Socs3 gene promoters. Moreover, we show that SOCS3 strongly regulates the capability of Tc17 cells to produce IL-17, in addition to regulating the expression of the type 17-master regulator RORγt. These findings elucidate the mechanisms underlying the conversion of Tc17 cells into Tc17/IFN-γ cells. As these cells are known to have potent antitumor activities, manipulation of these conversion mechanisms for therapeutic tumor immunity may be possible.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.