| Objective We aimed to elucidate the mechanism of cells evading immunosurveillance by examining the cell surface activating receptor NKG2D of NK cell, its specific ligands of leukemia cells and the levels of soluble ligands in sera of acute leukemia (AL) patients. Try to elucidate the mechanism of graft versus leukemia (GVL) effects after unrelated umbilical cord blood stem cell transplantation (UCBT) by observing the reconstitution of subsets and surface receptors of NK cells and the cytotoxicity of NK cells.Methods In the first section, thirty untreated patients diagnosed AL and ten healthy adults were studied. The flow cytometry analysis was used to detect the activating receptor NKG2D and its ligands, MICA/MICB and ULBP1,2,3 in the peripheral blood of the patients and the adults.ELISA was used to detect the levels of soluble MICA,MICB(sMICA,sMICB) and soluble ULBP1,2,3(s ULBP1,2,3) in the sera. In the second section, from October 2008 to January 2010, 14 cases with AL underwent UCBT in Anhui Provincial Hospital. The absulte count of T, B, NK cells, the reconstitution of NK cell subsets and surface receptors after UCBT was measured by flow cytometry in different intervals within six months. PBMCs was collected from the healthy adults and from patients monthly thereafter in the following half year after UCBT, NK cell cytotoxicity against K562 cell line was evaluated in a LDH release assay. All the same texts was compared with ten healthy adults.Results①Expression of NKG2D and its ligands in the periferal blood (PB) of AL patients.The expression of NKG2D on NK cells in patients was dramatically 1ower than that on the donors (P< 0.01) ((60.23土11.06) %VS(94.5±4.68)%).MICA/B and ULBP-1,2,3 were not expressed or expressed in very low levels on blast cells, in the sera of patients,but not of healthy donors,contained elevated 1evels of sMICA and sMICB(63.42±45.78pg/ml,121.57±63.8pg/ml) (P<0.01).However, we do not have the same finding for the ULBP1,2,3 in the sera.②Reconstitution of lymphocyte subsets after UCBT.The distribution of lymphocyte subsets after UCBT showed that B and T lymphocytes were barely detectable after transplantation and remained significantly lower than in adult controls during the first 4 months for T cells and during 6 months for B cells. In contrast, CD3-CD56+ NK cells reconstitute in all recipients soon after transplantation at similar level as in the control group, in percentage and absolute value. It was even slightly higher than control group in the second month as accounted (57.28±18.54)×107/L,while the normol level is (50.25±15.94)×107/L.③Analysis of NK cells subsets and NK-receptors after UCBT.The CD56bright subset was significantly higher, the most high as (63.4±16.5)%,than in NK cells from healthy adult(14.51±4.99)%during the first 6 months post-UCBT. Furthermore, an increase in the CD16+ CD56-NK cells in PB was observed in seven of 14 UCBT recipients, the average proportion is 12.42±10.47% (percentage of lymphocytes).The expression of C-type-lectin receptors NKG2A/CD94 were sharply higher after UCBT than in adult control samples in early stage after UCBT. By contrast, the level of NKG2D slightly decreased after UCBT, and it recovers to nomal level until the sixth month. The expression of major specific activating NK receptors increased after engraftment at 1 month post-UCBT, levels of NKp46 was significantly higher than in control group.This effect persisted for at least the first 2 months,then recovered. The expression of the expression of killer immunoglobulin-like receptors(KIR), CD158a, was significantly lower than that in adult controls during the first 6 months post-UCBT. In contrast, CD158b was similar to that in healthy adult controls.④Cytotoxicity analysis of NK cells after UCBT.Direct cytotoxicity against HLA class I-negative K562 target cells was similar for healthy adults and patients at all times tested after UCBT. Conclusions:The low expression of NKG2D on NK cells, the absence of MICA/B and ULBPs in malignant cells, the increase levels of sMICA/B in sero are all impair NKG2D-mediated immune surveillance of leukemias and may leads to the tumor escaping. Meanwhile, an early recovery of mature NK cells following UCBT, compared with other lymphocytic cell subsets, that represent the great majority of circulating lymphocytes, in early stage post-transplantation, were usually accompanied by highly expression of specific activating NK-cell receptors NKp46, even an increase in the CD16+ CD56-NK cells. so as we found, the direct cytotoxicity against K562 target cells was similar for healthy adults even in the first month after UCBT. This strongly suggested that UCBT creates optimal conditions for sufficient NK-cell recovery required for disease control. These findings imply the key role of NK cells on the clinical paradox observed following UCBT consisting of a strong GvL effect associated with a low incidence of GvH disease...
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