| ObjectiveTo investigate the quantitative and functional changes of natural killer (NK) cells and it's subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST),evaluate the relationships between these changes and hematopoietic function,and explore the role of NK cells in the the pathogenisis of SAA.MethodsBy means of four-color monoclonal antibody labeling technology, the percentag-es of NK cells (CD3-CD56+/CD16+) and it's subsets [CD56bright (CD3-CD56bright CD16neg/dim) CD56dim (CD3-CD56dimCD16bright),CD3-CD56negCD16bright] in peripheral blood were detected in 42 SAA patients,including 12 untreated patients and 30 recovered patients, and 13 normal controls respectively. NK cells inhibitory receptors (CD158a and CD158b), activating receptors (NKG2D and NKp46), perforrin and granzyme-βof patients and normal controls were also detected.The correlation between these changes and hematopoietic function, including percentage of neutron-phil granulocyte (ANC%), percentage of lymphocyte (Lym%) and reticulocyte absol-ute value (Ret) in peripheral blood,and hyperplasia degree,percentage of granulocytes, erythrocytes,lymphocytes and megakaryocytes absolute value in bone marrow were evaluated.Results1. The variations of NK cells and it's subsets percentages in peripheral blood lymphocytes in SAA patients①The change in the proportion of NK cells in SAA patients The percentage of NK cells in peripheral blood lymphocytes was (10.30±6.08)% in untreated SAA patients, (16.47±8.29)% in recovered patients, and (19.45±6.88)% in normal controls. The percentage of NK cells in untreated SAA patients was lower than that of recovered patients and normal controls (p<0.05),but there was no statistical difference between recovered patients and normal controls (p>0.05).②The change in the proportion of CD56bright NK cells subset in SAA patients The median percentage of CD56bright NK cells subset was 0.11% in untreated SAA patients,0.68% in recovered patients and 0.53% in normal controls.The median percentage of CD56bnght cells in untreated patients was lower than that of the other two groups (p<0.05), but there was no statistical difference between recovered patients and normal controls (p>0.05).③The change in the proportion of CD56dim NK cells subset in SAA patients The percentage of CD56dim NK cells subset was (9.62±6.04)% in untreated patients, (13.81±7.52)% in recovered patients and (18.21±7.16)% in normal controls.The percentage of CD56dim cells in untreated SAA patients was lower than that of normal controls (p<0.05),but there was no statistical difference between the other groups (p>0.05).④The change in the proportion of CD3-CD56negCD16bright NK cells subset in SAA patients The median percentage of CD3-CD56negCD16bright NK cells subset was 0.37% in untreated SAA patients,0.79% in recovered patients and 0.41% in normal controls.The percentage of CD3-CD56negCD16bright NK cells subset in recovered SAA patients was higher than that of the other two groups (p<0.05), but there was no statistical difference between untreated patients and normal controls (p>0.05).2. The variation of NK cells function in SAA patients①The changes in the proportion of receptors on NK cells surface in SAA patients The median expressions of CD158a,NKG2D and NKp46 on NK cells surface were 16.45%,95.68%,88.23% in untreated SAA patients,19.34%,96.85%, 82.97% in recovered patients and 21.59%,96.13%,40.99% in normal controls.The expression of NKp46 on NK cells surface of untreated and recovered SAA patients were higher than that of healthy individuals (p<0.05).The expression of CD 158b on NK cells surface was (34.66±16.02)% in untreated patients,(39.13±17.75)% in recovered patients,(41.43±17.43)% in controls,but there was no statistical difference between each groups (p>0.05).②The changes in the proportion of perforin and granzyme-B in NK cells cytoplasm in SAA patients The expression of perforin in NK cells cytoplasm was (66.14±20.73)% in untreated SAA patients,(64.97±21.61)% in recovered patients and (42.11±27.25)% in controls.The expression of perforn in untreated and recovered SAA patients were higher than that of controls (p<0.05),but there was no statistical difference between that of the other groups (p>0.05).The median expression of granzyme-B in NK cells cytoplasm is 96.43% in untreated SAA patients,96.97% in recovered patients,92.88% in controls,there was no statistical difference between each groups (p>0.05).3. The correlations between the percentage of NK cells subsets and hematopoietic function in SAA patients①The correlations between the variations of NK cells and it's subsets percenage and hematopoietic function in SAA patients The percentage NK cells was positively correlated with ANC%,hyperplasia degree of bone marrow,percentage of granuloc-ytes and erythrocytes in bone marrow (r was 0.423,0.455,0.357,0.340 respectively, p<0.05), but negatively correlated with Lym% in peripheral blood and percentage of lymphocytes in bone marrow(r was-0.411,-0.522 respectively, p<0.05).The percen-tage of CD56brightNK cells subset was positively correlated with ANC%, hyperplasia degree of bone marrow, percentage of granulocytes and megakaryocytes in bone marrow(r was 0.609,0.412,0.517,0.362 respectively,p＜0.05). Meanwhile, it was nega-tively correlated with the percentage of lymphocytes in bone marrow(r=-0.435, p<0.05).The percentage of CD56dim NK cells subset was positively correlated with hyperplasia degree of bone marrow (r=0.404, p<0.05), but negatively correlated with the percentage of lymphocytes in bone marrow (r=-0.411,p<0.05).The percentage of CD3-CD56negCD16bright NK cells subset was positively correlated with ANC%, Ret, hyperplasia degree of bone marrow, percentage of granulocytes in bone marrow (r was 0.468,0.328,0.451,0.434 respectively,p＜0.05).Yet, it was negatively correlated with Lym% in peripheral blood and the percentage of lymphocytes in bone marrow (r was-0.389,-0.547 respectively,p<0.05).②The correlation between the function of NK cells and hematopoietic function in SAA patients The expression of CD158a, CD158b, NKG2D, NKp46, perforrin and granzyme-βof NK cells has no correlation with hematopoiesis (p>0.05) in SAA patients.Conclusion The decrease in the percentage of NK cells, CD56bright, CD56dimNK cell subsets may have led to the over-function of T lymphocytes and the hematopoiesis failure in SAA. The higher expression of NKp46 and perforin of NK cells may be a compensation for the decrease of NK cells propotion.
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