In vitro effects of mesenchymal stem cells on secreting function of T lymphocytes and CD4⁺CD25⁺ T cells from patients with immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze in vitro the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes and ratio of CD4⁺CD25⁺ T cells from patients with immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells. Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column, and the ratio of CD4⁺CD25⁺ T cells was detected by flow cytometry. Then the different amounts of 1 × 10⁴, 5 × 10⁴, 2 × 10⁵ MSCs per well treated with mitomycin as stromal feeder layers were co-cultured with above-mentioned T lymphocytes, 5 days after cocultivation, the ratio of CD4⁺CD25⁺ T cells was detected by flow cytometry and the levels of IL-2, IFN-γ, IL-4, IL-10 were measured by enzyme- linked immune sorbent assay (ELISA).</p><p><b>RESULTS</b>After co-cultured with 2 × 10⁵ MSCs for 5 days, the ratio of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺/CD4⁺ were significantly higher than of separate T lymphocytes in ITP patients [(4.56 ± 0.70)% vs (2.24 ± 0.81)%, (9.91 ± 1.18)% vs (4.08 ± 1.17)%, respectively] (P<0.05). To compare with separate T lymphocytes in ITP patients, the cytokine concentrations of IL-2 and IFN-γ from the culture supernatants significantly reduced from (280.47 ± 17.33) pg/ml to (97.21 ± 12.07) pg/ml and from (129.33 ± 16.34) pg/ml to (72.75 ± 7.81) pg/ml, respectively. In contrast, the cytokine concentrations of IL-4 and IL-10 increased from (16.34 ± 2.60) pg/ml to (37.98 ± 4.05) pg/ml and from (54.78 ± 5.62) pg/ml to (113.77 ± 5.68) pg/ml, respectively.</p><p><b>CONCLUSION</b>MSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by Th1 cells and promoted the releases of IL-4 and IL-10 by Th2 cells in ITP , thereby regulating the balance between Th1 and Th2 reaction, as well as up-regulating the expression of CD4⁺CD25⁺ T cells in vitro,then induced the immunologic tolerance of ITP.</p>

Expression of CXCR3 and CCR5 chemokine receptor in spleens of patients with primary immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study CXCR3 and CCR5 chemokine receptor expression in spleens of patients with primary immune thrombocytopenia (ITP) and its clinical significance.</p><p><b>METHODS</b>The splenectomy specimens from 10 ITP patients (ITP group) and 8 patients with traumatic splenic rupture (normal control group) were studied. Immunohistochemistry (IHC) was used to study the positive rate of CXCR3 and CCR5. Western blot was performed to detect CXCR3 and CCR5 protein expression, while real-time polymerase chain reaction (RT-PCR) was conducted to analyze their mRNA expression.</p><p><b>RESULTS</b>The positive rate of CXCR3 and CCR5 were both higher in ITP group (90% and 100%, respectively) than those in control group (75% and 87.5%, respectively)(P < 0.05). The differences were statistically significant (P < 0.05). Protein and mRNA level of CXCR3 in ITP group were 3.0 and 3.5 times as high as those in control group, respectively. Those of CCR5 in ITP group were 1.2 and 1.7 times as high as those in control group, respectively.</p><p><b>CONCLUSION</b>High expression of CXCR3 and CCR5 may play a part in the splenic immune disorders in patients with ITP.</p>

In vitro activation of bone marrow natural killer T cells of aplastic anemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the quantitative and qualitative changes of TCRVα24(+)Vβ11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer).</p><p><b>METHODS</b>NKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry.</p><p><b>RESULTS</b>In AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\].</p><p><b>CONCLUSION</b>Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.</p>