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High STAT5A activity promotes CD8+ T-cell neoplasia
bodies against CD3, Ki67, pYSTAT5, STAT5A and STAT5B (Online Supplementary Table S4, Online Supplementary Methods). For immunohistochemical analysis of STAT5A and STAT5B expression in human samples, tissue microarrays were built including 14 AITL, 35 PTCL, NOS, 7 CTCL, and 6 mycosis fun- goides cases each represented by duplicate or triplicate core biopsies. In addition, paraffin-preserved sections of 29 CTCL samples (from 23 patients) were analyzed. Fivc non-neoplastic lymph nodes served as controls. Quantification of the staining is described in the Online Supplementary Methods. Images were taken with a Zeiss Imager Z1 microscope (Carl Zeiss, Oberkochen, Germany).
RNA sequencing
mRNA was isolated from CD8+ T cells harvested from lymph nodes [wildtype (wt) - 2-3 mice pooled per sample, cS5Alo, cS5Ahi, hSTAT5BN642H n=5, hSTAT5B n=4]. RNA sequencing was performed with Illumina HiSeq-2500 (Illumina, San Diego, CA, USA) at the VBCF next-generation sequencing unit (www.vbcf.ac.at). Details of the analysis are provided in the Online Supplementary Methods.
RNA-sequencing data can be found in the GEO database with the accession identities GSE124102 and GSE93847.
Statistical analysis
Data are reported as mean values ± standard error of mean and were analyzed by GraphPad Prism® 5 (San Diego, CA, USA) or RStudio Version 1.0.153 (Boston, MA, USA). In vitro data, western blots, quantitative reverse transcriptase polymerase chain reactions (qRT-PCR) and viability assays were repeated at least three times (unless indicated otherwise). The numbers of animals or patients are stated in each figure or figure legend. Applied statistical tests are mentioned in the respective figure legend. P values <0.05 were accepted as statistically significant and denoted as follows: *P<0.05; **P<0.01; ***P<0.001, and ****P<0.0001.
Results
STAT5A or STAT5B activation leads to a mature CD8+ T-cell disease in mice
To test whether hyperactive STAT5 signaling alone is sufficient to drive PTCL, we generated transgenic mice with graded expression of wildtype (wt) STAT5B or gain- of-function Stat5a or STAT5B. We used the well-charac- terized hyperactive Stat5aS710F (cS5F) variant37 expressed at
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Figure 1. Expression of a gain-of-function Stat5a or STAT5B variant leads to a polyclonal CD8+ T-cell disease. (A) Schematic representation of the FLAG-tagged STAT5 constructs for generation of transgenic mouse lines expressing hyperactive Stat5a (cS5Alo and cS5Ahi) or human STAT5B (hSTAT5B and hSTAT5BN642H). (B) Immunoblot on lymph node lysates from cS5Ahi, cS5Alo, wildtype (wt), hSTAT5B, and hSTAT5BN642H mice (n=2/genotype) using antibodies to FLAG, phosphotyrosine(Y694)-STAT5 (pYSTAT5) and STAT5. HSC70 was used as a loading control. Representative blot of four experiments. (C) Kaplan-Meier disease-free survival plot of wt (n=20), cS5Alo (n=12), cS5Ahi (n=37), hSTAT5B (n=20) and hSTAT5BN642H (n=34) mice; P≤0.0001 with the log-rank (Mantel-Cox) test. (D) White blood cell (WBC) count measured at 6-week intervals from wt (n≥6), cS5Alo (n≥8) and cS5Ahi (n≥10) mice for 66 weeks (cS5Ahi until 42 weeks, unpaired t-test wt vs. cS5Alo P≤0.0001, wt vs. cS5Ahi P=0.003). (E) Representative blood smears of 32-week old mice, scale bar 30 μm, and representative flow cytometry dot plots of CD4 and CD8 cells in peripheral blood of 32-week old wt, cS5Alo and cS5Ahi mice. (F) Macroscopic appearance of thymi, lymph nodes (LN), and spleens of representative age-matched wt, cS5Alo and diseased cS5Ahi mice.
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