Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses

Immunotherapy strategies targeting the programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) pathway in clinical treatments have achieved remarkable success in treating multiple types of cancer. However, owing to the heterogeneity of tumors and individual immune systems, PD-L1/PD-1 blockade still shows slow response rates in controlling malignancies in many patients. Accumulating evidence has shown that an effective response to anti-PD-L1/anti-PD-1 therapy requires establishing an integrated immune cycle. Damage in any step of the immune cycle is one of the most important causes of immunotherapy failure. Impairments in the immune cycle can be restored by epigenetic modification, including reprogramming the environment of tumor-associated immunity, eliciting an immune response by increasing the presentation of tumor antigens, and by regulating T cell trafficking and reactivation. Thus, a rational combination of PD-L1/PD-1 blockade and epigenetic agents may offer great potential to retrain the immune system and to improve clinical outcomes of checkpoint blockade therapy.

Adapting liposomes for oral drug delivery

Liposomes mimic natural cell membranes and have long been investigated as drug carriers due to excellent entrapment capacity, biocompatibility and safety. Despite the success of parenteral liposomes, oral delivery of liposomes is impeded by various barriers such as instability in the gastrointestinal tract, difficulties in crossing biomembranes, and mass production problems. By modulating the compositions of the lipid bilayers and adding polymers or ligands, both the stability and permeability of liposomes can be greatly improved for oral drug delivery. This review provides an overview of the challenges and current approaches toward the oral delivery of liposomes.

Effect of apigenin on dendritic cells maturation and function in murine splenocytes / 药学学报

This study was designed to explore the effect of apigenin (Api) on dendritic cell (DCs) maturation and function in murine spleen cells. The single spleen cell was isolated, and then cultured with lipopolysaccharide (LPS) in the present and absence of apigenin. After 24 h, the toxicity of Api and the T cell proliferation were determined by CCK8 kit. In addition, we collected the cell-free supernatants to measure cytokine production using ELISA, collected the cells to determine the DC maturation using flow cytometry. Finally, we purified Api and/or LPS-treated CD11c+ DCs which were pulsed with ovalbumin (OVA)323-339 and then were adoptive transferred into C57BL/6 mice to detect the OVA323-339-specific T cell proliferation and T helper (Th1) and Th2 cell secreting IFN-γ and IL-4 production, respectively. We found that Api did not affect splenocyte viability, but inhibited the production of pro-inflammatory cytokine IL-1β, IL-6 and TNF-α, not anti-inflammatory cytokine IL-10. In addition, Api inhibited the expression of co-stimulatory CD80, CD86 and MHCII of CD11c+ DCs. Finally, compared to LPS+OVA DCs group, DCs from Api and LPS co-treated splenocytes (Api+LPS+DCs) impaired OVA323-339-specific T cell proliferation and the production of IFN-γ and IL-4 in CD4+ T cells, which had the similar responses with OVA+DCs. These data suggest that Api exhibits anti-inflammatory properties via inhibiting DC activation and function, as a new immune-modulator, which may induce immune-tolerance with a benefit to those with chronic inflammation.

Mechanism of MOG i.p.for protecting mice from EAE / 中华微生物学和免疫学杂志

Objective To investigate mechanisms underlying the prevention of experimental autoimmune encephalomyelitis (EAE) in mice by intraperitoneal infusion of myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) (MOG i.p.).Methods C57BL/6 mice were immunized with MOG35-55 to establish the model of EAE and then were intraperitoneally injected daily with MOG35-55 or ovalbumin (OVA, served as control) from day 6 to day 16.EAE was evaluated daily using a general clinical scoring system and histological analysis.Numbers of lymphocytes in peripheral blood and central nervous system (CNS) were detected at different time points.Effects of MOG i.p.on the migration of MOG-T cells in vivo were analyzed by an adoptive transfer experiment.Maturation of splenic antigen-presenting cells (APCs) and migration of MOG-T cells in vitro were examined by fluorescence activated cell sorting (FACS) and a Transwell system, respectively.Results MOG i.p.protected the mice from development of EAE by blocking the lymphocyte recruitment to CNS.More effector T cells were trapped in the periphery of EAE and naive mice in adoptive transfer experiment after MOG i.p.treatment.MOG i.p.induced the maturation of splenic APCs and enhanced the expression of CD80, CD86 and major histocompatibility complex class Ⅱ (MHCⅡ) molecules.Mature APCs blocked the recruitment of effector T cells to CNS.Conclusion MOG i.p.protects mice from EAE by inducing the maturation of splenic APCs.

Tejido linfático asociado a mucosas: Células presentadoras de antígenos / Mucose associated lymphoid tissue: Antigen presenting cells

Se estudiaron al microscopio electrónico biopsias de mucosas normales y patológicas (cavidad bucal y cuello uterino), con especial atención a los sistemas de defensa existentes en las células epiteliales (CE) y en las células dendríticas (CD). Las CE, cuando están activadas, muestran su capacidad de fagocitar y procesar antígenos con la finalidad de presentarlos luego a las CD; los elementos implicados en esta función son vesículas de micropinocitosis, cuerpos multivesiculares, lisosomas, fagosomas, vesículas recubiertas por clatrina, gránulos de contenido denso recubiertos por una unidad de membrana, gránulos en cuyo interior se aprecian láminas que simulan hojas de cebolla, microcuerpos y gránulos con actividad de fosfatasa ácida. Las CD que recién han ingresado al interior del epitelio son de baja densidad electrónica y poseen grandes prolongaciones citoplasmáticas, que luego se reducen de tamaño, a la vez que aumenta la densidad de su citoplasma. Muestran vesículas de micropinocitosis, algunas recubiertas por clatrina, lisosomas y corpúsculos de Birberk. En este momento son reconocidas como células de Langerhans. Tanto en las CE como en las CD existen abundantes “pliegues marginales o de superficie“ (surface folds), conteniendo numerosas vesículas de micropinocitosis. Entre la CE y la CD se establecen íntimos contactos a través de los cuales las primeras presentan los antígenos fagocitados y tratados a las CD donde son terminados de procesar y se unen a las moléculas del complejo principal de histocompatibilidad y/o a moléculas con función similar (CD1). Las CD migran a los ganglios linfáticos donde presentan los antígenos a los linfocitos T y empieza el proceso de activación de estos, que conduce a la defensa frente a las noxas que han ingresado al organismo. De esta manera tanto las CD como las CE son un lazo de unión entre los sistemas de defensa innata y la adquirida.

We studied samples of normal and abnormal human mucosae, including oral tissue and uterine cervix, using electron microscopy. Special attention was given to the functions and mechanisms of defense carried out by the epithelial (EC) and dendritic cells (DC). Activated epithelial cells posses the capacity to uptake and process antigens, in order to present them, subsequently, to the dendritic cells. The structures and elements of the cells intervening on this function are: micropinocytic vesicles, multivesicular bodies, lysosomes, phagosomes, clathrin-covered vesicles, dense granules covered by a unit membrane, granules with onion likes leaves, microbodies, and dense granules with acid phosphatase activity. When they first arrive within the epithelial layers, the DC are clear with long cytoplasmic projections, which later become short, and the density of their cytoplasm increases. They possess mycropinocytic vesicles, some clathrine-covered vesicles, lysososmes and Birbeck granules. At this moment, they are known as Langerhans cells. EC and DC present many surface folds rich in micropynocytic vesicles. Between EC and DC there are many contacts (close junctions or tight junctions), through which antigens, phagocitized and processed by the EC, are given to the DC. These cells join them to major histocompatibility complex molecules or to other molecules with similar functions (CD1). Then the Langerhans cells travel to the lymphatic node to activate T cells and continue the immunologic task. So, in this way, both the EC and the DC are a link between the natural and the acquired immunological mechanisms.

Enhancement of Proliferation and Antigen Presentation of Human B Cells in Vitro by K562 Cells Expressing CD40L

BACKGROUND: CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the convenience to obtain from peripheral blood and expand in vitro. Moreover, CD40-B cells were found to be comparable with DCs in their capacity to raise antigen-specific CD8+ T cells. Here, we have established K562 cells expressing CD40L to expand CD40-activated B cells used for APCs. METHODS: After activation of B cell by K562/CD40L, CD40-B cells were examined by counting B cell numbers. Surface expression of CD54, CD80, CD86 and HLA class II was measured by flow cytometry. The CD40-B cells were tested for its function as APC by mixed lymphocyte reactions (MLR) and by induction of T cell responses specific for pp65 peptide in vitro. RESULTS: The expansion of B cells by K562/CD40L increased about 6-folds compared with anti-CD40 or K562. Furthermore, the expression of CD54, CD80, CD86 and HLA class II was up-regulated by K562/CD40L. B cells by K562/CD40L showed comparable antigen presentation activity with mature DCs as shown in MLR, INF-gamma ELISPOT assay. CONCLUSION: These results suggest that K562/CD40L could be used to generate activated B cells as potent APCs which could be useful for cellular vaccination and adoptive immunotherapy.

Age-related Changes of Antigen Presenting Cells in Rat Brain / 체질인류학회지

Activation of T cells for an immune response requires the participation of antigen presenting cells that express class II major histocompatibility complex gene products on their surface. As far as we know, there is no study on the agerelated changes of ED2 immunoreactive macrophages and MHC class II immunoreactive dendritic cells in the normal rat brain. The aim of the present study is to investigate the age-related changes of dendritic cells and macrophages in rat brain. The distribution and morphology of the macrophages and dendritic cells in the rat brain were studied from the 1 month-, 12 month- and 24 month-old rats by means of immunohistochemical methods using anti-rat MHC class II and anti-rat ED2 monoclonal antibodies. Antigen presenting cells were observed in choroid plexuses and white matter of the rat brain. The numbers of antigen presenting cells gradually increased with age. At all age stages and regions of the rat brain, the numbers of ED2 immunoreactive macrophages was higher than that of MHC class II immunoreactive dendritic cells. According as age increases, shapes of antigen presenting cells became more complex and aggregated together. In conclusion, the above results suggest that the increases of the number and the changes of the morphology in two kinds of the antigen-presenting cells, MHC class II-immunoreactive dendritic cells and ED2-immunoreactive macrophages, with age may influence on effects of cell-mediated immune responses.

Oral Tolerance: Not Simple But more Complex

The intestinal immune system can discriminate between harmful and unharmful antigens and do not provoke productive immunity to unharmful antigen. Thus oral administration of antigen is one of classical methods for inducing antigen-specific immune tolerance in the periphery. Furthermore, oral tolerance has been investigated for the treatment of autoimmune disorders in human clinical trials. However, the detail mechanism of oral tolerance and contributing factors are not defined clearly at this time. Recent studies demonstrate unique types of immune cell that suppressing immune response, such as regulatory T cell and tolerogenic dendritic cell. This article reviews the factors involved in oral tolerance and discusses our current understanding base on the recent literatures and our works.

CHANGE IN IL-12 EXPRESSION AND ITS ROLE IN IMMUNODISSONANCE IN MODS MICE / 解放军医学杂志

Objective To determine the dynamic change in IL-12 in MODS mice at different post-trauma time points and its relationship with immunodissonance. Methods 96 male C57BL/6 mice, 6-8 weeks old, were randomly divided into normal control, 3h, 24h, 3d, 7d and 12d post-trauma groups. MODS model were replicated by injection of zymosan into the peritoneal cavity of the mice. Spleen and blood were sampled at different time points. Pathological changes of spleen were observed by light microscope. Immunohistochemistry marker of IL-12p40 in spleen was produced by Powervision method. The levels of IL-12p40 and IL-12p70 in serum and homogenate of spleen were determined by enzyme-link immuno-sorbent assay,and T lymphocyte subsets were analyzed by flow cytometry, respectively. Results Atrophy of the white pulps, proliferation of monocytes and dendritic cells, infiltration of neutrophilic granulocytes in the red pulps could be seen in 12d post trauma group. During the "two hits" in early period (3-24h) and late period (12d) in MODS, the amount of IL-12p40 positive cells increased significantly in the spleen; the level of IL-12p40 increased, whereas the level of IL-12p70 decreased evidently, in serum and the homogenate of the spleen. In the early period, the amount of CD8 + T cells in peripheral blood increased, while the amount of CD4 + T cells reduced obviously in the late stage, thus CD4/CD8 ratio declined in both periods. Conclusions The results indicated that over expression of IL-12p40 by antigen presenting cells in immune organs was correlated with immunosuppression of MODS mice.

The mRNA expression of TLR4,TLR9 in experimental autoimmune neuritis induced by P0 180-199 and effects of TWP on it / 中国免疫学杂志

Objective:To observe the dynamic expression of mRNA of TLR4 and TLR9 in Lewis rats with experimental autoimmune neuritis(EAN) and the effect of TWP on the disease.Methods:Male Lewis rats were immunized with P0 180-199(100 microgram),TWP was profused into post-immunization rats’ stomach daily.The clinical signs of rats and pathological changes in the sciatic nerves were observed.TLR4 and TLR9 were detected by RT-PCR dynamically which spleens,sciatic nerves and peripheral blood lymphonodes as sample.Results:EAN group got the peak of clinical score at the 17 d.p.i,and ameliorated obviously at 33 d.p.i,and the mRNA expression of TLR4 got the peak at the 16 d.p.i,then reduced gradually(P

Immunohistochemical Characterization of Dendritic Cells after Systemic Administration of Staphylococcal Enterotoxin B / 대한해부학회지

This study was performed to investigate the effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) and other immune cells in the major lymphoid organs. A single dose of SEB (25 microgram/kg) was administered to BALB/c mice by intraperitoneal injection. After the mice were sacrificed in groups of three at 2 h, 6 h, 1 day, 2 days, 3 days, 1 week and 2 weeks, the spleen, lymph node and thymus were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. We demonstrated in this study the distribution patterns of DCs and their major costimulatory and adhesive molecules in the murine spleen, lymph node and thymus after SEB administration. We obtained the evidence for maturation of DCs in vivo in response to SEB. DCs were found in increased number in the periarterial lymphatitc sheath (PALS) of spleen, paracortex of lymph nodes and thymic medulla. CD86, ICAM-1 and MHC class II molecules were upregulated on the activated and matured DCs after SEB injection. The most salient feature of the present study was the differential expression pattern of the costimulatory and adhesive molecules on the activated DCs. In addition to DCs, T cells expressing T cell receptor Vbeta8 were increased in number after SEB treatment. In conclusion, SEB exhibited a potent and effective stimulative effect on DCs in vivo.

Antigen Presenting Cells in the Palatine Tonsils and Adenoids / 대한이비인후과학회지

The functions of the palatine tonsils and adenoids have been investigated for many years, but there are still a lot of debates on their immunological roles. The aim of this study was to investigate the distribution of the antigen presenting cells in the palatine tonsils and adenoids, and to reveal their possible association with middle ear effusion. Tissues were collected from 18 pediatric patients who underwent tonsillectomy and adenoidectomy. In 8 of 18 cases, middle ear effusions were coexistent. The immunohistochemical staining using monoclonal antibodies, major histocompatibility complex(MHC) class II, CD1a, CD35, intercellular adhesion molecule(ICAM)-1 and B7 were done. In 10 cases of them, the cells which express MHC class II and ICAM-1 molecules were quantified by flow cytometry. The distribution patterns of the antigen presenting cells between the palatine tonsils and adenoids were not so different, but CD1a-positive cells were more abundant in the tonsils than in the adenoids. Middle ear effusion had no effects on the distribution of the antigen presenting cells in the tonsils and adenoids. Our data suggested both of the palatine tonsils and adenoids might have the same immunological roles, but the tonsils are more active in the antigen-recognizing stage than the adenoids.