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B. Maurer et al.
low or high level, human (h)STAT5B served as a negative control and hSTAT5BN642H served as a positive T-cell neo- plastic model.32 All transgenes contain a C-terminal FLAG-tag driven under control of the vav-promoter enabling expression from the hematopoietic stem cell stage onwards throughout all blood lineages40 (hereafter referred to as cS5AF, cS5Alo, cS5Ahi, hSTAT5B and hSTAT5BN642H) (Figure 1A, Online Supplementary Figure S1A-D). We confirmed significantly elevated pYSTAT5 levels in hematopoietic organs of cS5Ahi and hSTAT5BN642H mice whereas the cS5Alo animals showed a modest increase in pYSTAT5 (Figure 1B, Online Supplementary Figure S1E). Kidney, colon and liver did not display signif- icant transgene expression (Online Supplementary Figure S1E). Transgene expression was verified by qRT-PCR in FACS-sorted CD4+, CD8+, CD19+, CD11b+ and NK cells of 8-week old cS5Alo and cS5Ahi mice, with CD8+ T cells showing highest transgene mRNA expression (Online Supplementary Figure S1F).
Enhanced STAT5 activation in the hematopoietic sys- tem led to development of neoplasia resulting in death between 25 to 45 weeks of age irrespective of gender in cS5Ahi mice, which was significantly later than in hSTAT5BN642H mice which develop a lethal disease within 10 weeks.32 cS5Alo were as long-lived as wt mice; hSTAT5B mice were also followed for more than 1 year without showing signs of disease (Figure 1C). The strong lymphoma phenotype of hSTAT5BN642H mice suggests a role of STAT5A as a possible balancer due to STAT5A/B heterodimerization, but only high pYSTAT5 levels drive the disease.
White blood cell counts rose steadily in cS5Ahi mice to a level comparable to that in diseased hSTAT5BN642H mice, whereas cS5Alo exerted only a subtle effect, resulting in doubling of the white cell count compared to that in wt mice (Figure 1D, Online Supplementary Figure S1H), also seen in the blood smears (Figure 1E). Like hSTAT5BN642H mice, adult cS5Ahi massively expanded CD8+ T cells and the phenotype in cS5Alo mice was intermediate (Figure 1E, Online Supplementary Figure S1G, H). At 32 weeks of age, cS5Ahi mice had massively enlarged lymph nodes at all lymphatic sites and severe splenomegaly, but cS5Alo mice did not show any pathological abnormalities. Interestingly, macroscopically, STAT5A transgenic thymi showed no obvious differences from those of age- matched controls (Figure 1F). Our further analysis focused on the comparison of cS5Ahi mice and their wt littermates.
Expanded CD8+ T cells exert a mature cytotoxic T-lymphocyte phenotype
At the age of 25-45 weeks all cS5Ahi mice developed ter- minal disease. CD8+ T cells were dominant in spleen (Figure 2A, Online Supplementary Figure S2A), peripheral blood, lymph nodes and bone marrow of cS5Ahi mice (Online Supplementary Figure S2B). While relative levels of CD4+ T, CD19+ B and CD11b+Gr1hi myeloid cells were decreased (Online Supplementary Figure S2A-C), absolute numbers of these cell types were elevated, although to a lesser extent than CD8+ T cells (Figure 2A). In contrast, both the relative and absolute numbers of NK cells were decreased (Figure 2A, Online Supplementary Figure S2A). Western blot analysis of lymphocyte subpopulations con- firmed the highest levels of cS5AF expression in CD8+ T cells (Online Supplementary Figure S2D). The cS5Alo mice did not display a phenotype despite increased CD8+ T-cell
numbers and pronounced pYSTAT5 levels (Online Supplementary Figure S2E-I).
To assess the functionality of the cytotoxic T cells (CTL), we injected the C57BL/6 isogenic CTL-responsive lymphoma cell line E.G7 or the colon carcinoma cell line MC-38 into flanks of 10-week old wt, cS5Alo or cS5Ahi mice. In comparison to tumors in wt hosts, tumors in cS5Alo and cS5Ahi mice were significantly smaller or absent and infiltrated more by CD8+ T cells, indicating that the expanded CD8+ T cells retained functionality (Figure 2B, Online Supplementary Figure S2J).
Next, we sought to further characterize the disease- causing cells. cS5Ahi CD8+ T cells retained CD2, CD3 and CD5 expression (Figure 2C, Online Supplementary Figure S2K). CD25+ and CD44+ CD8+ T cells were expanded in peripheral blood and hematopoietic organs of cS5Ahi ani- mals (Figure 2D, Online Supplementary Figure S2L, M), indicative of an activated/memory-like T-cell pheno- type.41 CD8+ T cells can be divided into effector (TEM) and central memory (TCM) subsets, either representing a rapid effector cell or exerting lymph node-homing properties with potent proliferative potential.42,43 cS5Ahi mice dis- played elevated levels of CD44+CD62L+CD8+ (TCM) and CD44+CD62L-CD8+ (TEM) T cells (Figure 2E, Online Supplementary Figure S2N, O). Gene set enrichment analy- sis on differentially expressed genes in CD8+ T cells of cS5Ahi compared to cS5Alo mice also correlated to a mem- ory/effector signature (Online Supplementary Figure S2P).44 The homing and activation marker CCR7 was also expressed on a subpopulation; CD8+CD62L-CD27-CCR7+ cells in particular were more frequent in cS5Ahi than in wt cases (Online Supplementary Figure S2Q, R). Furthermore, hyperactive STAT5A signaling led to more Treg and γδ T cells (Online Supplementary Figure S2S, T).
Together, these data indicate that hyperactivation of STAT5A in cS5Ahi transgenic mice induces mature T-cell neoplasia with an activated cytotoxic CD8+ memory phe- notype.
STAT5-driven CD8+ T cells infiltrate organs
Enlarged lymph nodes, splenomegaly and infiltration of T cells into organs such as the skin, liver, lung and bone marrow are hallmarks in human PTCL. Histological analysis of tissues from cS5Ahi mice revealed disrupted lymph nodes and spleen architecture with dense infiltra- tion of CD3+ T cells and increased proliferation (Ki67+) within lymphomas (Figure 3A, B, Online Supplementary Figure S3A). With regards to skin pathology, diseased mice displayed a thickened dermis with diffuse infiltra- tion of CD3+ T cells (Online Supplementary Figure S3B, C). Moreover, peribronchial and interstitial T-cell infiltrations were detected in lungs of cS5Ahi mice (Figure 3C). Hepatic T-cell infiltration in portal tracts and sinusoids as well as diffuse perivascular and interstitial renal infiltrates were prominent (Figure 3D, Online Supplementary Figure S3D). Higher numbers of Ki67+ cells indicated active prolifera- tion of infiltrating T cells (Figure 3C, D, Online Supplementary Figure S3B, D, quantification summarized in Online Supplementary Figure S3E).
Transferred cS5Ahi CD8+ T cells induce peripheral T-cell lymphoma in non-irradiated recipients
To establish the disease-initiating potency of the mature and differentiated cS5Ahi-CD8+ T cells, we transferred CD8+ Ly5.2+/CD45.2+ T cells from lymph nodes or spleens
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haematologica | 2020; 105(2)