[Inhibitory effects of tacrolimus on effector T cells from patients with severe aplastic anemia].

OBJECTIVE: To explore the inhibitory effects of tacrolimus (FK506) on effector T cells in vitro and examine the relationship between effector T cells and clinical features in patients with severe aplastic anemia (SAA) to elucidate its immune mechanism. METHODS: The CD8(+) HLA-DR(+) cells, sorted by immunomagnetic separation from bone marrow mononuclear cells (BMMNC) of 16 SAA patients, were cultured in different concentrations of interleukin-2 (IL-2) alone or with FK506 for 72 hours. The proliferation effect was measured with methyl thiazolyl tetrazolium (MTT) method. The T lymphocytes were sorted from the SAA patients by lymphocyte separation medium and cultured alone or with IL-2 or with FK506 or FK506 plus cyclosporin A (CsA) for 18 hours. The expression of tumor necrosis factor-ß (TNF-ß) in CD8(+) HLA-DR(+) T cells was analyzed by flow cytometry. The relationship between the expression of TNF-ß and the clinical data, including percentages of reticulocyte and lymphocytes in peripheral blood cell count and ratio of CD4(+) T cells and CD8(+)T cells, was also analyzed. RESULTS: At the concentration of IL-2 greater than or equal to 20 U/ml, the cell proliferation (A values, 0.538 ± 0.142) were significantly higher than that in the blank culture hole (0.505 ± 0.153) (P < 0.05). The A values significantly decreased (0.386 ± 0.124) after the addition of FK506 (P < 0.05). Compared with control group, the expression of TNF-ß was significantly higher in IL-2 group (73.36% ± 16.73% vs 66.61% ± 16.20%, P < 0.05), significantly lower in FK506 and FK506 plus CsA groups (P < 0.05). No significant differences existed between the FK506 and FK506 plus CsA groups (47.78% ± 20.09% and 42.23% ± 21.35%, P > 0.05). The expression of TNF-ß in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4(+) T cell and CD8(+) T cell, positively correlated with the percentage of lymphocyte in peripheral blood count (r = -0.86, -0.90, 0.77, all P < 0.05). CONCLUSIONS: IL-2 can enhance the proliferation and expression of TNF-ß of CD8(+)HLA-DR(+)T cells from SAA patients. Such an effect is inhibited by FK506. And FK506 and FK506 plus CsA have similar effects.

[Telomere length and gene expression of shelterin in CD3(+) T cell of severe aplastic anemia patients].

OBJECTIVE: To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA). METHODS: Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR). RESULTS: Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P < 0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P < 0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P < 0.05). CONCLUSION: Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

[Expression of phosphorylated STAT5 in bone marrow hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria before and after in vitro G-CSF or SCF stimulation].

OBJECTIVE: To study the expressions of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH) before and after in vitro G-CSF or SCF stimulation, then evaluate the functions of G-CSF and SCF receptors in PNH clone cells. METHODS: Bone marrow mononuclear cells (BMMNC) of 26 PNH patients and 14 normal controls were stimulated in vitro with G-CSF (100 ng/ml) or SCF (100 ng/ml) for 10 min. Before and after these stimulations, the mean fluorescence intensity (MFI) of P-STAT5 in CD34(+)CD59(+) BMMNC and CD34(+)CD59(-) BMMNC were measured by flow cytometry. RESULTS: (1) The P-STAT5 MFI was (24 ± 18) in unstimulated CD34(+)CD59(-) cells. And it was significantly lower than that in unstimulated CD34(+)CD59(+) cells of PNH patients (64 ± 49) and normal controls (61 ± 33) (both P < 0.01). No statistic difference existed between the latter two. (2) The P-STAT5 MFI was (36 ± 35) in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 84) and normal controls (116 ± 59) (both P < 0.01). There was no statistic difference between the latter two. (3) The P-STAT5 MFI was (34 ± 27) in SCF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 97) and normal controls (128 ± 62) (both P < 0.01). And no statistic difference existed between the latter two. (4) The increased P-STAT5 MFI in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. The increase of P-STAT5 MFI in SCF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. CONCLUSION: There is a lower expression of P-STAT5 expressed in G-CSF or SCF stimulated PNH clone cells compared to that in normal clone cells.

[Research of regulative factors on CD8(+)HLA-DR(+) effector T cells in severe aplastic anemia].

OBJECTIVE: To explore the regulative factors on CD8(+)HLA-DR(+) T cells in the patients with severe aplastic anemia (SAA) and examine the roles of these cells in the immunopathogenesis of SAA. METHODS: CD8(+)HLA-DR(+) T cells were sorted from bone marrow mononuclear cells of 13 SAA patients from July 2011 to March 2012 by magnetic activated cell sorting system and were divided into 3 groups: interleukin 2 (IL-2) group (0, 0.1, 1, 10, 100 and 1000 U/ml), cyclosporine A (CsA) group (addition of 400 ng/ml CsA in each IL-2-containing well),receptor antagonist group (addition of IL-2 receptor antagonist 8 µg/ml in each IL-2-containing well). Then cell proliferation rate was evaluated by MTT assay after a 72-hour culturing. Bone marrow mononuclear cells of the SAA patients were divided into CsA group, IL-2 group and control group and cultured for 18 hours and another 4 hours following the dosing of phorbol ester. The expression of tumor necrosis factor ß (TNF-ß) in CD8(+)HLA-DR(+) T cells was analyzed by flow cytometry. RESULTS: The cell proliferations of IL-2 wells at the concentrations of 10, 100 and 1000 U/L (0.36 ± 0.12, 0.41 ± 0.12, 0.46 ± 0.14) were significantly higher than those of the control wells (0.23 ± 0.11), CsA group (0.18 ± 0.05, 0.19 ± 0.00, 0.20 ± 0.04) and receptor antagonist group (0.18 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, all P < 0.05). No statistic difference existed between CsA and receptor antagonist groups (P > 0.05). The expressions of TNF-ß of CD8(+)HLA-DR(+)T cells of the IL-2 group were higher than those of the control group (64% ± 25% vs 46% ± 22%) whereas the CsA group (27% ± 20%) were lower than those of the control group (both P < 0.05). CONCLUSIONS: IL-2 can significantly stimulate the proliferation of CD8(+)HLA-DR(+) T cells and accelerate the in vitro secretion of TNF-ß in SAA patients. The proliferation may be inhibited by CsA and receptor antagonist. And the expression of TNF-ß is suppressed significantly by CsA.

[Expression of lymphokines in CD8(+)HLA-DR(+) T lymphocytes of patients with severe aplastic anemia].

OBJECTIVE: To examine the expression of lymphokines damaging hematopoietic cells by CD8(+)HLA-DR(+) effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia (SAA) and explore further the heterogeneous immunopathogenesis of SAA. METHODS: The CD8(+)HLA-DR(+) cells were sorted by immunomagnetic separation from the PB of 24 untreated SAA patients and 23 normal controls. The mRNA expressions of perforin, granzyme B, FasL and tumor necrosis factor ß (TNF-ß) of sorted cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mRNA levels of perforin, granzyme B of CD8(+)HLA-DR(+)T cells in untreated group were higher than those of the controls (0.66 ± 0.25, 0.56 ± 0.26 vs 0.53 ± 0.14, 0.40 ± 0.13, P = 0.042, 0.012). The mRNA level of FasL in CD8(+)HLA-DR(+) T cells of untreated SAA patients was higher than that of the controls (0.77 ± 0.24 vs 0.61 ± 0.16, P = 0.011). The mRNA of TNF-ß in CD8(+)HLA-DR(+) T cells of untreated SAA patients was also higher than that of the controls (0.58 ± 0.16 vs 0.46 ± 0.15, P = 0.011). CONCLUSIONS: CD8(+)HLA-DR(+) T cells may damage hematopoiesis through the actions of perforin, granzyme B, TNF-ß and FasL. And it thus contributes to the immunopathogenesis of SAA.

[Count and function of CD8(+)CXCR3(+) regulatory T cells in peripheral blood of patients with autoimmune hemolytic anemia].

OBJECTIVE: To explore the effects of CD8(+)CXCR3(+)T cells on autoimmune hemolytic anemia (AIHA). METHODS: Twenty-two AIHA patients, including 11 untreated and 11 recovered ones, and 23 normal controls were recruited from July 2010 to November 2010. The percentage of CD8(+)CXCR3(+)/CD8(+)T cells in peripheral blood and the expression of interleukin-10 (IL-10) in CD8(+)CXCR3(+)T cells were detected by flow cytometry. Their correlations with the count of CD3(+)CD4(+)cells and the percentage of CD5(+)CD19(+) in CD19(+) B cells were analyzed. The expression level of CXCR3 mRNA in PBMC was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The percentage of CD8(+)CXCR3(+)/CD8(+) of untreated AIHA patients was (39.80 ± 19.96)%. And it was lower than that of recovered patients [(58.76 ± 14.22)%, P < 0.05] and normal controls [(59.66 ± 12.62)%, P < 0.01]. The percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells of untreated patients was (22.98 ± 14.96)% and it was lower than that of normal controls [(38.15 ± 17.03)%, P < 0.05]. The expression level of CXCR3 mRNA for untreated AIHA patients was (0.51 ± 0.19) and it was lower than that of normal controls (1.67 ± 1.17, P < 0.01). The percentage of CD8(+)CXCR3(+)/CD8(+)T cells had a negative correlation with the count of CD3(+)CD4(+) cells and the percentage of CD5(+)CD19(+)/CD19(+) B cells (r = -0.571, -0.583, both P < 0.05). So did the percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells (r = -0.524, -0.523, both P < 0.05). CONCLUSION: The decreased count of CD8(+)CXCR3(+)T cells and the lowered level of IL-10 may disturb the immune tolerance and lead to the occurrence of AIHA.

[Percentages and functions of natural killer cell subsets in peripheral blood of patients with severe aplastic anemia].

OBJECTIVE: To analyze the percentage and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cells in the pathogenesis of SAA. METHODS: By flow cytometry, the percentages of NK cells (CD3(-)CD56(+)CD16(+)) and its subsets [CD3(-)CD56(bright)CD16(-)(CD56(bright)), CD3(-)CD56(dim) CD16(+)(CD56(dim)), CD3(-)CD56(-)CD16(+)] in peripheral blood lymphocytes were detected in 12 untreated patients, 30 recovered patients and 13 normal controls respectively from April 2010 to December 2010 in our hospital. NK cells activating receptors (NKG2D and NKp46), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte and reticulocyte absolute value in peripheral blood, and hyperplasia degree, percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes absolute value in bone marrow were evaluated. RESULTS: (1) The percentages of NK cells (10.30% ± 6.08%) and CD56 bright cells (0.11%) in untreated patients were significantly lower than those of recovered patients (16.47% ± 8.29%, 0.68%, both P < 0.05) or normal controls (19.45% ± 6.88%, 0.53%, both P < 0.05). The percentage of CD56(dim) cells in untreated patients was significantly lower than that of normal controls (9.62% ± 6.04% vs 18.21% ± 7.16%, P < 0.05). The percentage of CD3(-)CD56(-)CD16(+) cells was significantly higher in recovered patients than that of untreated patients or normal controls (0.79% vs 0.37%, 0.41%, both P < 0.05). (2) The expression of NKp46 and perforin of NK cells in untreated (88.23%, 64.97% ± 21.61%) and recovered patients (82.97%, 66.14% ± 20.73%) were significantly higher than those of healthy controls (40.99%, 42.11% ± 27.25%, all P < 0.05). (3) The percentage of NK CD56(bright) and CD3(-)CD56(-)CD16(+) cells of patients was positively correlated with ANC% (r = 0.423, 0.609, 0.468 respectively, all P < 0.05) and the percentage of granulocytes in bone marrow (r = 0.357, 0.517, 0.434 respectively, all P < 0.05). The percentages of NK, CD56(bright), CD56(dim) and CD3(-)CD56(-)CD16(+) cells were positively correlated with the hyperplasic degree of bone marrow (r = 0.455, 0.412, 0.404, 0.451 respectively, all P < 0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow (r = -0.522, -0.435, -0.411, -0.547 respectively, all P < 0.05). The expression of NKG2D, NKp46, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage of NK CD56(bright), CD56(dim) cells and a higher expression of perforin may cause the over-function of T lymphocytes and thus lead to hematopoietic failure in SAA.

[STAT5 phosphorylation levels of erythropoietin and thrombopoietin receptors in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of patients with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To study the STAT5 phosphorylation levels of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The bone marrow mononuclear cells (BMMNC) were extracted from 23 PNH patients treated at our department from April 2010 to February 2011 and 11 normal controls. The mean fluorescence intensity (MFI) of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(+) cells and CD34(+)CD59(-) cells with or without the stimulation of 10 U/ml EPO and 50 U/ml TPO were examined by flow cytometry. RESULTS: (1) Without stimulation, the P-STAT5 MFI in CD34(+)CD59(-) cells of PNH patients was significantly lower than that of CD34(+)CD59(+) cells (31 ± 15 vs 74 ± 47, P < 0.01). And it was 59 ± 23 in normal control CD34(+)CD59(+) cells (P < 0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. (2) Under the stimulations of EPO and TPO, the P-STAT5 MFI was significantly lower in CD34(+)CD59(-) cells of PNH patients than that of CD34(+)CD59(+) cells (49 ± 24 and 51 ± 41 vs 120 ± 82 and 124 ± 87, both P < 0.01). For the normal control CD34(+)CD59(+) cells, they were 79 ± 47 and 98 ± 53 respectively (P < 0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. P-STAT5 MFI was elevated after the stimulations of EPO and TPO. The increments of CD34(+)CD59(+) cells in PNH patients were significantly higher than those of CD34(+)CD59(-) cells (49 ± 11 and 54 ± 43 vs 17 ± 4 and 16 ± 6, both P < 0.01). CONCLUSION: Under the in vitro stimulations of EPO and TPO, the STAT5 phosphorylation levels of EPO and TPO receptors in normally cloned hematopoietic stem cells in PNH patients are obviously superior to those in abnormally cloned counterparts.

[Differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes].

OBJECTIVE: To detect the abnormalities of differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes (MDS). METHODS: Forty-five newly diagnosed MDS cases from July 2008 to March 2010 in our hospital and 30 normal controls were enrolled. There were 17 low-risk and 28 high-risk patients. The CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor (SCF-R), erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptors (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry. RESULTS: The mean percentage of CD34+ in karyocyte of MDS cases in high-risk patients [0.53% (0.10%-1.68%)] was significantly higher than that of control group [0.13% (0.08%-0.32%), P<0.01]. The mean percentages of CD34+CD38+ cells were significantly lower in low and high-risk groups (86.3%±8.5% and 82.6%±11.1%) than those in control group (92.3%±3.4%). And the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group (13.7%±8.5% and 17.4%±11.0%) than that in control group (7.7%±3.4%, both P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells than that in CD34+CD38- cells (18.7%±18.3% vs 63.6%±20.0%, P<0.01). The expressions of SCF-R, G-CSFR and TpoR were not significantly different between two cell populations. The expressions of EpoR on CD34+CD38+ cells of low and high-risk MDS groups [9.0% (1.4%-12.7%), 5.2% (1.1%-14.1%)] were significantly lower than those of control group [9.6% (5.1%-30.1%), both P<0.05]. The expressions of G-CSFR on CD34+CD38+ cells of low and high-risk MDS groups (29.8%±19.1%, 28.7%±21.1%) were significantly lower than those of control group (44.4%±23.4%, both P<0.05). The quantities of EpoR on CD34+CD38- cells of low and high-risk MDS groups (42.2%±21.9%, 25.7%±15.6%) were significantly lower than those of control group (63.6%±20.0%, both P<0.01). The expressions of TpoR on CD34+CD38- cells of low and high-risk MDS groups (5.4%±4.7%, 4.1%±4.0%) were significantly lower than those of control group (10.1%±8.3%, both P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells was higher than that of MDS with high expression rates. CONCLUSION: The abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS are associated with MDS cytopenia and may be useful for the diagnosis of MDS.

[Abnormalities of CD34+ cells differentiation and bone marrow cell cycle in myelodysplastic syndrome].

OBJECTIVES: To detect the abnormalities of CD(34)(+) cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS). METHODS: Fifty newly diagnosed MDS (17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD(34)(+)CD(38)(+), CD(34)(+)CD(38)(-) bone marrow cells and bone marrow cell cycle were measured with flow cytometry. RESULTS: The mean percentages of CD(34)(+) cells in bone marrow karyocyte of high risk [(2.29 ± 2.17)%] and MDS-AML groups [(18.69 ± 17.47)%] were significantly higher than that of control group [(0.36 ± 0.49)%, P < 0.05]. The mean percentages of CD(34)(+)CD(38)(+) cells were significantly lower in low risk, high risk and MDS-AML groups [(86.09 ± 7.79)%, (81.68 ± 11.82)% and (82.88 ± 2.60)%, respectively] than that in control group [(92.21 ± 3.85)%, P < 0.05], thus the percentages of CD(34)(+)CD(38)(-) cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [(13.91 ± 7.79)%, (18.32 ± 11.82)% or (17.13 ± 2.60)%, respectively] than that in control group [(7.79 ± 3.85)%, P < 0.05]. The percentages of CD(34)(+)CD(38)(-) cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r = 0.493, P = 0.001) and WHO Adapted Prognostic Scoring System (WPSS) (r = 0.586, P = 0.000) scores. The percentages of bone marrow mononuclear cells (BMMNCs) in G(0)/G(1) phase of in low risk, high risk and MDS-AML groups [(94.52 ± 4.32)%, (96.07 ± 3.88)% and (94.65 ± 4.55)%, respectively] were significantly higher than that in control group [(88.94 ± 7.30)%, P < 0.01], thus the percentages of BMMNCs in S and G(2)/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P < 0.05). MDS patients with low percentages of CD(34)(+)CD(38)(-) cells presented higher therapeutic efficacy than those with high percentages of CD(34)(+)CD(38)(-) cells, while without significant differences (P > 0.05). CONCLUSIONS: There are abnormalities of differentiation of CD(34)(+) bone marrow cells and high proportion of G(0)/G(1) cells which indicates a G(1) phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD(34)(+) bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.