A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1).

BACKGROUND: Proinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific pro-inflammatory milieu. METHODS: Peripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation. RESULTS: IDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation. CONCLUSION: IDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells.

Aryltetralin lignans from Hyptis brachiata inhibiting T lymphocyte proliferation.

Increased activation and proliferation of T lymphocytes plays an essential role in the development of chronic inflammation and autoimmune diseases. Currently used immunosuppressive drugs often do not provide long-lasting relief of symptoms and show a gradual loss of efficacy over time, and are accompanied by various side effects. Therefore, novel immunosuppressive lead substances are needed. For this purpose, an in-house library consisting of 600 extracts of plants from Panama was screened for inhibition of human T lymphocyte proliferation. As one of the hits, an ethyl acetate extract from the aerial parts of Hyptis brachiata (Lamiaceae) exhibited strong inhibitory effects. Subsequent investigation resulted in the isolation of seven aryltetralin lignans, five arylnaphthalene lignans, two flavonoids, three triterpenes, and cinnamyl cinnamate. Aryltetralin lignans inhibited T lymphocyte proliferation in a concentration-dependent manner without induction of apoptosis. No relevant inhibition was observed for the arylnaphthalene lignans, flavonoids, and triterpenes. Additional cell cycle arrest investigations revealed that isolated aryltetralin lignans potently inhibited cell division in G2/M phase similarly to podophyllotoxin. Multifluorescence panel analyses of the extract also showed weak suppressive effects on the production of IL-2 and TNF-α. Therefore, preparations made out of H. brachiata could be further explored as an interesting herbal alternative in the treatment of autoimmune diseases.

Immunosuppressant flavonoids from Scutellaria baicalensis.

Some plants used in Traditional Chinese Medicine serve as treatment for disease states where a suppression of the cellular immune response is desired. However, the compounds responsible for the immunosuppressant effects of these plants are not necessarily known. The immunosuppressant compounds in the roots of Scutellaria baicalensis, one of the most promising plants identified in a previous screening, were tracked by HPLC activity profiling and concomitant on-line spectroscopic analysis. Compounds were then isolated by preparative chromatography, and structures elucidated by spectroscopic methods. Twelve flavonoids (5-16) were identified from the active time windows, and structurally related flavones 2, 4, and 17, and flavanones 1 and 3 were isolated from adjacent fractions. All flavonoids possessed an unusual substitution pattern on the B-ring, with an absence of substituents at C-3 and C-4. Compounds 11, 13, 14, and 16 inhibited T-cell proliferation (IC50 values at 12.1-39 µM) at non-cytotoxic concentrations. The findings may support the use of S. baicalensis in disorders where a modulation of the cellular immune response is desirable.

In vitro influence of Theileria annulata on the functions of bovine dendritic cells for stimulation of T lymphocyte proliferation.

The present study was performed on antigen-presenting cells (APCs) of Theileria annulata transformed dendritic cells (TaDCs) and monocyte-derived dendritic cells (MoDCs) to compare differences in antigen presentation and stimulation of T lymphocyte proliferation. Antigen presentation for T lymphocyte proliferation was analysed by flow cytometry. Additionally, the level of mRNA transcription of small GTPases of the Rab family expressed in the TaDC cell line was analysed by quantitative real-time polymerase chain reaction (Q-RT-PCR). The endocytosis rate of TaDCs was significantly (P < 0.01) lower than in MoDCs. In contrast, when T lymphocytes were co-cultured with TaDC-APCs T cell proliferation was similar, while co-culture with MoDC-APC stimulated proliferation of CD4+ cells to a greater degree than CD8+ cells. However, the efficacy of TaDC-APCs to stimulate T lymphocytes dropped as the number of passages of TaDC-APC increased. Likewise, the transcription level of Rab family genes also significantly (P > 0.001) declined with progressive passages (>50) of the TaDC cell line. We conclude that initially the TaDC cell line efficiently presents antigen to stimulate T lymphocyte proliferation to produce a cellular immune response against the presented antigen.

Effect of the melatonin nuclear receptor RORα on monochromatic light-induced T-lymphocyte proliferation in chicken thymus.

Present study clarified role of melatonin nuclear receptor RORα in monochromatic light-induced T-lymphocyte proliferation in chicks. Green light elevated plasma melatonin level and organ index, T-lymphocyte proliferation and IL-2 production in thymus, but decreased RORα, p-P65 and p-IκB expressions relative to red light. By contrast, pinealectomy decreased the melatonin content and reversed the stimulatory effect of green light, and resulted in that these thymus parameters were not significantly different among the light-treated groups. Exogenous melatonin supplementation enhanced T-lymphocyte proliferation and IL-2 production in cultured thymocytes. This stimulatory effect of melatonin was reversed by RORα agonist but was enhanced by RORα antagonist. In contrast to RORα antagonist, RORα agonist decreased cytoplasmic P65 level and increased nuclear P65 level. Supplementation with P65 antagonist increased T-lymphocyte proliferation. We conclude that RORα could negatively regulate green light-enhanced T-lymphocyte proliferation in chick thymus by upregulating IκB phosphorylation, which promotes P65 nuclear translocation and NF-κB activation.

A benzoxazole derivative PO-296 inhibits T lymphocyte proliferation by the JAK3/STAT5 signal pathway.

Immunosuppressants have shown striking achievements in treating autoimmune diseases in recent years. It is urgent to develop more immunosuppressants to provide more options for patients. PO-296 [2-(6-chlorobenzo[d]oxazol-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol] was identified as a novel benzoxazole derivative. We observed that it exhibits an obvious immunosuppressive activity to T lymphocytes. PO-296 significantly inhibited the proliferation of activated human T lymphocyte without cytotoxicity. Moreover, PO-296 did not affect the expression of cluster of differentiation (CD)-25 or CD69 but induced T lymphocyte cycle arrest in the G0/G1 phase. Furthermore, PO-296 inhibited interleukin (IL)-6, IL-17, and interferon gamma expression but had no effect on IL-2, IL-4, or IL-10. Yet, importantly, PO-296 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5), increased the phosphorylation of p70S6K, but did not affect the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mitogen-activated protein kinase pathway. In conclusion, these findings indicate that PO-296 inhibits human activated T-lymphocyte proliferation by affecting the janus kinase 3 (JAK3)/STAT5 pathway. PO-296 possesses a potential lead compound for the design and development of new immunosuppressants for the treatment of autoimmune diseases.

NDV entry into dendritic cells through macropinocytosis and suppression of T lymphocyte proliferation.

Newcastle disease virus (NDV) causes major economic losses in the poultry industry. Previous studies have shown that NDV utilizes different pathways to infect various cells, including dendritic cells (DCs). Here, we demonstrate that NDV gains entry into DCs mainly via macropinocytosis and clathrin-mediated endocytosis. The detection of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-4 (IL-4) and interleukin-10 (IL-10) indicates that NDV significantly induces Th1 responses and lowers Th2 responses. Furthermore, NDV entry into DCs resulted in the upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and cleaved caspase-3 proteins, which in turn activated the extrinsic apoptosis pathway and induced DCs apoptosis. Transwell® co-culture demonstrated that direct contact between live NDV-stimulated DCs and T cells, rather than heated-inactivated NDV, inhibited CD4+ T cell proliferation. Taken together, these findings provide new insights into the mechanism underlying NDV infections, particularly in relation to antigen presentation cells and suppression of T cell proliferation.

In vitro T lymphocyte proliferation by carboxyfluorescein diacetate succinimidyl ester method is helpful in diagnosing and managing primary immunodeficiencies.

BACKGROUND: Functional studies besides routine laboratory tests for the definitive diagnosis of T lymphocyte disorders with isolated T or combined T/B-cell immunodeficiencies are important. We hereby summarized our experience with a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay for the assessment of mitogenic T-cell proliferation responses in primary immunodeficiency (PID) patients who have not been diagnosed yet or genetically analyzed, but classified as probably having T-cell defects. METHODS: Unclassified patients (n=46) and controls (n=25) were evaluated for T-cell disorders with CFSE-based assay. RESULTS: CD3+ blast cells after PHA-L stimulation were significantly lower in patients (31.1±28.8) than controls (67.9±8.79; P<.001). Nine patients with low and four patients with normal CD3 values had severely decreased blastic transformation. The proliferation response decreased mostly in combined immunodeficiency group. Sixteen of them had impaired proliferation responses. Appropriate molecular genetical analyses were planned after thorough evaluation of each patient. CONCLUSIONS: In vitro lymphocyte cell proliferation analysis by CFSE method is a reliable and practical choice for the assessment of mitogenic T lymphocyte responses in yet unclassified PID patients for targeting further genetical analyses.

Impaired Immunosuppressive Effect of Bronchoalveolar Mesenchymal Stem Cells in Hypersensitivity Pneumonitis: Preliminary Findings.

BACKGROUND: Bronchoalveolar mesenchymal stem cells (MSCs) play an important role in the maintenance of lung integrity. Therapeutic application of bone marrow-derived MSCs reduced chronic bronchial inflammation in idiopathic pulmonary fibrosis, and improved the ratio of survivors in sepsis with pneumonia. This study investigated the effect of MSCs from bronchoalveolar lavage fluid (BALF) of hypersensitivity pneumonitis (HP) on T-cell function under in vitro conditions. METHODS: Bronchoalveolar MSCs were obtained via bronchoscopy with BAL from children with severe subacute HP. As control, BALF MSCs were assessed from children without any inflammatory lung disease. Isolated MSCs were characterized via immunophenotyping by flow cytometry and confocal laser scanning microscopy. HP-derived and healthy separated peripheral blood mononuclear cells (PBMCs) were stimulated by 5 µg/mL phytohemagglutinin in the presence of HP-derived or control MSCs in 5-day cultures. Proliferation and activation of T-cells were characterized by the mean fluorescence intensity (MFI) of 5,6-carboxyfluorescein-diacetat succinimidyl ester (CFSE) and CD25, CD69 as well as HLA-DR surface positivities, respectively. RESULTS: HP-derived MSCs showed significantly lower level of CD73, CD90, and CD105 expression compared to control MSCs in both flow cytometric and confocal microscopic experiments. MSCs from HP did not reduce T-cell proliferation based on CFSE MFI values, while the level of CD25 expression on both control and HP-derived CD4+ and CD8+ T-cells was significantly reduced by normal MSCs, while HP-derived MSCs did not have any significant effect. The level of other activation markers was not markedly modulated by MSCs. CONCLUSIONS: BALF MSCs from HP are unable to downregulate the proliferation and activation of T-cells that may support the development of recurrent intrapulmonary inflammation in HP. © 2016 International Clinical Cytometry Society.

Physiological crosstalk between the AC/PKA and PLC/PKC pathways modulates melatonin-mediated, monochromatic-light-induced proliferation of T-lymphocytes in chickens.

Previous study has demonstrated that melatonin plays a critical role in monochromatic-light-induced lymphocyte proliferation in response to T cell mitogen concanavalin A (ConA). However, its intracellular mechanism is still unclear. In this study, we investigate the intracellular signal pathways of melatonin receptor-mediated T-lymphocyte proliferation in the spleens of chicks exposed to different light wavelengths. Results showed that green light enhanced T-lymphocyte proliferation by 2.46-6.83% and increased splenic mRNA and protein expressions of melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) by 16.05-40.43% compared with the white, red and blue light groups. However, pinealectomy resulted in a decrease in T-lymphocyte proliferation and melatonin receptor expression with no statistically significant differences between the different light groups. In vitro experiments showed that the Mel1b selective antagonist 4P-PDOT, the Mel1c selective antagonist prazosin and the mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 suppressed both melatonin-induced lymphocyte proliferation in response to ConA and melatonin- and ConA-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) activity but that the Mel1a/Mel1b non-selective antagonist luzindole did not. In addition, pretreatment with forskolin (FSK, the adenylyl cyclase activator), H89 (the PKA inhibitor), U73122 (the PLC inhibitor) or Go6983 (the broad spectrum PKC inhibitor) markedly attenuated melatonin- and ConA-stimulated T-lymphocyte proliferation and ERK1/2 activity. These results demonstrate that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways followed by ERK1/2 activation.

Staphylococcus enterotoxin profile of China isolates and the superantigenicity of some novel enterotoxins.

The genus of staphylococcus widely distributes in environments and contributes to a variety of animal and human diseases. The enterotoxins (SEs) secreted by this type of pathogen have been the leading cause of bacterial toxic shock syndrome and food poisoning, and thus present a substantial concern to public health. In this study, we analyzed the superantigen profile of 122 staphylococcus strains isolated from diverse sources. When screened for the presence and prevalence of 17 known se or se-like (sel) genes, except selj, all other genes were detected in these isolates. In particular, 95.9% of the isolates harbored at least one se/sel gene. Moreover, 47.5% of them bore at least 5. Remarkably, several non-pathogenic species of animal- and environment-origin were also found to carry multiple se/sels. The most frequent genes detected were tsst (62.3%), sei (54.1%), and seb (46.7%), followed by some sel genes (selo, selu, and selm), which also were present at relatively high frequency (20-30%). The generated data improved understanding of strain-specific differences in enterotoxin expression. The gene products of the latter (selo and selu) were subsequently analyzed for their antigenicity in a mouse model using purified E. coli-based recombinant proteins. The studies revealed a strong activity for SEO in induction of T-lymphocyte proliferation and production of various inflammatory cytokines either in vivo or in vitro. In contrast, SEU exhibited little superantigenic effects. The molecular basis for the difference in antigenicity was analyzed by 3D homology remodeling, which revealed a difference in binding and affinities for MHC-II molecules and TCR Vß region.

Effects of dexmedetomidine on cellular immune function of immunosuppression juvenile rats / 临床麻醉学杂志

Objective To explore the effects of dexmedetomidine on peripheral blood T lymphocyte proliferation and T lymphocyte subsets of juvenile rats with splenectomy.Methods Twenty-four healthy male Sprague-Dawley rats,weighing 130-150 g,aged six weeks were enrolled in this study.Half of the rats received splenectomy to make an immunosuppressive model,then they were randomly divided into 2 groups (n=6 each): splenectomy+normal saline group (group SN) and splenectomy+dexmedetomidine group(group SD).The another half of the rats without splenectomy were randomly divided into 2 groups: normal saline group(group S) and dexmedetomidine group(group D).After one week of normal feeding,normal saline 10 ml/kg was injected intraperitoneally (ip) in groups S and SN,dexmedetomidine 50 μg/kg was injected ip in groups D and SD respectively.Two hours after the injection,blood samples were collected.MTT was utilized to examine the peripheral blood T lymphocyte proliferative capability.T lymphocyte subsets CD4+,CD8+ were determined by flow cytometry.CD4+/CD8+ was calculated.Results Compared with group S,T lymphocyte proliferative capability,the percentages CD4+,CD8+ and CD4+/CD8+ ratio were significantly decreased in group SN (P<0.05);T lymphocyte proliferative capability in group D was decreased (P<0.05),but no significant changes was found in the percentages CD4+,CD8+ and CD4+/CD8+ ratio.Compared with the group D,T lymphocyte proliferative capability,the percentages CD4+,CD8+ and CD4+/CD8+ ratio in group SD were significantly decreased (P<0.05).Compared with the group SN,T lymphocyte proliferative capability in group SD was significantly decreased (P<0.05).Conclusion Cellular immune function of juvenile rats with or without splenectomy is suppressed by dexmedetomidine,and the suppressive function is more severe in splenectomy rats than that in normal juvenile rats.

Effects of exercise on spleen T lymphocyte proliferation and T-lymphocyte subsets of SD rats / 中国免疫学杂志

Objective:To explore the effects of exercise on the spleen T lymphocyte proliferation and T lymphocyte subsets of SD rats.Methods:24 SD rats were divided into 3 groups randomly:control group,30 min exercise group,60 min exercise group;MTT and flow cytometry ( FCM ) were utilized to examine the spleen T lymphocyte proliferative capability and T lymphocyte subsets, respectively.Results:Compared with the control group,the T lymphocyte proliferation,levels of CD4+and CD4+/CD8+T lymphocytes in 30 min exercise group rats were significantly increased(P<0.05),levels of CD8+T lymphocytes in 30 min exercise group rats had no statistically significant change;the T lymphocyte proliferation,levels of CD4+,CD8+and CD4+/CD8+T lymphocytes in 60 min exercise group rats had no statistically significant changes.Conclusion: Suitable loaded exercise could enhance the cellular immune function, this is probably related with the mechanisms of improving the T lymphocyte proliferative capability and regulating the CD4+/CD8+T lymphocyte subsets.

Effects of peripheral blood derived dendritic cells on T cell activity in colorectal cancer patients / 中国实用内科杂志

0.05).The stimulative index(SI)tested by MTT in MLR at stimulator-to-reactor cell ratio 1∶1,1∶10,1∶20,1∶40 and 1∶80 was 2.7,4.1,3.1,2.5 and 1.6,respectively.The proliferation of T cells stimulated by DCs could be detected at all stimulator-to-reactor ratio.Conclusion DCs could be successfully induced from peripheral blood monocyte in the patients with colorectal cancer by AIM-V serum-free media or by traditional FBS-contained media.AIM-V is more suitable for clinical immunotherapy because it can avoid the risk of allergic reaction for clinical purpose.AIM-V might take the place of serum-contained media to culture DCs.According to the results of MLR,DCs could stimulate allogenetic T cells proliferation at all stimulator-to-effector ratio,furthmore the optimal ratio is 1∶10 .