A novel mouse model for the aggressive variant of NK cell and T cell large granular lymphocyte leukemia

Abstract

Murine models of disease are vital to the understanding of pathogenesis and the development of novel therapeutics. We have previously established interleukin (IL)-15 transgenic (tg) mice that demonstrate rapid proliferation of natural killer (NK) and T cells, followed by spontaneous transformation to lethal leukemia. Herein, we have characterized this model, which has many features in common with the aggressive variants of NK and T large granular lymphocyte leukemia (LGLL) in humans. The LGLL blasts are cytolytic and produce IFN-gammaex vivo. Cytogenetic analysis revealed trisomy of chromosome 17 and/or 15. This model should provide opportunities to develop effective standard therapies for this fatal disease.

Figure 1. Flow cytometry analysis of LGLL

(A) Splenocytes from leukemic mice were stained with anti-NK1.1, anti-CD3ε and anti-CD49b, and analyzed by flow cytometry; isotype antibody control is in grey. (B) Expression of CD62L and CD69 on NK1.1+ cells is altered in leukemic mice versus WT controls.

Figure 2. Leukemia mice have reduced survival and enhanced lymphoid proliferation

(A) Blood smear staining with Wright-Giemsa stain shows a large number of leukemic blasts in peripheral blood of a representative moribund mouse. (B) Transmission electron micrograph (TEM) showing LGL leukemia within the spleen of leukemic mouse. The LGL leukemia cells show characteristic electron dense granules in the cytoplasm and LGL morphology. Bar = 2 µm. (C) H&E staining of bone marrow and spleen of leukemia mouse reveals massive infiltration of blasts in bone marrow and spleen (40X magnification).

Figure 3. SKY and FISH analysis reveal trisomy 15 and/or 17 in both NK and T LGL blasts

(A) SKY analysis of T LGL leukemia reveals an extra copy of chromosome 9 (red oval), 15 (white oval) and 17 (blue oval). (B) Dual-color interphase FISH of independent NK (right) and T LGL (left) leukemia. Chromosome 15 probe (in green), chromosome 17 probe (in red), and DAPI counter-stain (blue) were used for detection of trisomies. The left image illustrates gain of both chromosomes 15 and 17, whereas the right image shows a gain of chromosome 17 only.

Figure 4. Cytotoxicity and IFN-γ release by NK and T LGL blasts

(A) Freshly isolated whole blood leukocytes from two mice with NK LGL (○) and T LGL (●) leukemia that contained approximately 90% blasts were incubated with ⁵¹Cr labeled YAC-1 target cells as described in ‘Materials and Methods’ at the indicated effector:target in 96-well plates in triplicate. The data represents mean ± SD of triplicates for all the effectors to target ratios. (B) IFN-γ production in leukemia mice was determined (n = 6) by overnight stimulation of total splenocytes with IL-12 and IL-18 in vitro. Supernatants were harvested and analyzed for IFN-γ production using an ELISA. Data are expressed as mean ± SD of duplicates and are pooled from four independent experiments.

Figure 5. Transplantability of primary leukemia cells in vivo

(A) Kaplan-Meier overall survival analysis shows a significant difference between WT controls (-*-) and leukemia mice (NK LGL leukemia and T LGL leukemia ). (B) Kaplan Meier survival curve of SCID mice after transfer of leukemia cells to secondary recipients. Groups of 4 mice were injected intravenously with 1 × 10⁵ peripheral blood cells (-□-) or injected with same number of spleen cells (-◊-) or bone marrow cells (-Δ-) on days 0. Mice transplanted with different sources of LGL leukemic blasts showed mortality within the same period of time irrespective of the source of LGL leukemic blasts. Results shown are representative of two independent experiments that were performed.