Single-Cell Multi-Omics Reveals Immune Microenvironment Alterations in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that predominantly occurs in children and young adults. Although intensified multi-agent chemotherapy improved overall survival rates, relapsed/refractory T-ALL remains difficult to treat. In addition, these intensive chemotherapies carry a risk for short- and long-term side effects, especially in children. There is an urgent need for novel therapeutic strategies to improve patient outcome. Targeting the tumor microenvironment has been recognized as a promising strategy in many adult tumors with high neoantigen load, but it is thought to be less effective in pediatric and hematologic malignancies as these carry few genetic mutations. However, comprehensive studies of the immune microenvironment are lacking in many pediatric malignancies, including T-ALL, but are urgently required to identify novel therapeutic targets. The aim of this study is to unravel mechanisms of immune evasion in T-ALL and to establish novel immunotherapeutic targets for the treatment of T-ALL.

Methods We performed 5’ single cell transcriptome, surface proteome of ~200 proteins and immune repertoire analyses of ~54,000 cells from 10 primary T-ALL pediatric patient samples at initial diagnosis and 4 normal donors using the 10x Genomics platform. Sequencing data was processed using Cell Ranger (reference genome hg38). Quality control was performed using Scrublet (doublet removal), DropletQC (quality control damaged cells) and Seurat (percentage mtDNA < 10% and genes expressed > 200). RNA counts were normalized with sctransform and surface protein antibody counts were centered log-ratio normalized using Seurat. Non-linear dimensional reduction (UMAP) and differential gene/protein expression was done using Seurat and cell type annotation was done using Azimuth in combination with known marker genes. In addition, we analyzed differential cell type abundance (MiloR), the immune repertoire (Immunarch), transcription factor activity (pyScenic) and ligand-receptor activity (NicheNet).

Results Our multi-omics analysis revealed changes in the immune cell type composition in T-ALL associated with an immunosuppressive microenvironment. Specifically, we identified a significant enrichment of non-classical monocytes, atypical memory B-cells and double-negative T-cells (CD3+, CD4-, CD8-) in the T-ALL immune microenvironment compared to the normal immune microenvironment. These CD21-, CD11c+ atypical memory B-cells showed high expression of multiple inhibitory receptors (including PD-1, FCRL3/5 and ILT2), suggesting muted BCR-signaling. Transcription factor analysis showed transcriptional activity of known B-cell regulators SOX5, TBX21 and POU2F2, of which SOX5 is known to be highly expressed during late B-cell differentiation stages including atypical memory B-cells. Double-negative (DN) T-cells, which were oligoclonal, were also overrepresented in the T-ALL microenvironment. Correspondingly, immune repertoire analysis of normal T-cells showed decreased TCR diversity (Chao1 index, p = 0.016, Wilcoxon rank sum test) compared to normal donors. Surprisingly, these DN T-cells displayed features of CD8+ T-cell dysfunction, such as high expression of PD-1, TIM3 and LAG3, as well as features of regulatory T-cells, such as high expression of IL10, ICOS and CTLA4. These cells also demonstrated high transcription factor activity of EOMES, NFATC1 and ETV1, which are all associated with a CD8+ T-cell dysfunctional transcriptional network, as well as FOXP3 transcription factor activity, the master transcription factor in regulatory T-cells.

Conclusions Multi-omics profiling of the immune microenvironment in T-ALL revealed the presence of several populations of dysfunctional cells associated with an immunosuppressive microenvironment. Studying ligand-receptor interactions analysis between malignant and immune cells will provide the basis for future (pre)clinical studies leading to the development of novel immunotherapeutic strategies for the treatment of T-ALL.

Place:Abbvie: Consultancy, Research Funding; Novartis: Consultancy, Other: Meal, Research Funding; Servier: Consultancy, Research Funding. Silverman:Servier: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Lohr:Bristol Myers Squibb: Other: Research funding outside submitted work.