AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

Despite major advances in human to mouse xenografts, most of the human acute myeloid leukemia (AML) samples fail to adequately engraft available strains of mice. The reasons for the disparate results with AML samples remain unclear, but likely include elements of homing, survival in a foreign niche, expansion in the absence of specific human growth factors and supporting stromal cells, escape from immune surveillance and residual innate immunity in the mice, as well as intrinsic differences among AML samples. One general strategy for improving engraftment has been the development and use of increasingly immunodeficient mouse strains (reviewed in¹). For example, targeting of the IL2RG gene in the NOD/SCID (NS) strain by either complete knockout or truncation of the intracellular portion (NOD/LtSz-scid IL2RG: NSG and NOD/shi-scid IL2RG, respectively) was shown to diminish innate immunity, in particular macrophage function and NK activity, thereby improving engraftment by normal human CD34+ cells and AML cells.^2,3 Alternatively, transgenic expression of hSCF, hGM-CSF and hIL-3 (three non- or poorly cross-reacting cytokines) in the NOD/SCID background (NOD/LtSz-scid -SGM3, NSS) led to improvements in expansion of normal human myeloid cells, as well as slight gains in engraftment of AML samples.^4,5

In this study, we have generated a new mouse strain by crossing the NSG mouse with the NSS mouse to generate the NOD/LtSz-scid IL2RG–SGM3 mouse (NSGS) (Figure 1a). Tail clip gDNA PCR and Enzyme-linked immunosorbent assay were used to identify mice with IL2RG knockout and human cytokine expression. Enzyme-linked immunosorbent assay assays for IL-3 and granulocyte macrophage-colony stimulating factor were well correlated and clearly differentiated cytokine homozygotes from heterozygotes and, therefore, were used to identify suitable breeder pairs for further colony expansion (Figure 1b). Although our Enzyme-linked immunosorbent assay measurements differed somewhat from those reported for the NSS mouse², we observed very similar levels of expression between the newly generated NSGS mice and our line of NSS mice (Figure 1c). Peripheral blood (PB) complete blood counts yielded no consistent, meaningful differences among strains, indicating that no major changes in myeloid, lymphoid, erythroid or platelet levels occurred in the PB of these mice in response to the human cytokines (Figure 1d).

Figure 1.

Characterization of the NSGS mouse. (a) Breeding scheme. NOD/SCID mice previously modified by IL2RG knockout (NSG) or expression of human cytokines (NSS) were crossbred to yield NOD/SCID IL2RG knockout with cytokine expression (NSGS). (b) Enzyme-linked immunosorbent assay assays with NSGS and NSS serum showed a high degree of correlation between hIL-3 and hGM-CSF expression. Cytokine heterozygotes and homozygotes were easily distinguished by this analysis. IL2RG status was determined by tail clip gDNA PCR. (c) Average cytokine expression in PB serum from NSGS and NSS mice. hIL-3, hGM-CSF and hSCF levels are shown. n = 15–21 mice per group, error bars = +/−s.d. (d) PB complete blood counts were determined by a Hemavet 950. n = 8–17 per group, error bars indicate s.d. No consistent differences were observed between strains. Top panel shows average total counts of specific cell types. Middle panel shows measures of red blood cell characteristics. Lower left and right panels show platelet numbers and volume, respectively. All mouse experiments were conducted according to an IRB approved protocol.

In this study, we focused on the possible improvements that could be realized in human AML and pre-leukemia models, given the dependency of many of these cells on cytokine stimulation for growth and the already proven superiority of mice lacking IL2RG for AML xenografts. For these experiments, we first used a cord blood-derived AML driven by retroviral expression of the MLL-AF9 oncogene⁶ into which we subsequently transferred an activated NRas cDNA containing a codon 12 (G to D) point mutation (MA9-NRas). We have found that the NRas ‘second hit’ significantly and reproducibly decreases latency of disease and allows quick development of AML in non-irradiated NS and NSG mice (MW and JCM, unpublished observations). We monitored MA9-NRas injected mice for signs of illness. Although nearly all mice eventually developed lethal AML, a marked difference was observed in latency between the four strains of mice, with cytokine expression the dominant factor in rapid disease development (Figure 2a). NSGS mice developed terminal AML slightly sooner than the NSS group and both of the cytokine-expressing strains showed a narrow window in which all mice succumbed to disease. This narrow range of disease manifestation represents an advantage over the wide range observed in NS and NSG mice and is ideal for several applications in which survival is a key readout, including therapeutic testing. The effect of IL2RG loss was apparent whether cytokines were present or not, reproducibly shortening disease latency.

Figure 2.

The NSGS mouse shows improved xenograft efficiency. (a) One million MA9.3-NRas(G12D) leukemic cells were injected into the tail vein of non-irradiated mice. Survival is shown over time. NSGS mice succumbed to AML faster than all other strains, including NSS (log rank test, P<0.0004). NS mice were consistently the least efficient (log rank test, P=0.065 vs NSG). (b) Homing assay. 10 million MA9 leukemic cells were injected by tail vein into sublethally irradiated (280 Rad) recipients. Human CD45 + CD33 + cells in the femurs were quantified 24h later by flow cytometry. Results are normalized to those obtained with NS hosts. (c) Mice injected with human leukemic cells as in ‘A’ were subjected to BM aspiration (top panel) and tail bleeds (bottom) to assess the xenograft status (percentage of CD45 + CD33 + cells by flow cytometry) on day 30. NSGS mice had significantly higher grafts than all others by Student's t-test (P<0.05). (d) Long-term cord blood cultures expressing AML1-ETO or CBFb-MYH11 were transferred to the left femurs of sublethally irradiated hosts by intrafemoral injection. The contralateral femur was analyzed at 8 weeks by BM aspiration. At 16 weeks, both the injected femur and a non-injected tibia were analyzed for the presence of human CD45 +CD33+ cells by flow cytometry. (e) Patient AML samples were injected into tail veins of sublethally irradiated NSG or NSGS mice. Xenografts were determined by BM aspiration and flow cytometry at 12–16 weeks. Shown are five samples with engraftment in the NSGS mice. When all these data were subjected to Student's t-test, NSGS grafts were significantly higher than NSG grafts (P= 0.05). Three additional samples were unable to engraft either mouse strain. No sample that engrafted NSG mice failed to engraft NSGS mice. (f) Flow cytometry plots showing significant human CD45 + cells in the BM of NSGS mice. AML cells were also detected in the spleen and PB. (g) Lineage stains indicate a myelomonocytic phenotype and significant CD34 + CD133 + population in the BM of primary mice as well as secondary recipients 16 or more weeks after transplant. All error bars represent s.d. *P<0.05 by Student's t-test. Flow cytometry performed on a BD FacsCantoII with BD antibodies (except anti CD133, Miltenyi Biotech) and analyzed with FlowJo software (Tree Star).

Multiple mechanisms could be involved in these differences. To initiate a successful graft, cells must first properly home to the BM. To determine the variation of homing in the four mouse strains under investigation, we injected 10 million leukemia cells and analyzed the BM 24 h after injection. The level of engraftment was significantly higher in IL2RG-deficient mice (NSG and NSGS) compared with the other two strains (Figure 2b). Although absolute homing efficiency was low (ranging from approximately 0.04% in NS and NSS mice to 0.1% in NSG and NSGS), these levels were sufficient to initiate lethal disease in NSGS and NSS mice within 4–5 weeks (Figure 2a). These data highlight the critical role of innate immunity for the initial stage of engraftment, whereas expression of human myeloid cytokines alone does not significantly alter it. In addition, we examined engraftment by measuring human AML cells in the BM and PB 1 month after injection. BM aspiration showed the highest chimerism in NSGS mice (Figure 2c, top). NSG and NSS mice also clearly outperformed the NS mice in this test, with the NS mice seeming to be essentially negative for engraftment. PB measurement of the grafts showed essentially the same results (Figure 2c, bottom). Moreover, engraftment is significantly increased in human cytokine-expressing mice (NSGS and NSS) compared with their counterparts (NSG and NS, respectively). Thus, the reduced innate immune response increases initial homing efficiency to the marrow and may support subsequent growth/survival of the cells. In contrast, human cytokines do not affect homing efficiency but provide cell proliferation and survival signals that become more measurable over time. These effects seem additive, as evidenced by the superior engraftment observed in NSGS mice relative to NSG or NSS mice.

We also analyzed the engraftment of long-term pre-leukemic myeloid cultures established by transduction of cord blood CD34+ cells with the Core Binding Factor oncogenes AML1-ETO (AE) and CBFβ-MYH11 (CM) that have historically been very difficult to study in vivo.^7–9 We consider these cultures to represent a pre-leukemic state, as these clonal cultures retain an abnormally high percentage of cells positive for the progenitor cell marker CD34 and the ability to differentiate into cells of erythroid, myeloid and B-cell lineage in a context-dependent manner. We have observed poor engraftment of these cells when using NS, NSG or NSS mice (unpublished observations).^7,9 To determine whether NSGS mice could significantly increase engraftment of pre-leukemic samples, three separate AE cultures and one CM culture were injected into femurs of NSG and NSGS mice. At 8–10 weeks and 14–16 weeks, BM aspirations were performed on the non-injected femur and tibia to measure the spread and expansion of the injected cells (Figure 2d). NSGS mice reproducibly showed significantly higher levels of human CD45+/CD33+ myeloid cells compared with NSG mice at both time points, with approximately 10 times the number of cells in both the injected bone, as well as in distant bones (Figure 2d). The graft also increased over time, implying that the cells were expanding in the mice. Thus, NSGS mice offer the opportunity to perform xenotransplant studies for these pre-leukemic myeloid cells.

Finally, we evaluated the combined effect of human cytokine expression and IL2RG knockout for xenografts of human AML patient samples. We compared engraftment in age-matched NSG and NSGS mice using a number of samples we recently identified as either ‘low engrafters’ or ‘high engrafters’ in NSG mice.¹⁰ A paired analysis considering all AML engraftment data together showed that NSGS gave significantly improved grafts relative to NSG, even with this small sample size (Figure 2e, P=0.05). Furthermore, samples that failed to engraft NSG mice were readily detectable in NSGS mice (samples 180 and 501). Sample no. 501 engrafted NSG mice in a previous study,¹⁰ but failed to engraft in this study probably because of a limiting cell dose (125 000 cells per mouse, compared with 5 million in the previous study). However, in NSGS mice, engraftment was robust even at these limiting cell numbers. Another sample, no. 990, was previously shown to be a poor engrafter in NSG mice,¹⁰ but showed enhanced engraftment in the PB, spleen and BM of the NSGS mice (Figure 2f). AML#990 presented as a myelomonocytic blast population with a substantial proportion of primitive CD34+CD133+ cells in the BM (Figure 2g). Moreover, secondary transplant into NSGS mice resulted in a similar phenotype of the AML cells including the primitive cell population, indicating retention of the leukemia initiating cells in the NSGS mouse (Figure 2g). These results suggest that the NSGS mouse is a significantly better host for at least a subset of AML samples relative to NSG mice, the current standard. In summary, the combination of human cytokine expression and IL2RG knockout in NOD/SCID strains results in enhanced engraftment of pre-leukemic myeloid cell cultures, as well as primary human AML samples. The novel NSGS mouse provides optimal conditions for engraftment and expansion of these cells, and opens the door to in vivo studies that would otherwise be impossible with pre-existing immunodeficient mice.