| Objective:The purpose of the present study was to investigate the immune reconstitution of lymphocyte subsets after allogeneic hematopoietic stem cell transplantation(allo-HSCT), and analyse the influencing factors, and to provide important clinical data for monitoring of immune reconstitution before and after transplantation, the application and adjustment of immunosuppressive agents.Methods:Seventy-one patients who had undergone allo-HSCT between June 2011 and May 2015 in the first Bethune Hospital of Jilin University were enrolled in this study. The subsets of peripheral blood lymphocytes were determined before transplantation, on day 14, 28, 42 and at 2, 3, 6, 9, 12, 15, 18, 24 months after allo-HSCT. Flow cytometric immunophenotyping was performed to evaluate T cells(CD3+), helper T cells(CD3+CD4+), suppressor/cytotoxic T cells(CD3+CD8+), activated T cells(CD3+HLA-DR+), inactivated T cells(CD3+HLA-DR-), regulatory T cells(CD4+CD25high+Foxp3+), B lymphocytes(CD19+), natural killer cells(CD3-CD56+), natural killer like T cells(CD3+CD56+). Absolute counts for each subset were calculated at the product of the absolute WBC count and lymphocyte differential percentage and the cell subset percentage. Normal values of each cell subset were determined from 16 healthy donors. The influencing factors were divided into different groups and compared.Results:(1) The number of CD3+T cells returned to the value of(1521.41±617.95)/μl within the normal range at 9 months after allo-HSCT; and that of CD3+CD4+T cells remained abnormal at 24 months after allo-HSCT; while that of CD3+CD8+T cells returned to the value of(723.18±469.31)/μl within the normal range at 2 months after transplantation. There was thus a characteristic inversion of the CD4/CD8 ratio up to a long time after allo-HSCT. The ratio of CD4/CD8 was 0.56±0.26 near the lowest of the normal range 0.68 at 24 months after allo-HSCT. The number of CD3+DR+ T cells returned to the normal range nearly after engraftment, then even over the normal range, and gradually returned to normal at 12 months after allo-HSCT; and that of CD3+DR-T cells returned to the value of(1368.77±623.38)/μl within the normal range at 12 months after allo-HSCT. CD4+CD25high+Foxp3+ regulatory T cells(Treg) recovered to(91.69±61.74)/μl within the normal range at 15 months after allo-HSCT.(2)The number of B cells returned to the value of(307.04±127.57)/μl within the normal range at 18 months after allo-HSCT, while the percentage of B cells recovered at 9 months after allo-HSCT.(3)The number of natural killer(NK) cells returned to the value of(220.20±117.89)/μl near the normal range at 3 months after allo-HSCT, while the percentage of NK cells recovered nearly after engraftment. The number of NK like T cells returned to the value of(84.33±55.64)/μl near the normal range at 6 months after allo-HSCT and returned to the value of(113.23±69.29)/μl within the normal range at 12 months after allo-HSCT, while the percentage of NK like T cell recovered after engraftment.(4)All of the patients were divided into two groups by the patients’ ages: <35 years and ≥35 years, the differences of B cells and NK cells at different time points after allo-HSCT between the two age groups were of no significance. All of the patients were divided into two groups by the donors’ ages: <35 years and >35 years, the differences of CD4+T cells on day 28 and at 15 months after allo-HSCT between the two groups were significant, the number of CD4+T cells of the <35 years group was more than the ≥35 years group(P=0.011, 0.042); while the differences of CD8+T cells between the two groups were of no significance. Patients of haploidentical HSCT were divided into two groups by the patients’ ages: <35 years and ≥35 years, the differences of CD4+T cells and CD8+T cells at different time points after allo-HSCT between the two groups were of no significance, although the number of cells of the <35 years group was more than the ≥35 years group. Patients of identical HSCT had the same outcome.(5) All of the patients were divided into two groups by the conditioning with or without anti-thymocyte globulin(ATG). The number of CD3+T cells of no ATG group was significantly higher than that of ATG group on day 14 and at 2 months after allo-HSCT(P=0.012, 0.043); and that of CD4+T cells of no ATG group was significantly higher than ATG group on day 14, 28, 42 after allo-HSCT(P=0.008, 0.001, 0.012); and that of CD8+T cells of no ATG group was significantly higher than ATG group at 2 months after allo-HSCT(P=0.020). The ratio of CD4/CD8 of no ATG group was significantly higher than ATG group on day 14 and at 2 months after allo-HSCT(P=0.027, 0.030). The number of NK cells of no ATG group was significantly higher than ATG group on day 42 after allo-HSCT(P=0.027). The number of B cells of no ATG group was significantly higher than ATG group at 3 months after allo-HSCT(P=0.029). The differences between the two groups of Treg were of no significance at different time points after allo-HSCT.(6) All of the patients were divided into two groups by the source of stem cell. The number of CD4+T cells of peripheral blood stem cell group was significantly higher than that of peripheral blood plus bone marrow stem cell group on day 14, 28, 42 and at 2, 3, 6, 9, 12, 15 months after allo-HSCT(P=0.013, 0.001, 0.004, 0.000, 0.039, 0.045, 0.001, 0.045, 0.042). The ratio of CD4/CD8 of peripheral blood stem cell group was significantly higher on day 14, 28, 42 and at 2, 6, 12, 15, 18 months after allo-HSCT(P=0.017, 0.003, 0.029, 0.026, 0.008, 0.022, 0.019, 0.043). The difference of NK cells of the two groups was significant on day 42 after allo-HSCT(P=0.007). The difference of B cells of the two groups was significant at 6 months after allo-HSCT(P=0.012).(7) At 15 months after allo-HSCT, the number of CD34+ cells and the number of CD3+ T cells showed a highly significant positive correlation(r=0.800, P=0.003), and that of CD34+ cells and CD8+ T cells showed a highly significant positive correlation(r=0.791, P=0.004), and that of CD34+ cells and NK cells showed a highly significant positive correlation(r=0.721, P=0.019), and that of CD34+ cells and CD3+DR-T cells showed a highly significant positive correlation(r=0.809, P=0.003). The number of CD34+ cells and NK like T cell showed a lowly significant positive correlation(r=0.0.367, P=0.043).(8) All of the patients were divided into two groups by the match degree of HLA: haploidentical HSCT and identical HSCT. The number of CD4+T cells of identical HSCT group was significantly higher than that of haploidentical HSCT group on day 14, 28, 42 and at 2, 6, 9, 15, 18 months after allo-HSCT(P=0.000, 0.000, 0.000, 0.000, 0.037, 0.001, 0.042, 0.030). The number of CD8+T cells of haploidentical HSCT group was significantly higher on day 28 and at 6 months after allo-HSCT(P=0.034, 0.027). The ratio of CD4/CD8 of identical HSCT group was significantly higher than that of haploidentical HSCT group on day 14, 28, 42 and at 6, 12, 15, 18 months after allo-HSCT(P=0.010, 0.014, 0.001, 0.000, 0.041, 0.012, 0.005). The number of NK cells of identical HSCT group was significantly higher than that of haploidentical HSCT group on day 42 after allo-HSCT(P=0.001). The number of B cells of identical HSCT group was significantly higher than that of haploidentical HSCT group on day 28 and at 6 months after allo-HSCT(P=0.027, 0.005).(9) All of the patients were divided into two groups by the occurrence of acute graft-versus-host disease(a GVHD) or not. The number of CD4+T cells of no a GVHD group was significantly higher than that of a GVHD group at 2 months after allo-HSCT(P=0.013); the number of CD8+T cells of a GVHD group was significantly higher than that of no a GVHD group on day 28 after allo-HSCT(P=0.038); the ratio of CD4/CD8 of no a GVHD group was significantly higher than that of a GVHD group on day 28 after allo-HSCT(P=0.049). The number of B cells of no a GVHD group was significantly higher than that of a GVHD group on day 28 and at 2, 3, 6 months after allo-HSCT(P=0.021, 0.025, 0.005, 0.006). Besides, the number of B cells of 0-I a GVHD group was significantly higher than that of II-IV a GVHD group at 3 and 6 months after allo-HSCT(P=0.013, 0.019).(10) All of the patients were divided into three groups by the occurrence and the degree of chronic graft-versus-host disease(c GVHD): no c GVHD, limited c GVHD and extensive c GVHD. The number of CD3+T cells of no c GVHD group was significantly higher than that of extensive c GVHD group at 2 months after allo-HSCT(P=0.031), and CD3+T cells of limited c GVHD were significantly higher than extensive c GVHD(P=0.007) at 3 months after allo-HSCT. The number of CD4+T cells of no c GVHD group was significantly higher than that of extensive c GVHD group at 15 months after allo-HSCT(P=0.048). The number of CD8+T cells of limited c GVHD group was significantly higher than that of extensive c GVHD group(P=0.036), and no c GVHD was significantly higher than extensive c GVHD(P=0.005) at 2 months after allo-HSCT. The number of NK like T cells of no c GVHD group was significantly higher than that of c GVHD group(including limited and extensive c GVHD)(P=0.036), and no c GVHD group was significantly higher than extensive c GVHD(P=0.008) at 2 months after allo-HSCT, the number of NK like T cells of limited c GVHD group was significantly higher than that of extensive c GVHD at 3 months after allo-HSCT(P=0.011). The number of B cells of limited c GVHD group was significantly higher than that of extensive c GVHD group on day 42 after allo-HSCT(P=0.008), and B cells of no c GVHD were significantly higher than extensive c GVHD(P=0.048) at 15 months after allo-HSCT. The number of CD3+DR-T cells of no c GVHD gro up was significantly higher than that of extensive c GVHD group at 2 months after allo-HSCT(P=0.018), and CD3+DR-T cells of limited c GVHD were significantly higher than extensive c GVHD(P=0.020) at 3 months after allo-HSCT.(11) All of the patients were divided into two groups by the infection of cytomegalovirus(CMV) or not. The number of CD4+T cells of CMV- group was significantly higher than that of CMV+ group on day 28, 42 and at 2, 3, 6, 9, 15, 24 months after allo-HSCT(P=0.000, 0.011, 0.000, 0.008, 0.007, 0.006, 0.042, 0.024). The ratio of CD4/CD8 of CMV- group was significantly higher than that of CMV+ group on day 28 and at 3, 6, 9, 12, 15, 24 months after allo-HSCT(P=0.001, 0.010, 0.001, 0.032, 0.029, 0.012, 0.017). The number of B cells of CMV- group was significantly higher than that of CMV+ group on day 42 and at 3, 6 months after allo-HSCT(P=0.034, 0.028, 0.023). The infection of CMV had no significant influences on the number of other lymphocytes(P>0.05).Conclusions:(1)The number of CD3+T cells returned to normal range at 9 months after allo-HSCT; the number of CD3+CD4+T cells returned to the value near the normal range at 24 months after allo-HSCT; the number of CD3+CD8+T cells returned to normal at 2 months after allo-HSCT. There was thus a characteristic inversion of the CD4/CD8 ratio up to over 24 months after allo-HSCT. CD3+DR+T cells returned to normal nearly after engraftment; CD3+DR-T cells returned to the value near normal range at 12 months after allo-HSCT. Treg returned to normal at 12 months after allo-HSCT. B cells returned to normal at 18 months after allo-HSCT. NK cells returned to normal at 3 months after allo-HSCT, and NK like T cells returned to normal at 12 months after allo-HSCT.(2)Ages of patients had no significant influences on the immune reconstitution(IR) after allo-HSCT, while younger donors contributed to the IR of CD4+T cells. Peripheral blood hematopoietic stem cell transplantation contributed to the IR of CD4+T, B and NK cells comparing to bone marrow plus peripheral blood hematopoietic stem cell transplantation. More CD34+ cells helped the recovery of CD3+T, CD8+T, CD3+DR-T and NK like T cells. Identical HSCT contributed to the IR after allo-HSCT. ATG conditioning could delay the reconstitution of lymphocytes, the occurrence of a GVHD had the same function, and especially a GVHD of severe degree delayed the reconstitution of B cells worse. The occurrence of c GVHD would delay the reconstitution of T and B cells, and extensive c GVHD delays worse. The infection of CMV delayed the reconstitution of CD4+T and B cells. |
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