Yan Ge

A novel approach to induce human DCs from monocytes by triggering 4-1BBL reverse signaling

Dendritic cells (DCs) are responsible for the initiation of immune responses. Our study demonstrates a new pathway for generating a large quantity of stimulatory monocyte-derived DCs (Mo-DCs) from human monocytes using anti-4-1BB ligand (4-1BBL) mAb to trigger reverse signaling. The anti-4-1BBL-driven Mo-DCs (DCs(alpha-4-1BBL)) not only express higher levels of CD86, CD83 and HLA-DR, when compared with the Mo-DCs matured by tumor necrosis factor alpha, but also exhibit a unique phenotype that expresses lower levels of PD-L1. High levels of GM-CSF, M-CSF and Flt3 ligand (FL) were found in the anti-4-1BBL-differentiation culture. Neutralizing M-CSF, GM-CSF and FL inhibited Mo-DC proliferation stimulated by anti-4-1BBL mAb, suggesting that M-CSF, GM-CSF and FL are involved in cell proliferation stimulated by anti-4-1BBL. Further analysis of the DCs(alpha-4-1BBL) showed increased secretion of T(h)1-type cytokines IL-12 and IFN-gamma and decreased secretion of IL-10. DCs(alpha-4-1BBL) induced much stronger proliferative responses in the mixed lymphocyte reaction assay when compared with DCs derived by GM-CSF. Moreover, DCs(alpha-4-1BBL) preferentially induced T(h)1 responses. We have further demonstrated that anti-4-1BBL antibody stimulated nuclear translocation of NF-kappaB from the cytoplasm in monocytes, suggesting that reverse signaling by 4-1BBL is likely responsible for mediating DC differentiation. Collectively, we have found that reverse signaling of 4-1BBL promotes the differentiation of potent T(h)1-inducing DCs from human monocytes.

Blockade of PD-1/PD-L1 immune checkpoint during DC vaccination induces potent protective immunity against breast cancer in hu-SCID mice

Immune checkpoints, such as the PD-1/PD-L1 pathway, negatively interfere in the efficiency of dendritic cell (DC) vaccination in cancer immunotherapy. In this study, we demonstrated that the blockade of PD-L1 signaling could promote DC maturation, proliferation, and IL-12 secretion, augment DC primed T cell response and reverse tumor cell dampened T cell impairment. Blockade of PD-L1 signaling during DC vaccination showed better therapeutic effects than classic DC vaccination by preventing tumor growth and prolonging survival times in a breast tumor-bearing hu-SCID model. Taken together, suppressing immune checkpoints during DC vaccination might be a more efficient strategy for cancer therapy.

CD13+CD4+CD25hi regulatory T cells exhibit higher suppressive function and increase with tumor stage in non-small cell lung cancer patients.

Regulatory T cells (Tregs) in peripheral blood and tumor infiltrating lymphocytes (TILs) play crucial roles in suppressing anti-tumor immune responses in cancer patients, and correlate with clinical outcomes. We identified an important subpopulation, CD13+CD4+CD25hiTreg cells, among CD4+CD25hiTreg cells in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with NSCLC (n=72) or from healthy donors (n=30). Flow cytometric analyses were performed to study the expression of cell-surface or intracellular markers on the CD4+CD25hiTreg cells. The immune suppressive function of CD13+CD4+CD25hiTreg cells was evaluated by co-culturing with CD4+CD25-T cells that were activated by PHA. Our data showed that, compared with CD4+CD25Low/-T cells, CD13 expression was enriched on CD4+CD25hiTreg cells. The CD13+CD4+CD25hiTreg cells also expressed higher levels of Foxp3, CTLA-4, membrane-bound transforming growth factor β1 (mTGF-β1) and B7-H1, and are more suppressive to CD25 expression and proliferation of CD4+CD25-T cells. Additionally, we showed that the expression of Foxp3, CTLA-4, B7-H1, mTGF-β1 and the secretion of TGF-β1 and IL-10 on CD13+CD4+CD25hiTreg cells was significantly suppressed by anti-CD13 mAb (WM15), and the ability of these cells to suppress CD25 expression and proliferation of CD4+CD25-T cells was inhibited by WM15 as well. Interestingly, the percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg population increased significantly and correlated with pathological stage in NSCLC: healthy donor (9.84%±2.23%) < stage I (21.64%±2.78%) < stage II (31.86%±3.01%) < stage III (45.64%±6.12%) < stage IV (58.78%±12.89%). Moreover, the percentage of CD13+CD4+CD25hiTreg cells decreased dramatically after surgical removal of tumors. CD13 is a new surface molecule for identifying a CD4+CD25hiTreg cell subpopulation with higher suppressive ability. The percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg cell population correlated with the pathological stage in NSCLC and tumor burden. CD13 represents a potential target to suppress Treg cells in anti-tumor therapy.

Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro.

AbstractAim:To explore the biofunctions of human B7-H4 generated from prokaryotic system.Methods:The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [3H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA.Results:We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion.Conclusion:The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.

Fine-Tuned Expression of Programmed Death 1 Ligands in Mature Dendritic Cells Stimulated by CD40 Ligand Is Critical for the Induction of an Efficient Tumor Specific Immune Response

During maturation, murine myeloid dendritic cells (DCs) upregulated the expressions of CD11c, CD25, CD40, CD80, CD86, MHC II and programmed death 1 ligands 1 and 2 (PD-L1 and PD-L2). Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-α. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-α stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-ligated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation.

CD40-activated apoptotic tumor cell-pulsed dendritic cell could potentially elicit antitumor immune response: involvement of up-regulation of B7-H3 expression.

Dendritic cells (DCs) initiate and direct immune responses. Previous in vitro and in vivo studies have showed that DCs matured with CD40 linking signal could potentially elicit and boost antitumor immunity, however, its molecular mechanism remain elusive. This study demonstrates that expression of B7-H3 on apoptotic cell-loading DCs is up-regulated markedly by CD40 activation but not by tumor necrosis factor-α stimulation. There was no significant difference found with CD40, CD80, or CD86 expressions when activated by CD40 or tumor necrosis factor-α stimulation. In tumor-bearing mice, T cells conditioned with B7-H3–blocked on CD40-activated apoptotic tumor cell-pulsed DCs had a decreased ability to inhibit tumor growth. Therefore, it is hypothesized that high levels of B7-H3 expression contributes to the ability of CD40-activated tumor associated DCs in eliciting efficient antitumor immune response, given this fact the potentially significant clinical implications, CD40-activated DCs merit further considerations when preparing DCs for clinical application.

Dietary quercetin ameliorates experimental colitis in mouse by remodeling the function of colonic macrophages via a heme oxygenase-1-dependent pathway

ABSTRACT Inflammatory bowel disease (IBD) results from a chronic intestinal inflammation and tissue destruction via an aberrant immune-driven inflammatory response towards an altered gut microbiota. Dietary intervention is becoming an attractive avenue for the therapy of colitis because diet is a key determinant of the mucosal immune response. Quercetin (QCN) is the most common in nature and the major representative of dietary antioxidant flavonoids, which has been demonstrated to influence the progression of colitis. However, the underlying mechanism of QCN on intestinal immunomodulation remains unclear. Here, our study demonstrated dietary QCN could ameliorate experimental colitis in part by modulating the anti-inflammatory effects and bactericidal capacity of macrophages via Heme oxygenase-1 (Hmox1, HO-1) dependent pathway. It suggested that QCN might restore the proper intestinal host-microbe relationship to ameliorate the colitis via rebalancing the pro-inflammatory, anti-inflammatory and bactericidal function of enteric macrophages. Hence, modulating the function of intestinal macrophages with dietary administration of QCN to restore the immunological hemostasis and rebalance the enteric commensal flora is a potential and promising strategy for IBD therapy.

An Anti-Human CD13 Monoclonal Antibody That Suppresses the Suppressive Function of Treg Cells

CD13 (CD13/aminopeptidase N, APN, or CD13/APN) is a widely expressed type II membrane-bound metalloprotease. It is often overexpressed on cancer cells and expressed on CD4(+)CD25(hi) Treg cell subpopulation with higher suppressive ability. It has been determined to be a promising target in cancer diagnosis and therapy. In this study, a functional anti-human CD13 monoclonal antibody, MAb 9E4, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the CD13 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 9E4 suppresses the suppressive function of Treg cells. This functional anti-human CD13 MAb provides a valuable tool for further study targeting the CD13 positive Treg cells.

Directional delivery of RSPO1 by mesenchymal stem cells ameliorates radiation-induced intestinal injury.

HighlightsMSCs exhibit a negative immunoregulatory phenotype.MSCs directionally migrate to the radiation‐damaged intestinal epithelium.RSPO1 is critical for the survival and proliferation of intestinal crypts.Directional delivery of RSPO1 by MSCs promotes ISCs survival following irradiation.Directional delivery of RSPO1 by MSCs rescues the abdominal irradiated mice. Abstract Radiation‐induced intestinal injury (RIII) commonly occurs in patients who received radiotherapy for pelvic or abdominal cancer, or who suffered from whole‐body irradiation during a nuclear accident. RIII can lead to intestinal disorders and even death given its integrity damage that results from intestinal stem cell (ISC) loss. Recovery from RIII relies on the intensity of supportive treatment, which can attenuate lethal infection and give surviving stem cells an opportunity to regenerate. It has been reported that RSPO1 is a cytokine with potent and specific proliferative effects on intestinal crypt cells. MSCs have multiple RIII‐healing effects, including anti‐inflammatory and anti‐irradiation injury properties, due to its negative immune regulation and its homing ability to the damaged intestinal epithelia. To combine the comprehensive anti‐injury potential of MSCs, and the potent ability of RSPO1 as a mitogenic factor for ISCs, we constructed RSPO1‐modified C3H10 T1/2 cells and expected that RSPO1, the ISC‐proliferative cytokine, could be delivered to the site of injury in a targeted manner. In this study, we transferred C3H10/RSPO1 intravenously via the retro‐orbital sinus into mice suffering from abdominal irradiation at lethal dosages. Our findings demonstrated that C3H10/RSPO1 cells are able to directionally migrate to the injury site; enhance ISC survival, proliferation, and differentiation; and effectively repair the radiation‐damaged intestinal epithelial cells. This study suggests that the directional delivery of RSPO1 by MSCs is a promising strategy to ameliorate, and even cure, RIII.

Immune checkpoint molecule PD-1 acts as a novel biomarker for the pathological process of gestational diabetes mellitus

AIMnAccumulating evidence suggested that challenge of the maternal-fetal interaction during pregnancy might cause the impairment of immunological hemostasis and lead to gestational diabetes mellitus (GDM) pathological process. Immune checkpoint molecule PD-1 is one of the critical molecule balancing immune response and immunological tolerance.nnnMETHODSnPD-1 expressions on T-cell subsets of GDM patients and control groups were measured via flow cytometric analysis and followed up.nnnRESULTSnDownregulation of PD-1 acted as an indicator for GDM occurrence in the third trimester of pregnancy. With the recovery of GDM, PD-1 expression restored to normal level.nnnCONCLUSIONnPD-1 expression on T-cell subsets is a novel biomarker for the occurrence and recovery of GDM.