Pre-transplant αTREC diversity reflects thymic function and independently predicts mortality following allogeneic hematopoietic cell transplantation

Abstract

Abstract Introduction: Reliable biomarkers to predict mortality after allogeneic hematopoietic cell transplantation (allo-HCT) are lacking. Although delayed T cell recovery is associated with poor outcomes, the extent to which pre-transplant thymic function can predict survival remains unclear. The most widely used surrogate of thymic output, δRec–ψJα, a single T cell receptor excision circle (TREC), has shown limited prognostic value when measured in whole blood. However, δRec–ψJα represents only one of many TRECs generated, and it remains unknown whether other TRECs or their combined diversity can predict outcomes after allo-HCT. We hypothesized that broader TREC diversity may serve as a functional biomarker of thymic reserve and predict post-transplant mortality. Methods: We developed a novel multiplex droplet digital PCR assay to quantify full δRec–to–Jα and Dβ–to–Jβ recombination events, encompassing all 61 α- and 13 βTRECs. Overall TREC diversity was calculated using the Shannon diversity index. To validate gene assembly and representation of intrathymic T cell development, we used synthetic DNA standards and sorted thymocyte subsets from thymi obtained from pediatric open-heart surgeries (female donors, n=4). Furthermore, the impact of age-associated thymic involution was analyzed in whole blood samples from 100 healthy donors aged 15 to 80 years paired with flow cytometric analysis of recent thymic emigrants. Next, to evaluate the clinical relevance of TREC diversity and its association with age and transplant outcomes, we analyzed 172 allo- and 162 auto-HCT patients treated at City of Hope. Kaplan-Meier survival curves were generated using the Shannon α- and βTREC Index, with the median (2.7) and upper quartile (2.97) serving as cutoff values. Cumulative incidence curves were used to estimate rates of all-cause mortality (ACM), non-relapse mortality (NRM), and relapse-related mortality (RRM), stratified by Shannon α- and βTREC Index using the median as the cutoff. Patients with index values below the median were designated as the reference group. Multivariate Cox regression was used to analyze ACM and Fine-Gray regression for NRM and RRM, treating the other outcome as competing risk. All models were adjusted for age, sex, HCT-comorbidity index, CMV serostatus, and disease type. Adjusted Hazard ratios (aHRs) and 95% confidence intervals (CIs) were estimated. Results: The multi-TREC assay detected rare TREC events (<1 copy/μL) with a turnaround time under 12 hours. TREC diversity increased across thymocyte maturation stages and declined with age in healthy individuals. In the allo-HCT cohort, the two-year post-transplant mortality rate was 20.9%. The median age at transplant was 56.9 years (range 18.3–81.6), 57.0% were male; race/ethnicity: White not Hispanic (55.2%), White Hispanic (27.9%), other (16.9%); 29.1% had lymphoma and 69.8% had myeloma; CMV positive status (64.5%); intensity of conditioning regimen: Ablative (44.8%) and non-Myeloablative (55.2%). In this cohort, pre-transplant αTREC diversity, but not δRec–ψJα quantification, was strongly associated with outcomes. In adjusted Cox regression, individuals with αTREC diversity values below the median had a significantly higher risk of ACM (aHR = 3.94, 95% CI: 1.7–9.2, p = 0.0016) independent of HCT-comorbidity index, CMV serostatus, age, and sex. Cumulative incidence analyses showed that patients with high αTREC diversity had significantly lower ACM compared to those with low diversity (9.2% vs. 34.7%, p < 0.0001). Similar differences were observed for NRM (6.9% vs. 20.4%, p = 0.013) and RRM (2.3% vs. 14.3%, p = 0.0049). Kaplan–Meier survival curves further confirmed these findings for ACM using both median and 75th percentile cutoffs (p < 0.0001 and p = 0.0037, respectively) over a 730-day follow-up period. Conclusions: This study provides the first comprehensive profiling of the α- and βTREC repertoires across thymic development, healthy aging, and HCT. We demonstrate that pre-transplant αTREC diversity is a strong and independent predictor of survival following allo-HCT, outperforming δRec–ψJα quantification while remaining independent of age, sex, CMV status, HCT-comorbidity index, and disease type. Our findings establish αTREC diversity as a novel functional biomarker of thymic reserve, enabling risk stratification beyond current indices and informing personalized conditioning or early immune interventions to improve transplant outcomes.