P592: single-cell rna sequencing reveals the spatial heterogeneity of exhausted t cells in btki-resistant richter transformation patients

Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research Background: The transformation of CLL to aggressive B-cell lymphoma (Richter transformation, RT) has remained a clinical challenge, with poor prognosis to standard treatment. The underlying mechanisms of such aggressive transformation has not been fully disclosed yet, especially the heterogeneity of immune microenvironment when transformed after novel targeted agents like Bruton’s Tyrosine Kinase inhibitors (BTKi). Aims: To explore the spatial heterogeneity of exhausted T cells in BTKi-Resistant Richter Syndrome patients Transformation patients Methods: Single-cell RNA sequencing (scRNA-seq) of paired lymph node (LN) and peripheral blood (PB) was performed among 2 RT (P1, P2) and 2 accelerated CLL (P3, P4) patients (pathologically diagnosed in LN) and bioinformatics analysis was conducted. Patient 1 and Patient 2 were treated with BTKi (P1 ibrutinib for 3 months and P2 zanubrutinib for 19 months) and then progressed to RT. Patient 3 were initially diagnosed as accelerated CLL and Patient 4 were treated with zanubrutinib for 54 months and then progressed to accelerated CLL. Results: Distribution of T cell subsets between PB and LN showed great spatial heterogeneity, especially in those patients who transformed to DLBCL after BTKi progression. PB of all four patients showed relatively similar T cells distribution, with CD4+ naïve T cells and CD8+ effector memory T cells being two dominant clusters. However, LN of two RT patients displayed significant enrichment of CD8+ exhausted T cells while CD4+ naïve T cells were dominant cluster in LN of treatment-naïve accelerated CLL. The heterogeneity of CD8+ exhausted T cells were further explored and we found four clusters of exhausted T cells manifesting heterogeneous characteristics. Herein, exhausted CD8+ T cells in PB were mostly cluster 1, showing highest cytotoxicity and lowest exhausted feature. Cluster 3 was LN specific exhausted T cells, showing highest exhausted score and lowest cytotoxicity score. Cluster 2 was distributed scantly in PB while mostly in LN, showing highest cycling and proliferation score. Trajectories analysis of exhausted CD8+ T cells indicated that cluster 1 were precursors of exhausted cells while cluster 3 were terminally exhausted cells. Construction of cell-cell interaction displayed that high proliferating transformed B cells showed significantly higher number of interactions with T cell subsets than treatment-naïve CLL cells, especially the interactions with CD8+ effective and exhausted T cells. Further analysis of receptor-ligand interactions between T cell subsets and tumor cells were conducted and activation of CD70-CD27 and LGALS9 (galectin-9)-HAVCR2 (TIM3) were found between BTKi-resistant tumor cells and CD8+ effective and exhausted T cells, indicating the underlying mechanism contributing to immune escape of Richter transformation. Summary/Conclusion: scRNA-seq analysis of paired PB and LN in BTKi-resistant Richter transformation and accelerated CLL patients indicated great heterogeneity of T cell subsets distribution. LN of Richter transformation were enriched in CD8+ terminally exhausted T cells. High proliferating transformed B cells showed strong interaction with CD8+ effective and exhausted T cells and contributed to T cell exhaustion through the activation of CD70-CD27 and LGALS9-HAVCR2 crosstalk.Keywords: Microenvironment, T cell, B-CLL