Delay of Regulatory T Cell Reconstitution in Elderly Patients Increases the Frequency of Chronic Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Regulatory T cells (Treg) play important roles in onset of graft-versus-host disease (GVHD) and chronic GVHD (cGVHD) in elderly people is more frequent than in younger people after allogeneic hematopoietic stem cell transplantation (HCT). However, little is known about the differences in the number and function of Treg and natural killer (NK) cells in elderly and young patients. In this study, we examined the cause of the difference in incidences of cGVHD in these patient populations by measuring Treg and NK cells over time after HCT.

Patients and Methods: The subjects were 65 consecutive patients aged >18 years who underwent HCT for different hematologic diseases at our hospital between December 2008 and December 2018 and survived for >100 days after HCT. The median age was 57 years (range: 19-73 years), and those aged ≥58 (n=33) and ≤57 (n=32) were classified as elderly and younger patients, respectively. Evaluated variables included recipient and donor characteristics, and transplant-related factors, including conditioning regimen, donor type, degree of match, and GVHD prophylaxis. After obtaining informed consent, blood was drawn from all patients on day 100 and at 12-month intervals thereafter. Analysis of CD4⁺CD25⁺Foxp3⁺ Treg cells, CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, CD19⁺ B cells, and CD3^-CD56⁺ NK cells was performed using flow cytometry, and the absolute numbers of these cells were calculated. NK activity was measured by the standard ⁵¹Cr release assay at an effector:target (E:T) ratio of 20:1.

Results: The incidence of grades II-IV acute GVHD was 29.2% at 100 days. There was no difference in the incidence of acute GVHD between elderly and younger patients [HR=0.85, 95% CI 0.34-2.09, p=0.72]. However, the overall incidence of cGVHD was 37.1%. In univariate analysis, the incidence of cGVHD in elderly patients was significantly higher than that in younger patients [HR=3.61, 95% CI 1.13-11.59, p=0.031]. Elderly patients had a significantly lower percentage of B cells than younger patients at 24 months (13.4±9.6% vs. 27.0±15.6%, p=0.024) and 36 months (16.4±10.3% vs. 31.1±11.2%, p<0.01); and also had a lower percentage and absolute number of Treg cells at 12 months (0.24±0.10% vs. 0.47±0.32%, p=0.010; 14.8±7.7 /µl vs. 24.0±18.4 /µl, p=0.061) and 36 months (0.17±0.15% vs. 0.37±0.20%, p<0.01; 12.7±8.54 /µl vs. 20.9±12.9 /µl, p=0.046). The percentage and absolute number of CD8⁺ cells were higher in elderly patients at 12 months (50.4±17.2% vs. 33.5±12.9%, p<0.01; 1130.5±588.5 /µl vs. 611.8±365.9 /µl, p=0.016) and 24 months (46.2±10.8% vs. 26.1±9.7%, p<0.01; 1115.7±484.2 /µl vs. 584.2±372.2 /µl, p<0.01). However, elderly patients had a lower percentage of NK cells at 12 months (11.9±8.0% vs. 20.1±14.5%, p=0.045) and lower NK activity at 24 months (24.0±14.0% vs. 47.6±16.3%, p<0.01). The same results were obtained in 50 cases after exclusion of patients who developed cGVHD (≥58, n=22; ≤57, n=28). Thus, the 22 elderly patients had a lower percentage and absolute number of Treg cells at 12 months (0.22±0.16% vs. 0.47±0.32%, p=0.015; 14.1±8.3 /µl vs. 24.0±18.4 /µl, p=0.071) and 36 months (0.16±0.11% vs. 0.40±0.20%, p<0.01; 12.2±9.2 /µl vs. 22.7±12.7 /µl, p=0.023), and a higher percentage and absolute number of CD8⁺ cells at 12 months (49.0±18.8% vs. 33.5±12.9%, p=0.031; 1181.9±619.0 /µl vs. 611.8±365.9 /µl, p=0.015) and 24 months (45.6±11.4% vs. 25.4±9.6%, p<0.01; 1067.6±494.1 /µl vs. 587.1±382.9 /µl, p=0.012).

Conclusion: The percentage and absolute number of Treg cells in elderly patients were significantly lower than those in younger patients, whereas CD8⁺ T cells were significantly higher in elderly patients after HCT. The absolute number of Treg cells tended to reconstitute one year after HCT in younger patients, but tended to decrease gradually in elderly patients, with no recovery even three years after HCT. In contrast, there was no change in NK cells. Similar results were obtained in an analysis excluding patients who developed cGVHD. These results suggest that a difference in immunological reconstitution between elderly and younger patients may explain the difference in the incidence of cGVHD between these patients. This may be useful in determining the choice of therapy with consideration of the immunologic mechanism.