Dysregulation of the IFN-γ-STAT1 signaling pathway in a cell line model of large granular lymphocyte leukemia

<div><p>T cell large granular lymphocyte leukemia (T-LGLL) is a rare incurable disease that is characterized by defective apoptosis of cytotoxic CD8+ T cells. Chronic activation of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway is a hallmark of T-LGLL. One manifestation is the constitutive phosphorylation of tyrosine 701 of STAT1 (p-STAT1). T-LGLL patients also exhibit elevated serum levels of the STAT1 activator, interferon-γ (IFN-γ), thus contributing to an inflammatory environment. In normal cells, IFN-γ production is tightly controlled through induction of IFN-γ negative regulators. However, in T-LGLL, IFN-γ signaling lacks this negative feedback mechanism as evidenced by excessive IFN-γ production and decreased levels of suppressors of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ. Here we characterize the IFN-γ-STAT1 pathway in TL-1 cells, a cell line model of T-LGLL. TL-1 cells exhibited lower IFN-γ receptor protein and mRNA expression compared to an IFN-γ responsive cell line. Furthermore, IFN-γ treatment did not induce JAK2 or STAT1 activation or transcription of IFN-γ-inducible gene targets. However, IFN-β induced p-STAT1 and subsequent STAT1 gene transcription, demonstrating a specific IFN-γ signaling defect in TL-1 cells. We utilized siRNA targeting of STAT1, STAT3, and STAT5b to probe their role in IL-2-mediated IFN-γ regulation. These studies identified STAT5b as a positive regulator of IFN-γ production. We also characterized the relationship between STAT1, STAT3, and STAT5b proteins. Surprisingly, p-STAT1 was positively correlated with STAT3 levels while STAT5b suppressed the activation of both STAT1 and STAT3. Taken together, these results suggest that the dysregulation of the IFN-γ-STAT1 signaling pathway in TL-1 cells likely results from low levels of the IFN-γ receptor. The resulting inability to induce negative feedback regulators explains the observed elevated IL-2 driven IFN-γ production. Future work will elucidate the best way to target this pathway, with the ultimate goal to find a better therapeutic for T-LGLL.</p></div

Working model for JAK-STAT signaling pathway in TL-1 cells.

<p>(A) Based on our findings, TL-1 cells have a decreased expression of IFNGR1 and IFNGR2, rendering the cells unresponsive to IFN-γ-induced signaling. This allows uncontrolled production of IL-2 induced IFN-<b>γ</b> production due to lack of induction of negative feedback regulators. (B) However, TL-1 cells are responsive to IL-2 leading to activation of STAT1, STAT3, and STAT5. STAT1 activation positively correlates with STAT3 while the activation of these proteins is enhanced upon knockdown of STAT5b. STAT5b and STAT3 promote transcription of IFN-γ and IL-10, respectively.</p

STAT5b is required for maximal production of IL-2-induced IFN-γ mRNA in TL-1 cells.

<p>STAT1 (A), STAT3 (B), or STAT5b (C) were knocked down using siRNA in TL-1 cells supplemented with IL-2. Protein lysates and RNA were harvested as well as conditioned media collected 48 h after siRNA transfection. Representative western blots of the knockdowns are shown. (D) Supernatant was analyzed to determine changes in secreted IFN-γ as a result of each knockdown. (E) The effect of knockdown on IFN-γ transcript levels was determined using qPCR. Results were normalized to the UBC gene and further normalized to the scrambled siRNA control. (F) Changes in IL-10 production were also assessed in each knockdown group using Luminex. (G) Viability following knockdown was measured by MTS assay at 48h. All results were normalized to scrambled siRNA treated cells. Student’s T test was used to determine significance compared to scrambled siRNA. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001. Data are presented as mean +/- Stdev (n = 3 biological replicates).</p

TL-1 cells exhibit lower expression of the IFNGR1 and IFNGR2 compared to Jurkat T cells.

<p>The surface expression of the IFNGR1 and IFNGR2 was determined using flow cytometry in the TL-1 cell line compared to Jurkat T cells, an IFN-γ responsive cell line. Representative flow cytometry histograms of (A) IFNGR1 and (B) IFNGR2 and (C) median fluorescence intensity (MFI) of IFNGR1 and IFNGR2 in TL-1 and Jurkat T cells are shown. (D) IFNGR1 and IFNGR2 transcripts were quantified using qPCR. Results were normalized to ubiquitin C (UBC), a housekeeping gene. Student’s T test was used to determine significance of TL-1 cells compared to Jurkat T cells. All data are presented as mean +/- Stdev (n = 3 biological replicates).</p

JAK2 is unresponsive to IFN-γ in TL-1 cells.

<p>Jurkat T cells (A) or TL-1 cells (B) were treated with 10 ng/mL IFN-γ or water (vehicle control) for the indicated time. TL-1 cells were also treated with IL-2 as a positive control for induction of p-JAK2. p-JAK2, total JAK2, and β actin were measured using western blot. The ratio of p-JAK2:JAK2 is shown for each condition. (C) TL-1 cells were pre-treated with 5 μM ruxolitinib (Ruxo) or DMSO for 2 h prior to the addition of IL-2. Protein lysates were created 1 h after the addition of IL-2. Western blots were probed for p-STAT1, total STAT1, and β actin. p-STAT3, total STAT3, p-STAT5 and total STAT5 were used as controls for the functionality of the JAK inhibitors.</p

STAT5b inhibits STAT1 and STAT3 activation in TL-1 cells.

<p>STAT1 (A, B), STAT3 (C, D), and STAT5b (E, F) were knocked down using siRNA in IL-2 supplemented TL-1 cells. Protein lysates were harvested 48 h after siRNA transfection. Each blot was probed for p-STAT1, STAT1, p-STAT3, STAT3, p-STAT5, STAT5, and β actin. p-STAT proteins were normalized to their respective total STAT proteins. STAT1, STAT3, and STAT5 were normalized to β actin. Representative western blots are shown (A, C, E) as well as quantification of experimental replicates (B, D, F). Student’s T test was used to determine significance compared to scrambled siRNA. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001. Data are presented as mean +/- Stdev (n = 3 biological replicates).</p

TL-1 cells are responsive to IFN-β, supporting a type II interferon-specific signaling defect.

<p>Jurkat T cells (A) or IL-2-starved TL-1 cells (B) were treated with 5000 U/mL IFN-β or water (vehicle control) for the indicated time. TL-1 cells were also treated with 200 U/mL IL-2 as a positive control for induction of p-STAT1. p-STAT1, total STAT1, and β actin were measured using western blot. (C) Jurkat or TL-1 cells were treated with 5000 U/mL IFN-β or water for 6 h prior to RNA extraction. Induction of IFN-γ and STAT1 transcripts was quantified using qPCR. Results were normalized to UBC (a housekeeping gene) and then to the water control to demonstrate fold change. Student’s T test was used to determine significance compared to vehicle control. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001. Data are presented as mean +/- Stdev (n = 3 biological replicates).</p

Schematic of whole-body BCAA metabolism.

<p>Ketoacids are formed by reversible transamination catalyzed by the mitochondrial or cytosolic isoforms of branched chain amino acid transaminase (<i>BCAT</i>). The action of the branched chain keto acid dehydrogenase complex (<i>BCKDC</i>) in the mitochondrial matrix leads to the evolution of CO₂ from the 1-carbon of the keto acids including KIC, which was ¹⁴C labeled and measured from the expired air in these studies. Subsequent intramitochondrial metabolism leads to the formation of various acyl-coenzyme A (R-CoA) esters that can reversibly form acylcarnitines (not displayed). Neither FAD and NAD Cofactors nor CO₂ and H₂O substrates are displayed. Bold font indicates metabolites or corresponding acylcarnitines that were detected and measured quantitatively in the 24 h urines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone-0059443-t004" target="_blank">Table 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone-0059443-t005" target="_blank">5</a>). AA, amino acids.</p

Food, fluid, protein and Leu intakes with urine outputs in lean and obese Zucker rats^*.

*<p>Data from cohort C (the cohort in which no radioactivity was used for metabolomics/enzyme activity assays). Values are means ± SE, n = 9–10 per group. NS indicates not significant (P>0.05). Leucine and protein intake are determined from the values provided by the manufacturer of the diet. Small differences in percent changes (+98 vs +97%) are due to removal of significant figures and rounding.</p

Body weight, organ weights and plasma glucose and insulin concentrations of lean and obese rats^*.

*<p>Weights in g were obtained from cohort B (the cohort used for protein synthesis using the [³H]-Phe) and represent the sum of the weight of the left and right side where applicable. Plasma values are from cohort A (the cohort used for [¹⁴C]-Leu metabolism studies). Values are means ± SE, n = 9–10 per group. Percent differences were determined before rounding. NS indicates not significant (P>0.05). Data on amount of protein per g wet weight for some tissues is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone-0059443-t007" target="_blank">Table 7</a>.</p