Transcriptomic Signatures of T and CAR T Cells Aspredictors of Treatment Outcomes Inrelapsed/Refractory Large Diffuse B Cell Lymphoma
Authors: De La Iglesia San Sebastián Ismael , Fernández de Córdoba-Oñate Sara , Bastos-Oreiro Mariana , Fernández González Raquel , Anguita Javier , Gómez-Centurión Ignacio , Bailen Rebeca , Kwon Mi , Garcia-Sanz Ramon , Buño Ismael , Martinez-Laperche Carolina 
Published: December 2024

INTRODUCTION
More than a third part of patients with relapsed or refractory large diffuse B cell lymphoma (r/r LDBCL) progress after 6-months upon infusion. Transcriptomic profiles of T cells pre-infusion and of CAR T cells in expansion moment could be fundamental for gain insights in transcriptional signatures that could identify poor treatment outcomes.
MATERIAL & METHODS
Peripheral blood (PB) samples was collected from 26 patients treated with anti-CD19 CAR T cells. Progressed patients (PP) were considered those that did not achieve complete response in PET-TC. Among them, 24 PB samples (PP: n = 11; not-PP: n = 11) were selected from apheresis appointment and 9 PB samples (PP: n=6; not-PP: n = 3) were chosen from the CAR T cell expansion moment (+14 day). T cells and CAR T cells were sorted using immunomagnetic separation device (autoMacs Pro, Milteny Biotec) with CD3 antibodies for T cells sorting and FITC-CD19 anti-FITC antibodies for CAR T cells isolation. RNA extraction was performed with Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) and RNA libraries were synthetized using SMARTer Stranded Total RNA-Seq kit v3 (Takara Bio). Each sample was sequenced to 25-30M of clusters in a 150PE configuration. Reads were trimmed and aligned to reference genome (hg38) using trim galore and STAR. Differentially expressed (DE) genes were identified with DESeq2 and considered DE when P &lt; 0.01. Tertiary analysis was performed with gene set enrichment analysis (GSEA) and regulatory gene network analysis to identify transcription factors (TF) that may affect CAR T or T cells differentiation or activation.
RESULTS
Pre-apheresis T cells analysis
Once T cell transcriptome were analyzed, 136 genes were down-regulated and 64 genes were up-regulated in progressed patients. When analyzing these genes separately depending on its over- or under-expression, interleukin-2 (IL-2) signaling pathway and clathrin adaptor complex were enriched terms in the down-regulated gene list, indicating a lower activation of these pathways. IL-2 signaling has been previously defined as a fundamental cytokine for T cell activation and function, its down-regulation could reduce tumor clearance and T cell activation. On the other hand, clathrin is a protein that moves T cell receptors (TCR) from the membrane to immune synapsis (IS) zone once T cells recognize an antigen, its down-regulation could imply lower amount of TCR or other receptors in the IS, and thus impairing T cell activation. Moreover, a RGN was constructed using DE genes to look for TF that modulates gene expression in progressed patients. FOSB, E2F4 and NFE2L were DE TF in T cells pre-infusion that potentially determines CAR T cell response through modulating CAR T cell function.
CAR T cells analysis
Similarly, CAR T cells exhibit 150 DE genes between progressed and non-progressed patients. 47 genes were up-regulated and 103 genes down-regulated in progressed-patients. Some of them were non-coding RNAs that might play regulatory roles in gene expression, methylation and post-transcriptional modifications. Among coding genes, GSEA analysis demonstrated two pathways enriched in our DE gene list: reactive oxygen species (ROS) induced genes and TP53 target pathways, both under-represented in progressed patients, indicating a lower oxidative stress which conduct to a better Th1 response and a higher proliferation rate of CAR T cells of non-progressed patients.
CONCLUSION
This is the first study to use bulk RNAseq to look for differences in CAR T cell and T cell transcriptome to better understand progression. Transcriptomic profiles of CAR T and T cells could be potential indicators of cell activity and correct function. Oxidative stress and TP53 pathway down-regulation in CAR T cells, and IL-2 signaling and clathrin complex up-regulation in T cells, could be fundamental for an optimal differentiation and activation of CAR T cells. At this time, we are validating this results in a new patient cohort.