by: Megan M. MacBride, PhD | Published: June 10th, 2024
Key Takeaways
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).
The two most commonly used human cytokine transgenic models on super immunodeficient background strains are the NOG-EXL and 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/ml1,2. It is commercially available pre-engrafted with human immune cells from the Jackson Laboratory.
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.
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
Graft exhaustion was reported in NOD scid mice carrying the same cytokine transgene as in the NSG-SGM3 in the first report on this transgene: "This suggests that the mechanism underlying this failure of primitive cell accumulation in the BM [bone marrow] was due to a direct effect of their exposure to excessive levels of human IL-3, GM-CSF, and SF causing an increased probability of their differentiation and premature extinction (exhaustion)."1 It is thus not surprising that NSG-SGM3 mice would display a similar phenotype upon human HSC engraftment.
"We found that NSGS mice developed a severe, eventually fatal anemia upon humanization with umbilical cord blood (UCB) CD34+ cells."
Wunderlich, et al.4
Mulloy and coauthors followed up with a paper in 2016 characterizing the phenotype of humanized NSG-SGM3 mice as "a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc [red blood cell] numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis."5
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
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.
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
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
NSG-SGM3 suffer from severe, progressive anemia, human immune graft failure and fatal myeloid cell hyperactivation syndrome.
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.
Figure 1: Reduced lifespan in human HSC-engrafted NSG-SGM3 mice.5 Used with permission.
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.
Figure 2. Survival curve for huNOG-EXL mice across 23 donors (n=5-9 per donor), generated in the Taconic Biosciences humanization core during 2022-2023. Internal Taconic data.
huNOG-EXL mice have been used extensively in immuno-oncology drug discovery and development. They can successfully engraft key human immune cell types and support immuno-oncology studies with sufficient timelines for tumor growth and treatment. For longer studies, the huNOG-EXL EA model option provides access to humanized mice soon after engraftment and may be preferred. Post-engraftment human immune cell differentiation and QC takes place at the customer site, maximizing the potential study window.
| Model | huNOG-EXL | Hu-CD34+ NSG-SGM3 |
|---|---|---|
| Strain | NOG-EXL | NSG-SGM3 |
| Also Known As | hGM-CSF/hIL-3 NOG | NSGS |
| Nomenclature | NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3,CSF2)10-7Jic/JicTac | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ |
| Background | NOG (NOD strain background) | NSG (NOD strain background) |
| Cytokines Expressed | Human GM-CSF (CSF2) Human IL-3 | Human GM-CSF (CSF2) Human IL-3 Human KITLG (SF) |
| Promoter | SV40 | CMV |
| Increase in Myeloid Cells Over Base Model | ~3 fold relative to NOG3 | ~1.5 to 5 fold relative to NSG8,9 |
| Lifespan Unengrafted | Expected normal lifespan | Expected normal lifespan |
| Lifespan After CD34+ HSC Engraftment | Up to 50 weeks.10 Taconic recommends researchers should plan for studies to be complete by 23-26 WPE. Mice develop anemia and mild to moderate myeloid cell hyperactivation syndrome.6 Lifespan can be impacted by donor effects. | Up to 4 months reported. Mice develop anemia, mast cell hyperplasia, and severe, fatal myeloid cell hyperactivation syndrome. 4-7 |
| Other Comments | Stable engraftment through lifespan of mouse | Loss of human graft after 3–5 months1,10 |
| Terms of Use | Label license — no signatures or license fees required. May be used for contract or sponsored studies when purchased under for-profit terms and price. | Research institutions require an MTA, companies require a license prior to shipping. Commercial users are prohibited from engrafting naïve NSG-SGM3 mice with CD34+ HSCs derived from common sources such as cord blood.2 |
| Available From | Taconic Biosciences | The Jackson Laboratory |
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
An important note for those who prefer to humanize in-house: license restrictions on the NSG-SGM3 prohibit commercial researchers (or nonprofit researchers engaging in commercial uses) from engrafting in-house with common CD34+ HSC sources such as cord blood, limiting the application of these mice.
Figure 3: Success rates for engraftment of human umbilical cord blood-derived CD34+ HSCs are better in NOG-EXL mice compared to NOG mice within individual donor lots. Data from Champions Oncology.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:
