No abstract available.
Allografts* ; Animals ; Blood Transfusion* ; Cyclosporine* ; Lymphocyte Culture Test, Mixed* ; Rats*

Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-PD-L1 Abs, have shown efficacy for the treatment of various cancers. Although research has actively sought to develop new ICIs and immunomodulators, no efficient in vitro assay system is available to evaluate their functional activities. In the present study, we established a two-round MLR with human PBMCs for evaluation of the T cell-activating capacity of anti-PD-1 and other immunomodulators. We initially performed conventional MLR for this purpose. However, anti-PD-1 blocking Abs could not increase the proliferation of allo-reactive T cells in conventional MLR because PD-L1+ and PD-L2+ cells disappeared gradually during MLR. Therefore, we re-applied the same stimulator PBMCs to the allo-stimulated responder cells as a second-round MLR on day 6 when anti-PD-1 or immunomodulators were also added. In this two-round MLR, the proliferation of allo-reactive T cells was enhanced by anti-PD-1 in a dose-dependent manner or by immunomodulators, such as lenalidomide and galunisertib, a TGF-β receptor-1 inhibitor. Proliferation was further increased by the combination of immunomodulators with anti-PD-1. Here, we established a modified two-round MLR method with human PBMCs for evaluation of the functional activities of anti-PD-1 and immunomodulators.
Humans ; Immunologic Factors* ; In Vitro Techniques ; Lymphocyte Culture Test, Mixed* ; Methods ; T-Lymphocytes

OBJECTIVE: To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy. METHODS: Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay. RESULTS: Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner. CONCLUSION An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.
Cell Culture Techniques ; Cell Separation ; Cells, Cultured ; Humans ; Interleukin-2 ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Regulatory ; cytology

We studied host immune parameters which might be related to the activity and the pathogenetic mechanism of chronic active hepatitis. The subjects consisted of 45 cases with hepatitis B virus surface antigen (HBsAg)-positive chronic active hepatitis (CAH), 44 HBsAg-negative CAH, 22 with inactive chronic hepatitis, and 45 cases of normal persons, hepatitis B virus (HBV) carriers, or the patients with acute myocardial infarction. The in vitro assay for the in vivo activated lymphocytes was performed by measuring spontaneous thymidine uptake (SLT) of lymphocytes isolated from peripheral blood. SLT was significantly (p less than 0.001) elevated in cases with HBsAg-positive (1227 +/- 806 cpm) and-negative CAH (1017 +/- 559 cpm) compared to the patients with inactive chronic hepatitis (347 +/- 79 cpm) and to the control group (320 +/- 106 cpm). SLT values observed in 7 cases with active disease (group I and II), in which remission and relapsing phase could be assessable, were elevated from 648 +/- 121 cpm in remission phase to 1548 +/- 606 cpm one to two weeks before the appearance of biochemical evidence (SGPT) of relapse. This pattern of SLT elevation, however, was not observed in patients with inactive hepatitis. Neither the abnormal distribution of T-celi subsets nor the presence of conventional HBV markers were related to the elevated SLT value. Our findings may therefore indicate that SLT might be useful in assessment of the disease activity in patients with CAH.
Adult ; Alanine Transaminase/blood ; Female ; Hepatitis, Chronic/*immunology ; Humans ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Male ; Middle Aged ; T-Lymphocyte Subsets/immunology

Objective：To investigate the promotive effect of dendritic cells(DCs) on proliferation of CRTH2 (CD4(+)CD294(+)Th2) cells and the influence of CRTH2 cells on secretion of immunoglobulin from B cells so as to provide a new approach for amplification and sorting of Th2 cells. Methods：DCs were induced from peripheral blood mononuclear cells, then the loaded-BCGV-Ag-DCs were cocultured with T cells, and the mixed lymphocyte reaction(MLR) was performed by CCK8 method. The phenotypes of DCs and CRTH2 cells were detected by flow cytometry. CRTH2 cells sorted by MACS were co-cultured with B cells for 5 days to detect the secretion of immunoglobulin. Results：The subsets and absolute number CRTH2 cells were significantly increased by loaded-BCGV-Ag-DCs. The levels of IgG, IgA and IgE were higher increased in supernatant of CRTH2 and B cell co-culture system than that in control group or that in transwell group(P<0.05). Conclusion：The proliferation of CRTH2 cells can be greatly promoted by loaded-BCGV-Ag-DCs, and the CRTH2 cells can help B cells to secrete IgG, IgA and IgE.
B-Lymphocytes ; Cell Proliferation ; Coculture Techniques ; Dendritic Cells ; Flow Cytometry ; Humans ; Immunoglobulins ; Lymphocyte Culture Test, Mixed ; Th2 Cells

Jeotgal (salted seafood) has been one of major fermented foods in Korea for long time. Although there are many studies about Jeotgal in various aspects of food, its immunological importance on hosts has not been elucidated yet. In this study, we investigated if several bacteria isolated from Jeotgal may modulate the function of dendritic cells (DCs), powerful antigen-presenting cells equipped with special immunological capabilities. 4 Jeotgal bacteria were selected as representatives and used for experiments. To treat viable DCs, those bacteria were killed at 60degrees C for 30 min. The viability of DCs treated with Jeotgal bacteria was verified and two isolates significantly induced high production of interleukin-12, a representative cell-mediated cytokine of DCs. Surface activation and maturation markers (MHC class II, CD40, CD86) of DCs were analyzed by flow cytometer. In addition, the treated DCs showed significantly high lymphocyte stimulatory capability compared to control DCs based on allogeneic mixed lymphocyte reactions. These observations suggest that Jeotgal isolates can function as immunostimulating bacteria in hosts, like Lactobacillus. Taken together, these experimental evidences may broaden the use of Jeotgal isolates in immunological fields in addition to as a fermented food.
Animals ; Antigen-Presenting Cells ; Bacteria ; Dendritic Cells* ; Immunization ; Interleukin-12 ; Korea ; Lactobacillus ; Lymphocyte Culture Test, Mixed ; Lymphocytes ; Mice* ; Probiotics

Annexin-1 (ANX1) is a 37 kDa protein that is induced and secreted by glucocorticosteroid hormone. The secreted ANX1 has been believed to exert its function by binding to its putative rnembrane receptor. In this report we demonstrate that ANXl receptor (ANX1R) signal blocks the interleukin-1B (IL-1B) receptor signal pathway in human peripheral blood mononuclear cells (PBMCs). When PBMCs were treated with both IL-1B (100 ng/ml) and PMA (10 ng/ml) in the absence or presence of dexamethasone for 5 days, dexamethasone (100 nM) suppressed lymphocyte proliferation to 24% of the control. However addition of anti-ANX1 polyclonal antibody of 1:200 and 1:1,000 dilution to this system induced recovery of proliferation to 80% and 40%, respectively, when compared to the control. In the mixed lymphocyte reaction, dexamethasone suppressed lymphocyte proliferation to 9% of that of control when stimulated with IL-1B (100 ng/ml) and phorbol myristate acetate (10 ng/ml). Addition of anti-ANX1 polyclonal antibody (1:1,000) to this system also recovered the proliferation to 20% of that of the control system. In the ANX1 receptor induction experiment using flow cytometry, ANX1 receptor expression on lymphocytes, CD4+ T cells, CD8+ T cells and monocytes increased depending on the externally added IL-1B ranging from 10 to 1,000 ng/ml. From these results, it is evident that dexamethasone induces ANX1 secretion into the culture medium and anti-ANX1 polyclonal antibody abolishes the effects of dexamethasone. Furthermore these results imply that extracellular ANX1 exerts its effects by binding to the receptor on the cell membrane and the activated signal(s) of ANX1R block IL-1B receptor signal in the lymphocytes.
Cell Membrane ; Dexamethasone ; Flow Cytometry ; Humans ; Interleukin-1* ; Lymphocyte Culture Test, Mixed ; Lymphocytes* ; Monocytes ; Signal Transduction ; T-Lymphocytes ; Tetradecanoylphorbol Acetate*

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.
Acetylcholine ; Autoimmune Diseases ; Humans ; Immunosuppression ; Inflammation ; Lymphocyte Culture Test, Mixed ; Lymphocytes ; Mesenchymal Stromal Cells ; Methods ; Phenotype ; Receptors, Nicotinic

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.
Bone Marrow* ; Dendritic Cells ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocytes* ; Immunosuppression ; Lymphocyte Culture Test, Mixed ; Major Histocompatibility Complex ; T-Lymphocytes

OBJECTIVETo investigate the effect of SAHA on the maturation of human dendritic cells (DC) and to explore its underlying mechanism.

METHODSPeripheral blood mononuclear cells (PBMNC) were isolated from human peripheral blood and cultured in RPMI 1640 medium with 100 ng/ml rhGM-CSF and 500 U/ml rhIL-4. In the LPS induced maturation process, dendritic cells treated with or without SAHA were used as test group, and dendritic cells treated without LPS or SAHA were used as control group. DC was observed under inverted microscope. Flow cytometer was used to detect the surface antigen molecules expressed by DC. The mixed lymphocyte culture (MLC) was used to observe the allogeneic lymphocyte stimulation. The NF-κB signaling pathway was detected by electrophoretic mobility shift assay (EMSA).

RESULTSThe SAHA could effectively suppress the maturation of DC induced by LPS, the DC treated with SAHA+LPS had immature morphological characteristics; the expression of CD80, CD83 and HLA-DR in SAHA+LPS group and control group were significantly down-regulated as compared with single LPS group (P<0.01); the ability of DC to stimulate the proliferation of allogeneic T lymphocytes in SAHA+LPS group and control group was significantly weaker than that in single LPS group (P<0.01); EMSA results showed that NF-κB activity decreased after SAHA and LPS treatment and was significantly lower than that of single LPS group.

CONCLUSIONSAHA can effectively suppress DC maturation induced by LPS and also weaken the ability to stimulate allogeneic T lymphocyte. NF-κB signaling pathway is involved in regulating DC maturation.