Immune Derangements in Patients with Myelofibrosis- the Role of Treg, Th17, and sIL2α

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1763-1763
Author(s):  
Hemant Sindhu ◽  
Ajay Kundra ◽  
Chi Chen ◽  
Jen-Chin Wang
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Th17 Cells ◽  
Interleukin 2 ◽  
Treg Cells ◽  
Cd4 Cells ◽  
Treg Function ◽  
Significant Difference ◽  
Normal Controls

Abstract Abstract 1763 Primary myelofibrosis (PMF), myelofibrosis post essential thrombocytosis (MF-ET), and myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomena, such as Coombs positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex, etc. Regulatory T cells (Treg) and IL-17 producing cells (Th17) have been known to play important roles in modulating immune responses. Hence we studied Treg cells in 38 patients with MF including PMF (25), MF-ET (8), MF-PV (5) and compared them to other MPD patients including ET (7), PV (14), and normal controls (16). Patients on lenalidomide or Pomalidomide were excluded. Blood ( 106 MNCs) were stained for flow cytometry analysis using the Treg Detection Kit (Miltenyi Biotec). The number of Treg cells was calculated as the percentage of positive CD4+ CD25+ FoxP3+ T cells from the number of gated CD4+ cells. Treg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of Treg to T-effector cells at 1:1 in the presence of anti-CD3 and CD28 micro-beads (Invitrogen). The results (mean + SE) showed numbers of Treg cells in MF were 0.79 + 0.080, other MPD were 1.27 + 0.20, and normal controls were 1.21 + 0.30. No significant difference was found among the three groups. Treg function was evaluated in MF (18 patients). MPD (21) and controls (16). No significant difference was found among the three groups. Th17 cell assays were performed by culturing Blood CD4+ cells in IMDM medium and stimulated with PMA (25ng/ml), ionomycin (1 ug/ml ) and monensin ( 500ng/ml) for 4h at 37°C with 5% CO2. Then Th17 was stained with Th17 Flow Kit (Biolegend) and analyzed by flow cytometry. The results were expressed as % of isolated CD4+ cells (mean ± SE) as follows: MF (2.31 ± 0.65) (n=15), MPD (1.31 ± 0.32) (n=7), and controls (1.89 ± 0.44) (n=10). No significant difference was found among the three groups. We further studied the soluble interleukin 2 alpha subunit (sIL2α) levels. sIL2 α were measured in plasma by ELISA kits, results were expressed as (mean± SE ) ( pg/ml) as follows: MF ( 3534± 298) (n=18), MPD ( 2303 ± 171) (n=22) and controls ( 1734 ± 115) (n=16), P values were <0.05 in MF vs MPD and MF vs Controls, and P=NS in MPD Vs controls. These results confirm our previous observation (Br J Haematol,1994), that sIL2 α levels were significantly elevated in patients with MF, compared with MPD patients and controls. We further studied the effects of the sIL2α on the immune function in MF patients: A) Effects on the Th1, Th17, and Treg cells. CD4+ cells after isolation were cultured for 7 days in IMDM containing IL-2 (100ug/ml) and anti- CD3CD28 micro-beads, with or without recombinant sIL-2Rα (100ng/ml). The cells then were stimulated with PMA (20ng/ml) ionomycin (1ug/ml) and monensin (1uM) for 4 hours before harvest. Then Th1 Th17 and Treg were numerated by flow cytometry. The results showed no difference in Th1 and Th17 cells in cultures with and without sIL2α, but sIL2α significantly increased the numbers of T reg (1.71 +.28,P=0.02) ( fold increase). B) Effects on the Treg function. Viable CD4+CD25− (106) cells were labeled with CFSE ( Invitrogen), then added unlabeled Tregs at 1:1 (responder: Treg) ratio and stimulated with 50 μl of anti CD3/CD28 micro-beads with and without sIL2α for 4 –7days at 37 °C in a 5% CO2 incubator. Cell proliferation was measured by counting the percentage of CFSEdim cells. The results showed that sIL2α significantly increased the CFSE dim cells. Therefore sIL2α suppressd the T reg cell function and increase the T responsive cell proliferation. C) Identification of cells producing sIL2α. CD4+, CD14+, CD8+, CD20+, and Treg were isolated using isolation kits (Miltenyi ), then cultured either with PHA or anti CD3/CD28 micro-beads. ELISA assay for sIL2α were then performed on the cultured supernatant. The results (mean ± SE) ( pg/ml) showed that CD4+ cells produced 342 ±152.6; CD8 +,71.6 ± 19.8; Treg, 306.9 ± 53.5, while CD14+ and CD20+ cells produced negligible quantity of sIL2α. Thus Treg cells were the cells predominantly producing sIL2α in patients with MF. We conclude that in patients with MF, numbers of Treg and Th17 cells were not different from controls or other MPD patients, but Treg cells produce significantly increased amount of sIL2α which further inhibits Treg function and results in autoimmune phenomenon observed in patients with MF. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3464-3464
Author(s):  
Yang Song ◽  
Yu-tong Wang ◽  
Xiao-jun Huang ◽  
Yuan Kong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Perivascular Cells ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

scholarly journals Melatonin, an Endogenous Hormone, Modulates Th17 Cells via the Reactive Oxygen Species/TXNIP/HIF-1α Axis to Alleviate Autoimmune Uveitis

2022 ◽  
Author(s):  
Dan Liang ◽  
Jun Huang ◽  
Zhuang Li ◽  
Yunwei Hu ◽  
Zuoyi Li ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Flow Cytometry ◽  
T Cells ◽  
Th17 Cells ◽  
Reactive Oxygen ◽  
Treg Cells ◽  
Oxygen Species ◽  
T Helper ◽  
Autoimmune Uveitis

Abstract Background Melatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis. Methods EAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1-20 (IRBP1−20). melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were employed to evaluate the disease progression. T lymphocytes accumulation, the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR. In vivo and in vitro experiments, T helper 1 (Th1), T helper 17 (Th17) and regulatory T (Treg) cells were detected via flow cytometry, the level reactive oxygen species(ROS) from CD4+ cells were tested via flow cytometry, and the expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α)proteins were also quantified via western blot analysis, to elucidate the mechanism of melatonin inhibiting EAU. Results Melatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells and potentiated Treg cells by suppressing their transcription factors. In vitro studies corroborated that melatonin restrains the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU. Conclusions Melatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

scholarly journals Plasticity of Treg and imbalance of Treg/Th17 cells in patients with systemic sclerosis modified by FK506

2021 ◽  
Vol 35 ◽  
pp. 205873842199808
Author(s):  
Xinjuan Liu ◽  
Yu Wu ◽  
Mengtao Li ◽  
Jianyu Hao ◽  
Qian Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Systemic Sclerosis ◽  
Th17 Cells ◽  
Treg Cells ◽  
Proliferation Capacity ◽  
Healthy Control ◽  
Immune Imbalance

To determine the effects of Tacrolimus (FK506) on Treg cells and subpopulations in SSc patients and assess the ability of FK506 to modify the immune imbalance of Treg/Th17 cells. We analyzed PBMC from five SSc patients and six healthy control by flow cytometry after cultured with 0, 0.1, 1, or 10 ng/ml FK506 in vitro. The number of Treg cells decreased in SSc patients treated with FK506. The number of FrI cells were decreased in SSc following FK506 treatment. The drug did increase the frequency of FrII/Treg cells, but not FrII cells. However, FK506 significantly decreased FrIII in both SSc patients and controls. FK506 clearly decreased the numbers of Th17 cells and FoxP3+IL-17+ cells. The proliferation capacity of cells was also inhibited by FK506, which had a greater effect on FoxP3− cells than FoxP3+ cells. FK506 did inhibit the proliferation of FrIII cells, but not FrI or FrII cells. Our study provides that FK506 reduced the number of FoxP3low CD45RA− T cells (FrIII) by inhibiting its proliferation. Therefore, FK506 modifies Treg cells and the immune imbalance between Tregs and Th17 cells in SSc patients.

scholarly journals Study on Quantity and Function of Regulatory T Cell Subsets in Multiple Myeloma and MGUS

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3154-3154
Author(s):  
Jinuo Wang ◽  
Jian Li ◽  
Xinxin Cao ◽  
Hao Cai ◽  
Ai-lin Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Healthy Controls ◽  
Newly Diagnosed ◽  
Inhibition Rate ◽  
Significant Difference ◽  
Normal Controls

Abstract Introduction Almost all multiple myeloma (MM) cases were progressed from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). So far, the pathogenesis of myeloma is not yet clear. The immune cells in the tumor microenvironment, such as regulatory T (Treg) cells with a unique immunosuppressive function, play an important role in myelomagenesis. Although there have been reports on Treg cells in MM patients, the results were still in debate. In this study, we performed a comprehensive analysis of peripheral blood (PB) and bone marrow (BM) Treg subsets and aging Treg-like cells in untreated MM patients and individuals with MGUS, which might help further elucidate mechanisms of immune dysfunction during myelomagenesis. Methods Our study included 20 MGUS patients and 26 newly diagnosed MM patients. Flow cytometry was applied to determine the proportion of Treg cell subsets and aging Treg-like cells in PB and BM. Flow sorting technology was used to separate Treg cell subsets and effector T cells in the bone marrow of newly diagnosed MM patients. The inhibitory function was indirectly calculated by detecting proliferation rate of CFSE-labelled effective T cells which were cocultured with different Treg cell subsets. Concentration of IL-10 from the culture supernatants of proliferation assay was measured using ELISA. Results In PB, the proportion of activated Tregs (aTregs, CD4+CD45RA-FoxP3hi) in CD4+ T cells was significantly higher in MGUS and untreated MM patients than healthy controls (P=0.01, P<0.001); there was no difference in the proportion of resting Tregs (rTregs, CD4+CD45RA+FoxP3lo) between MGUS and untreated MM patients compared with healthy adults (P=0.72, P=0.07). There was also no significant difference in the frequencies of non-Tregs (CD4+CD45RA-FoxP3lo) from MGUS and MM patients with normal controls (P=0.22, P=0.67). The proportion of CD4+CD28-FoxP3+ Treg-like cells in CD4+ T cells was gradually increased in MGUS, untreated MM patients than healthy controls (P<0.01, P<0.01); Treg-like cells in newly diagnosed MM patients were significantly higher than those in MGUS patients (P=0.01). In BM, the proportion of aTregs was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01); the proportion of rTregs in MGUS, untreated MM patients was significantly lower than that of controls (P=0.02, P<0.01). However, there was no significant difference in the frequencies of non-Tregs in BM from MGUS and MM patients with normal controls (P=0.14, P=0.88). The proportion of Treg-like cells in CD4+ T cells was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01, P<0.01). Treg-like cells in untreated MM patients were significantly higher than those in MGUS patients (P<0.01). The inhibition rate of aTreg in bone marrow of newly diagnosed MM patients was significantly higher than that of rTreg (P<0.01), while the inhibition rate of non-Treg was significantly lower than that of rTreg cells (P<0.01). The inhibition rates of aTreg (P=0.21), rTreg (P=0.08) and non-Treg (P=0.09) in healthy controls were no difference from those in MM patients. The level of IL-10 secreted by non-Treg in untreated MM patients was notably higher than that of aTreg and rTreg; the ability of cytokine secretion of Treg subsets in MM patients was similar with that of healthy controls. Conclusions There were significant changes in the frequencies of Treg cell subsets and Treg-like cells in peripheral blood and bone marrow of MGUS and MM patients, suggesting that immunomodulatory abnormality has existed in patients at premalignant stage. The immunosuppressive and cytokine secretory functions of Treg subsets in bone marrow of untreated MM patients were intact compared with that in healthy adults. Disclosures No relevant conflicts of interest to declare.

Study of the Quantity and Function of Regulatory T Cells In the Hemocytopenic Patients with Positive BMMNC-Coombs Test

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4875-4875
Author(s):  
Rong Fu ◽  
Jin Chen ◽  
Zonghong Shao ◽  
Jun Wang ◽  
Lijuan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Treg Cells ◽  
Cd4 Cells ◽  
Healthy Donors ◽  
Two Factors ◽  
And Function ◽  
Normal Controls

Abstract Abstract 4875 Objective To investigate the quantity and function of regulatory T cells in the IRP, then explore the significance of Treg cells in the pathogenesis of IRP. Methods Sixty-two patients with IRP and twenty-four healthy donors were enrolled in this study. The ratios of CD4+CD25+/CD4+ and CD4+CD25+CD127low/CD4+ in bone marrow were examined with flow cytometry. The levels of IL-2, ATGF-β were tested with ELISA. The expressions of FoxP3 and Galectin-10 mRNA in BMMNC were measured by semiquantitive RT-PCR. Results The levels of IL-2, ATGF-β in bone marrow of untreated or recovered IRP patients (5.64±1.70, 6.19±2.53; 1.79±0.67, 1.86±0.76) were significantly lower than them of normal controls (7.91±3.71,2.48±0.94) (p<0.05); The ratio of CD4+CD25+/CD4+ cells in bone marrow of untreated IRP patients (22.46±9.47) was significantly lower than that of recovered IRP patients or normal controls (27.10±7.08, 30.59±8.58) (p<0.05); The ratio of CD4+CD25+CD127low/CD4+ cells in bone marrow of untreated IRP patients (7.18±2.72) was significantly lower than that of recovered IRP patients or normal controls (9.07±4.67, 10.44±3.24) (p<0.05). The relative mRNA expressions of FoxP3 and galentin-10 were 0.34±0.25, 0.69±0.51, 0.82±0.65 and0.66±0.11, 0.74±0.11, 0.76±0.09 in three groups, respectively. The expressions of the two factors in untreated IRP patients were significantly lower than them in recovered IRP patients or normal controls (p<0.05). Conclusions There exist abnormalities in quantity and function of Treg cells in IRP patients which might play important role in the pathogenesis of IRP. Disclosures: No relevant conflicts of interest to declare.

T Regulator Cells (Treg) In Patients with Myelofibrosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5051-5051 ◽  
Author(s):  
Shams A Shakil ◽  
Chi Chen ◽  
Ali Surahio ◽  
Mariam B Mirza ◽  
Marisa Y Su ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Normal Volunteer ◽  
Primary Myelofibrosis ◽  
Treg Cells ◽  
T Effector Cells ◽  
Treg Function ◽  
Autoimmune Phenomenon ◽  
Normal Controls

Abstract Abstract 5051 Primary myelofibrosis (PMF) and myelofibrosis post essential thrombocytosis (MF-ET) or myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomenon, such as Coomb positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex etc. Regulatory T cells (Treg) also have known to play important roles in modulating immune responses. Therefore we studied Treg cells in 25 patients with MF including PMF (12), MF-ET (8), MF-PV (5) and compared with other MPD including ET (7), PV (8), and normal volunteer controls (17). Mononuclear cells (MNC) were separated from peripheral blood. 106 MNCs were stained for flow cytometry analysis using Treg Detection Kit from Miltenyi Biotec Inc (Auburn, CA). The numbers of Treg was calculated as the percentage of positive CD4+ CD25+ FoxP3+ T cells (Treg) from the numbers of gated CD4+ cells. Treg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of Treg vs. T-effector cells (CD4+ CD25-) at 1:1, 1:2, 1:4, and 0:1 in the presence of anti-CD3 CD28 microbeads (Invitrogen). The results (mean ± SE) showed numbers of Treg cells in MF were 0.73 + 0.08, other MPD were 1.37 ± 0.22, and normal controls were 0.96 ± 0.27 (p=NS). Treg function was evaluated in 12 patients with MF and 6 normal volunteer controls. Four MF patients were found to have significant lower values than controls. We concluded that in MF, quantitatively Treg were not different from other MPD or normal controls but Treg dysfunction was observed in 30–40 % of MF patients. This could explain why some patients with MF are prone to develop autoimmune phenomenon. Further studies with more patients are in progress. Disclosures: No relevant conflicts of interest to declare.

Depot Formulation of Interleukin-2 as an Immunotheraphy for B-Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1398-1398
Author(s):  
Sofia Grille ◽  
Andreina Brugnini Lic ◽  
Esteban Corley ◽  
Martha Nese ◽  
Jose Alejandro Chabalgoity ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
Interleukin 2 ◽  
B Cell Lymphoma ◽  
Treg Cells ◽  
Depot Formulation

Abstract Relapses occurring in most lymphoma patients after treatment highlight the need for effective immunotheraphy approaches. Induction of tumor-specific adaptive immunity in cancer patients is an ideal approach because its selectivity and memory for the same tumors eventually prevent the relapse after conventional therapies. Vaccination with cytokines and tumor cells represents an attractive therapeutic approach for patients with B-cell lymphoma. Interleukin-2 (IL-2) has a wide range of immunologic effects, including the activation of cytotoxic T cells and natural killer cells (NK). The therapeutic use of IL-2 has generated substantial interest based on its ability to induce the regression of metastatic renal cell carcinomas and malignant melanomas tumors in humans. However, toxicity associated with systemic administration of large doses of cytokine is a major drawback for clinical application. In this work, we investigated whether in vivo vaccination with a cytokine-based immunotherapy using IL-2 adsorbed in alum, as a depot system, with lysed lymphoma cells could stimulate lymphoma-specific immunity and improve survival. We developed a reproducible syngenic model of B-cell lymphoma in Balb/c mouse based upon the A20 cell line. In this model animals died between day 35 and 40 after tumor cells inoculation. Mice were first injected subcutaneously with 1 x 106 A20 cell in right flank. Four groups of mice received at days 3 and 7 after tumor cell inoculation by subcutaneous injections one of the following: lysed A20 cells with IL-2 in alumn (A20-IL-2); Lysed A20 cells in alumn (A20); IL-2 in alumn (IL-2); and phosphate-buffered saline (PBS) as control. Mice were followed for survival and immune response was evaluated. At day 22, 17% of A20-IL-2 group had systemic disseminated disease in comparison with 33% A20 group, 83% for IL-2 group and 100% of PBS group (p=0.009). Mice vaccinated with A20-IL-2 had longer survival compared with mice vaccinated with A20 or PBS (p=0.0019). Prolonged survival was related with a marked increase in the number of intratumoral CD4+ T cells (p=0.0001), CD8+ T cells (p=0.001) and NK cells (p=0.001). Aditionally, at day 22 groups of mice vaccinated with A20 showed a significantly lower percentages of intratumoral CD4+ CD25+ CD127- Treg cells in the tumor as compared with PBS group (p=0.0001). At day 39 the percentage of intratumoral Treg cells increased in the groups vaccinated with IL-2 (A20-IL-2 and IL-2 groups). Intratumoral CD4+, CD8+, NK and Treg cells were evaluated by flow cytometry. Interestingly, intracellular cytokine analysis showed a greater number of intratumoral INF-γ producing CD4+ T cells in mice vaccinated with A20-IL-2. Proliferation assays by flow cytometry (using carboxy-fluorescein diacetate, succinimidyl ester and propidium iodide) showed enhanced proliferation upon stimulation with irradiated A20 cells in splenocytes from A20-IL-2 vaccinated mice (p=0.004). In conclusion, the results of this study indicate that vaccination with A20 antigens combined with a depot formulation of IL-2 elicits strong anti-tumor specific immunity and extended survival. This approach may be an interesting strategy to promote systemic immunity against B-cell lymphoma with therapeutic value.

Study of the Quantity and Function of Th17 Cells In the Hemocytopenic Patients with Positive BMMNC-Coombs Test

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4874-4874
Author(s):  
Rong Fu ◽  
Honglei Wang ◽  
Zonghong Shao ◽  
Jun Wang ◽  
Lijuan Li ◽  
...  
Keyword(s):  
Th17 Cells ◽  
Conflicts Of Interest ◽  
Cd4 Cells ◽  
Rt Pcr ◽  
Healthy Donors ◽  
Significant Difference ◽  
Positive Correlations ◽  
And Function ◽  
Normal Controls

Abstract Abstract 4874 Objective To explore the role of Th17 cells in the pathogenesis of IRP. Methods Forty-three untreated IRP patients, 34 recovered IRP patients and 13 healthy donors were enrolled in this study. The ratio of IL-23R+CD4+/CD4+cells in bone marrow were examined with flow cytometry (FCM). The levels of IL-6, IL-23, IL-17 were measured with ELISA. The expressions of RORγt mRNA ASTAT3 mRNA in BMMNC were measured by semiquantitive RT -PCR. Results The ratio of IL-23R+CD4+/CD4+ cells, the levels of IL-6, IL-23, IL-17 and the expressions of RORγt mRNA and STAT3 mRNA in BMMNC of untreated IRP patients were significantly higher than those of recovered IRP patients (p<0.05); there was no significant difference between those of recovered IRP patients and normal controls (p>0.05). There were significantly positive correlations between the ratio of IL-23R+CD4+/CD4+ cells and CD5+CD19+/CD19+ (p <0.05). There were significantly positive correlations between the levels of IL-17 and CD5+CD19+/CD19+the quantity of BMMNC–antibody (p<0.05); There were significantly positive correlations between the expressions of RORγt mRNA and the ratio of CD5+CD19+/CD19+, the quantity of BMMNC-antibody (p <0.05). There were significantly positive correlations between the ratio of IL-23R+CD4+/CD4+ and the level of IL-17, the expression of STAT3 mRNA (p<0.05). Conclusions There exists increased quantity and hyperfunction of Th17 cells in the IRP patients which induce B cells hyperfunction and production of autoantibodies against the BM hematopoietic cells. Th17 cells might be regarded as new therapeutic target of IRP. Disclosures: No relevant conflicts of interest to declare.

T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression

1987 ◽  
Vol 7 (12) ◽  
pp. 4472-4481
Author(s):  
C H June ◽  
J A Ledbetter ◽  
M M Gillespie ◽  
T Lindsten ◽  
C B Thompson
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
T Cell Proliferation ◽  
Cd28 Molecule ◽  
T Cell Stimulation ◽  
Protein Kinase C Activation

CD28 is a homodimeric glycoprotein expressed on the surface of a major subset of human T cells that has recently been identified as a member of the immunoglobulin supergene family. The binding of monoclonal antibodies to the CD28 antigen on purified T cells does not result in proliferation; however, previous studies have shown that the combination of CD28 stimulation and protein kinase C activation by phorbol myristate acetate (PMA) results in T-cell proliferation that is independent of both accessory cells and activation of the T-cell receptor-CD3 complex. In the present study, effects of stimulation by anti-CD28 on cell cycle progression and on the interleukin 2 (IL-2) and IL-2 receptor system have been investigated on primary cultures of purified peripheral-blood CD28+ T cells. There was no measurable effect on cell size or on DNA synthesis after stimulation of resting (G0) cells by CD28 alone. After 3 h of activation of T cells by PMA alone, a slight (8%) increase in cell volume occurred that did not progress to DNA synthesis. In contrast, T-cell stimulation by CD28 in combination with PMA resulted in a progressive increase in cell volume in approximately 100% of cells at 12 to 14 h after stimulation. Northern blot (RNA blot) analysis revealed that CD28 stimulation alone failed to cause expression of the alpha chain of the IL-2 receptor or of IL-2 mRNA, and in accord with previous studies, stimulation by PMA alone resulted in the accumulation of IL-2 receptor transcripts but no detectable IL-2 mRNA. In contrast, T-cell stimulation by the combination of CD28 and PMA resulted in the appearance of IL-2 transcripts and enhanced expression of IL-2 receptor mRNA. Functional studies revealed that the proliferation induced by CD28 and PMA stimulation was entirely resistant to cyclosporine, in contrast to T-cell activation induced by the CD3-T-cell receptor complex. Cyclosporine was found not to affect the accumulation of IL-2 mRNA after CD28 plus PMA stimulation, although there was no detectable IL-2 mRNA after stimulation by CD3 in the presence of the drug. Furthermore, stimulation by CD28 in combination with immobilized CD3 antibodies caused a striking enhancement of IL-2 mRNA expression that was, in part, resistant to the effects of cyclosporine. These studies indicate that the CD28 molecule synergizes with protein kinase C activation to induce IL-2 gene expression and demonstrate that stimulation by the CD28 pathway can cause vigorous T-cell proliferation even in the presence of cyclosporine and that cyclosporine does not prevent transcription of 16-2 mRNA, as has been suggested previously. Moreover, these findings suggest that a potential role for the CD28 molecule in vivo may be to augment IL-2 production after stimulation of the CD3-T-cell receptor molecular complex and thereby to amplify an antigen-specific immune response. Finally, these results provide further evidence that the CD28 molecule triggers T-cell proliferation in a manner that differs biochemically from CD3-T-cell receptor-induced proliferation.

Abstract 51: Peripheral Regulatory T cells and Th17 Cells Is Associated With Pathogenesis of Moyamoya Disease

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Leihua Weng ◽  
Xiang Chen ◽  
Yun Xu
Keyword(s):  
Healthy Volunteers ◽  
Moyamoya Disease ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Treg Cells ◽  
Peripheral Blood Lymphocytes ◽  
Pathological Process ◽  
Medium Term ◽  
Significant Difference ◽  
And Gender

Background: Despite unclear pathogenesis, previous studies have suggested immune responses may play a pivotal role in the process of Moyamoya disease (MMD), a rare cerebrovascular occlusive disorder. The objective of this study is aimed to explore the change of peripheral Treg/Th17 in MMDpatients and whether the change is associated with pathogenesis of MMD. Methods: In the present study, we collected 26 MMD patients diagnosed by angiography according to the diagnostic criteria of definitive MMD and recruited 32 healthy volunteers. To explore the balance of peripheral Treg/Th17 in MMD patients, lymphocytes in peripheral blood were harvested and flow cytometry was used to measure the percentage of Treg and Th17in CD4+ Tcells, respectively. Meanwhile, relevant cytokines in serum were measured to evaluate the function of Treg and Th17 cells. Results: According to Suzuki’s angiographic staging of moyamoya disease, patients were divided into subgroups of the preliminary-term, medium-term and late-term. Cerebral hemorrhage is thefirstsymptom of onset occuringin half of patients, followed bycerebral ischemia.Our data revealed that both the percentage of Treg and Th17 cells in peripheral blood lymphocytes was increased in MMD patients compared with volunteer group. Meanwhile, the levels of IL-6, IL-10,IL-12, IL-17, TNF-α, VEGF and TGF-β in serum were significantly increased in MMD patients. In this study, the level of HMGB-1, a middle-late period inflammation biomarker, in serum of MMD patients is obviously elevated compared with volunteers. However, the ratio of Treg/Th17 had no significant difference in MMD patients compared to healthy volunteers. Intriguingly, our data revealed that ratio of Treg/Th17 was significantly increased in late-term MMD patients compared with medium-term patients as evidenced by elevated percentage of Treg cells.. In addition, TGF-β level in later-term MMD patients was significantly higher than this in medium-term MMD patients. No difference was observed in the way of onset and gender between two groups. Conclusion: Enhanced peripheral Treg and Th17 in MMD patients suggested that there may be an immunological component in the pathogenesis of MMD. Peripheral Treg may be associated with pathological process of MMD.

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