OBJECTIVE: To explore the relation of circulating follicular helper T cell (c Tfh) changes with B cell dysfunction in MDS patients. METHODS: 20 patients diagnosed as MDS from Auguct 2015 to October 2017 were enrolled in MDS group, and 20 healthy valuntears matching in age and sex were enrolled in healthy control (HC) group. The perepheral blood in 2 groups were collected, the mononuclear cells (PBMC) from which were isolated by densily gradient contrifugation, at the same time, the serum left in isolation process was reserved for further study. The flow cytometry was used to detect the ratio of cTfh such as CD4CXCR5 T cells and its subset CD4CXCR5ICOS T cells, CD4CXCR5PD-1 T cells in PBMC, as well as the ratio of plasmablast CD19CD20CD38 B cells. The ELISA was used to detect the concentration of IgA, IgM and IgG. The differences in ratio of cTfh cells and plasmablast B cells, as well as the concentration of IgA, IgM and IgG between MDS and HC groups were compared, at the same time, the correlation of cTfh cell ratio with the plasmablast B cell ratio and the concentration of IgA, IgM and IgG in MDS patient was analyzed. RESULTS: The ratio of CD4CXCR5T, CD4CXCR5ICOST cells and CD19CD20CD38B cells and the concentration of IgA, IgM and IgG decreased in MDS patients, while the ratio of CD4CXCR5PD-1T cells increased in MDS patients. The ratio of CD4CXCR5T cells, CD4CXCR5ICOST cells positively correlated with the ratio of CD19CD20CD38B cells, as well as with the concentration of IgA, IgM and IgG in MDS patients. However, the ratio of CD4CXCR5PD-1T cells negatively correlated with the ratio of CD19CD20CD38B cells, as well as with the concentration of IgA, IgM and IgG. CONCLUSION The ratio of circulating Tfh cells and their subsets showed significant changes, that correlate with B cell dysfunction in MDS patients.
B-Lymphocytes ; Humans ; Interleukins ; Leukocytes, Mononuclear ; Myelodysplastic Syndromes ; Plasma Cells ; Receptors, CXCR5 ; T-Lymphocytes, Helper-Inducer

<b>OBJECTIVEb>To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.

<b>METHODSb>The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration. Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry. CD4(+)T cells and CD4(+)CD25(+)CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) by Magnetic cell sorting. After exosomes-like vesicles cultured with CD4(+)T cells or CD4(+)CD25(+)CD127low Treg cells, cell proliferation and apoptosis were assayed. Phosphorylated β-catenin level in Wnt signaling by phosflow.

<b>RESULTSb>Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-II molecule, costimulatory molecules CD86 etc. After co-cultured with CD4(+) T cells, exosomes-like vesicles inhibited the proliferation of the T cells in a dose-dependent manner. After Treg cells cultured with exosomes-like vesicles for 14 days, the survival rate of the Treg cells was 57.07%, while that of the control Treg was 30.91%. Frizzled receptors 2, 3, 4and LRP6 gene mRNA expressed (the relative gray value was 48.50, 34.84, 23.85, 49.73) in the Treg cells by RT-PCR, and Wnt molecular expressed in exosomes-like vesicles. After Treg cells co-cultured with exosomes-like vesicles, the MFI of phosphorylated β-catenin decreased (from 20.06 ± 2.99 to 12.41 ± 2.08), and the expression of Bcl-2 mRNA was upregulated significantly (the relative gray value from 0.45 to 84.97).

<b>CONCLUSIONSb>Exosomes-like vesicles existed in human plasma and express immune regulatory molecules. They can suppress the proliferation of activated CD4(+) T cells induce their apoptosis and pro-long the survival of natural Treg cells via Wnt signaling pathway.

CD4-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Exosomes ; Flow Cytometry ; Humans ; Immunologic Factors ; Interleukin-2 Receptor alpha Subunit ; Leukocytes, Mononuclear ; immunology ; T-Lymphocytes, Regulatory ; immunology

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology

OBJECTIVES: Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4⁺ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease. METHODS: From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. RESULTS: In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI. CONCLUSIONS In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.
Humans ; Leukocytes, Mononuclear ; Lupus Erythematosus, Systemic/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; DNA Repair ; RNA, Messenger/metabolism*

<b>OBJECTIVEb>To study the effects of CpG oligodeoxyribonucleotide (ODN) as adjuvant on the immune responses in PBMC and CD4+ T cell with chronic hepatitis B virus.

<b>METHODSb>The selected 20 infections were averagely divided two groups. The frequency of IFN-gamma secreting PBMC and CD4+ T cell in immune tolerant phase and in the immune clearance phase that had stimulated by CpG ODN, HBsAg and Mixture [CpG ODN + HBsAg] were analyzed by enzyme linked immune spot (ELISOT).

<b>RESULTSb>The PBMC and CD4+ T cell were differently incubated by CpG ODN, HBsAg and M [CpG ODN + HBsAg]. The number of IFN-gamma spot differently are 3 +/- 8, 339 +/- 429, 375 +/- 496, 1 +/- 4, 5 +/- 16 and 5 +/- 12; the results of immume tolerance are 3 +/- 8, 361 +/- 153, 375 +/- 276, 0 +/- 2, 2 +/- 2 and 4 +/- 4; but the results of immune clearance are 3 +/- 21, 289 +/- 345, 405 +/- 656, 2 +/- 14, 8 +/- 40 and 7 +/- 30. The IFN-gamma spots statistical analysis of PBMC were differently incubated by HBsAg and M, the total is P = 0.720, The IFN-gamma spots statistical analysis of CD4+ T cell were differently incubated by HBsAg and M, the total is P = 0.890, The IFN-gamma spots statistical analysis of PBMC and CD4+ T cell were differently incubated by M, the total is P = 0.000.

<b>CONCLUSIONSb>The ability that CpG ODN can not significantly increase the IFN-gamma secreting of PBMC and CD4+ T cell that were incubated by HBsAg to the infection in immune tolerant phase and in the immune clearance phase, but the PBMC outweighed The CD4 T cell.

Adult ; CD4-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Female ; Hepatitis B Antigens ; immunology ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; immunology ; virology ; Humans ; Interferon-gamma ; immunology ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged ; Oligodeoxyribonucleotides ; immunology ; Young Adult

OBJECTIVE: To explore the role of follicular helper T cell (Tfh)/ follicular regulatory T cell (Tfr) imbalance in B-cell lymphoma (BCL). METHODS: Sixteen BCL patients who were admitted to the Department of Hematology of The First People's Hospital of Yichang and 20 healthy people from December 2019 to November 2020 were enrolled and respectively divided into observation group and control group. The levels of Tfh and Tfr in peripheral blood were detected by flow cytometry. The changes of Tfh, Tfr, and Tfh/Tfr ratio were compared and the relationship between Tfh/Tfr ratio and efficacy, prognosis was analyzed. RESULTS: Compared with the healthy controls, Tfh and Tfh/Tfr ratio in peripheral blood of the BCL patients increased (P<0.05, P<0.01), while levels of Tfr was decreased (P<0.01). After chemotherapy, Tfh and Tfh/Tfr ratio in peripheral blood of the BCL patients decreased significantly than before chemotherapy (P<0.01), but Tfr was no significant difference. Multivariate analysis showed that Tfh and Tfh/Tfr ratio were positively correlated with international prognostic index (IPI) score and Ann Arbor stage (r=0.626, 0.564, 0.573, 0.608, respectively), while Tfr negatively (r=－0.504, －0.542, respectively). According to the average value of Tfh/Tfr ratio at initial diagnosis, BCL patients were divided into Tfh/Tfr high ratio group and low ratio group. It was found that the complete remission (CR) rate, overall response rate (ORR), and survival time in the high ratio group were significantly lower than the low ratio group (P<0.01). CONCLUSION There is an imbalance of Tfh/Tfr ratio in peripheral blood of the BCL patients, and those with a high Tfh/Tfr ratio have lower CR, ORR and shorter survival time.
Flow Cytometry ; Humans ; Lymphoma, B-Cell ; T Follicular Helper Cells ; T-Lymphocytes, Helper-Inducer ; T-Lymphocytes, Regulatory

OBJECTIVE: To investigate the role of relationship between the expression of miRNA181a-5p and imbalance of Treg/Th17 in the pathogenesis of primary immune thrombocytopenia(ITP), which contributes to clarify the mechanism of T cell immune imbalance in ITP patients. METHODS: Peripheral blood was collected from 37 ITP patients, concluding 21 untreated patients and 16 effectively treated patients, and 19 healthy controls; Peripheral blood mononuclear cells (PBMC) were isolated and the expression of miRNA181a-5p and Notch1 was analyzed by RT-PCR. The proportion of Th17 subsets and Treg cells in the peripheral circulation was detected by flow cytometer (FCM). Clinical data of ITP group was collected, including age, platelet count and disease course. RESULTS: The expression of miR-181a-5p was significantly decreased in ITP group than that of healthy control group (P＜0.01). After effective treatment, the expression of miR-181a-5p was significantly higher than that of ITP group (P＜0.05), but still significantly lower than that of healthy control group (P＜0.01); The expression of Notch1 was significantly increased in ITP group and effectively treated group than that of healthy control group (P＜0.01). There was no significant difference in proportion of Treg cells in ITP group, effectively treated group and healthy control group (P＞0.05). The proportion of Th17 subsets in ITP group was significantly increased than that of healthy control group (P＜0.05), while the ratio of Treg/Th17 was significantly decreased (P＜0.05). There was a positive correlation between the expression of miR-181a-5p and ratio of Treg/Th17 in ITP group (r=0.555). CONCLUSION The expression of miR-181a-5p is significantly decreased in ITP patients, which is closely related to the imbalance of Treg/Th17 cells. After effective treatment, the expression of miR-181a-5p can be significantly corrected, but still failed to reach the level of healthy people. While the expression of Notch1 is significantly increased in ITP patients, and could not reach the level of healthy people after effective treatment.
Humans ; Leukocytes, Mononuclear ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; T-Lymphocytes, Regulatory ; Th17 Cells

OBJECTIVE: To investigate the effects of exosomes from human umbilical cord mesenchymal stem cells on the development of Treg and TH17 cells. METHODS: Exosomes from the serum-free-culture supernatants of hUC-MSC were harvested by ultracentrifugation. The electron microscopy, nanoparticle tracking analysis and western blot were used to identify the hUC-MSC-exosomes, such as the morphology, the paticle chameter, and the protein content. The PBMC stimulated with anti-CD3/CD28 were incubated with the exosomes for five days, and then the percentage changes of Treg and TH17 cells were analyzed by using flow cytometry. RESULTS: The hUC MSC-derived exosomes were saucer-like in morphology the averge diameter was approximately 142 nm. They were identified as positive for CD9 and CD63. Flow cytometry showed that the proportion of CD4CD25Foxp3 Treg cells in the PBMC were significantly higher, but the proportion of CD4IL17A T cells in the hUC-MSC-exosome group was obviously lower than that in the group without the hUC-MSC-exosom (control group) (P<0.05). CONCLUSION The hUC-MSC-exosomes have an immunomodulatory effect on T cells in vitro by increasing the ratio of Treg and reducing the ratio of TH17 cells, expecting the hUC-MSC-exosom as a novel cell-free target for immunotherapy.
Exosomes ; Humans ; Leukocytes, Mononuclear ; Mesenchymal Stem Cells ; T-Lymphocytes, Regulatory ; Th17 Cells ; Umbilical Cord

Since primary Sjögren's syndrome (pSS) is an autoummune disease of B cell hyperactivity and pathologic autoantibody response, follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are suggested to be key players in pSS. We examined subsets of Tfh and Tfr cells from the blood in pSS patients, and whether these subsets represent disease activity, glandular inflammation, or autoantibody responses in pSS. Circulating Tfh and Tfr cells, along with their specific subsets, were identified from the peripheral blood of 18 pSS patients and 14 age- and sex-matched healthy controls (HCs) using flow cytometry analysis. Blood Tfr and Tfh cell ratios were increased in pSS patients compared with HCs. The CCR7(lo)PD-1(hi) subset of circulating Tfh cells was increased in pSS patients with high degree of focal lymphocytic sialadenitis; whereas circulating Tfh cells did not differ between pSS patients and HCs. The frequency of CCR7(lo)PD-1(hi) Tfh cells was significantly correlated with disease activity scores and differentiated B cells. PD-1 expression on blood Tfh and Tfr cells showed positive correlations with IL-21 in pSS. Increasing trend of blood Tfr cells was observed in pSS patients, and blood Tfr cells (particularly Th1 and Th17 subsets) represented hypergammaglobulinemia in pSS. In summary, circulating CCR7(lo)PD-1(hi) Tfh cells indicated disease activity and glandular inflammation in pSS. Circulating Tfr cells, shifted toward Th1 and Th17 subsets, indicated ongoing IgG production in pSS. Subsets of circulating Tfh or Tfr cells could be biomarkers for disease monitoring and patient stratification in pSS.
Autoantibodies ; B-Lymphocytes ; Biomarkers ; Flow Cytometry ; Humans ; Hypergammaglobulinemia ; Immunoglobulin G ; Inflammation ; Sialadenitis ; T-Lymphocyte Subsets ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer ; T-Lymphocytes, Regulatory

PURPOSE: To evaluate the changes of differential counts and lymphocyte subsets in cancer patients' leukocyte before and after radiotherapy. METHODS AND MATERIALS: From Dec. 1994 to May 1995, the changes of leukocyte and its subsets in 16 patients who received radiotherapy in the Dept. of Radiation Oncology of Dong-A University Hospiatal were investigated. Radiation was delivered from 2700 cGy to 6660 cGy with median dose of 5400 cGy. The results of pre- and post-radiotherapy were analyzed by paired T-test. The results of patients who received < 50 Gy and > or = 50 Gy were analyzed by wilcoxon test. RESULTS: Before and after radiotherapy, there was not any significant differences in the counts of leukocyte, granulocyte and monocyte. A remarkable decrease was noted in lymphocyte counts after radiotherapy(p=0.015). T cells, B cells and natural killer cells were also decreased in number after radiotherapy but it was not significant statistically. T helper cells and T suppressor cells were also decreased in number(p>0.05). The ratio of T helper/suppressor cell was decreased from 1.52 to 1.11 and it was significant statistically(p=0.016). The portion of T suppressor cell among all T cells was increased after radiotherapy (p=0.0195). No significant difference was observed in the analysis of leukocyte and its subsets between patients who reveived < 50 Gy and > or = 50 Gy. CONCLUSION: Radiotherapy caused remarkable decrease in lymphocyte count and its subsets. Among all lymphocyte subsets, T helper cell might be the most vulnerable to radiation, considering decreased ratio of T helper/surppressor cell count after radiotherapy.
B-Lymphocytes ; Cell Count ; Granulocytes ; Humans ; Killer Cells, Natural ; Leukocytes ; Lymphocyte Count ; Lymphocyte Subsets* ; Lymphocytes* ; Monocytes ; Radiation Oncology ; Radiotherapy* ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer