Interleukin-15 Deficiency Promotes the Development of T-Cell Acute

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Letters to the Editor 1749 Interleukin-15 deﬁciency promotes the development of T-cell acute lymphoblastic leukemia in non-obese diabetes mice with severe combined immunodeﬁciency

Leukemia (2016) 30, 1749–1752; doi:10.1038/leu.2016.28 within 12 weeks after adoptive transfer (Supplementary Figure S4, upper panel). Surprisingly, purified CD4+ T cells from 8.3-NOD donors caused leukemia within 6 weeks, whereas CD8+ T cells Interleukin-15 (IL-15) is critical for the development and home- failed to cause disease (Supplementary Figure S4, lower panel). ostasis of natural killer and memory CD8+ T lymphocytes.1 CD4+ T cells, but not total splenocytes, from non-TCR transgenic By promoting activation of CD8+ T cells, IL-15 can boost anti- diabetic NOD mice also induced leukemia, albeit at a lower tumor immune responses, and is undergoing clinical trials as frequency and delayed kinetics (Supplementary Figure S4, upper cancer therapy.2 However, when expressed as transgene, IL-15 can and lower panels). Similar to leukemic cells induced by total cause fatal leukemia.3,4 IL-15 also facilitates the activation of splenocytes, those arising after the transfer of 8.3-NOD CD4+ − autoreactive CD8+ T cells and promotes inflammatory and T cells showed the CD4+CD8int TCR phenotype (Supplementary autoimmune diseases.1 We have recently shown using the Figure S5). non-obese diabetes (NOD) mouse model that IL-15 plays a We maintained the leukemic cells that developed in NOD/SCID. − − pathogenic role in autoimmune type 1 diabetes by activating Il15 / mice in NOD/SCID recipients by serial passage, which diabetogenic CD8+ T cells.5 Adoptive transfer of splenocytes from increased their pathogenic potential, causing rapid mortality even diabetic 8.3-NOD mice, which express a transgenic major in wild-type NOD mice (Supplementary Figures S6a and b). These histocompatibility complex class-I-restricted autoreactive T-cell leukemic cells rapidly colonized lymph nodes, liver and kidneys antigen receptor (TCR) called 8.3-TCR, induced type 1 diabetes in and occasionally the thymus in recipient mice (Supplementary NOD mice with severe combined immunodeficiency (NOD/SCID) Figures S6b and c). Cells colonizing the thymus also displayed − but not in IL-15-deficient NOD/SCID (NOD/SCID.Il15−/−) mice5 the CD4+CD8loCD3/TCR phenotype and expressed CD25 (Supplementary Figure S1a). Intriguingly, the NOD/SCID.Il15−/− (Supplementary Figures S6c and S6d (upper panel)). These cells recipients showed massive enlargement of lymph nodes and readily grew in vitro, from which we established the SID-T-cell line spleen with high cellularity and became sick within 12 weeks, (Supplementary Figure S6e; Figure 1e) that retained the surface characterized by kyphosis and piloerection (Supplementary phenotype (Supplementary Figure S6d, lower panel). We estab- Figure S1b; Figures 1a and b). These NOD/SCID.Il15−/− recipients lished several other leukemic cell lines directly from lymph nodes − − showed massive mononuclear cell infiltration of lymph modes, and spleen of NOD/SCID.Il15 / recipients without passaging liver and kidneys, and abundant lymphoblasts with large nuclei in in NOD/SCID mice (Figure 1e). the peripheral blood (Figure 1c). These findings indicated that The leukemic cell lines grew exponentially without IL-15 plays an important role in preventing leukemia requiring any cytokines or growth factors (Figure 2a). Therefore, development. we examined whether these leukemic cells displayed constitutive Adoptively transferred splenocytes from 8.3-NOD donors activation of signaling molecules. Murine T-leukemic cell populate the lymphoid organs of NOD/SCID recipients with lines EL4 and P1798, IL-2-dependent CTLL-2 and naïve CD3+ CD8+ and CD4+ T cells, which express the transgenic 8.3-TCR T cells served as controls. All the leukemic cell lines displayed (Figure 1d, upper panels). In contrast, leukemic cells accumulating increased protein tyrosine phosphorylation and elevated in NOD/SCID.Il15−/− mice showed CD4hiCD8lo phenotype and did expression of LCK and FYN kinases (Figure 2b). As CTLL-2 not express the 8.3-TCR (Figure 1d, lower panels), suggesting that cells showed STAT5 phosphorylation (induced by IL-2), STAT3 the IL-15-deficient lymphopenic environment in NOD/SCID.Il15−/− and ERK, the leukemic cell lines only showed moderate activation mice gave rise to abnormal CD4+CD8loTCR− cells with leukemic of STAT3. However, these cell lines (except SID-T) showed AKT properties. To test whether these leukemic cells outnumbered phosphorylation similar to EL4 and P1798. These findings autoreactive CD8+ T cells, the leukemic cells were adoptively suggested that constitutive activation of diverse signaling transferred to NOD/SCID mice along with splenocytes from molecules might contribute to leukemogenesis in NOD/SCID. − − diabetic NOD donors (Supplementary Figure S2a). The leukemic Il15 / recipients. cells not only prevented type 1 diabetes in NOD/SCID recipients Human T-acute lymphoblastic leukemia (T-ALL) cells often but also expanded robustly, causing lymphadenopathy, spleno- overexpress the MYC proto-oncogene downstream of NOTCH1, megaly, kidney enlargement and death within 4–6 weeks which is activated by gain-of-function mutations.6 NOTCH1 (Supplementary Figure S2b). The expanding cells retained the activation also occurs in mouse T-ALL with concomitant upregula- CD4+CD8int phenotype, expressed CD5 but not major histocom- tion of MYC.7 MYC gene activation can also occur by t(12:15) patibility complex-I or B220 (Supplementary Figure S3). These chromosomal translocation in T-ALL.8 Among the leukemic observations indicated that the leukemic cells outnumbered the cell lines that we have established, only SID-T showed elevated diabetogenic CD8+ T cells and retained aggressive growth MYC expression comparable to EL4 thymoma (Figure 2b), potential even in an IL-15-sufficient environment. suggesting that Myc gene activation is unlikely to be a key As the leukemic cells showed CD4+CD8int phenotype, we mediator of leukemogenesis in NOD/SCID.Il15−/− recipients. Next, examined whether they originated from CD4+ or CD8+ donor we carried out karyotype analyses by multiplex fluorescence in T cells. For this, we compared total splenocytes and purified situ hybridization (Figure 2c). Even though many of the leukemic CD4+ or CD8+ T cells for their ability to induce leukemia in NOD/ cell lines showed t(12:15) translocation, they also harbored other SCID.Il15 −/− recipients. Total splenocytes from pre-diabetic chromosomal abnormalities such as 1p+ and t(10:13) transloca- (3 week-old) and diabetic 8.3-NOD donors induced leukemia tion. Clearly, the t(12:15) translocation did not always result

Accepted article preview online 15 February 2016; advance online publication, 1 March 2016

Figure 1. Lymphoid cell infiltration of parenchymatous organs and leukemia development in NOD/SCID.Il15−/− mice following adoptive transfer of diabetogenic T cells. Total splenocytes from diabetic 8.3-TCR transgenic NOD mice were adoptively transferred to NOD/SCID or NOD/SCID.Il15−/− mice. (a) Two months after adoptive transfer, the recipient mice were sacrificed and total cell numbers in the spleen and the indicated lymph nodes (LN) were estimated. ANOVA: ****Po0.0001. (b) Proportion of recipient mice that became overtly ill with kyphosis and piloerection, and showed enlargement of spleen and LN upon examination. The curves were compared by log-rank (Mantel–Cox) test. (c) Histologic sections of lymph node, liver and kidney, and peripheral blood smear were stained with hematoxylin and eosin. Representative images from eight mice in each group are shown. (d) Leukemic cells accumulating in NOD/SCID.Il15−/−mice show an abnormal CD4+CD8lo phenotype and do not express the transgenic TCR. Two months after adaptive transfer of splenocytes from 8.3-NOD mice, spleen and pooled peripheral lymph nodes of NOD/SCID and NOD/SCID.Il15−/− recipient mice were evaluated for CD4 and CD8 markers. The expression of the transgenic 8.3-TCR was evaluated on gated CD4+CD8lo cells. Representative data from two mice for each genotype out of 6–8 recipients are shown. (e) Summary of the leukemic cell lines established from NOD/SCID mice lacking IL-15 or IL-15 Rα that developed leukemia following adoptive transfer of T cells. The first cell line (SID-T) was derived following serial in vivo passaging in NOD/SCID mice as detailed in Supplementary Figure S5. Some cell lines were established from spontaneously arising leukemia from NOD/SCID.Il15−/− and 8.3-NOD.Il15−/− mice (shown in Figure 2e).

in increased MYC expression (Figures 2b and c). These data might have originated from host cells. To test this idea, we suggest that multiple genetic and signaling abnormalities may examined the leukemic cell lines for the presence or absence the contribute to leukemia induction in NOD/SCID.Il15−/− mice. Il15 knockout allele, due to the lack of donors and recipients Defective thymic maturation in NOD mice has been reported to bearing different allelic markers. Surprisingly, all the leukemic cell facilitate thymoma development under lymphopenic conditions lines harbored the Il15 knockout allele (Figure 2d), indicating that caused by SCID mutation (67% at 40 weeks) or recombination they arose from the recipient’s cells. Further supporting the host activating gene deﬁciency (44% at 25 weeks),9,10 raising the origin of leukemic cells, long-term observation of NOD/SCID mice possibility that the leukemic cells in NOD/SCID.Il15−/− recipients lacking either IL-15 revealed that they all developed spontaneous

Figure 2. Leukemic cell lines derived from NOD/SCID.Il15−/− recipients show diverse show activation of diverse signaling molecules and multiple cytogenetic abnormalities, and are endogenous in origin. (a) Leukemic cell lines derived from NOD/SCID.Il15−/− recipients show exponential growth without the addition of cytokines or growth factors. Cells (3 × 106) of the indicated cell lines were seeded in 100 cm petri dishes in RPMI-1640 medium containing 10% fetal bovine serum. The cells were counted and the culture medium replenished every day. Representative data from triplicate cultures are shown (mean ± s.e.m.). (b) Cells from exponentially growing cultures were lysed and protein tyrosine phosphorylation and expression of the indicated proteins were evaluated by western blot. Representative data from three separate experiments are shown. (c) Mitotic spread from the indicated cell lines were analyzed by multiplex ﬂuorescence in situ hybridization. Representative data from three independent experiments are shown. The chromosomal abnormalities observed in each cell line are circled and indicated below the images. (d) Polymerase chain reaction analysis of the targeted allele of the Il15 gene in the DNA isolated from leukemic cell lines. DNA from NOD/SCID.Il15+/ − and NOD/SCID.Il15−/− mice served as control for wild-type and the targeted allele. (e) NOD/ SCID.Il15−/− and NOD/SCID.Il15ra−/− mice followed for overt illness and development of splenomegaly and lymph node enlargement. Log- rank (Mantel–Cox) test: ***Po0.005. (f) TdT expression in the leukemic cell lines. TdT staining on C57Bl/6 thymocytes gated into CD4+CD8+ double positive and CD4+ or CD8+ single positive cells were used as positive and negative controls, respectively, for TdT staining. leukemia over a period of 32 weeks, whereas control NOD/SCID binding α-subunit of the IL-15R complex also developed mice did not (Figure 2e). These spontaneously developing spontaneous leukemia that was accelerated by adoptively leukemia also gave rise to leukemic cell lines (Figure 1e). It is transferred of splenocytes from diabetic 8.3-NOD mice noteworthy that NOD/SCID.Il15ra−/− mice lacking the ligand (Figure 2e and Supplementary Figure S7). The SID-15RaL cell line

© 2016 Macmillan Publishers Limited Leukemia (2016) 1742 – 1792 Letters to the Editor 1752 − − derived from NOD/SCID.Il15ra / recipients (Figure 1e), harbored assisted in performing the experiments. HK and FB participated in data analysis the wild-type Il15 allele as expected (Figure 2d). Finally, we and in writing the manuscript. examined the expression of terminal deoxynucleotidyl transferase 11 (TdT), a widely used marker for T-ALL. TdT, involved in DNA D Bobbala1, R Kandhi1, X Chen1, M Mayhue1, E Bouchard2, J Yan2, + + repair during TCR gene rearrangement, is expressed in CD4 CD8 H Knecht3, F Barabé4,5, S Ramanathan1,6,7 and S Ilangumaran1,6,7 + + double positive thymocytes, but is repressed in CD4 or CD8 1Immunology Division, Faculty of Medicine and Health Sciences, 12 single positive T cells (Figure 2f). Similarly to EL4 and P1798 cells, Université de Sherbrooke, Sherbrooke, QC, Canada; all leukemic cells expressed TdT (Figure 2f). Collectively, our 2Genetics Division, Department of Pediatrics, Faculty of Medicine and findings indicate that lack of IL-15 signaling in NOD/SCID mice due Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada; to the absence of IL-15 or IL-15Rα facilitated leukemogenesis of 3Department of Hematology, Faculty of Medicine and Health T-cell precursors within the recipients. Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada; Animal models play an important role in deciphering molecular 4Department of Medicine, Université Laval, Quebec City, QC, Canada; mechanisms of leukemogenesis, and in developing new drugs 5CHU de Québec-Université Laval, Quebec City, QC, Canada and and treatment methods.13 In this study, we have shown that NOD/ 6CR-CHUS, Sherbrooke, Québec, Canada SCID mice lacking IL-15 or IL-15Rα develop leukemia with 100% 7SR and SI share senior authorship. penetrance within 32 weeks, and this process is accelerated by E-mail: [email protected] or adoptively transferred CD4+ T cells (100% within 12 weeks). [email protected] Moreover, the leukemic cells from NOD/SCID.Il15−/− mice readily grow in vitro without requiring in vivo passage. Overall, the leukemia model described in our study provides a unique REFERENCES opportunity to characterize the genetic determinants and 1 Fehniger TA, Caligiuri MA. 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Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu).