hGM-CSF/hIL3-NOG Mouse Questions - We Have the Answers!

Michael Seiler, PhD
Monday, February 23rd, 2015
Albino Mouse
Taconic recently hosted a webinar titled The hGM-CSF/hIL3-NOG mouse: Enhanced differentiation of multiple human cell lineages in humanized mice, presented by Dr. Takeshi Takahashi. Dr. Takahashi, Chief Scientist in the Immunology Laboratory at the Central Institute for Experimental Animals (CIEA), introduced this new model and presented data from humanization and some early tumor immunology experiments. The Q&A session at the end of the presentation was lively, with far more questions submitted than could be answered during the event. We have thus included an expanded Q&A section in this Taconic Insight.
The hGM-CSF/hIL3-NOG mouse is a super immunodeficient NOG mouse expressing human GM-CSF and human IL-3 cytokines to support myeloid lineage engraftment. The model has higher overall engraftment levels of human hematopoietic stem cells (HSC) compared to the NOG mouse. It provides higher levels of myeloid cell differentiation following human HSC engraftment compared to the NOG mouse. This novel mouse model is useful for various research applications including cancer, infectious disease, immunology and allergy. The hGM-CSF/hIL3-NOG mouse is available now for purchase from Taconic. Please note that research on this model is ongoing, including process optimization for humanization protocols.

General characteristics of the hGM-CSF/hIL3-NOG mouse

Q. What's the strain of these mice? Same as the NOG?
A. The hGM-CSF/hIL3-NOG (formal nomenclature NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3,CSF2)10-7Jic/JicTac) is based on the NOG mouse. The NOG mouse is on the NOD inbred strain background and carries the scid mutation and a knockout of Il2rg. The hGM-CSF/hIL3-NOG mouse carries a transgene which expresses the human cytokines GM-CSF and IL-3.

Q. Do the mice develop spontaneous lymphomas?
A. No spontaneous murine lymphoma development has been observed in the hGM-CSF/hIL3-NOG mouse.

Q. Do the mice develop leakiness as is seen in nude and scid mice?
A.Like the NOG mouse, the hGM-CSF/hIL3-NOG mouse is not leaky.

Q. Do lymph nodes develop in the hGM-CSF/hIL3-NOG mouse? How does this compare to what is seen in the NOG mouse?
A. The hGM-CSF/hIL3-NOG mouse has almost no lymph nodes except small mesenteric lymph nodes. Axillary lymph nodes can sometimes be observed. They have no inguinal or popliteal lymph nodes.

Q. Do the mice develop liver tumors over time?
A. No. We have not observed the development of spontaneous liver tumors in the hGM-CSF/hIL3-NOG mouse.

Humanization experiments in the hGM-CSF/hIL3-NOG mouse

Q. Does the expression level of human GM-CSF and IL3 vary from mouse to mouse and does this affect engraftment? / Do you see variation in IL-3 and does it affect variation in mast cells/basophils?
A. There is some variation in expression levels, but this does not appear to affect engraftment level. Even mice with lower expression levels still induce good differentiation of human cells.

Q. Can you pool CD34+ HSC donors to produce more mice for a single experiment?
A. This is possible, particularly if you are not concerned with HLA types. hGM-CSF/hIL3-NOG mice can be engrafted with a low cell dose such as 20,000-25,000 HSCs/mouse. CIEA typically uses 50,000 HSCs per NOG mouse, but it's possible to use fewer cells per hGM-CSF/hIL3-NOG mouse. This effectively doubles in number the cohort size that can be produced from a single donor.

Q. How much variation is seen between mice engrafted using cells from the same donor?
A. There is small variation among mice engrafted from a single donor when engrafted at the same time. If you freeze cells down and engraft batches of mice at different times, this will increase variability among the mice.

Q. How does the donor to donor variation affect the level of engraftment of the different myeloid populations?
A. There is variation from donor to donor in engrafted NOG and hGM-CSF/hIL3-NOG mice. Variation is seen in chimerism level, but frequency of each cell type is relatively consistent from mouse to mouse, even when the overall engraftment level differs.

Q. Is there any data from the mast cells of lung tissue?
A. Various human cells have been detected in bronchoaveolar lavage fluid from HSC-engrafted hGM-CSF/hIL3-NOG mice. Human mast cells have been detected in the lung by immunohistochemistry.

Q. Do you see any improvement in erythrocyte and megakaryocyte lineages engraftment in your transgenic model?
A. To date, no one has developed a humanized model that successfully produces mature megakaryocytes and red blood cells. There is a report that immature erythrocytes and megakaryocytes can be detected in bone marrow of HSC-engrafted conventional NOG mice, but these are not found in circulation in the peripheral blood. Differentiation of erythrocytes and megakaryocytes has not been assessed in HSC-engrafted hGM-CSF/hIL3-NOG mice.

Q. Have you ever used a myelodepletion method prior to engraftment to eliminate residual mouse immune cells (such as busulfan), and what effect does that have on human immune cell engraftment?
A. We have tested busulfan in conventional NOG mice. It works as nicely as x-ray irradiation., but it appears there may be a slight difference in the composition of reconstituted human cells between the two myelodepletion methods. We have not tested busulfan treatment in hGM-CSF/hIL3-NOG mice.

Q. Do hGM-CSF/hIL3-NOG mice show improved mature cell transfer such as PBMCs? If so, is the engraftment of CD8+ T cells better than just CD8+ transfer with IL2 and IL15?
A. PBMC engraftment has not been assessed in the hGM-CSF/hIL3-NOG mice. Conventional NOG mice engraft PBMCs very well, so NOG mice would be the recommended model to use.

Q. What is the lifespan of HSC-engrafted hGM-CSF/hIL3-NOG mice?
A. After HSC transplantation, the mice survive around 5 months. Depending on the protocol used for HSC transplantation, the lifespan can vary. The irradiation level is critical. Higher radiation doses can produce higher engraftment levels, but can also accelerate the development of anemia (i.e. within 3-4 months post-engraftment). Taconic recommends users perform an irradiation dose study starting at 1 Gy to determine the most appropriate irradiation dose for your experimental setup.

Q. How long does the engrafted human immune population last for? What kind of window of time do you have to work with these mice?
A. HSC-engrafted hGM-CSF/hIL3-NOG mice have good human cell engraftment levels and are healthy in the period of 3-4 months after engraftment. After 4 months post-engraftment, the mice can develop slight anemia. T cells and B cells develop very well and earlier in HSC-engrafted hGM-CSF/hIL3-NOG mice compared to HSC-engrafted NOG mice, so experiments can often be started earlier. This window of time should be sufficient for many types of experiments.

Q. Do the percentages of immune cells change over time, in that 3-4 month window?
A. Human cell levels in HSC-engrafted hGM-CSF/hIL3-NOG mice peak around 4 months post-engraftment and then gradually fall. Engraftment levels are higher compared to those seen in conventional NOG mice. The frequency of myeloid cells falls ~2-3 months after HSC transplantation, as lymphoid cells begin to rapidly proliferate at that point and become the dominant cell type. But this does not mean that the absolute number of myeloid cells drops. As in NOG mice, the composition of human cell types changes over time.

Q. Do you see age related major changes in these mice?
A. Unengrafted hGM-CSF/hIL3-NOG mice should have a normal lifespan if kept under conditions suitable for immunodeficient mice, however a formal longevity study has not been completed. Once engrafted with human HSCs, the hGM-CSF/hIL3-NOG mice remain healthy for ~4-5 months, but gradually become anemic. The average lifespan for HSC-engrafted hGM-CSF/hIL3-NOG mice is 5-6 months.

Q. Is there a residual presence of any mouse immune cells, and what is the ratio of human engraftment over residual mouse cells? Do residual mouse cells affect the activity of engrafted human immune cells?
A. HSC-engrafted hGM-CSF/hIL3-NOG mice often have 50-60% human cells in peripheral blood. We do not know whether the remaining mouse innate immune cells are able to affect the human cell function. If these innate mouse cells produce cross-reactive cytokines, it is possible for them to interfere with human cells. Work is underway at CIEA to develop next generation NOG models in which the remaining mouse immune cells have been eliminated.

Q. Did you monitor antibody production such as IgG, IgM in the hGM-CSF/hIL3-NOG mice?
A. HSC-engrafted hGM-CSF/hIL3-NOG mice have been assessed for production of human antibodies. So far, no IgG response has been observed. Further modification to introduce human HLA transgenes may be necessary.

Q. Do these mice develop GvHD and when? Also, have you checked antigen-specific T cell responses?
A. In HSC-engrafted hGM-CSF/hIL3-NOG mice, we have not observed the symptoms of GvHD (T cell-mediated). Antigen-specific T cell responses may be possible, if HLA genes are further introduced and the mice engrafted with HLA-matched HSC.

Q. What is the % of mast cells in BALF?
A. By flow, about 20% of human cells in bronchoalveolar lavage fluid were FceR+CD203c+. But we have not stained them with anti-c-kit to distinguish mast cells and basophils. We have no precise answer.

Q. Have you assessed the microbiota composition of humanized mice?
A. This is an area of interest, but no assessment of microbiota has been done to date.

Oncology experiments in the hGM-CSF/hIL3-NOG mouse

Q. What is the take rate of human tumor grafts such as cell lines or PDX in the hGM-CSF/hIL3-NOG mouse?
A. Tumor immunology experiments are in early stages with the hGM-CSF/hIL3-NOG mouse, so there is little data available on tumor take rate for cell lines or PDX.

Q. Do you see evidence of immune cell infiltration in the HSC4 tumor model?
A. Human immune cells can be detected in the engrafted HSC4 tumor in HSC-engrafted hGM-CSF/hIL3-NOG mouse.

Q. Do human myeloid cells infiltrate into human xenotransplant tumors in HSC-engrafted hGM-CSF/hIL3-NOG mouse?
A. In an experiment in which the HSC4 head/neck cancer cell line was implanted into HSC-engrafted hGM-CSF/hIL3-NOG mice, various human cell types could be detected infiltrating into the tumor, including human T and B cells as well as macrophages. Both CD4+ and CD8+ T cells were identified, as well as some double positive cells. Some macrophages in tumor were negative for HLA-DR and positive for ILR4a, which is reminiscent of tumor-associated macrophages. More characterization work of this population is underway.

Q. In the HSC4 tumor engraftment model, are there differences if the HSCs are HLA-matched to the tumor line versus not HLA-matched?
A. The data shown represented non-HLA matched tumor and immune cells. The experiment was not tried with matched samples.

Q. In the slide deck that was presented, the final experiment describes the implantation of HCS4 head and neck squamous tumor into hGM-CSF/hIL3 transgenic mice and non-transgenic mice. All of the subsequent data is from the hGM-CSF/hIL3 transgenic mouse alone. How does the infiltration of T cells and macrophages in the tumor compare between the hGM-CSF/hIL3 transgenic and non-transgenic CD34+ engrafted mice?
A. Even in conventional NOG mice, infiltration of T cells and macrophages can be detected. But it is dependent on the tumor cell lines engrafted. Since many tumors produce a significant amount of GM-CSF, it may help the differentiation of some part of human myeloid cells. But we are not sure whether they contain various subsets which are seen in the hGM-CSF/hIL3-NOG mouse. This topic is under investigation.

Other questions

Q. Can the hGM-CSF/hIL3-NOG mouse be used for engrafting human skin?
A. This has not been tried in the hGM-CSF/hIL3-NOG mouse. NOG mice engrafted with human skin rejected the graft within ~two weeks. Some technical improvements may be required to support human skin engraftment.

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