Targeting JMJD1C to selectively disrupt tumor Treg cell fitness enhances antitumor immunity.

Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.

Perivascular Lymphocyte Clusters Induced by Gastric Subserous Layer Vaccination Mediate Optimal Immunity against Helicobacter through Facilitating Immune Cell Infiltration and Local Antibody Response.

BACKGROUND: In situ vaccination-induced local inflammatory response resulted in the establishment of a pool of tissue-resident memory T (TRM) cells and new vessels after the resolution of inflammation. TRM cells have received increasing attention; however, the role of new vessels in protective response is still unknown. MATERIALS AND METHODS: We performed the laparotomy to access the stomach and injected alum-based vaccine into the gastric subserous layer (GSL). At 28 days post vaccination, a parabiosis mouse model along with depletion of anti-CD90.2 antibody was employed to explore the function of perivascular lymphocyte clusters in recall responses. The composition of the gastric lymphocyte clusters was analyzed by immunofluorescence staining. Antibody responses were detected using ELISA. Gastric lymphocytes were analyzed using flow cytometry. RESULTS: GSL vaccination induced the formation of new vessels in the inflamed region. These new vessels were different from native vessels in that they were generally accompanied by perivascular lymphocyte clusters that mainly consisted of CD90-expressing cells. Additionally, histological analysis revealed the presence of CD4+ and CD8+ T cells in the perivascular lymphocyte clusters. Administration of a dose of an anti-CD90.2 antibody to GSL-vaccinated mice resolved these clusters. The efficacy of protection was compared in the parabiosis mice. Upon challenge, the presence of perivascular lymphocyte clusters was responsible for the fast recall response, as depletion of these clusters by CD90.2 antibody administration resulted in decreased expressions of VCAM-1, Madcam-1, and TNF-α, as well as lower recruitment of proinflammatory immune cells, decreased antibody levels, and poor protection. CONCLUSIONS: Our research demonstrates that in situ vaccination-induced regional inflammatory response contributes to optimal recall response not only by establishing a CD4+ TRM pool but also by creating an "expressway," i.e., perivascular lymphocyte cluster.

Vaccine-induced gastric CD4+ tissue-resident memory T cells proliferate in situ to amplify immune response against Helicobacter pylori insult.

BACKGROUND: Tissue-resident memory T cells accelerate the clearance of pathogens during recall response. However, whether CD4+ TRM cells themselves can provide gastric immunity is unclear. MATERIALS AND METHODS: We established a parabiosis model between the enhanced green fluorescent protein and wild-type mice that the circulation system was shared, and the wild-type partner was vaccinated with H pylori vaccine composed of CCF and silk fibroin in gastric subserous layer to induce gastric EGFP+ CD4+ TRM cells. Antigen-specific EGFP+ CD4+ T cells and proliferous TRM cells were analyzed by flow cytometry. The colonization of H pylori was detected by quantitative real-time PCR. EGFP+ CD4+ TRM cells and the inflammation of the stomach were observed by histology. RESULTS: A parabiosis animal model was employed to identify the cells that introduced by vaccination in GSL. Antigen-specific EGFP+ CD4+ T cells could be detected at day 7 post-vaccination. Thirty days later, EGFP+ CD4+ TRM cells were established with a phenotype of CD69+ CD103- . Of note, we found that when circulating lymphocytes were depleted by FTY720 administration, these TRM cells could proliferate in situ and differentiate into effector Th1 cells after H pylori challenge. A decrease in H pylori colonization was observed in the vaccinated mice but not unvaccinated mice. Further, we found that although FTY720 was administrated, mounted pro-inflammatory myeloid cells still emerged in the stomach of the vaccinated mice, which might contribute to the reduction of H pylori colonization. CONCLUSIONS: Our study reveals that H pylori vaccine-induced CD4+ TRM cells can proliferate and differentiate in situ to enhance gastric local immunity during recall response.

Limonin ameliorates ulcerative colitis by regulating STAT3/miR-214 signaling pathway.

Ulcerative colitis (UC) is a major inflammatory bowel disease (IBD) which has become a global public health problem. Limonin is a triterpenoid extracted from citrus which possesses the capacities to against inflammations and cell apoptosis. However, the efficacy and the underlying mechanisms of limonin in the treatment of UC remain unclear. In this study, we first investigated the therapeutic effects of limonin on dextran sodiumsulfate (DSS)-induced UC in vivo by examining the changes of disease activity index (DAI), the colon length, the colon histology, and cyto/chemokine levels. We found that limonin markedly reduced DAI, intestinal damages, and the levels of pro-inflammatory cytokines, such as TNF-α and IL-6. In vitro, limonin significantly repressed the productions of pro-inflammatory cytokines in cultured normal colonic epithelial cells. Mechanistically, we demonstrated that limonin improved the prognosis of UC mainly through downregulating p-STAT3/miR-214 levels. Collectively, our results suggested that limonin was a novel therapeutic agent and it was expected to be translated into the clinic to improve the prognosis of UC.

Gastric Subserous Vaccination With Helicobacter pylori Vaccine: An Attempt to Establish Tissue-Resident CD4+ Memory T Cells and Induce Prolonged Protection.

Tissue-resident memory T (Trm) cells are enriched at the sites of previous infection and required for enhanced protective immunity. However, the emergence of Trm cells and their roles in providing protection are unclear in the field of Helicobacter pylori (H. pylori) vaccinology. Here, our results suggest that conventional vaccine strategies are unable to establish a measurable antigen (Ag)-specific memory cell pool in stomach; in comparison, gastric subserous injection of mice with micro-dose of Alum-based H. pylori vaccine can induce a pool of local CD4+ Trm cells. Regional recruitment of Ag-specific CD4+ T cells depends on the engagement of Ag and adjuvant-induced inflammation. Prior subcutaneous vaccination enhanced this recruitment. A stable pool of Ag-specific CD4+ T cells can be detected for 240 days. Two weeks of FTY720 administration in immune mice suggests that these cells do not experience the recirculation. Immunohistochemistry results show that close to the vaccination site, abundant CD4+T cells locate on epithelial niches, independent of lymphocyte cluster. Paradigmatically, Ag-specific CD4+ T cells with a phenotype of CD69+CD103- are preferential on lymphocytes isolated from epithelium. Upon Helicobacter infection, CD4+ Trm cells orchestrate a swift recall response with the recruitment of circulating antigen-specific Th1/Th17 cells to trigger a tissue-wide pathogen clearance. This study investigates the vaccine-induced gastric CD4+ Trm cells in a mice model, and highlights the need for designing a vaccine strategy against H. pylori by establishing the protective CD4+ Trm cells.

Shape of gastrointestinal immunity with non-genetically modified Lactococcus lactis particles requires commensal bacteria and myeloid cells-derived TGF-ß1.

Heat-killed probiotics or microbial autologous components show multiple activities on modulating host immune responses towards tolerance or vice versus aggressiveness. Gram-positive enhancer matrix particles (GEMs), the non-genetically modified particles which composed of the cell wall derived from Lactococcus lactis (L. lactis), were used as a typical microbial molecule to investigate the mechanism of opposite immune responses generated in disparate scenarios. The results of stool 16S rRNA Illumina sequencing suggested that the overwhelming number of mice pre-administered with GEMs showed the expansion of Bacteroidetes but contraction of Verrucomicrobia. Co-administration GEMs and antibiotics could preserve the microbial diversity, even though the abundance of gut microbes was largely depleted by antibiotics. Additionally, dendritic cells (DCs) from mice receiving GEMs rather than DCs that in vitro treated with GEMs induced the expansion of regulatory T cells (Tregs), witnessing the critical role of gut flora alteration. Importantly, this alteration provided protection to alleviate dextran sulfate sodium (DSS)-induced intestinal inflammation. On the other hand, in the context of Helicobacter felis (H. felis) infection, the mice pre-administrated with GEMs exhibited a comparably potent gastric immunity with the elevated expression of IFN-γ, IL-17, and multiple anti-microbial factors, leading to the reduced burden of H. felis. However, tolerance for both DSS-induced intestinal inflammation and immunity against H. felis was depleted in a mice model lacking of transforming growth factor-ß1 (TGF-ß1) in myeloid cells. These findings suggest that GEMs can modulate host immune responses bidirectionally according to context, and may serve as a supplement for antibiotic treatment.

Toxic adjuvants alter the function and phenotype of dendritic cells to initiate adaptive immune responses induced by oral Helicobacter pylori vaccines.

BACKGROUND: Toxic adjuvant is considered as an indispensable constituent for oral Helicobacter pylori (H. pylori) vaccines. However, the elaborate role of toxic adjuvant in the initiation of adaptive immune response is largely undescribed. MATERIALS AND METHODS: We employed an acid-resistant HP55/PLGA nanoparticles (NPs) delivery system encapsulating three antigens (Hsp, Nap, and Lpp20) from H. pylori and accompanied with three adjuvants (LPS, CpG, and chimeric flagellum (CF)) to explore the underlying mechanism of the adjuvant constituent. H. pylori-specific antibody responses were detected by ELISA. Gastric inflammatory and Th1/Th17 responses were analyzed by flow cytometry. Expressions of inflammatory cytokines were measured by quantitative real-time PCR. RESULTS: In bone marrow-derived dendritic cells' (BMDCs) model, the addition of toxic adjuvants is responsible for the proinflammatory function, but not the mature phenotype of BMDCs. In vivo, intestinal loop injection with NPs + LPS, rather than NPs alone, altered the dendritic cell (DC) phenotypes in mesenteric lymph nodes and drove a local proinflammatory microenvironment. In a prophylactic vaccination model, mice immunized with NPs + adjuvants significantly reduced the gastric colonization of H. pylori, induced antigen-specific antibody responses and Th1/Th17 cell responses. After H. pylori challenge, these mice showed potent recall responses involving both neutrophil and inflammatory monocyte infiltration. Additionally, TLR4 knockout mice were immunized with NPs + LPS and NPs + CF, respectively; only the recipients of NPs + CF orchestrated a protective response to control bacterial infection. CONCLUSIONS: Our study indicated that toxic adjuvants within oral H.pylori vaccines altered the function and phenotype of dendritic cells and facilitated the establishment of proinflammatory microenvironment to initiate adaptive immune responses.

Promoting Immune Efficacy of the Oral Helicobacter pylori Vaccine by HP55/PBCA Nanoparticles against the Gastrointestinal Environment.

The immunogenicity of oral subunit vaccines is poor partly as a result of the harsh milieu of the gastrointestinal (GI) tract. For some pathogens that restrictedly inhabit the GI tract, a vaccine that works in situ may provide more potent protection than vaccines that operate parenterally. Yet, no appropriate delivery system is available for oral subunit vaccines. In this study, we designed HP55/poly( n-butylcyanoacrylate) (PBCA) nanoparticles (NPs) to carry Helicobacter pylori ( H. pylori) subunit vaccine CCF for oral administration in a prophylactic mice model. These NPs, which are synthesized using an interfacial polymerization method, protected the CCF antigen not only from the acidic pH in simulated gastric fluid (SGF, pH 1.2) but also from the proteolysis in simulated intestinal fluid (SIF, pH 7.4). Oral vaccination of mice with HP55/PBCA-CCF NPs promoted the production of serum antigen-specific antibodies, mucosal secretory IgA, and proinflammatory cytokines. Moreover, a Th1/Th17 response and augmented lymphocytes were found in the gastric tissue of HP55/PBCA-CCF NP-immunized mice, which might eventually limit H. pylori colonization. Collectively, these results indicate that HP55/PBCA NPs are promising carriers against the severe situation of the GI tract and thereby may be further utilized for other orally administrated vaccines or drugs.

Nongenetically modified Lactococcus lactis-adjuvanted vaccination enhanced innate immunity against Helicobacter pylori.

BACKGROUND: Gram-positive enhancer matrix particles (GEM) produced by Lactococcus lactis can enhance vaccine-induced immune response. However, the mechanism under which this adjuvant mounts the efficacy of orally administered vaccines remains unexplored. MATERIALS AND METHODS: We used a prophylactic mice model to investigate the mechanism of GEM-adjuvanted vaccination. Helicobacter pylori urease-specific antibody response was monitored and detected in murine serum by ELISA. Urease-specific splenic cytokine profile was examined. Gastric inflammatory responses were measured on day 43 or 71 by quantitative real-time PCR, flow cytometry and histology. RESULTS: We found that GEM enhanced the efficiency of oral H. pylori vaccine by promoting innate immunity. The vaccine CUE-GEM composed of GEM particles and recombinant antigen CTB-UE provided protection of immunized mice against H. pylori insult. The protective response was associated with induction of postimmunization gastritis and local Th1/Th17 cell-medicated immune response. We showed that innate inflammatory responses including neutrophil chemokines CXCL1-2, neutrophils, and antimicrobial proteins S100A8 and MUC1 were significantly elevated. Within all infected mice, S100A8 and MUC1 levels were negatively correlated with H. pylori burden. Strikingly, mice receiving GEM also show reduction of colonization, possibly through natural host response pathways to recruit CD4+ T cells and promote S100A8 expression. CONCLUSIONS: These findings suggest that GEM-based vaccine may impact Th1/Th17 immunity to orchestrate innate immune response against H. pylori infection.