Th2 Cytokine Production Research Articles

Bias of the Immune Response to Pneumocystis murina Does Not Alter the Ability of Neonatal Mice to Clear the Infection.

Newborn mice are unable to clear Pneumocystis (PC) infection with the same efficiency as adults due, in part, to their inability to develop a robust immune response to infection until three weeks of age. It is known that infants tend develop a Th2 skewed response to antigen so we sought to determine whether a biased cytokine response altered the clearance of PC infection in neonatal mice. P. murina infection in neonatal mice resulted in increased IL-4 expression by CD4 T cells and myeloid cells, augmented IL-13 secretion within the airways and increased arginase activity in the airways, indicative of Th2-type responses. P. murina-infected IL-4Rα−/− neonates had a shift towards Th1 cytokine production and increased numbers of CD4 and CD8 T cells within the lung as well as elevated levels of P. murina-specific IgG. IFNγ−/− and IL-23 p19−/− mice had altered CD4-T cell-dependent cytokine and cell responses. Though we could alter the T helper cell environment in neonatal knockout mice, there was no loss in the ability of these pups to clear infection. It is possible that the Th2 phenotype normally seen in neonatal mice protects the developing lung from pro-inflammatory immune responses without compromising host defense against P. murina.

Ding Chuan Tang Attenuates Airway Inflammation and Eosinophil Infiltration in Ovalbumin-Sensitized Asthmatic Mice.

Asthma is a T helper 2 (Th2) cell-associated chronic inflammatory diseases characterized with airway obstruction, increased mucus production, and eosinophil infiltration. Conventional medications for asthma treatment cannot fully control the symptoms, and potential side effects are also the concerns. Thus, complement or alternative medicine (CAM) became a new option for asthma management. Ding Chuan Tang (DCT) is a traditional Chinese herbal decoction applied mainly for patients with coughing, wheezing, chest tightness, and asthma. Previously, DCT has been proved to improve children airway hyperresponsiveness (AHR) in a randomized and double-blind clinical trial. However, the mechanisms of how DCT alleviates AHR remain unclear. Since asthmatic features such as eosinophil infiltration, IgE production, and mucus accumulation are relative with Th2 responses, we hypothesized that DCT may attenuate asthma symptoms through regulating Th2 cells. Ovalbumin (OVA) was used as a stimulant to sensitize BALB/c mice to establish an asthmatic model. AHR was detected one day before sacrifice. BALF and serum were collected for immune cell counting and antibody analysis. Splenocytes were cultured with OVA in order to determine Th2 cytokine production. Lung tissues were collected for histological and gene expression analyses. Our data reveal that DCT can attenuate AHR and eosinophil accumulation in the 30-day sensitization asthmatic model. Histological results demonstrated that DCT can reduce cell infiltration and mucus production in peribronchial and perivascular site. In OVA-stimulated splenocyte cultures, a significant reduction of IL-5 and IL-13 in DCT-treated mice suggests that DCT may alleviate Th2 responses. In conclusion, the current study demonstrates that DCT has the potential to suppress allergic responses through the reduction of mucus production, eosinophil infiltration, and Th2 activity in asthma.

The STE20 kinase TAOK3 controls the development of house dust mite–induced asthma in mice

The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an invivo mouse model. Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by invivo and invitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.

IL-37 regulates allergic inflammation by counterbalancing pro-inflammatory IL-1 and IL-33.

Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation. IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1β, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1β, IL-33, and IL-37levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy. IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37led to differential expression of >90genes induced by IL-1β and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1β- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1β and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls. IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1β and IL-33.

The extract of black cumin,\xa0licorice, anise, and black tea alleviates OVA-induced allergic rhinitis in mouse via balancing activity of helper T cells in lung

BackgroundA formulation of black cumin (Nigella sativa L.), licorice (Glycyrrhiza glabra L.), anise (Pimpinella anisum L.) and tea (Camellia sinensis (L.) Kuntze) (denoted BLAB tea) is traditionally used to relief allergy reaction including allergic rhinitis. However, little is known about its underlining mechanism of anti-allergic effects.MethodsTo investigate the anti-allergenic mechanism of BLAB tea, we treated ovalbumin (OVA)-induced allergic rhinitis (AR) model of mice with BLAB tea, and elucidated its possible mechanism of action. Mice in the control group were treated with phosphate-buffered saline only. Subsequently, the infiltration of different inflammatory cells was measured. In addition, histopathological changes in the nasal mucosa, and the levels of allergen-specific cytokines and OVA-specific immunoglobulins were measured.ResultsThe aqueous extract of BLAB significantly alleviated the nasal symptoms and reduced the accumulation of inflammatory cells in the nasal mucosa and nasal lavage fluid of AR model of mice.ConclusionThe aqueous extract of BLAB induced the production of Th1 and Treg cytokines and inhibited the release of Th2 cytokines and histamine in nasal mucosa and serum of mice while decreasing the serum levels of OVA-specific IgE, IgG1, and IgG2a. These results suggest the potential of the aqueous extract of BLAB as a treatment option for allergic diseases.

Monophosphoryl lipid A directly regulates Th1 cytokine production in human CD4+ T-cells through Toll-like receptor 2 and 4

BackgroundThe monophosphoryl lipid A (MPLA) is a detoxified LPS derivative and an emerging safe immune adjuvant in human vaccine development. The adjuvant MPLA promotes antigen-presenting cell (APC) function and preferentially induces a Th1 response following vaccination. However, the mechanism by which the MPLA detoxicates and exerts its adjuvants effect on T-cell, particualrly the Th1 response is unknown. AimsWe assessed the direct effects of MPLA on murine and human CD4+ T-cell proliferation and the profile of cytokine production ex vivo. ResultsWe report that CD4+ T-cells only express functional TLR2 and TLR4 when activated by TCR stimulation, in particularly in the presence of IFNα. The activated T cells thereafter can respond directly to MPLA. MPLA does not affect T-cell proliferation in human T cells, but can induce a balanced Th1 cytokine profile in CD4+ T-cells by reducing the production of Th1 cytokines and enhancing the production of the regulatory cytokine IL-10. The MPLA-mediated regulatory effect on activated CD4+ T-cells is TLR2 and TLR4 dependent and can be abolished by the lipid A blocker polymyxin B. ConclusionThese data provide evidence, at least in part, for the safe induction of an appropriate level of Th1 response by adjuvant MPLA in human vaccine development.

Gekko gecko extract attenuates airway inflammation and mucus hypersecretion in a murine model of ovalbumin-induced asthma

Ethnopharmacological relevanceGekko gecko is used as a traditional medicine for various diseases including respiratory disorders in northeast Asian countries, mainly Korea, Japan, and China. Aim of the studyAllergic asthma is a chronic respiratory disease caused by an inappropriate immune response. Due to the recent spread of coronavirus disease 2019, interest in the treatment of pulmonary disorders has rapidly increased. In this study, we investigated the anti-asthmatic effects of G. gecko extract (GGE) using an established mouse model of ovalbumin-induced asthma. Materials and methodsTo evaluate the anti-asthmatic effects of GGE, we evaluated histological changes and the responses of inflammatory mediators related to allergic airway inflammation. Furthermore, we investigated the regulatory effects of GGE on type 2 helper T (Th2) cell activation. ResultsAdministration of GGE attenuated asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and expression of Th2 cytokines. Furthermore, GGE treatment reduced Th2 cell activation and differentiation. ConclusionsThese results indicate that GGE alleviates allergic airway inflammation by regulating Th2 cell activation and differentiation.

Lack of WDFY4 Aggravates Ovalbumin-Induced Asthma via Enhanced Th2 Cell Differentiation

Background: Asthma is a chronic inflammatory airway disease, and Th2 cells play an important role in asthma. WDFY4 (WDFY family member 4) is a susceptibility gene in several autoimmune diseases. Objective: In this study, the roles of WDFY4 in Th2 cell differentiation and Th2-dependent asthma were investigated. Methods: Naïve CD4⁺ T cells were isolated from wild-type and WDFY4-deficient mice and induced to differentiate in vitro. Subsequently, a mouse model of asthma was established by sensitization with ovalbumin. Results: Study results showed that WDFY4 deficiency could promote the differentiation of Th2 cells and the production of Th2 cytokines. WDFY4-deficient asthmatic mice showed higher levels of Th2 cytokines in the lungs and bronchoalveolar lavage fluid than wild-type mice. Moreover, infiltration of inflammatory cells, hyperplasia of goblet cells, production of mucus, and deposition of collagen were enhanced in WDFY4-deficient asthmatic mice. Conclusions: Our study demonstrates the pivotal role of WDFY4 in the pathogenesis of asthma and in Th2 cell differentiation.

Arf6 exacerbates allergic asthma through cell-to-cell transmission of ASC inflammasomes.

Asthma is a chronic inflammatory disease of the airways associated with excess production of Th2 cytokines and lung eosinophil accumulation. This inflammatory response persists in spite of steroid administration that blocks autocrine/paracrine loops of inflammatory cytokines, and the detailed mechanisms underlying asthma exacerbation remain unclear. Here, we show that asthma exacerbation is triggered by airway macrophages through a prion-like cell-to-cell transmission of extracellular particulates, including ASC protein, that assemble inflammasomes and mediate IL-1β production. OVA-induced allergic asthma and associated IL-1β production were alleviated in mice with small GTPase Arf6-deficient macrophages. The extracellular ASC specks were slightly engulfed by Arf6–/– macrophages, and the IL-1β production was reduced in Arf6–/– macrophages compared with that in WT macrophages. Furthermore, pharmacological inhibition of the Arf6 guanine nucleotide exchange factor suppressed asthma-like allergic inflammation in OVA-challenged WT mice. Collectively, the Arf6-dependent intercellular transmission of extracellular ASC specks contributes to the amplification of allergic inflammation and subsequent asthma exacerbation.

Fasting impairs type 2 helper T cell infiltration in the lung of an eosinophilic asthma mouse model.

Eosinophilic asthma is a form of bronchial asthma that is caused by the pulmonary infiltration of eosinophils and accounts for approximately half of the patients with severe asthma. Several cell types of the immune system in synergy with the epithelial cells of the lung provoke an inflammatory response in patients with asthma. Recently, the effect of fasting on immune cells and inflammation has attracted considerable attention. Therefore, we examined whether fasting may serve as novel preventive strategy in patients with asthma. In our study, we employed a previously established mouse model of eosinophilic asthma. C57BL/6 mice were inoculated intranasally with interleukin‐33 and ovalbumin (OVA) in order to induce eosinophil infiltration in the lung and subjected to a 48‐h long fasting period directly after or 7 days postinoculation. We used flow cytometry to characterise infiltrated immune cells in the lung and measured the quantity of inflammatory cytokines as well as antigen‐specific immunoglobins (Ig) by ELISA. Our results indicated that fasting lowered the number of eosinophilic pulmonary infiltrates in the eosinophilic asthma model mice. Furthermore, fasting suppressed anti‐OVA IgG1 production. Fasting suppressed Th2 cytokine production by impairing Th2 accumulation in the lung. The findings suggest that fasting may be a novel preventive strategy for eosinophilic asthma.

Di-(2-ethylhexyl) phthalate enhances cytokine release from group 2 innate lymphoid cells in the presence of interleukin-33

Epidemiological and experimental studies have shown that di-(2-ethylhexyl) phthalate (DEHP), a plasticizer, can aggravate allergic diseases. DEHP promotes adaptive immune responses, although its effect on the innate immune system remains largely unknown. The present study investigated the effects of DEHP on group 2 innate lymphoid cells (ILC2) that produce Th2 cytokines in response to epithelial cell-derived cytokines, such as interleukin (IL)-33. ILC2 (lineage-negative, CD45.2+, Sca1+, KLRG1+) were isolated from the lungs of C57BL/6 J mice. Co-exposure to DEHP and IL-33 significantly increased IL-5 release from ILC2, whose level was higher than that of the vehicle and IL-33 alone. The effects of DEHP in the presence of IL-33 showed an inverted-U dose-response. The present is the first report showing that DEHP exacerbates allergy through the innate immune system.

IL23R on myeloid cells is involved in murine pulmonary granuloma formation

Purpose of the Study The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4+ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Infection with Hymenolepis diminuta Blocks Colitis and Hastens Recovery While Colitis Has Minimal Impact on Expulsion of the Cestode from the Mouse Host.

Two experimental paradigms were adopted to explore host–helminth interactions involved in the regulation of colitis and to understand if colitis affects the outcome of helminth infection. First, male BALB/c mice infected with H. diminuta were challenged 4 days later with dinitrobenzene sulphonic acid (DNBS) and necropsied 3 days later. Second, mice were infected with H. diminuta 3 days after DNBS treatment and necropsied 11 or 14 days post-DNBS. Mice were assessed for colitic disease severity and infectivity with H. diminuta upon necropsy. Supporting the concept of helminth therapy, mice are protected from DNBS–colitis when infected with H. diminuta only 4 days previously, along with parallel increases in splenic production of Th2 cytokines. In the treatment regimen, H. diminuta infection produced a subtle, statistically significant, enhanced recovery from DNBS. Mice regained body weight quicker, had normalized colon lengths, and showed no overt signs of disease, in comparison to the DNBS-only mice, some of which displayed signs of mild disease at 14 days post-DNBS. Unexpectedly, colitis did not affect the hosts’ anti-worm response. The impact of inflammatory disease on helminth infection is deserving of study in a variety of models as auto-inflammatory diseases emerge in world regions where parasitic helminths are endemic.

Plasmacytoid Dendritic Cells Facilitate Th Cell Cytokine Responses throughout Schistosoma mansoni Infection.

Plasmacytoid dendritic cells (pDCs) are potent producers of type I IFN (IFN-I) during viral infection and respond to IFN-I in a positive feedback loop that promotes their function. IFN-I shapes dendritic cell responses during helminth infection, impacting their ability to support Th2 responses. However, the role of pDCs in type 2 inflammation is unclear. Previous studies have shown that pDCs are dispensable for hepatic or splenic Th2 responses during the early stages of murine infection with the trematode Schistosoma mansoni at the onset of parasite egg laying. However, during S. mansoni infection, an ongoing Th2 response against mature parasite eggs is required to protect the liver and intestine from acute damage and how pDCs participate in immune responses to eggs and adult worms in various tissues beyond acute infection remains unclear. We now show that pDCs are required for optimal Th2 cytokine production in response to S. mansoni eggs in the intestinal-draining mesenteric lymph nodes throughout infection and for egg-specific IFN-γ at later time points of infection. Further, pDC depletion at chronic stages of infection led to increased hepatic and splenic pathology as well as abrogated Th2 cell cytokine production and activation in the liver. In vitro, mesenteric lymph node pDCs supported Th2 cell responses from infection-experienced CD4+ T cells, a process dependent on pDC IFN-I responsiveness, yet independent of Ag. Together, these data highlight a previously unappreciated role for pDCs and IFN-I in maintaining and reinforcing type 2 immunity in the lymph nodes and inflamed tissue during helminth infection.

TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization

BackgroundTo explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization.MethodsTLR2−/− and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iμ-Cμ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iμ-Cγ, Iμ-Cα or Iμ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2−/− mice (with or without IL21 treatment).ResultsThe lungs of TLR2−/− mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2−/− compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2−/− mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency.ConclusionThese results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

The role of bcsE gene in the pathogenicity of Salmonella

The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the 'rdar' (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1>1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.

Alpha-Fetoprotein- and CD40Ligand-Expressing Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma.

Simple SummaryIn first clinical trials, vaccinations against tumor-associated antigens (TAA), such as Alpha-Fetoprotein (AFP) using antigen presenting cells, such as dendritic cells (DC), failed to achieve effective immune responses towards hepatocellular carcinoma (HCC). CD40Ligand is a potent immune checkpoint, which can increase the antitumoral immune response of DC. In this study, a subcutaneous vaccination with DCs, which were transduced with AFP-coding adenoviruses and an intratumoral treatment with DCs, which were transduced with CD40L-coding adenoviruses, induced an antitumoral immune response and led to complete remissions and long-term survival in 62% of mice with established HCC. Combined strategy causes rapid and profound changes in the tumor environment with enhanced Th1-cytokine expression, strong tumor infiltration of cytotoxic T lymphocytes and DC, and higher tumor apoptosis, leading to effective tumor regression of HCC. Thus, intratumoral CD40L co-stimulation represents a promising tool for improving tumor-antigen DC-based immunotherapy of HCC.Dendritic cells (DC) as professional antigen presenting cells are able to prime T-cells against the tumor-associated antigen α-fetoprotein (AFP) for immunotherapy of hepatocellular carcinoma (HCC). However, a strong immunosuppressive tumor environment limits their efficacy in patients. The co-stimulation with CD40Ligand (CD40L) is critical in the maturation of DC and T-cell priming. In this study, the impact of intratumoral (i.t.) CD40L-expressing DC to improve vaccination with murine (m)AFP-transduced DC (Ad-mAFP-DC) was analyzed in subcutaneous (s.c.) and orthotopic murine HCC. Murine DC were adenovirally transduced with Ad-mAFP or Ad-CD40L. Hepa129-mAFP-cells were injected into the right flank or the liver of C3H-mice to induce subcutaneous (s.c.) and orthotopic HCC. For treatments, 106 Ad-mAFP-transduced DC were inoculated s.c. followed by 106 CD40L-expressing DC injected intratumorally (i.t.). S.c. inoculation with Ad-mAFP-transduced DC, as vaccine, induced a delay of tumor-growth of AFP-positive HCC compared to controls. When s.c.-inoculation of Ad-mAFP-DC was combined with i.t.-application of Ad-CD40L-DC synergistic antitumoral effects were observed and complete remissions and long-term survival in 62% of tumor-bearing animals were achieved. Analysis of the tumor environment at different time points revealed that s.c.-vaccination with Ad-mAFP-DC seems to stimulate tumor-specific effector cells, allowing an earlier recruitment of effector T-cells and a Th1 shift within the tumors. After i.t. co-stimulation with Ad-CD40L-DC, production of Th1-cytokines was strongly increased and accompanied by a robust tumor infiltration of mature DC, activated CD4+-, CD8+-T-cells as well as reduction of regulatory T-cells. Moreover, Ad-CD40L-DC induced tumor cell apoptosis. Intratumoral co-stimulation with CD40L-expressing DC significantly improves vaccination with Ad-mAFP-DC in pre-established HCC in vivo. Combined therapy caused an early and strong Th1-shift in the tumor environment as well as higher tumor apoptosis, leading to synergistic tumor regression of HCC. Thus, CD40L co-stimulation represents a promising tool for improving DC-based immunotherapy of HCC.

A Defective CXCL16/CXCR6 Axis Increases the Risk of Pregnancy Loss via the Abnormal Crosstalk between Decidual γδ T Cells and Trophoblasts

Abstract Objective: The maternal-fetal interface undergoes dynamic changes to allow the fetus to grow and develop in the uterus. The interaction between decidual γδ T cells and trophoblasts plays a pivotal role during successful pregnancy; however, their physiological functions in early-term human pregnancy are still not completely illustrated. This study was undertaken to illustrate the functional roles of CXCL16/CXCR6 to prevent pregnancy loss via the crosstalk between decidual γδ T cells and HTR8/SVneo trophoblast cells. Methods: The percentile of CXCR6+ γδ T cells in the peripheral blood from normal female and recurrent spontaneous abortion (RSA) patients was analyzed by flow cytometry. The expression of CXCR6 was detected in decidual immune cells via flow cytometry, and the expression of CXCL16 was analyzed in HTR8/SVneo trophoblast cells and lentivirus (LV)-HTR8/SVneo trophoblast cells via enzyme-linked immunosorbent assay. Reverse transcriptase-polymerase chain reaction was used to verify the expression of the CXCL16 gene in LV-HTR8/SVneo trophoblast cells. Expression of granzyme B and cytokines and proliferation of decidual γδ T cocultured with HTR8/SVneo trophoblast cells were analyzed by flow cytometry. Invasion of HTR8/SVneo trophoblast cells was assessed via Matrigel transwell assay. Adoptive transfer was induced in vivo further to illustrate that the normal expression of CXCL16/CXCR6 could prevent pregnancy loss. Results: The percentile of CXCR6+ γδ T cells in the peripheral blood from RSA patients was lower than normal pregnancies. The expression of CXCR6 was highest in the decidual γδ T cells among decidual immune cells, and the expression of CXCL16 increased as the amount of HTR8/SVneo trophoblast cells increased. Expression of granzyme B in the decidual γδ T cells was downregulated by cocultured with HTR8/SVneo cells dependent of CXCL16, and HTR8/SVneo trophoblast cells induced the Th2 cytokines production in the decidual γδ T cells. Both the expression of CXCR6 in the decidual γδ T cells and proliferation of the decidual γδ T cells were promoted by HTR8/SVneo trophoblast cells. On the other hand, decidual γδ T cells enhanced the invasion of HTR8/SVneo trophoblast cells and thus promoted embryo implantation. In vivo study was taken further and shown that low expression of CXCL16/CXCR6 results in pregnancy loss because of dialog disorder between decidual γδ T cells and trophoblasts. Conclusions: Low expression of CXCL16/CXCR6 results in pregnancy loss because of the dialog disorder between decidual γδ T cells and trophoblasts, and it showed a light on the effective strategy of adoptive transfer of CXCR6+ γδ T cells on the treatment of RSA. This observation provides a scientific basis on which a potential strategy can be applied to the early-detect and treatment of RSA.

Polymorphisms of Monocyte-Activating Cytokine/Chemokine Genes Affect the Susceptibility to, and Severity of Chronic ITP

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the production of platelet antibodies, resulting in the destruction of platelets and inhibition of their production. A shift toward Th1 and Th17 cells together with impaired Tregs has been reported using quantitative RT-PCR and flow cytometry method. Further, recent reports have shown that CD16 (+) monocytes in ITP patients are polarized to secrete the Th1 cytokine IL-12, promoting Th1 responses. Enhanced phagocytosis of splenic macrophages was also observed in ITP patients. Thus, monocytes/macrophages are also involved in the pathogenesis of ITP. However, it remains unclear whether genetic factors of cytokines/chemokines activating monocytes increase the risk of chronic ITP. We investigated the impact of monocyte-related gene polymorphisms: TNF-α -857C/T, IFN-γ +874T/A, and MCP1+2518G/A on the susceptibility and clinical feature of chronic ITP (cITP) and Th1/Th2 ratio in peripheral blood of cITP patients,Patients and methods: In this study, 126 Japanese cITP patients (92 females and 34 males with a median age of 47.7 [range: 2.4-82.3] years), as well as 202 race-matched healthy subjects were examined. The diagnosis of cITP was defined according to International Working Group criteria. Genotyping of the polymorphisms was determined by PCR based technique and direct sequencing. We evaluated Th1/Th2 ratio in peripheral blood of 15 normal donors and 25 ITP patients. Intracellular IL-4 (Th2 cytokine) and IFN-γ (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin by flow cytometry.This study was approved by the Institutional Review Board of Gunma University Hospital.Results: The platelet count ranged from 1×109/L to 98×109/L with a mean count of 32×109/L at the initial diagnosis. Steroid treatment was given to 86 patients (58.1%), and splenectomy was performed in only 18 patients (12.2%). The genotype distributions of these three polymorphisms in healthy subjects were similar to those observed in previous studies of Japanese populations. Patients with cITP had a significantly higher frequency of the TNF-α -857 TT genotype (high producer type) and MCP1+2518 G allele (high-producer type) compared to healthy controls (14.0% vs 5.4%, p < 0.01; 67.6% vs. 60.6%, P < 0.01). However, no significant difference in the genotype frequencies of IFN-γ was observed between cITP patients and healthy controls. Thus, it was suggested that the TNF-α and MCP-1 polymorphisms shifted toward monocyte activation in cITP patients. We examined the association between these polymorphisms and the clinical characteristics of cITP patients. cITP patients with IFN-γ +874 non-AA genotype (high expression type) showed a lower minimum platelet count than those with an AA genotype (12.9 × 109/L ± 10.7 ×109/L vs 19.4×109/L ± 18.9 ×109/L, P < 0.05). We also examined the association between these polymorphisms and Th1/Th2 ratio in both normal donors and cITP patients. The median Th1/Th2 ratio in patients with cITP was significantly higher than that of healthy controls (31.4, range 0.6-98.8 vs. 17.8, range, 2.2-52.5, P < 0.001). Patients with the IFN-γ +874 non-AA genotype (high expression type) had a significantly higher Th1/Th2 ratio compared to those with the IFN-γ +874 AA genotype (low expression type) (71.5, range 29.4-92.8 vs. 27.5, range 0.6-98.8, P < 0.05). However, there was no significant association between the Th1/Th2 ratio and other polymorphism in cITP patients.Conclusion: These findings suggested that the high expression type of monocyte-activating cytokine/chemokine genes increased the susceptibility to, and severity of chronic ITP DisclosuresMurakami:Ono: Honoraria; BMS: Honoraria; Fujimoto: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Celgene: Honoraria.

Venous Thromboembolism in Patients with Steroid-Refractory Chronic Graft-Versus-Host Disease Receiving Synthetic Retinoid Am80

Chronic graft-versus-host disease (GVHD) remains a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation, and the treatment of chronic GVHD remains challenging. All-trans retinoic acid (ATRA), a potent vitamin A derivative, can regulate immune responses. Synthetic retinoid Am80, which shows biological activity approximately 10 times more potent than that of ATRA, directly inhibits Th1 cytokine production. We previously reported in an experimental mouse model that Am80 down-regulated both Th1 and Th17 differentiation in donor T cells, resulting in attenuation of chronic GVHD (Nishimori H. et al. Blood 2012). Based on this background, we conducted an open-labeled, multicenter phase II study of Am80 in patients with steroid-refractory chronic GVHD (Maeda Y. et al. Acta Med. Okayama. 2016). Six facilities are participating in this study. Patients take Am80 orally (4 mg/day) for 24 weeks. If any unexpected adverse events with Grade 2 or expected adverse events with Grade 3 develop, the daily dose will be decreased to 2 mg/day, 2 mg every other day, or discontinued.Eighteen patients were enrolled and 5 out of 18 (27.8%) patients experienced venous thromboembolism (VTE). Table 1 shows the patient characteristics of those developed VTEs.It is controversial whether retinoid would enhance the incidence of VTE. Only one patient reported thrombosis when Am80 orally (6 mg/m2) for 2-8 weeks was administrated to approximately 800 patients with acute promyelocytic leukemia. In this study, 5 out of 18 (27.8%) patients experienced thrombosis and the safety committee in this study has stopped the trial due to safety concerns. Recently, a meta-analysis reported the incidence of VTE in chronic GVHD was 35 % (20-54 %). Further studies are needed to confirm the causal relationship between Am80 orally (4 mg/day) for 24 weeks and VTE. And also, alternative treatment schedule in addition to anticoagulants for the prevention of VTE will be investigated in subsequent studies. [Display omitted] DisclosuresNishimori:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.. Maeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.