Unique characteristics of lung-resident neutrophils are maintained by PGE2/PKA/Tgm2-mediated signaling.

Lung-resident neutrophils need to be tightly regulated to avoid degranulation- and cytokine-associated damage to fragile alveolar structures that can lead to fatal outcomes. Here we show that lung neutrophils (LNs) express distinct surface proteins and genes that distinguish LNs from bone marrow and blood neutrophils. Functionally, LNs show impaired migratory activity toward chemoattractants and produce high levels of interleukin-6 (IL-6) at steady state and low levels of tumor necrosis factor-α in response to lipopolysaccharide (LPS) challenge. Treating bone marrow neutrophils with bronchoalveolar lavage fluid or prostaglandin E2 induces LN-associated characteristics, including the expression of transglutaminase 2 (Tgm2) and reduced production of inflammatory cytokines upon LPS challenge. Neutrophils from Tgm2-/- mice release high levels of inflammatory cytokines in response to LPS. Lung damage is significantly exacerbated in Tgm2-/- mice in an LPS-induced acute respiratory distress syndrome model. Collectively, we demonstrate that prostaglandin E2 is a key factor for the generation of LNs with unique immune suppressive characteristics, acting through protein kinase A and Tgm2, and LNs play essential roles in protection of the lungs against pathogenic inflammation.

A unique population of neutrophils generated by air pollutant-induced lung damage exacerbates airway inflammation.

BACKGROUND: Diesel exhaust particles (DEPs) are the main component of traffic-related air pollution and have been implicated in the pathogenesis and exacerbation of asthma. However, the mechanism by which DEP exposure aggravates asthma symptoms remains unclear. OBJECTIVE: This study aimed to identify a key cellular player of air pollutant-induced asthma exacerbation and development. METHODS: We examined the distribution of innate immune cells in the murine models of asthma induced by house dust mite and DEP. Changes in immune cell profiles caused by DEP exposure were confirmed by flow cytometry and RNA-Seq analysis. The roles of sialic acid-binding, Ig-like lectin F (SiglecF)-positive neutrophils were further evaluated by adoptive transfer experiment and in vitro functional studies. RESULTS: DEP exposure induced a unique population of lung granulocytes that coexpressed Ly6G and SiglecF. These cells differed phenotypically, morphologically, functionally, and transcriptionally from other SiglecF-expressing cells in the lungs. Our findings with murine models suggest that intratracheal challenge with DEPs induces the local release of adenosine triphosphate, which is a damage-associated molecular pattern signal. Adenosine triphosphate promotes the expression of SiglecF on neutrophils, and these SiglecF+ neutrophils worsen type 2 and 3 airway inflammation by producing high levels of cysteinyl leukotrienes and neutrophil extracellular traps. We also found Siglec8- (which corresponds to murine SiglecF) expressing neutrophils, and we found it in patients with asthma-chronic obstructive pulmonary disease overlap. CONCLUSION: The SiglecF+ neutrophil is a novel and critical player in airway inflammation and targeting this population could reverse or ameliorate asthma.

A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis.

Formyl peptide receptors (FPRs) are G protein-coupled receptors mainly expressed in inflammatory myeloid cells. Previous reports demonstrated that human neutrophils express only FPR1 and FPR2 but not FPR3. Here, we found that FPR3 is expressed in sepsis patient derived neutrophils and Fpr3 is expressed in the mouse neutrophils. To test the role of Fpr3 in neutrophil activity, we synthesized Fpr3 pepducins and successfully developed an agonistic pepducin that stimulates Fpr3, eliciting calcium increase and chemotactic migration of neutrophils. We also found that administration of an Fpr3 pepducin in an experimental mouse sepsis model significantly increased the survival rate. The pepducin markedly inhibited lung injury, splenocyte apoptosis, and inflammatory cytokine production. Bacterial counts were significantly decreased by the pepducin in septic mice. Based on these results, we suggest that FPR3 can be regarded as a new target to control sepsis, and the newly generated Fpr3-based pepducin can be used for the development of anti-septic agents.

Dendritic Cell-Mediated Th2 Immunity and Immune Disorders.

Dendritic cells (DCs) are the professional antigen-presenting cells that recognize and present antigens to naïve T cells to induce antigen-specific adaptive immunity. Among the T-cell subsets, T helper type 2 (Th2) cells produce the humoral immune responses required for protection against helminthic disease by activating B cells. DCs induce a Th2 immune response at a certain immune environment. Basophil, eosinophil, mast cells, and type 2 innate lymphoid cells also induce Th2 immunity. However, in the case of DCs, controversy remains regarding which subsets of DCs induce Th2 immunity, which genes in DCs are directly or indirectly involved in inducing Th2 immunity, and the detailed mechanisms underlying induction, regulation, or maintenance of the DC-mediated Th2 immunity against allergic environments and parasite infection. A recent study has shown that a genetic defect in DCs causes an enhanced Th2 immunity leading to severe atopic dermatitis. We summarize the Th2 immune-inducing DC subsets, the genetic and environmental factors involved in DC-mediated Th2 immunity, and current therapeutic approaches for Th2-mediated immune disorders. This review is to provide an improved understanding of DC-mediated Th2 immunity and Th1/Th2 immune balancing, leading to control over their adverse consequences.

p53-derived host restriction of HIV-1 replication by protein kinase R-mediated Tat phosphorylation and inactivation.

UNLABELLED: Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53(-/-) and eIF2α(CA) cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat-Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCE: HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.

Highly activated p53 contributes to selectively increased apoptosis of latently HIV-1 infected cells upon treatment of anticancer drugs.

BACKGROUND: Despite the successful inhibition of human immunodeficiency virus type 1 (HIV-1) replication by combination antiretroviral therapy, cells latently infected with HIV-1 remaining in patients are a major obstacle for eradication of HIV-1 infection. The tumor suppressor factor p53 is activated by HIV-1 infection, and restricts HIV-1 replication. However, a therapeutic strategy based on p53 activity has not been considered for elimination of latently infected cells. METHODS: Apoptotic cells were analyzed using flow cytometry with anti-annexin A5-FITC Ab and PI staining upon treatment of anticancer drugs. The expression and activation of p53 and apoptotic molecules in latently HIV-1-infected T cells were compared using Western blot analysis. The role of p53 in the anticancer drug treatment-induced apoptosis of cells latently infected with HIV-1 was determined by knock-down experiment using siRNA against p53. RESULTS: Upon treatment with 5-fluorouracil (5-FU), apoptosis was increased in latently infected ACH2 cells encoding competent p53 compared with uninfected parent A3.01 cells, while the apoptosis of latently infected p53 null J1.1 cells was less than that of uninfected cells. Treatment with 5-FU increased the levels of cleaved caspase-3 and PARP in ACH2 cells compared with uninfected and latently infected p53 null J1.1 cells. The levels of expression and activation of p53 were higher in both latently infected ACH2 and NCHA2 cells than in uninfected cells. Furthermore, the activation levels of p53 in both cells were further increased upon 5-FU treatment. Consistent with p53 status, apoptosis was markedly increased in ACH2 and NCHA2 cells compared with uninfected and latently infected J1.1 cells upon treatment with other anticancer drugs such as doxorubicin and etoposide. Inhibition of p53 in cells with latent HIV-1 infection diminished apoptosis upon 5-FU treatment. CONCLUSION: Evidence described here indicate that when treated with anticancer drugs, apoptosis of cells with latent HIV-1 infection was increased via the p53 activation pathway and may provide information for application of anticancer drugs to selectively eliminate HIV-1 reservoirs.

A phase I/IIa study of adjuvant immunotherapy with tumour antigen-pulsed dendritic cells in patients with hepatocellular carcinoma.

BACKGROUND: To date, no adjuvant treatment has been shown to have a clear benefit in patients with hepatocellular carcinoma (HCC). In this prospective phase I/IIa study, we evaluated the safety and efficacy of adjuvant dendritic cell (DC) therapy in HCC patients who received primary treatment for HCC. METHODS: Twelve HCC patients who had no viable tumour after primary treatments were included. Dendritic cell vaccines pulsed with cytoplasmic transduction peptide-attached alpha-fetoprotein, glypican-3 and melanoma-associated antigen 1 recombinant fusion proteins were injected subcutaneously near to inguinal lymph nodes. Adverse effects, time to progression (TTP), and associated immune responses were evaluated after DC vaccination. RESULTS: Nine of 12 patients had no tumour recurrence up to 24 weeks after DC vaccination. Among a total of 144 adverse events, 129 events (89.6%) were regarded as adverse drug reactions, all of which were grade 1 or 2. The majority of patients showed enhanced anti-tumour immune responses after DC vaccination. Recurrence-free patients exhibited relatively stronger anti-tumour immune responses than patients who developed recurrence after DC vaccination, as evidenced by lymphocyte proliferation and IFN-γ ELISPOT assays. The median time of TTP was 36.6 months in the DC-vaccination group and 11.8 months in the control group (hazard ratio, 0.41; 95% confidence interval, 0.18-0.95; P=0.0031 by log-rank test). CONCLUSIONS: Adjuvant DC vaccine for HCC was safe and well tolerated in phase I/IIa study, and preliminary efficacy data are encouraging to warrant further clinical study in patients with HCC after primary treatments.

Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity.

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.

Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes.

HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes.

Adenovirus expressing both thymidine kinase and soluble PD1 enhances antitumor immunity by strengthening CD8 T-cell response.

Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.

IL-33 and IL-33-derived DC-based tumor immunotherapy.

Interleukin-33 (IL-33), a member of the IL-1 family, is a cytokine released in response to tissue damage and is recognized as an alarmin. The multifaceted roles of IL-33 in tumor progression have sparked controversy within the scientific community. However, most findings generally indicate that endogenous IL-33 has a protumor effect, while exogenous IL-33 often has an antitumor effect in most cases. This review covers the general characteristics of IL-33 and its effects on tumor growth, with detailed information on the immunological mechanisms associated with dendritic cells (DCs). Notably, DCs possess the capability to uptake, process, and present antigens to CD8+ T cells, positioning them as professional antigen-presenting cells. Recent findings from our research highlight the direct association between the tumor-suppressive effects of exogenous IL-33 and a novel subset of highly immunogenic cDC1s. Exogenous IL-33 induces the development of these highly immunogenic cDC1s through the activation of other ST2+ immune cells both in vivo and in vitro. Recognizing the pivotal role of the immunogenicity of DC vaccines in DC-based tumor immunotherapy, we propose compelling methods to enhance this immunogenicity through the addition of IL-33 and the promotion of highly immunogenic DC generation.

The protective roles of integrin α4ß7 and Amphiregulin-expressing innate lymphoid cells in lupus nephritis.

Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.

Bioconjugated Antibody-Trojan Immune Converter Enhance Cancer Immunotherapy with Minimized Toxicity by Programmed Two-Step Immunomodulation of Myeloid Cells.

Current immune checkpoint blockade therapy (ICBT) predominantly targets T cells to harness the antitumor effects of adaptive immune system. However, the effectiveness of ICBT is reduced by immunosuppressive innate myeloid cells in tumor microenvironments (TMEs). Toll-like receptor 7/8 agonists (TLR7/8a) are often used to address this problem because they can reprogram myeloid-derived suppressor cells (MDSCs) and tumor-associated M2 macrophages, and boost dendritic cell (DC)-based T-cell generation; however, the systemic toxicity of TLR7/8a limits its clinical translation. Here, to address this limitation and utilize the effectiveness of TLR7/8a, this work suggests a programmed two-step activation strategy via Antibody-Trojan Immune Converter Conjugates (ATICC) that specifically targets myeloid cells by anti-SIRPα followed by reactivation of transiently inactivated Trojan TLR7/8a after antibody-mediated endocytosis. ATICC blocks the CD47-SIRPα ("don't eat me" signal), enhances phagocytosis, reprograms M2 macrophages and MDSCs, and increases cross-presentation by DCs, resulting in antigen-specific CD8+ T-cell generation in tumor-draining lymph nodes and TME while minimizing systemic toxicity. The local or systemic administration of ATICC improves ICBT responsiveness through reprogramming of the immunosuppressive TME, increased infiltration of antigen-specific CD8+ T cells, and antibody-dependent cellular phagocytosis. These results highlight the programmed and target immunomodulation via ATICC could enhance cancer immunotherapy with minimized systemic toxicities.

Tacrolimus-loaded chitosan-based nanoparticles as an efficient topical therapeutic for the effective treatment of atopic dermatitis symptoms.

Atopic dermatitis (AD) is a chronic cutaneous disease with a complex underlying mechanism, and it cannot be completely cured. Thus, most treatment strategies for AD aim at relieving the symptoms. Although corticosteroids are topically applied to alleviate AD, adverse side effects frequently lead to the withdrawal of AD therapy. Tacrolimus (TAC), a calcineurin inhibitor, has been used to treat AD, but its high molecular weight and insolubility in water hinder its skin permeability. Herein, we developed and optimized TAC-loaded chitosan-based nanoparticles (TAC@CNPs) to improve the skin permeability of TAC by breaking the tight junctions in the skin. The prepared nanoparticles were highly loadable and efficient and exhibited appropriate characteristics for percutaneous drug delivery. TAC@CNP was stable for 4 weeks under physiological conditions. CNP released TAC in a controlled manner, with enhanced skin penetration observed. In vitro experiments showed that CNP was non-toxic to keratinocyte (HaCaT) cells, and TAC@CNP dispersed in an aqueous solution was as anti-proliferative as TAC solubilized in a good organic solvent. Importantly, an in vivo AD mouse model revealed that topical TAC@CNP containing ~1/10 of the dose of TAC found in commercially used Protopic® Ointment exhibited similar anti-inflammatory activity to that of the commercial product. TAC@CNP represents a potential therapeutic strategy for the management of AD.

CISH is induced during DC development and regulates DC-mediated CTL activation.

The cytokine inducible SH2-domain protein (CISH) is a well-known STAT5 target gene, but its role in the immune system remains uncertain. In this study, we found that CISH is predominantly induced during dendritic cell (DC) development from mouse bone marrow (BM) cells and plays a crucial role in type 1 DC development and DC-mediated CTL activation. CISH knockdown reduced the expression of MHC class I, co-stimulatory molecules and pro-inflammatory cytokines in BMDCs. Meanwhile, the DC yield was markedly enhanced by CISH knockdown via cell-cycle activation and reduction of cell apoptosis. Down-regulation of cell proliferation at the later stage of DC development was found to be associated with CISH-mediated negative feedback regulation of STAT5 activation. In T-cell immunity, OT-1 T-cell proliferation was significantly reduced by CISH knockdown in DCs, whereas OT-2 T-cell proliferation was not affected by CISH knockdown. CTLs generated by DC vaccination were also markedly reduced by CISH knockdown, followed by significant impairment of DC-based tumor immunotherapy. Taken together, our data suggest that CISH expression at the later stage of DC development triggers the shutdown of DC progenitor cell proliferation and facilitates DC differentiation into a potent stimulator of CTLs.

JAK3 inhibitor suppresses multipotent ILC2s and attenuates steroid-resistant asthma.

Steroids are the standard treatment for allergic airway inflammation in asthma, but steroid-refractory asthma poses a challenge. Group 2 innate lymphoid cells (ILC2s), such as T helper 2 (TH2) cells, produce key asthma-related type 2 cytokines. Recent insights from mouse and human studies indicate a potential connection between ILC2s and steroid-resistant asthma. Here, we highlight that lung ILC2s, rather than TH2 cells, can develop steroid resistance, allowing them to persist and maintain their disease-driving activity even during steroid treatment. The emergence of multipotent IL-5+IL-13+IL-17A+ ILC2s is associated with steroid-resistant ILC2s. The Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT) 3, 5, and 6 pathways contribute to the acquisition of steroid-resistant ILC2s. The JAK3 inhibitor reduces ILC2 survival, proliferation, and cytokine production in vitro and ameliorates ILC2-driven Alternaria-induced asthma. Furthermore, combining a JAK3 inhibitor with steroids results in the inhibition of steroid-resistant asthma. These findings suggest a potential therapeutic approach for addressing this challenging condition in chronic asthma.

Discovery of highly immunogenic spleen-resident FCGR3+CD103+ cDC1s differentiated by IL-33-primed ST2+ basophils.

Recombinant interleukin-33 (IL-33) inhibits tumor growth, but the detailed immunological mechanism is still unknown. IL-33-mediated tumor suppression did not occur in Batf3-/- mice, indicating that conventional type 1 dendritic cells (cDC1s) play a key role in IL-33-mediated antitumor immunity. A population of CD103+ cDC1s, which were barely detectable in the spleens of normal mice, increased significantly in the spleens of IL-33-treated mice. The newly emerged splenic CD103+ cDC1s were distinct from conventional splenic cDC1s based on their spleen residency, robust effector T-cell priming ability, and surface expression of FCGR3. DCs and DC precursors did not express Suppressor of Tumorigenicity 2 (ST2). However, recombinant IL-33 induced spleen-resident FCGR3+CD103+ cDC1s, which were found to be differentiated from DC precursors by bystander ST2+ immune cells. Through immune cell fractionation and depletion assays, we found that IL-33-primed ST2+ basophils play a crucial role in the development of FCGR3+CD103+ cDC1s by secreting IL-33-driven extrinsic factors. Recombinant GM-CSF also induced the population of CD103+ cDC1s, but the population neither expressed FCGR3 nor induced any discernable antitumor immunity. The population of FCGR3+CD103+ cDC1s was also generated in vitro culture of Flt3L-mediated bone marrow-derived DCs (FL-BMDCs) when IL-33 was added in a pre-DC stage of culture. FL-BMDCs generated in the presence of IL-33 (FL-33-DCs) offered more potent tumor immunotherapy than control Flt3L-BMDCs (FL-DCs). Human monocyte-derived DCs were also more immunogenic when exposed to IL-33-induced factors. Our findings suggest that recombinant IL-33 or an IL-33-mediated DC vaccine could be an attractive protocol for better tumor immunotherapy.

Sphingosylphosphorylcholine inhibits plasma cell differentiation and ameliorates experimental autoimmune encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a potentially disabling disease that damages the brain and spinal cord, inducing paralysis of the body. While MS has been known as a T-cell mediated disease, recent attention has been drawn to the involvement of B cells in its pathogenesis. Autoantibodies from B cells are closely related with the damage lesion of central nervous system and worse prognosis. Therefore, regulating the activity of antibody secreting cell could be related with the severity of the MS symptoms. Methods: Total mouse B cells were stimulated with LPS to induce their differentiation into plasma cells. The differentiation of plasma cells was subsequently analyzed using flow cytometry and quantitative PCR analysis. To establish an experimental autoimmune encephalomyelitis (EAE) mouse model, mice were immunized with MOG35-55/CFA emulsion. Results: In this study, we found that plasma cell differentiation was accompanied by upregulation of autotaxin, which converts sphingosylphosphorylcholine (SPC) to sphingosine 1-phosphate in response to LPS. We observed that SPC strongly blocked plasma cell differentiation from B cells and antibody production in vitro. SPC downregulated LPS-stimulated IRF4 and Blimp 1, which are required for the generation of plasma cells. SPC-induced inhibitory effects on plasma cell differentiation were specifically blocked by VPC23019 (S1PR1/3 antagonist) or TY52159 (S1PR3 antagonist), but not by W146 (S1PR1 antagonist) and JTE013 (S1PR2 antagonist), suggesting a crucial role of S1PR3 but not S1PR1/2 in the process. Administration of SPC against an EAE mouse model significantly attenuated the symptoms of disease, showing decreased demyelinated areas of the spinal cord and decreased numbers of cells infiltrated into the spinal cord. SPC markedly decreased plasma cell generation in the EAE model, and SPC-induced therapeutic effects against EAE were not observed in µMT mice. Conclusion: Collectively, we demonstrate that SPC strongly inhibits plasma cell differentiation, which is mediated by S1PR3. SPC also elicits therapeutic outcomes against EAE, an experimental model of MS, suggesting SPC as a new material to control MS.

A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion.

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.