Altered Collagen Production by Dysfunctional Mesenchymal Stem Cells Is Linked to T Cell Large Granular Lymphocyte Leukemia Pathophysiology.

Abstract 2595 Large Granular Lymphocyte (LGL) leukemia is a chronic lymphoproliferative syndrome that can be broadly classified into two groups depending on whether the expanded cells are T-cells or NK-cells. The clinical characteristics of the disease include lymphocytosis, neutropenia, anemia, that can be associated with rheumatoid arthritis and pulmonary arterial hypertension (PAH). Hematologic improvement with immunosuppressive agents such as cyclosporine and low-dose methotrexate has lead to the widely accepted theory that cytopenias are mediated by autoimmune destruction of the hematopoietic stem cell (HSC) compartment or lysis of mature myeloid cells in circulation. We found, however, that autologous HSCs and mature granulocyte populations fail to be recognized or lysed ex vivo by T-LGL leukemia cells suggesting that an alternate mechanism may be involved. In contrast to research done on the T-LGL cells themselves, the role of the bone marrow microenvironment and HSC compartment in T-LGL leukemia patients is completely unexplored. Therefore, bone marrow core biopsies, aspirates, and peripheral blood smears were obtained from 22 patients with LGL leukemia and 14 patients with non-hematological malignancies to serve as controls. Morphology and extracellular matrix composition were examined by HE p=0.0002), indicating a possible link between reticulin fibrosis and the quality of hematopoiesis. To explore the pathogenesis of medullary fibrosis, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of 6 LGL leukemia patients and 5 healthy controls and then expanded ex vivo under non-differentiating conditions. During expansion, healthy MSCs produce cytokines and growth factors necessary for self renewal and for the support of hematopoiesis. However, MSCs from T-LGL patients displayed severely reduced self-renewal potential, reaching a mean of 7 population doublings compared to a mean of 23 for normal MSCs, and were unable to support the proliferation of healthy HSCs in a co-culture proliferation assay. Microarray analysis (H6 V133 plus 2.0) was performed on the MSCs from both control and T-LGL patients with analysis focused on genes regulating basement membrane composition. For normal MSCs, significant reductions in the expression of numerous collagen genes occured as the cells underwent expansion in self-renewal conditions. However, MSCs from T-LGL patients failed to downregulate these genes despite months of culture. The most prominent collagen genes following this pattern were types I (α1, α2), III (reticulin), IV (α1, α2), and V (α1, α2). A combination of qRT-PCR and immunflourescent staining (Figure 2) were utilized to confirm these gene expression changes. Collectively, these results implicate aberrant MSC self-renewal capacity and skewed basement membrane protein expression in the pathogenesis of T-LGL leukemia and suggest that these abnormalities may represent novel targets for future drug discovery. Disclosures: No relevant conflicts of interest to declare.