Pre-Transplant Lymphocyte Levels Influence Anti-Thymocyte Globulin Exposure, Affecting Graft-versus-Host Disease Rates and Post-Transplant Thymic Recovery: Insights from a Single-Center Retrospective Analysis

Introduction

Despite advances in hematopoietic stem cell transplantation (HSCT), graft-versus-host disease (GVHD) continues to pose substantial morbidity and mortality [1, 2]. Immunosuppressive prophylaxis remains the primary strategy to reduce GVHD incidence and severity [3, 4]. While these interventions decrease GVHD-related deaths, relapse and infection-related mortality remain significant contributors to overall survival (OS) in HSCT recipients [5, 6].

The use of anti-thymocyte globulin (ATG) in conditioning regimens has become standard for unrelated or mismatched donor transplants, yet optimal dosing and timing remain unclear, particularly regarding minimizing delays in T-cell reconstitution while maximizing GVHD protection and graft success [7, 8]. Most data derive from adult studies employing high ATG doses due to the higher GVHD burden in adults [9–11]. Standard dosing is weight-based, but patient-specific responses can vary significantly [12–15]. Additionally, the anti-leukemic effects of ATG and its potential impact on relapse rates are not fully understood [16].

Given ATG’s mechanism of T-cell depletion, some studies suggest adjusting its use according to the recipient’s absolute lymphocyte count, as higher pre-transplant lymphocyte levels may reduce ATG’s immunosuppressive effect on donor lymphocytes, potentially increasing GVHD risk [17, 18].

Evidence linking ATG exposure with T-cell reconstitution and transplant outcomes is limited, particularly in pediatric populations, where these relationships are most critical [15, 17, 19, 20]. In this study, we retrospectively assessed pediatric HSCT patients receiving prophylactic ATG, stratifying them based on the ratio of total ATG dose to lymphocyte count on the day of administration.

Successful reconstruction of adaptive immunity post-HSCT relies on thymic activity, as active thymopoiesis is essential for generating a fully functional naïve T-cell pool with broad antigenic diversity [21, 22]. We evaluated thymus-dependent T-cell recovery by measuring thymic volume via MRI and quantifying recent thymic emigrants (RTEs) through immunophenotyping. Finally, we explored potential links between thymic function and transplant outcomes, including GVHD incidence, OS, event-free survival (EFS), and relapse rates.

Materials and Methods

This study was a retrospective analysis conducted at the Pediatric Transplant Center of the Institute for Maternal and Child Health–IRCCS “Burlo Garofolo” in Trieste, Italy. Ethical approval was granted by the IRCCS Burlo Garofolo Institutional Review Board (RC 08/19) and the Unique Regional Ethics Committee (2869), and the study was retrospectively registered on ClinicalTrials.gov (NCT04869254). Written informed consent for research use of clinical data was obtained from all parents.

We reviewed medical records of patients aged 18 years or younger who underwent allogeneic hematopoietic stem cell transplantation (HSCT) between April 2005 and April 2020 and received anti-thymocyte globulin (ATG; Thymoglobulin, Genzyme Europe BV, Amsterdam, The Netherlands) as part of myeloablative conditioning (ATG-exposed group). Only first HSCT procedures were included. No restrictions were placed on transplant indication, cell source, or ATG dose. Patients were excluded if they had received ATG within three months prior to HSCT or had documented ATG hypersensitivity. ATG total doses ranged from 2.5 to 12 mg/kg, reflecting changes in institutional dosing practices over the 15-year period. ATG was usually administered intravenously over three days starting four days before the transplant.

For analysis, the ATG-exposed cohort was divided into two subgroups based on the ratio of total ATG dose (mg) to pre-ATG lymphocyte count (cells × 10²/μL), using 0.01 as the cutoff to balance group sizes.

All patients underwent standard myeloablative conditioning regimens [23]. GVHD prophylaxis included a calcineurin inhibitor and mycophenolate mofetil for matched unrelated donors, with post-transplant cyclophosphamide added from 2013 for haploidentical transplants. Supportive care, including infection prevention and management, followed institutional protocols. Follow-up extended from HSCT until last contact or death, with surviving patients monitored for at least 12 months. Disease risk indices were calculated using refined criteria based on disease type, stage, and cytogenetic risk [24], while thalassemia risk classification followed published guidelines [25]. Ewing sarcoma was considered very high risk when requiring allogeneic transplant.

The primary outcome was GVHD incidence, defined as any episode necessitating systemic immunosuppressive therapy. Acute and chronic GVHD were defined by therapy initiation before or after 100 days post-HSCT, respectively, using standard diagnostic criteria [26, 27]. NIH GVHD scoring could not be applied due to the retrospective design. Secondary endpoints included overall survival (OS), defined as time from HSCT to death from any cause or last follow-up, and relapse-related mortality (RRM), defined as time to death due to disease recurrence. Viral reactivation was recorded if antigen positivity was detected in blood, urine, stool, or surface swabs. Immune reconstitution was defined as recovery of CD4+ T cells to ≥500 cells/μL on two consecutive measurements within 100 days post-HSCT; patients who died before day 100 were analyzed up to death.

Thymic recovery was assessed via MRI at 1, 3, and 12 months post-HSCT, and by quantifying recent thymic emigrants (RTEs) at 100 days and 1 year. RTEs were identified as naïve T cells expressing CD45+CD3+CD4+CD45RA+CD31+ by flow cytometry. For comparison, thymic volumes were obtained from 250 MRI scans of children aged 0–18 years with no hematologic, oncologic, inflammatory, or infectious diseases, stratified by year of age. Additionally, patients undergoing first-time allogeneic HSCT without ATG during the same period were evaluated to compare thymic recovery with controls.

Statistical analysis

Descriptive statistics summarized patient demographics and clinical characteristics. Categorical variables were compared using Fisher’s exact or chi-square tests, while continuous variables were analyzed with the Wilcoxon rank-sum test. Continuous data are reported as medians with interquartile ranges (25th–75th percentiles), and categorical data as counts or percentages. Box-and-whisker plots depicted distributions. The Mann–Whitney test and two-tailed Fisher exact test were applied as appropriate. OS and RRM were estimated using Kaplan–Meier methods and compared by log-rank test. Statistical significance was defined as p < 0.05. Analyses were performed using WinStat v.2012.1 and MedCalc v.18.9.1.

Results and Discussion

Between April 2005 and April 2020, 176 patients underwent HSCT at our center. After applying exclusion criteria, 102 patients were included in the ATG-exposed group and 69 in the ATG-unexposed group. Both groups were comparable in terms of sex distribution, median age at transplant, transplant indication, disease risk index, conditioning regimen, and graft source. Baseline characteristics for both cohorts are summarized in Table 1.

Abbreviations: MCHT, myeloablative chemotherapy; TBI, total body irradiation; SD, standard deviation; ATG, anti-thymocyte globulin.

To assess the impact of ATG exposure on transplant outcomes, the ATG-exposed cohort was further divided into two subgroups based on the ATG/lymphocyte ratio: a low-ratio subgroup (<0.01) and a high-ratio subgroup (>0.01). This stratification was guided by recent studies highlighting the significance of both the recipient’s lymphocyte count at the initiation of ATG infusion and the total cumulative ATG dose [28, 29]. Both subgroups were well-matched with respect to sex, age at transplantation, and disease risk index (Table 2).

Abbreviations: ATG, anti-thymocyte globulin; SD, standard deviation; GVHD, graft-versus-host disease; VOD, veno-occlusive disease; CMV, cytomegalovirus; EBV, Epstein–Barr virus.

*Patients deceased prior to lymphocyte recovery.

**Endothelial complications include VOD, capillary leak syndrome, engraftment syndrome, transplant-associated thrombotic microangiopathies, and diffuse alveolar hemorrhage.

Patients in the subgroup with an ATG/lymphocyte ratio below 0.01 exhibited a markedly higher lymphocyte count before ATG administration and received a considerably lower cumulative ATG dose compared with those in the subgroup with a ratio above 0.01 (p < 0.001; (Table 2)).

GVHD incidence

Acute GVHD developed in 36 patients within the ATG-exposed cohort, comprising 29 individuals (59%) in the low-ratio group and 7 individuals (16.6%) in the high-ratio group (p < 0.001). Within 100 days post-transplant, the cumulative incidence of grade I–II acute GVHD was 34.4 percent in the low-ratio subgroup compared to 9.5% in the high-ratio subgroup (p < 0.001), whereas grade III–IV acute GVHD was observed in 24.5% versus 7.1 percent, respectively (p < 0.05; (Figure 1)). The occurrence of chronic GVHD, either limited or extensive, did not differ significantly between 2 subgroups (p = 0.096).

Survival and relapse

Overall survival was significantly better in the low-ratio subgroup compared with the high-ratio subgroup (80.4 percent vs. 58.0 percent, p = 0.031), whereas non-relapse mortality was notably higher in patients with a high ATG/lymphocyte ratio (33.0% vs. 13.1%, p = 0.035). No statistically significant differences were observed for event-free survival (64% vs. 47%, p = 0.11) or relapse-related mortality (5.9% vs. 9.8%, p = 0.7) between 2 groups. Detailed outcomes, including the specific causes of death in each subgroup, are summarized in Table 2. The OS curves for both subgroups are illustrated in Figure 2. Multivariate Cox regression analysis supported the univariate findings, indicating that primary diagnosis, conditioning regimen, and stem cell source did not significantly influence OS, and that only the ATG/lymphocyte ratio remained an independent predictor of survival (p < 0.03, HR = 2.4, 95 percent CI 1–5.4; (Table 3)).

Abbreviations: HR, hazard ratio; CI, confidence interval; ATG, anti-thymocyte globulin; AML/MDS, acute myeloid leukemia/myelodysplastic syndrome; TBI, total body irradiation; PBSC, peripheral blood stem cells.