Global T-cell functionality evaluated by whole blood interferon-gamma release assay as a valuable indicator for immune reconstitution monitoring in pediatric allo-HSCT

Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a cornerstone therapeutic approach for treating a diverse range of hematological disorders, congenital rare diseases, and malignancies in pediatric patients (1,2). This procedure offers the potential for a definitive cure by replacing or restoring the damaged or depleted immune system with donor-derived hematopoietic stem cells, thereby re-establishing normal hematopoiesis and immune function. However, the success of allo-HSCT relies not only on the engraftment of donor cells but also critically on the subsequent immune reconstitution (IR) in the recipients. IR following allo-HSCT is a complex and dynamic process involving the recovery of both innate and adaptive immune systems, spanning several months to years to achieve optimal immune function (3). Notably, the reconstitution of adaptive immunity, particularly T-cell IR, significantly impacts transplant-related outcome parameters such as graft-versus-host disease (GvHD), infections, and relapse. Therefore, comprehensive monitoring and assessment of T-cell IR are paramount in optimizing patient outcomes post-allo-HSCT. The process of IR encompasses the recovery of immune cell populations and restoration of their cellular functionality. Currently, in the clinical settings, immune system assessments rely heavily on routine assays such as cell counting and flow cytometry-based measurement of cell surface biomarkers (4,5). However, these conventional assays mainly quantify viable immune cells but offer limited insights into their functional capabilities.

Recently, whole blood interferon-gamma release assay (WB-IGRA) has emerged as a promising method for evaluating T-cellular functionality (6). It accurately quantifies the concentration of cell-released interferon-gamma (IFN-γ) as a measure of T-cellular response to a given stimulation. By utilizing a nonspecific stimulation, this assay exhibits the potential to assess the global T-cell functionality. In a previous proof-of-concept study of WB-IGRA application in T-cell functionality of adult patients with septic shock, a significant decrease in IFN-γ release capacity was observed, which was associated with lower mHLA-DR expression and decreased CD8⁺ T cell count (7). These findings suggest that WB-IGRA has the potential to be a valuable tool for monitoring T-cell immune alterations in adults. However, the application of WB-IGRA in children, especially immunodeficient ones like those following HSCT, remains an area requiring further evaluation and exploration.

The goal of the current study is to provide a novel tool and strategy for assessing and monitoring T-cell IR after allo-HSCT using the VIDAS^® solution (bioMérieux) based on WB-IGRA. By doing so, we hope to contribute to improving the understanding of T-cell function recovery and facilitate the immune monitoring and management in children after allo-HSCT. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-80/rc).

Methods

Study design and participants

This single-center study was conducted at the Hematology & Oncology department of Shanghai Children’s Medical Center between November 2022 and August 2023. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board and the Ethics Committee of Shanghai Children’s Medical Center (SCMCIRB-K2022115-1). Routine follow-up was required for pediatric patients before transplantation and at 3, 6, 9, 12, 24, 36, 48, and 60 months after transplantation. During the follow-up, the attending physician sought the opinions of the parents on whether they were willing to participate in this clinical study. Written informed consent was obtained from the parent or legal guardian of the participants (and themselves in the case of children ≥8 years of age). A total of 126 pediatric patients (<18 years old) who underwent allo-HSCT were enrolled. The patients recruited could be at several months to several years post-allo-HSCT [median =9.3 months; interquartile range (IQR), 5.7–23.8 months]. Detailed patient characteristics, including age, gender, underlying disease, conditioning regimen, donor type, GvHD prophylaxis and history, transplant time, intravenous immunogloblin (IVIG) history and immunosuppressant usage status, were collected from electronic patient records. Additionally, cellular phenotype (DURAClone IM Phenotyping BASIC Tube RUO, Beckman coulter, USA) results, characterizing classical T cell subsets CD3⁺, CD4⁺ and CD8⁺, were obtained from the patients’ regular examinations at various follow-up times post-allo-HSCT.

Global T cell functionality

The VIDAS^® solution kits (bioMérieux, Marcy-l'Étoile, France) based on a standardized WB-IGRA were performed to evaluate the global T cell functionality. Briefly, fresh heparinized whole blood samples were stimulated with a concentration of 100 µg/mL reagent containing mitogen (VIDAS^® STIMM^TM BASIC RUO-bioMérieux), which served as a nonspecific T cell stimulant in this study. A blood sample stimulated with a blank stimulation reagent (VIDAS^® STIMM^TM BASIC RUO-bioMérieux) acted as a negative control. The mixed samples were then incubated at 37 ℃ under 5% CO₂ for 22 hours. Following the incubation period, the supernatant of each sample was collected to quantify the concentration of IFN-γ released due to nonspecific T cell activation, performed on the miniVIDAS^® platform (bioMérieux) with ready-to-use reagents based on enzyme-linked immunofluorescent assay (ELFA) (VIDAS^® IFN-γ RUO-bioMérieux). The measured results are expressed as the relative fluorescent value (RFV).

Statistical analysis

The demographic characteristics of the enrolled patients were summarized using median and IQR for continuous data, while absolute values and percentages were used for categorical data. Chi-squared test for categorical variables and Mann-Whitney test for quantitative data were performed. The results of IFN-γ and cell numbers were presented as individual values and using boxplots, which depict the 25^th to 75^th percentile range along with the median. The Mann-Whitney test was utilized for comparing two groups and Kruskal-Wallis test was used for comparing three or more groups. Mantel-Haenszel test was examined for linear trends. Spearman correlation analysis was used to assess the correlation between two factors. P value less than 0.05 was considered statistically significant. And P value for linear trend below 0.05 was considered as statistically significant. The statistical analyses were conducted using OriginPro 2017 (OriginLab Corporation, Massachusetts, USA) and SPSS R27.0.1.0 (IBM Corp., New York, USA) for Windows, which was also utilized to generate graphs and visualize the results.

Results

Clinical characteristics

The study cohort comprised 126 pediatric allo-HSCT patients. The clinical characteristics of these patients are shown in Table 1. The cohort was categorized based on the duration between transplant and recruitment. Specifically, 49 (39%) subjects were classified as post-transplant ≤6 months, 22 (17%) subjects fell in >6 and ≤12 months post-transplant period, and 55 (44%) subjects were categorized as post-transplant >12 months. Meanwhile, at the time of enrollment, 42 (33%) patients were actively using immunosuppressants, predominantly cyclosporin (82%). Furthermore, another 15 (12%) patients were in the process of gradually reducing the dosage of the immunosuppressants, while 69 (55%) had completely ceased their usage. The immunosuppressant usage exhibited a significant correlation with their post-transplant time. The patient proportion of immunosuppressant usage (including in use and decrease in use) was highest ≤6 months post-transplant (88%), gradually decreasing >6 and ≤12 months (45%), and mostly discontinued >12 months post-transplant (4%) (Table S1). Most subjects had recovered from GvHD (54%) or had no history (43%) at inclusion, while only 4 (3%) subjects showed active GvHD.

Evaluation and monitoring of global T-cell functionality

Here, a standardized WB-IGRA assay was employed to evaluate the global T-cell functionality of pediatric patients following their allo-HSCT. The results presented in Figure 1A exhibit a significant increase in the RFV of IFN-γ, which reflected the elevated level of global T-cell functionality across different post-transplant time periods (≤6 months, >6 and ≤12 months and >12 months post-allo-HSCT). Furthermore, our WB-IGRA results clearly indicated an increasing trend in T-cell functionality among pediatric allo-HSCT patient groups, from immunosuppressants use (in use), to reduced usage (decrease in use) and ultimately discontinuation (out of use) (Figure 1B). Considering the recovery time, pediatric allo-HSCT patients within the same post-transplant period who were receiving immunosuppressant medication (including those with reduced usage) exhibited a lower level of T-cell functionality compared to those not using immunosuppressants, though the difference was not statistically significant (Figure S1). However, among patients not using immunosuppressants, the level of global T cell functionality increased with recovery time, consistent with the overall trend. These findings suggest that this WB-IGRA assay shows promise as a valuable approach for assessing cellular immune functionality during post-allo-HSCT recovery in children.

Figure 1 The global T-cell functionality of allo-HSCT children in different post-transplant periods (≤6 months post-trans, >6 and ≤12 months post-trans and >12 months post-trans) (A) and those with different immunosuppressant usage status (in use, decrease in use and out of use) (B). allo-HSCT, allogeneic hematopoietic stem cell transplantation; IFN-γ, interferon-gamma; RFV, relative fluorescent value.

T-cellular IR in children of different ages

Clinically, counting T cell subset numbers always serves as a crucial indicator for monitoring IR. Notably, the quantity of immune cells varies among children of different ages because of varying degrees of immune system development (8,9). Indeed, within the same post-transplant period, we observed that most children ≤10 years old generally exhibited higher cell count levels of CD3⁺, CD4⁺ and CD8⁺ cells compared to those above 10 years old (Figure 2A-2C). In children ≤10 years old, there were statistically significant increases in CD3⁺, CD4⁺, and CD8⁺ cell numbers over the post-transplant period. In contrast, CD4⁺ cell numbers showed a statistically significant increase over time in children >10 years old, whose elevated trends of CD8⁺ cell numbers were not even significant. We further examined global T-cell functionality in these two age groups (Figure 2D). Our findings indicate a significant increase in the level of global T-cell functionality over time in children >10 years old. However, younger children showed no statistically significant differences in pairwise comparisons between consecutive timepoints, with some still exhibiting low levels of T-cell functionality even 12 months after transplantation. Subsequent correlation analysis revealed that, in children ≤10 years old, CD3⁺, CD4⁺ and CD8⁺ cell counts were more strongly correlated with post-transplant time compared to the level of global T-cell functionality (Figure 3A-3D). Conversely, in children >10 years old, both CD4⁺ cell number and the level of global T-cell functionality displayed greater relevance to post-transplant time (Figure 3E-3H). When analyzing the correlation between cell subset counting with T-cell functionality, all cell subset counts were significantly correlated with T-cell functionality in the older age group, whereas no such correlation was observed in the younger group (Figure 4). These results underscore the crucial importance of considering age-specific indicators and monitoring strategies for IR in pediatric allo-HSCT recipients. Compared to younger allo-HSCT children ≤10 years old, assessing T-cell functionality may be a more appropriate metric for evaluating IR in older children, whose immune systems exhibit greater maturity.

Figure 2 The numbers of T cell subsets, including CD3+ (A), CD4+ (B), CD8+ (C), and the global T cell functionality (D) of pediatric allo-HSCT patients in different post-transplant periods (≤6 months post-trans, >6 and ≤12 months post-trans and >12 months post-trans). The patients were divided into those ≤10 (blank) and >10 (twill) years of age. allo-HSCT, allogeneic hematopoietic stem cell transplantation; IFN-γ, interferon-gamma; RFV, relative fluorescent value.

Figure 3 The correlation between the numbers of T cell subsets, including CD3+ (A,E), CD4+ (B,F), CD8+ (C,G), and global T cell functionality (D,H), along with the post-transplant time in pediatric allo-HSCT patients ≤10 (upper) and >10 (bottom) years of age. allo-HSCT, allogeneic hematopoietic stem cell transplantation; IFN-γ, interferon-gamma; RFV, relative fluorescent value.

Figure 4 The correlation between global T cell functionality and the numbers of T cell subsets, including CD3⁺ (A,D), CD4⁺ (B,E), CD8⁺ (C,D), in pediatric allo-HSCT patients ≤10 (upper) and >10 (bottom) years of age. allo-HSCT, allogeneic hematopoietic stem cell transplantation; IFN-γ, interferon-gamma; RFV, relative fluorescent value.

Discussion

Immune functional assays, which measure the ability of immune cells to perform specific responses, face challenges due to a lack of standardization. Recent findings suggest that using whole blood samples for functional testing can yield more stable results compared to assays that require isolated peripheral blood mononuclear cells (PBMCs) (10,11). This approach could potentially improve the reliability and consistency of functional assays in monitoring T-cell IR post-allo-HSCT. In our current study, we introduced a new research use only (RUO) WB-IGRA assay to evaluate T-cell functionality in pediatric patients following allo-HSCT. Our findings demonstrate that the WB-IGRA method can effectively monitor the recovery process of T-cell functionality at various stages post-transplantation and distinguish between patients receiving immunosuppression and those who are not. This longitudinal assessment is critical for understanding the dynamics of IR in pediatric allo-HSCT recipients. It potentially provides clinicians with actionable insights into the effectiveness of treatment strategies and the immune status of transplant recipients, and therefore contributes to improving the outcomes after allo-HSCT.

Previous investigations have consistently emphasized recipient age as the paramount factor influencing IR post-transplant (12,13), with pediatric patients typically undergoing a more rapid T-cells reconstitution than adults (14). However, within the pediatric population, the specific characteristics of IR across different age brackets remain largely underexplored. Through a comparative assessment of children >10 and ≤10 years of age, we have observed distinct age-specific patterns in T-cell IR. Among the younger cohort ≤10 years of age, the process is characterized by a marked increase in the diversity and abundance of immune cell subtypes. In contrast, older children display a more substantial improvement in immune function assessment relative to their younger peers. It is noteworthy that within our cohort, the occurrence of primary immunodeficiency disease (PID) and the umbilical cord blood (UCB) transplantation (in which the lymphocyte content was less than in peripheral blood stem cells), both of which probably increase the difficulty in IR process (15,16), were confined solely to children ≤10 years of age (Table S2). The T-cell functionality of these UCB/PID children indeed showed a lower level (albeit non-significant) than that of children ≤10 years old during the same post-transplant period (Figure S2). However, even when excluding the UCB/PID children, older children still displayed an elevated level of T-cell functionality compared to their younger peers. The observed disparity in IR patterns between younger and older children is likely rooted in the varying degrees of immune system maturity across these age groups. As evidenced by research conducted by Knolle et al., the functional complexity of CD4⁺ and CD8⁺ T-cells increases with age in children, whereas younger children are dominated by a higher proportion of T-cells with less functional capacity (17). Additionally, previous studies have documented a decline in lymphocyte populations as children grow older (18,19), consistent with our observation of higher T cell counts in younger versus older children. These findings highlight the importance of considering age-specific differences in immune ontogeny and function when evaluating post-transplant IR in pediatric patients, thereby necessitating the adoption of tailored monitoring and evaluation strategies tailored to the varying ages of pediatric transplant recipients.

There were indeed several limitations in this study. Firstly, owing to the difficulty in recruiting healthy children as a control group, this study was unable to incorporate a definitive healthy control group for cellular immune analysis. In our study, those allo-HSCT children who have discontinued immunosuppressant usage and have been in recovery for over years (median =23.9 months; IQR, 19.5–29.2 months) may be considered relatively cellular immune reconstituted in certain aspects (20,21). But they still do not fully represent the immunological profile of healthy children, thereby limiting the comprehensiveness of our interpretation of the observed results. Incorporating age-stratified healthy control groups in future studies would enrich our understanding of IR process in pediatric patients of various ages. Secondly, the single-center design and modest sample size limited statistical power for subgroup analyses and multivariate modeling. This constraint likely contributed to the non-significant trends observed in some comparisons and precluded comprehensive evaluation of critical IR modifiers, such as recent thymic emigrants (RTEs), infection burden, and GvHD severity (22-24). Larger multi-center cohorts will be essential to disentangle these multifactorial influences and validate our findings. Finally, the lack of extended prognostic data (e.g., 5-year survival, late-effect incidence) prevented analysis of functional immune parameters as predictors of long-term outcomes (25,26). Subsequent investigations should integrate longitudinal immune monitoring with clinical endpoints to establish clinically meaningful immune recovery thresholds.

Conclusions

In conclusion, this study presented the application potential of the WB-IGRA in evaluating and monitoring T-cell immune functionality in allo-HSCT children. Combining the assessment of T-cell immune functionality with cellular phenotypes could provide a more comprehensive understanding of the age-specific IR among pediatric patients, which may help establish a theoretical basis for developing optimal immune monitoring and management after allo-HSCT.

Acknowledgments

We would like to thank the patients and their parents for their support and participation in this study.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-80/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-80/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-80/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-80/coif). L.M., A.F., F.B. and J.L. are bioMérieux employees. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board and the Ethics Committee of Shanghai Children’s Medical Center (SCMCIRB-K2022115-1). Written informed consent was obtained from the parent or legal guardian of the participants (and themselves in the case of children ≥8 years of age).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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