Abstract

Large granular lymphocyte leukemia (LGL) is chronic clonal lymphoproliferation of CTL. The association of LGL with autoimmune conditions imply that it arises in the context of polyclonal immune response but many features point towards viral etiology. Cytopenias suggest that LGL cells mediate inhibition of hematopoietic progenitors; possible mechanisms include direct cytotoxicity and secretion of inhibitory cytokines. In many aspects, LGL cells resemble normal, terminally differentiated CTL, including lack of CD28 expression, a high content of perforin and granzymes and expression of CD57. Our experiments were designed to gain insight into the gene expression profile of clonal CTL in LGL. First, we analyzed the expression profile of normal mature effector CTL defined by CD57 positivity. Due to low numbers of these cells in the blood of healthy individuals, flow sorted CD57+CTL from 14 controls were pooled. cRNA from patients and pooled controls was hybridized to total human genome U133+ 2.0 microarray covering 47.000 transcripts. For the analysis of LGL gene expression profile we took advantage of VB chain utilization pattern established in previous studies (Wlodarski et al, Blood 2005) and purified malignant LGL based on their VB chain usage and CD57 expression. Each LGL patient harbored an expanded VB clone comprising of 95%, 23% or 83% of the total CTL repertoire (VB1; VB20 and VB13.2, respectively). When expression pattern of LGL cells was compared to that of normal CD8+CD57+ cells, globally, in LGL concordant results were obtained for a total of 761 genes that were differentially expressed ≥2X (259 genes showed an increased expression and 503 were downregulated). Our analysis was in agreement with the previously described features of T-LGL including downregulation of CD28, and overexpression of CD16, FasL, IL8, IL-1Ra, HLA-DR and IL10. Some of the upregulated genes indicate important novel phenotypic differences between LGL and normal CTL cells. For the purpose of this study we focused our analysis on cytokine proteome of T-LGL and found a surprisingly consistent overexpression of transripts encoding cyto-/chemokines (IFN-g, IL-7, IL-8, IL-10, IL-18, CCL2, CCL3, CXCL2, CXCL10, CXCL16). Moreover, LGL clones were characterized by an increased expression of chemokine receptors that are known to facilitate viral infections (CCR1, CCR2). All these changes were described in the context of cellular reaction to intracellular pathogens. Similarly, genes highlighting a nonspecific reaction to stressors (e.g. HSP70) were upregulated in LGL. The elevated expression of IFN-g, Fas-L and IL-18, capable of inhibiting hematopoiesis is consistent with cytopenias seen in the patients studied. Based on the findings of expression arrays, we focussed our analysis on diagnostically useful soluble factors that can be measured in serum and correlated with clinical course. ELISA assays for a selection of cytokines were performed on plasma samples from 24 LGL patients and confirmed their elevated levels in vivo. For the genes for which protein assays were not available (e.g. CCR1, CCR2, CD164, CD302, CD31, CD38, MCL1, TNFRS9) Taqman PCR was performed. Our studies suggest that LGL clones, although phenotypically similar to terminal effector CTL show significantly altered cytokine proteome indicative of an ongoing viral infection. Overexpression of inhibitory cytokines may explain hematopoietic inhibition.

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