Evaluation of Donor CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells and CD3^-CD56⁺ NK Cells on Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

Background: It is important to determine the changes in both donor T cells and B cells on immune reconstitution following allogeneic hematopoietic stem cell transplantation (HCT). It is well-known that Treg cells play important roles in the prevention of T-cell-mediated graft-versus-host disease (GVHD) and in promoting tumor escape from T-cell-dependent immunosurveillance. However, very little is known about the number and function of both CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and CD3^-CD56⁺ natural killer (NK) cells and about NK activity during immune reconstitution. In this study, we examined changes in the number of circulating Treg and NK cells in the peripheral blood (PB) and the NK activity of HCT patients at various times after HCT, and we evaluated the clinical significance of these findings relative to patient survival.

Patients and Methods: We evaluated 29 consecutive patients (ages >18) without GVHD who underwent HCT for different hematologic diseases between December 2008 and April 2017. Treg and NK cells were characterized and quantified by flow cytometry from these 29 patients and 15 healthy controls. Evaluated variables included recipient and donor characteristics, transplant-related factors, including conditioning regimen (myeloablative conditioning or reduced-intensity conditioning), donor type (matched sibling donor, unrelated donor, other relative (haploidentical), or cord blood transplant), degree of match (8/8, 7/8, 6/8, or haploidentical), and GVHD prophylaxis. Patients who were followed up for more than 12 months were enrolled in the study. After obtaining informed consent, blood was drawn from all patients on the day of engraftment and at day 100 and at 12-month intervals thereafter. CD4⁺CD25⁺Foxp3⁺ Treg cells, CD3⁺CD4⁺T cells, CD3⁺CD8⁺ T cells, and CD3^-CD56⁺ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard ⁵¹Cr release assay at an effector: target (E: T) ratio of 20:1.

Results: The median patient age was 58 years (range: 19-71 years), and 21 out of 29 of the patients were male. At 100 days, the percentage of B cells (2.5±6.0%) and absolute numbers of CD8⁺ T cells (269.7±284.8/μL), CD4⁺T (22.83±292.4/μL) cells and NK cells (248.3±229.1/μL) were significantly lower than those at 2 years (20.9±11.6%, 747.2±648.4/μL, 588.0±607.3/μL, 558.1±336.2/μL, p<0.01, p<0.01, p<0.01, p=0.027, respectively) ,and at 3 years (18.2±11.2%, 554.1±343.3/μL, 488.1±210.3/μL, 500.1±451.3/μL, p<0.01, p=0.020, p=0.017, p=0.043, respectively). The changes in Treg% values in peripheral lymphocytes within 100 days to 3 years after HCT significantly decreased from 11.57 ±11.39 to 3.65 ±2.59% (p=0,039). The % of NK cells in the PB at 1 year after HCT (13.95±10.34%) was significantly lower than that within 100 days after HCT (27.1±16.8%, p=0.01). However, the absolute number of NK cells did not differ. In comparison with the normal control group, significant difference was observed in the Treg cells. Both percentage and absolute number of Treg cells (0.70±0.29%, 40.2±17.3/μL) in the normal control group were significantly higher than those at 1 year (0.33±0.16%, 20.6±15.4/μL, p<0.01, p<0.01, respectively), at 2 years (0.44±0.16/%, 25.1±15.4/μL, p=0.35, p<0.01, respectively), and at 3 years (0.29±0.23%, 15.5±11.8/μL, p<0.01, p<0.01, respectively). In contrast, the absolute number of CD8⁺ T cells (380.3±128.5/μL) in the normal control group were significantly lower than those at 1 year (935.1±648.4/μL, p<0.01), at 2 years (747.2±648.4/μL, p<0.01), and then did not differ at 3 years (554.1±343.3/μL).

Conclusion: The percentage and the absolute number of Treg cells in HCT patients were significantly lower than those in the normal control group, whereas CD8⁺ T cells was significantly higher in the patients within 2 years after HCT than that in the normal control group. The % Treg cells did not recover even at 3 years after HCT, rather it tended to be somewhat lower. In contrast, there was no change in the NK cells. These results suggest that the immunological reconstitution has not been achieved even at 3 years after transplantation and that the immunological consequences of GVHD are maintained. The antitumor effect is also maintained. On the other hand, NK cells recover at an early stage. The roles of these different immune cells after HCT require further investigation.