Donor and recipient age-related differences in immunological reconstitution affect development of chronic graft-versus-host disease

Abstract Background: Donor source (p=0.032) and patient age (p=0.015) were associated with onset of chronic graft-versus-host disease (cGVHD) in 81 cases of allogeneic hematopoietic cell transplantation (HCT) with survival for more than one year at our hospital since 2008. In this study, we divided older and younger HCT patients into those treated with umbilical cord blood (CB) and bone marrow/peripheral blood (BM/PB) to determine if differences in immunological reconstitution affect onset of cGVHD. Methods: The study included 40 consecutive patients aged ≥18 years who underwent HCT for various hematological diseases at our hospital between December 2008 and December 2023 and survived for ≥3 years after HCT without developing cGVHD. Patients aged ≤59 years were classified as young (n=23) and those aged ≥60 years (n=17) were classified as old. The patients were evaluated in four groups: CB-young (n=5), CB-old (n=8), BM/PB-young (n=18), and BM/PB-old (n=9). Evaluated variables included recipient characteristics and transplant-related factors, including conditioning regimen, degree of match, and GVHD prophylaxis. After obtaining informed consent, blood was drawn on day 200 and at 24 and 36 months after HCT. Analyses of CD4+CD25+Foxp3+ Treg cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ B cells, and CD3-CD56+ NK cells were performed using flow cytometry, and the absolute numbers of these cells were calculated. NK activity was measured by a standard 51Cr release assay at an effector:target (E:T) ratio of 20:1. T cells were isolated at the same time using EasySep kits and cultured at 37℃ in 5% CO2 in air for 4 days with 10% heat-inactivated fetal calf serum as controls, interleukin-2 (IL-2) and transforming growth factor-β1 (TGFβ1) as samples, or concanavalin A (ConA) (10 µg/ml) at a density of 1×106 cells/ml. Treg cells were analyzed by flow cytometry and ConA-stimulated culture supernatants were tested for levels of IL-4, IL-5, IL-6, IL-10, IL-13, TGFβ1, tumor necrosis factor α (TNFα) and interferon-ɤ (IFNɤ). Results: Peripheral blood CD4/8 improved in the CB-young group (0.30 ± 0 at 200 days, 0.59 ± 0.18 at 2 years, and 1.84 ± 0.27 at 3 years) (R = 0.913, p = 0.0016), but did not improve over these periods in young or old patients after BM/PB transplantation. Treg cell percentages, absolute numbers, and expansion rates after stimulation cultures on day 200 after transplantation were significantly higher in young patients (6.37 ± 4.02%, 3759.4 ± 1963 /mL, 12.4 ± 11.5-fold) than in old patients (3.03 ± 1.4%, 1783.9 ± 1133 /mL, 5.7 ± 3.8-fold) (p = 0.027, p = 0.003, p = 0.023). In particular, the Treg cell percentage proliferation rates in the CB-young group of 2.4±0, 2.8±0.6 and 5.8±2.1 times at 200 days, 2 years, and 3 years, respectively, were significantly higher than those in other groups (R=0.711, p=0.048). Regarding cytokine production, IL-5 in stimulation culture tended to increase over time in all young patients, from 4.75±8.89 pg/mL at 200 days, to 20.04±27.02 pg/mL at 2 years, and 59.51±104.93 pg/mL at 3 years (R=0.294, p=0.087), whereas there was no improvement in cytokine production in old patients, regardless of the donor source. Conclusion: In younger patients, T cell reconstitution and cytokine production improved over time from the early post-transplant period. Additionally, Treg cell reconstitution was higher in younger patients early after transplantation, and improved over time in younger patients after CB transplantation. Older patients showed slower recovery of T cell function than younger patients. However, there were no differences in immunological reconstitution in older patients according to donor source. This single-center study suggests that development of chronic GVHD is influenced by patient age, and that immunological reconstitution according to donor source is influential in younger patients.