儿童癌症长期生存者中治疗相关克隆性造血影响的T细胞转录组

Our previously published work on the St. Jude Lifetime Cohort Study (SJLIFE), a retrospectively constructed cohort with prospective follow-up of long-term survivors of pediatric cancer, unveiled the phenomenon of accelerated clonal hematopoiesis (CH) associated with prior exposure to radiotherapy and chemotherapy. STAT3 Y640F, a hotspot mutation specifically linked to prior exposure to procarbazine in Hodgkin Lymphoma (HL) survivors, was most frequent in the SJLIFE cohort. Based on joint profiling of DNA amplicons and cell-surface proteins at single cell resolution using the MissionBio platform, we found STAT3 Y640F mutation in blood leukocytes from 6 out of 10 HL survivors. Importantly, STAT3 Y640F was enriched in cells clustered within CD8 T-cells, indicating that mutation-positive cells have reprogramed the transcriptome that gave selective growth advantage to a subset of those cells. To investigate this, we performed PacBio scKinnex assay to generate full-length transcriptomes at single cell level, enabling integration of CH mutations with gene expression profiles. An initial analysis using blood samples of HL survivors yielded limited information as few mutant cells were detected due to the low mutation frequency (≤1%) and STAT3 expression. To enrich for the mutant cells, we performed sorting for CD8+ T-cells followed by scKinnex for three samples which yielded sufficient number of mutant cells (14 to 26 cells per sample) for gene expression analysis. For each sorted sample 4,396 to 5,416 cells were profiled, and clustering by gene expression revealed 11-14 subpopulations per sample. Guided by the expression status of marker genes, we performed cell annotation and identified multiple CD8 T-cell subsets, including naive, central memory, effector memory, and terminally differentiated effector memory cells. STAT3 mutant cells were present only in a few of these subpopulations: 3 out of 13 (23%), 1 out of 14 (7%), and 2 out of 11 (18%) in these three samples with an average frequency of 40% in each subpopulation. Nearly all mutant-positive clusters showed increased expression of STAT3 gene; four out of the five (80%) such clusters exhibited high expression of cytotoxic markers, including GZMB , GZMH , FGFBP2 and NKG7 , while the remaining one showed high expression of stress response markers from FOS/JUN pathway ( JUN , JUNB , and FOS ). To our best knowledge, this is the first study that shows CH mutation can confer selective advantage to specific functional cell subsets of CD8 T-cells and analysis of gene-regulatory network changes related to STAT3 Y640F mutation in those cell subpopulations is currently ongoing. Our study also demonstrates that cell sorting followed by scKinnex can be an effective strategy for studying the functional impact of rare CH mutations at the single cell resolution in relevant cell types.