Challenges in Models of Myeloid Cell Engraftment in Humanized Mice

Humanized immune system (HIS) mice are revolutionary research tools for the development of novel immune cell-targeting oncology therapeutics, but the use of first-generation models such as the huNOG (NOG mice engrafted with human CD34+ hematopoietic stem cells) has been limited for some applications by impaired human myeloid cell differentiation. Differences between mouse and human cytokine growth factors and receptors underpin these limitations in human immune cell differentiation. In response to this problem, multiple super immunodeficient mouse strains which express key human cytokines have been generated. These strains serve as hosts to generate HIS models which support both human myeloid and lymphoid cells, which are critical tools for immuno-oncology research on myeloid cell targets such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs).

Commonly used myeloid-supportive host strains

The two most commonly used human cytokine transgenic models on super immunodeficient background strains are the NOG-EXL and NSG-SGM3.

NSG™-SGM3

The NSG™-SGM3, also referred to as the NSGS, expresses three different human cytokines, GM-CSF, IL-3, and KITLG, on the NSG™ background. The transgene uses the relatively stronger viral CMV promoter and results in cytokine expression levels at supraphysiologic levels of ~2000-4000 pg/ml^1,2. It is commercially available pre-engrafted with human immune cells from the Jackson Laboratory.

NOG-EXL

The NOG-EXL strain expresses human GM-CSF and IL-3 under control of the SV40 promoter on the NOG background, with relatively lower cytokine expression levels³. It is commercially available pre-engrafted with human CD34+ stem cells from Taconic Biosciences as huNOG-EXL. A version of this strain is now available with knockout of murine Fc gamma receptors to remove potential confounding interference by residual murine immune cells as the FcResolv® NOG-EXL and FcResolv® huNOG-EXL.

Both cytokine transgenic models described above successfully engraft human HSCs with higher human chimerism and they develop more human myeloid cells compared to the parent NOG or NSG strains.

Anemia and Graft Exhaustion in Humanized NSG-SGM3 Mice

The developer of the NSG-SGM3, James Mulloy, first reported in a poster at the 2013 American Society of Hematology meeting that the model develops anemia and bone marrow failure after human CD34+ HSC engraftment: "we found that NSGS mice developed a severe, eventually fatal anemia upon humanization with umbilical cord blood (UCB) CD34+ cells... Bone marrow sections from anemic mice clearly showed a general hypocellularity with abundant fatty tissue filling the majority of the intramedullary space." ⁴

Fatal Phenotype Characterized as Macrophage Activation Syndrome

Macrophage activation syndrome is a feature of several human autoimmune diseases, and thus humanized NSG-SGM3 mice may be of interest for study of these disorders.

Semi-Quantitative Comparison of Myeloid Cell Hyperactivation Syndrome in NSG-SGM3 vs. NOG-EXL

Willis et al. contributed to characterization of these strains with a comprehensive pathological analysis of humanized NSG-SGM3 and NOG-EXL mice, including development of a semi-quantitative clinicopathological scoring system to characterize what the authors termed myeloid cell hyperactivation (MCH) syndrome. This study found significant differences in severity of MCH and lifespan between the two strains. The humanized NSG-SGM3 mice displayed mast cell hyperactivation and lethal MCH by 16 weeks post-engraftment. The humanized NOG-EXL mice developed less severe MCH, and all but one mouse survived to the study endpoint of 22 weeks post-engraftment. This led Willis et al. to conclude, “The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.” ⁶

Short Lifespan of Humanized NSG-SGM3 Challenging for Immuno-Oncology Studies

huNSG and huNOG mice can survive one year or more after engraftment, which is more than sufficient time for immuno-oncology studies. In contrast to that, the early-onset anemia, graft exhaustion, and fatal myeloid cell hyperactivation seen in humanized NSG-SGM3 mice is a challenge for immuno-oncology researchers, as it limits the useable study window. Wunderlich et al. reported that "Analysis of a large number of hu-NSGS showed that 100% of mice died between 10 and 27 weeks of engraftment, while all nonengrafted control mice remained alive and well." ⁵

A poster presentation from the 44th Annual Scientific Meeting of the International Society for Experimental Hematology confirmed other reports of short lifespan in humanized NSG-SGM3 mice. Yoshihara et al. from the Hyogo College of Medicine and the Columbia University Center for Translational Immunology reported that NSG-SGM3 mice engrafted with human CD34+ HSCs, either with or without additional human thymus tissue, "had fatal outcome 18 to 22 weeks after transplantation" and described the phenotype as resembling hemophagocytic lymphohistiocytosis in humans.⁷  

For therapeutics which rely on human T cells, dosing should be initiated after T cells have fully entered the periphery, which is around 16 weeks post-engraftment, and continue for 30-60 days (to ~20-24 weeks post-engraftment). The useable lifespan of a humanized NSG-SGM3 may not support that type of immuno-oncology study.

huNOG-EXL Have Extended Lifespan Compared to huNSG-SGM3 and Are Suitable for IO Studies

In contrast, lifespan for huNOG-EXL exceeds that of other myeloid-supportive HIS models. This varies by donor and can be impacted by environmental and experimental factors. While Taconic has observed that huNOG-EXL mice can often survive for 25+ weeks post-engraftment (WPE), researchers should plan for studies to be complete by 23-26 WPE.

Comparison of the huNOG-EXL and Hu-CD34+ NSG-SGM3 models

NOG-EXL as a Preferred Host for Humanization

It's important to understand the performance of any immune system humanized model can be affected by properties inherent to the donor cells. Entire single donor lots can fail to engraft, and the rate of failures within a single donor lot (defined as <25% chimerism following humanization) varies across donors. For investigators seeking to minimize failed engraftments when establishing their own humanized mouse platform, NOG-EXL mice have beneficial properties.

CD34+ humanized NOG-EXL mice tend to have much lower rates of intra-lot failures compared to first-generation hosts such as NSG and NOG. In data from Champions Oncology, close to zero failed humanizations were observed when NOG-EXL mice were engrafted with stem cells from one of four donors. Lacking supporting human cytokines, similarly engrafted conventional NOG mice resulted in some failures within all lots produced.¹¹

The huNOG-EXL is a humanized model with long term viability and enhanced myeloid lineage engraftment. These properties help make NOG-EXL mice a preferred host to consider when establishing internal humanized model production. For researchers who prefer to purchase pre-engrafted mice for immediate use in studies, the huNOG-EXL represents a humanized model which supports both human lymphoid and myeloid cells and has a longer lifespan compared to the humanized NSG-SGM3.

References:

1. Nicolini FE, Cashman JD, Hogge DE, Humphries RK, Eaves CJ. NOD/SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration. Leukemia. 2004 Feb;18(2):341-7. doi: 10.1038/sj.leu.2403222.
2. https://www.jax.org/strain/013062
3. Ito R, Takahashi T, Katano I, Kawai K, Kamisako T, Ogura T, Ida-Tanaka M, Suemizu H, Nunomura S, Ra C, Mori A, Aiso S, Ito M. Establishment of a human allergy model using human IL-3/GM-CSF-transgenic NOG mice. J Immunol. 2013 Sep 15;191(6):2890-9. doi: 10.4049/jimmunol.1203543.
4. Wunderlich M, Mizukawa B, Chou FS, Sexton C, Shrestha M, Saunthararajah Y, Mulloy JC. AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model. Blood. 2013 Mar 21;121(12):e90-7. doi: 10.1182/blood-2012-10-464677.
5. Wunderlich M, Stockman C, Devarajan M, Ravishankar N, Sexton C, Kumar AR, Mizukawa B, Mulloy JC. A xenograft model of macrophage activation syndrome amenable to anti-CD33 and anti-IL-6R treatment. JCI Insight. 2016 Sep 22;1(15):e88181. doi: 10.1172/jci.insight.88181.
6. Willis E, Verrelle J, Banerjee E, Assenmacher CA, Tarrant JC, Skuli N, Jacobson ML, O'Rouke DM, Binder ZA, Radaelli E. Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice. Vet Pathol. 2024 Jan 10:3009858231222216. doi: 10.1177/03009858231222216.
7. Yoshihara, S.; Li, Y.; Xia, J.; Li, W.; Fujimori, Y.; Yang, Y-G. Experimental Hematology, 2015, 43 (9):S48.
8. Miller PH, Cheung AM, Beer PA, Knapp DJ, Dhillon K, Rabu G, Rostamirad S, Humphries RK, Eaves CJ. Enhanced normal short-term human myelopoiesis in mice engineered to express human-specific myeloid growth factors. Blood. 2013 Jan 31;121(5):e1-4. doi: 10.1182/blood-2012-09-456566.
9. Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A. Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rγ(null) humanized mice. Blood. 2011 Mar 17;117(11):3076-86. doi: 10.1182/blood-2010-08-301507.
10. Sippel TR, Radtke S, Olsen TM, Kiem HP, Rongvaux A. Human hematopoietic stem cell maintenance and myeloid cell development in next-generation humanized mouse models. Blood Adv. 2019 Feb 12;3(3):268-274. doi: 10.1182/bloodadvances.2018023887.
11. Verma, B.; Mancini, M.; Davies, A.; Sidransky, D.; Wesa, A.; Goodwin, N. Poster at 30th EORTC-NCI-AACR Symposium, Nov 13-16, 2018, Dublin, Ireland.