| Background and purposeAs the graft of allogeneic hematopoietic stem cell transplantation (allo-HSCT), peripheral blood stem cell harvests mobilized by granulocyte colony stimulating factor (G-CSF) have been widely used. This measure not only met the number request of collections of hematopoietic stem/progenitor cells, but also brought us a phenomenon:patients who received the G-CSF mobilized peripheral blood stem cell harvests reached hematopoietic and immune reconstitution faster than that received the bone marrow transplant material harvests, although peripheral blood stem cell harvests'T cells were several times as bone marrow harvests', the incidence of graft-verse-host disease (GVHD), in particular, the incidence of acute GVHD (aGVHD) did not increase, the mechanism of which has not yet been fully explained. Several reports revealed that low GVHD incidence could be related to changes in the T cells subsets, CD34+ cell, HLA-G expression, cytokines expression and so on.Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cells, derived from bone marrow progenitor cells and immature myeloid cells. Its surface markers are CD33+/CDllb+/CD14- in man, or Gr-l+/CDllb+ in mouse. In healthy adult mice, there were many Gr-1+/CDllb+ cells in the bone marrow (BM), and the proportion of Gr-1+/CDllb+ cells were less than 4% in peripheral blood (PB) and spleen. In healthy adults there is no relevant report. In the normal physiological conditions, MDSCs can differentiate into dendritic cells, macrophages and granulocytes. In some pathological situations such as cancer, inflammation and trauma, MDSCs can be detected in expansion in vivo, particularly in the tumor tissue, the numbers of MDSCs have increased substantially, and there is a certain correlation between the number of MDSCs and tumor size. A large number of reports found that, in tumor development and progression, MDSCs play a role of immunosuppressive effect. Recently, researchers found that G-CSF could mobilize MDSCs from BM to PB, but there is no report about the effect of G-CSF to MDSCs in the human body and the relationship between MDSCs and GVHD. In this study, we conducted an analysis on G-CSF's effect on MDSCs in healthy donors' BM, PB and the relationship between MDSCs and the incidence of GVHD.MethodsThe samples of BM and PB were obtained before the G-CSF mobilization and BM, PB and peripheral blood stem cell grafts (PBSC) were obtained after the G-CSF mobilization. Flow cytometry was used to detect the level of MDSCs and lymphocyte subsets, and adhesion molecules on MDSCs, including CD11a, CD44, CD49d and CD49e. Enzyme-linked immunosorbent assay (ELISA) was used to detect the arginase (ARG) and inducible nitric oxide synthase (iNOS) expression in BM and PB before and after mobilization. On the basis of the median value of infusion of MDSCs cells,30 patients received allo-HSCT wew divided into two groups, the high and low MDSCs group. The indicators of hematopoietic reconstitution, immune reconstitution, GVHD incidence, remission, relapse, and long life were analyzed. The relationgship of the number of MDSCs cells and aGVHD occurrence was analyzed. Statistical analysis:SPSS 13.0 statistical package was used for processing, the data were showed as mean±standard deviation(x±s). The data before and after mobilization, between two independent groups were compared with independent sample or paired sample t test. The ranked data were compared with Chi-square test. The non-parametric analysis (Spearman correlation coefficient) was used to analyze non-bivariate normal distribution data. P<0.05 was considered statistically significant.Results1. After G-CSF mobilization, healthy donors' peripheral blood leukocyte (WBC) number was significantly increased (P<0.001), hemoglobin (HGB) and platelet(PLT) had no significant change (P=0.205,0.939).2. In the normal physiological conditions, MDSCs could be detected in healthy donors' PB and BM, the proportion of MDSCs in nuclear cells in the PB and BM was 0.35±0.07% and 0.65±0.13%, respectively. The proportion of MDSCs in BM was higher than that in PB (P<0.001). After G-CSF mobilization, the proportion of MDSCs in nuclear cells in the PB and BM was 0.79±0.16% and 0.72±0.13%, respectively.There was no significant change in the proportion of MDSCs between PB and BM (P=0.114). MDSCs in PB after mobilization were significantly higher than that before mobilization (P<0.001), and there was no significant change in bone marrow before and after mobilization (P=0.136).3. The expression of CD4+CD25+ Treg cells in PB after mobilization was significantly higher than that before mobilization (P=0.048). There was no significantly difference in other lymphocytes (P>0.05). MDSCs values and CD4+CD25+ Treg cells before and after mobilization were not significantly correlated (Pearson correlation coefficient rs=0.627, P=0.096).4. The expression of adhesion factor CD11a, CD44, CD49d and CD49e on MDSCs surface in PB before mobilization were 0.72±0.23%,0.63±0.17%,0.34±0.11% and 0.59±0.09%, respectively. The expression of adhesion factor CD11a, CD44, CD49d and CD49e on MDSCs surface in BM before mobilization were 1.06±0.16%, 1.08±0.13%,0.87±0.10% and 1.18±0.19%, respectively. The expression of adhesion factor CD11a, CD44, CD49d and CD49e on MDSCs surface in PB after mobilization were 0.44±0.21%,0.34±0.10%,0.44±0.11% and 0.41±0.12%, respectively. The expression of adhesion factor CD11a, CD44, CD49d and CD49e on MDSCs surface in BM after mobilization were 0.75±0.13%,0.52±0.14%,0.50±0.12% and 0.69±0.15%, respectively. The expression of adhesion molecule on MDSCs were significantly higher than that after mobilization both in PB and BM (P values were less than 0.05).5. The expression of Arg in PB and BM before mobilization was 0.66±0.15mIU/ml and 2.20±0.40 mIU/ml, respectively. The expression of Arg in PB and BM after mobilization was 1.89±0.19mIU/ml and 2.16±0.40 mIU/ml, respectively. The expression of iNOS in PB and BM before mobilization was 0.92±0.13mIU/ml and 2.68±0.36 mIU/ml, respectively. The expression of iNOS in PB and BM after mobilization was 2.10±0.13mIU/ml and 2.79±0.23 mIU/ml, respectively. The expression of Arg and iNOS in PB after mobilization were significantly increased, which had statistical significance (P values were less than 0.01). There was no significant change in BM before and after mobilizaion (P values were more than 0.05).6. The median follow-up time was 227 days (range,48-363 days), of 30 patients received allo-HSCT patients,18 patients survived and 12 died. The incidence of aGVHD in high MDSCs group was 20.00% (3/15), the incidence ofⅢ-Ⅳ°aGVHD and cGVHD was 0.00% (0/15) and 30.77% (4/13), respectively. The incidence of aGVHD in low MDSCs group was 66.67%(10/15), the incidence ofⅢ-Ⅳ°aGVHD and cGVHD was 20.00%(3/15) and 45.45%(5/11). The incidence of aGVHD in high MDSCs group was significantly lower than that in low MDSCs group (P=0.011), while the incidence ofⅢ-Ⅳ°aGVHD and cGVHD between two groups has no significant difference (P=0.073, P=0.469).7. The linear correlation analysis (Bivariate Correlations) was used to analyze the relationship between the number of transplanted MDSCs and aGVHD, the results show that MDSC transplant values and the incidence of aGVHD were significantly negatively correlated (Spearman correlation coefficient rs=-0.445, P=0.014). It suggested that MDSCs and GVHD incidence has a significantly negative correlation.8. The incidence of recurrence was 26.67%(4/15),20.00%(3/15) in high MDSCs group, low MDSCs group, respectively. There was no significant difference (P=0.671). The disease-free survial rate was 46.7±14.2% and 38.9±15.8% in high MDSCs group and low MDSCs group, respectively. The overall survial rate was 70.9±12.4% and 50.6±15.0% in high MDSCs group and low MDSCs group, respectively. There were no significant difference in disease-free survial and overall survial rates between two groups (P= 0.833 and 0.442, respectively).ConclusionThe G-CSF can mobilize MDSCs from BM to PB, have the number of MDSCs and the expression of ARG and iNOS increased. The mechanisms of G-CSF mobilized MDSCs from BM to PB may be with the change of adhesion molecule on MDSCs surface. The increase of MDSCs in G-CSF mobilized peripheral blood stem cell grafts has a significantly negative correlation with the low incidence of aGVHD. MDSCs cell infusion does not affect the function of GVL and immune reconstitution. |
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