STAT5BN642H drives transformation of NKT cells: a novel mouse model for CD56+ T-LGL leukemia

The signal transducer and activator of transcription 5B (STAT5B), downstream of IL-15 signaling and Janus kinase (JAK)1, and 3-mediated activation, is a master regulator of development, survival, and function of innate and innatelike lymphocytes (including natural killer (NK) and NKT cells) [1–3]. Gain-of-function mutations in the SH2 domain of human STAT5B, especially STAT5B, are associated with aggressive forms of CD56 T cell (NKT) and NK cell lymphomas/leukemias [4–6]. We described a mouse model expressing human (h)STAT5B under the Vav-1 promoter, which develops severe CD8 T cell neoplasia [7]. Here, we explore the ability of hSTAT5B to serve as an oncogenic driver in innate lymphocyte neoplasms. We found an increase in absolute NK cell numbers in the spleen of T cell-diseased hSTAT5B transgenic compared to wild-type (WT) mice (Fig. 1a), despite the relative decrease of the proportion of NK cells among splenic lymphocytes (Fig. S1A), while nonmutant hSTAT5B control mice showed intermediate NK cell numbers (Fig. 1a, Fig. S1A). No significant differences in NK cell numbers were observed in the bone marrow (BM) between genotypes (Fig. S1B). In addition, both hSTAT5B and hSTAT5B mice showed a similar increase in the proportion of mature NK cells (CD27CD11b and KLRG1) in the spleen (Fig. S1C, D). These findings suggest that the enforced expression of nonmutant hSTAT5B is sufficient to boost NK cell maturation, which is not further enhanced by introducing the activating hSTAT5B mutation. We hypothesized that any phenotypic alterations affecting innate lymphocytes might be masked by the hSTAT5B-driven aggressive CD8 T cell disease established in hSTAT5B mice at the age of 6–8 weeks [7]. To explore the potential of hSTAT5B to promote NK cell expansion in vivo, we transplanted CD3depleted BM from hSTAT5B or hSTAT5B mice in immune-deficient Rag2γc recipient mice. Using this approach, we observed an enhanced expansion of NK cells in the blood of hSTAT5B compared to hSTAT5Btransplanted recipients over a time course of 4 weeks (Fig. 1b). Long-term analysis of NK cells in hSTAT5Btransplanted mice was not possible due to expansion of residual CD8 T cells (Fig. S1E, F), which forced us to terminate the experiment. However, after 4 weeks, increased numbers of NK cells were also detected in the spleen upon transplantation of CD3-depleted hSTAT5B compared to hSTAT5B BM (Fig. S1G). To investigate long-term effects of hSTAT5B on innate lymphocytes, we sorted Lineage (Lin) (CD3/B220/ Ter119/Gr1/CD11b) Sca1c-KitCD127CD8 cells. This cellular fraction is devoid of hematopoietic stem cells, common lymphoid progenitors, and CD8 T cells and was obtained from hSTAT5B or hSTAT5B BM for further transplantation into Rag2γc recipient mice. We monitored NK and T cell numbers in the blood over a period of 5 weeks. Again, a modest increase but subsequent drop in NK cell numbers in mice transplanted with hSTAT5B compared to hSTAT5B BM cells was observed (Fig. S1H), whereas we failed to detect CD8 T cells in the blood of the recipient animals (Fig. S1I). This indicated that our sorting * Veronika Sexl veronika.sexl@vetmeduni.ac.at

and BMWFW-68.205/0112-WF/V/3b/2016 and were conducted according to the guidelines of FELASA and ARRIVE. Sex and age-matched (7-9 weeks) WT, hSTAT5B and hSTAT5B N642H mice were used for data presented in Fig. 1 and Fig. S1. Recipient mice for serial transplantation were age-matched (8-12 weeks) and both sexes were used.

Hematocytometry, organ preparation and flow cytometry
Mouse blood was collected in EDTA-tubes from the facial vein or from euthanized mice via cardiac puncture. White blood cell (WBC) count was measured using an animal blood counter (sciI Vet abc). For blood analysis by flow cytometry, erythrocytes were lysed using BD FACS Lysing Solution according to manufacturer's protocol (BD Bioscience, San Diego, CA, USA).
Single cell suspensions were prepared from spleen, liver and bone marrow (BM). Isolation of hepatic leucocytes was performed by separation with 37.5% percoll (GE Healthcare, Chicago, Illinois, USA). Prior to flow cytometric analysis, lysis of erythrocytes from single cell suspension was performed with Red blood cell Lysis Buffer (10 mM KHCO3 and 75 mM NH4Cl, pH 7.4).
The antibodies (clones) targeting following proteins were purchased from eBioscience (San
For serial BM transplantation of hSTAT5B N642H NKT cell-driven disease established in recipient mouse #1 (Fig. 2 and S2), BM was isolated from diseased mice and frequency of transformed NKT cells was determined by flow cytometry. Whole BM, containing 1*10 6 NKT cells was serially transplanted into Rag2 -/γc -/or NSG recipient mice for a total of six rounds. From the 3 rd round on only NSG mice were used for serial transplants. Of note, in the 6 th round a titration of the number of transplanted NKT cells was performed, with two NSG mice receiving 1*10 6 NKT cells, two NSG mice receiving 0.3*10 6 NKT cells and two NSG mice receiving 0.1*10 6 NKT cells. However, disease severity at the end point was similar 3 independent of initially injected cell numbers. Therefore, data from all mice of the 6 th round were pooled for analysis of disease severity, except for depicting days of survival (Fig. S2B), where only data from mice injected with 1*10 6 NKT cells were included. The mice were sacrificed at the first signs of disease (humane endpoint). For serial transplantation of NKT cell disease established in recipient mice #2 and #3 (Fig. S3), BM containing 0.3*10 6 NKT cells was serially transplanted into NSG recipient mice for a total of four rounds.

In vivo Ruxolitinib treatment
NSG mice were transplanted with BM containing 1*10 6 hSTAT5B N642H NKT cells (from 5 th round of serial transplant from recipient #1) and treated with Ruxolitinib (Jakavi®, Novartis) (85mg/kg body weight) as a powder in a Nutella® ball or Nutella® alone (control) twice daily, starting one day after transplant for 21 days.