Differential Gene Expression in Circulating T-Cells in Long-Term Adolescent/Young Adult Hodgkin Lymphoma (AYAHL) Survivors and Their Unaffected Twins
Authors: Cozen Wendy , Epeldegui Marta , Conti David V. , Hwang Amie E. , Wang Jun , Magpantay Larry , Mack Thomas M. , Martinez-Maza Otoniel , Cole Steve W 
Published: November 2021

Abstract
Background: We and others have demonstrated persistent differences in T cell function and cytokine responses in adolescent/ young adult Hodgkin lymphoma (AYAHL) survivors and their family members. To identify the transcription control pathways involved, we examined mRNA profiles from 17 pairs of long term AYA HL survivors and their unaffected twins.
Methods: Blood samples were collected remotely from AYAHL survivors and their like-sex unaffected co-twin controls and shipped to the Epeldegui laboratory at UCLA. B and T cells were negatively separated using magnetic beads. RNA was extracted and sequenced using a high-efficiency mRNA-targeted assay system (Lexogen QuantSeq 3' FWD), with an average 6.7 million reads per sample mapped to the hg38 human transcriptome (99%) using the STAR aligner. Raw read counts for each gene transcript were normalized to transcripts per million total mapped reads and log2 transformed for analysis using linear statistical models including a random effect of twin pair. Analyses examined average expression of pre-specified sets of 17 genes in general immunologic activation (i.e., AP-1- and NF-kB-family transcripts) and 28 Type I IFN-responsive genes (e.g., IFI-, OAS-, and MX-family), as well as TELiS promoter-based bioinformatics analyses of all genes showing &gt;2-fold empirical differences in gene expression (focusing on activation-induced transcription factors, NF-kB and AP-1, and Type 1 interferon-related IRF transcription factors). Body mass index, history of therapeutic radiation, zygosity, sex, age at diagnosis and age at blood collection were included in the model.
Results: The average age at diagnosis was 25 years with blood collected 33 years post-diagnosis. In T-cells, AYAHL survivors showed marginally lower expression of 12 activation-induced genes (average 0.41-fold, p = .038) and markedly lower expression of 28 Type 1 interferon-related genes (average 0.33-fold, p &lt; .001) relative to their unaffected co-twins. B cells showed similar reductions in activation-induced genes (0.33-fold, p &lt; .001) but no difference in Type 1 interferon genes (1.02-fold, p =. 942). Promoter-based bioinformatics analyses of all 151 genes found to be differentially expressed by &gt;2-fold in T cells between AYAHL survivors and their unaffected co-twin controls implicated IRF-family transcription factors in mediating observed differences, with IRF3 showing the most significant signal of decreased activity (0.68-fold, p = .059). We also identified marked up-regulation of multiple GATA family transcription factors (GATA3: p &lt; .001; GATA1: p = .009; GATA2: p = .032).
Conclusions: Transcriptional indicators of immunologic activation and Type 1 interferon activity show persistent down-regulation in T lymphocytes from long-term adult AYAHL survivors compared to their unaffected like-sex co-twin controls. Incidental findings also identified persistent activation of other gene regulatory pathways mediating T-cell differentiation (e.g., GATA family). We have previously demonstrated higher Epstein-Barr virus (EBV) copy number in AYAHL long-term survivors compared to their unaffected co-twin controls, as well as lower levels of circulating interleukin-12, consistent with a decrease in the ability to control viral replication. The GATA family plays a major role in commitment to T cell lineage (GATA3) and upregulation of the T-helper-type 2 response, and possibly in control of EBV viral infection (GATA2), processes relevant to AYAHL. These differences further support the premise that abnormalities in T-cells play a strong role in etiology, pathogenesis, and survivorship.

Disclosures
No relevant conflicts of interest to declare.