Altered Collagen Production by Dysfunctional Mesenchymal Stem Cells Is Linked to T Cell Large Granular Lymphocyte Leukemia Pathophysiology.
Authors: Mailloux Adam W. , Bai Fanqi , Zhang Ling , Padron Eric , Yang Lili , Wei Cody , Schmidt Amber , Sokol Lubomir , Chen Dung-Tsa , Loughran Thomas P. , List Alan F. , Epling-Burnette P. K. 
Published: October 2019

Abstract
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 H&E and reticulin stains, respectively. Utilizing the European consensus guidelines, grading scale (0 – 3), we determined that bone marrow reticulin fibrosis is present in patients with T-LGL leukemia (Figure 1). The mean fibrosis grade for the LGL group was 2.32 (median of 2.5), whereas, the mean fibrosis grade for the control group was 1.46 (median of 1.50), p-value of 0.01. Our analysis revealed that reticulin fibrosis in LGL leukemia bone marrow was particularly associated with the interstitial stroma and lymphoid aggregates. The degree of fibrosis in T-LGL bone marrow showed no relation to absolute neutrophil counts. However, an in depth analysis of neutrophil morphology revealed several dysplastic features within the neutrophil compartment in T-LGL patients. These features include decreased segmentation, increased numbers of pseudo-Pelger-Huet forms, and an increase percentage of immature neutrophils. Of these, the proportion of immature neutrophils positively correlated with fibrosis grade in T-LGL patients (Spearman r=0.7302; 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.
Figure 1: T-LGL patients have increase reticulin fibrosis in bone marrow stroma. Paraffin embeddeb from control (a) or T-LGL patient (b) bone marrow biopsies with reticulin. Figure 1:. T-LGL patients have increase reticulin fibrosis in bone marrow stroma. Paraffin embeddeb from control (a) or T-LGL patient (b) bone marrow biopsies with reticulin.
Figure 2: MSCs from T-LGL patients display altered basement membrane composition following ex vivo expansion. Immunofluorescent staining for collagen type I (green), collagen type II (red), and DAPI (blue) was performed on primary MSCs isolated from the bone marrow of normal donors at early passage (a), late passage (b), or T-LGL patients at late passage. Figure 2:. MSCs from T-LGL patients display altered basement membrane composition following ex vivo expansion. Immunofluorescent staining for collagen type I (green), collagen type II (red), and DAPI (blue) was performed on primary MSCs isolated from the bone marrow of normal donors at early passage (a), late passage (b), or T-LGL patients at late passage.

Disclosures:
No relevant conflicts of interest to declare.