Profiling the tissue microenvironment of Large Granular Lymphocyte Leukemia through cellular dynamics and genetic signatures

/Abstract Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder that affects approximately 1,000 people in the U.S. annually. LGLL is characterized by a clonal expansion of cytotoxic CD8+ T-cells (T-LGLL) or natural killer cells (NK-LGLL). LGLs account for approximately 10% of lymphocytes in healthy samples but can account for up to 90% in LGLL patients. As a result, LGLL patients experience a variety of clinical complications including various cytopenias, splenomegaly, and other autoimmune disorders. The pathogenesis of LGLL is not fully understood, and with no FDA-approved therapies available, there is a need for a deeper understanding of disease's molecular complexities and impact on the tissue microenvironment (TME). LGLL cells accumulate in sinusoidal spaces in bone marrow (BM) and other tissues in patients, including spleen and liver. These cytotoxic lymphoid aggregates formed clusters in BM and further organized into linear arrays, which are not typically seen in histopathology of healthy BM. Understanding the key components of the TME, in the context of LGLL cell accumulation and aggregation, is essential to uncover how cell-cell interactions influence pathways that lead to increased proliferation and cell survival. The interactions between LGLL cells and neighboring cells remain to be fully defined, which may significantly influence disease initiation, progression, and immune dysregulation. In addition to these histopathological findings, the Loughran lab has generated single-cell RNA (scRNA) sequencing data from peripheral blood mononuclear cells from LGLL patients, which revealed the upregulation of genes related to cell adhesion pathways. We will utilize spatial transcriptomics to better define these histopathological findings as well as the transcriptional profiles of LGLL patients in various tissues and microenvironments. We hypothesize that the LGLL cells 1) directly interact with antigen presenting cells specifically dendritic cells to maintain chronic cytotoxic activation and 2) upregulate adhesion-related genes including VCAM1 and ICAM1 that contribute to LGLL cell accumulation and adhesion compared to normal CD8+ T- or NK-cells in patient tissues. Aim 1 will identify cell populations that interact with LGLL cells in T- and NK-LGLL patient tissues utilizing Nanostring's CosMx to conduct spatial transcriptomics. We will validate the results by conducting ex vivo co- culture experiments to examine the impact of neighboring cell populations on LGLL cell proliferation and cell-cell communication. In Aim 2, we will distinguish gene expression profiles of LGLL cells versus non-leukemic CD8+ T- or NK-cells and their influence on LGLL accumulation and adhesion in various tissue types. We will focus on genes we have previously seen to be upregulated by LGLLs including VCAM1 and ICAM1 and conduct a CRISPR-Cas9 knockout of these genes. Successful completion of this project will uncover biomarkers and novel therapeutic targets to disrupt pathways that promote cell proliferation and halt antitumor immune responses. Project Number: 1F31CA301956-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Madison Turner | Institution: UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, VA | Award Amount: $39,498 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F09C-Z (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11315010