PMC0
10.1038%2Fnbt.2858
results
Newborn MISTRG mice and their littermates MITRG (lacking the hSIRPA transgene) were sub-lethally irradiated and transplanted with human fetal liver-derived CD34+ cells, following a standard protocol>>27<< (see also Methods). Rag2-/-Il2rg-/- mice that share the same genetic background (129xBalb/c N2) but lack all the humanized alleles, and commercially available NOD-Scid Il2rg-/- (NSG) mice served as controls.
1a) in NSG recipients was higher than in Rag2-/-Il2rg-/- recipients, as previously reported >>28<<,30. The percentage of blood hCD45+ cells was similar in MISTRG, MITRG and in NSG, suggesting that the humanization of the four cytokine genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity28,29,31,
1a) in NSG recipients was higher than in Rag2-/-Il2rg-/- recipients, as previously reported 28,>>30<<. The percentage of blood hCD45+ cells was similar in MISTRG, MITRG and in NSG, suggesting that the humanization of the four cytokine genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity28,29,31,
The percentage of blood hCD45+ cells was similar in MISTRG, MITRG and in NSG, suggesting that the humanization of the four cytokine genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity>>28<<,29,31, possibly due to defects in the mouse innate response caused by the absence of mouse cytokines.
The percentage of blood hCD45+ cells was similar in MISTRG, MITRG and in NSG, suggesting that the humanization of the four cytokine genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity28,>>29<<,31, possibly due to defects in the mouse innate response caused by the absence of mouse cytokines.
The percentage of blood hCD45+ cells was similar in MISTRG, MITRG and in NSG, suggesting that the humanization of the four cytokine genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity28,29,>>31<<, possibly due to defects in the mouse innate response caused by the absence of mouse cytokines.
In humans, three subsets of monocytes have been phenotypically and functionally described based on differential expression of CD14 and CD1619,>>32<< (CD14+CD16-, CD14+CD16+ and CD14dimCD16+). All three subpopulations were present in the blood as well as lymphoid and non-lymphoid tissues (e.g., lung and liver) of MI(S)TRG mice (Fig.
3c), reflecting the physiological properties of the corresponding subpopulations in human blood>>32<<. After injection with LPS or infection with Listeria monocytogenes or influenza A we detected higher amounts of human TNFα, IL-6 and interferon-β (IFNβ) in the tissues of MI(S)TRG compared to NSG mice (Fig. 3d-f). These results
Based on these findings, we analyzed the development of human immune cells dependent on IL-15 trans-presentation, such as NK cells, in MI(S)TRG mice33,>>34<<. Efficient development of human NK cells in existing humanized mouse models requires injection of exogenous human IL-15/IL-15Rα 7,12; because mouse IL-15/IL15Rα is not sufficient to support human NK cells in vivo.
Efficient development of human NK cells in existing humanized mouse models requires injection of exogenous human IL-15/IL-15Rα >>7<<,12; because mouse IL-15/IL15Rα is not sufficient to support human NK cells in vivo.
Efficient development of human NK cells in existing humanized mouse models requires injection of exogenous human IL-15/IL-15Rα 7,>>12<<; because mouse IL-15/IL15Rα is not sufficient to support human NK cells in vivo.
As previously reported>>7<<,8,12, we observed very few human NK cells (hNKp46+hCD3-) in engrafted NSG mice (Fig.
As previously reported7,>>8<<,12, we observed very few human NK cells (hNKp46+hCD3-) in engrafted NSG mice (Fig.
As previously reported7,8,>>12<<, we observed very few human NK cells (hNKp46+hCD3-) in engrafted NSG mice (Fig.
With the exception of the bone marrow, MITRG mice contained fewer human NK cells than MISTRG mice, most likely due to the previously reported requirement for human SIRPα for the survival of human NK cells in the periphery>>31<<. The hNKp46+hCD3- cells in MISTRG mice represented bona fide NK cells because like human NK cells they expressed the NK cell surface markers CD94, CD161, and killer inhibitory receptors (KIRs) (Supplementary Fig. 10a, b). Expression of
It is not known which cell(s) are responsible for trans-presentation of IL-15 in vivo in humans; however, human myeloid cells can support human NK cell proliferation in vitro>>7<<. To test if trans-presentation of human IL-15 by human monocytes/macrophages underlies the improved human NK cell development in MISTRG mice, we treated MISTRG mice with liposome-encapsulated clodronate to deplete phagocytic cells.
NK cells defend against pathogens by killing cells that lack surface MHC class I expression>>35<<, and by producing the IFN-γ 36.
NK cells defend against pathogens by killing cells that lack surface MHC class I expression35, and by producing the IFN-γ >>36<<. Compared to human NK cells from NSG mice, NK cells from MISTRG mice expressed higher amounts of the lytic granule protein perforin (Fig. 5a-b) and exhibited significantly enhanced cytotoxic activity against human cells lacking MHC class
These anti-inflammatory properties, if exhibited by tumor-infiltrating macrophages, can provide a survival advantage to evolving tumors>>22<<,23. We thus examined the influence of human myeloid cells on tumor development in MI(S)TRG mice. We used the human melanoma cell line Me290 as a tumor model38. In agreement with clinical observations showing that myeloid cells infiltrate
These anti-inflammatory properties, if exhibited by tumor-infiltrating macrophages, can provide a survival advantage to evolving tumors22,>>23<<. We thus examined the influence of human myeloid cells on tumor development in MI(S)TRG mice. We used the human melanoma cell line Me290 as a tumor model38. In agreement with clinical observations showing that myeloid cells infiltrate
In agreement with clinical observations showing that myeloid cells infiltrate tumors in several solid tumors>>23<<-26, we detected more human myeloid cell infiltration in tumors in MI(S)TRG than in NSG mice, as shown by the expression of human PTPRC mRNA (encodes CD45) and ITGAM mRNA (encodes CD11b) in subcutaneously grafted Me290 tumors (Fig.
VEGF is one such molecule>>39<<,40, and to test whether it was involved in tumor growth in MISTRG mice, we treated the mice with the human-VEGF inhibitor Avastinâ„¢.
VEGF is one such molecule39,>>40<<, and to test whether it was involved in tumor growth in MISTRG mice, we treated the mice with the human-VEGF inhibitor Avastinâ„¢.
methods
codon) of individual loci (encoding TPO, IL-3/GM-CSF or M-CSF) in the Rag2-/- Il2rg-/- 129xBalb/c (N2) genetic background was performed using Velocigene Technology in collaboration with Regeneron Pharmaceuticals, as reported previously>>16<<-18. Human SIRPα expression was achieved by BAC-transgenesis in the same genetic background28.
Human SIRPα expression was achieved by BAC-transgenesis in the same genetic background>>28<<. We intercrossed these strains by conventional breeding to obtain MITRG (M-CSFh/h IL-3/GM-CSFh/h TPOh/h Rag2-/- Il2rg-/-) and MISTRG (M-CSFh/h IL-3/GM-CSFh/h hSIRPAtg TPOh/h Rag2-/- Il2rg-/-) mice. We maintained them under specific
Recipient mice were engrafted with human hematopoietic stem and progenitor cells as previously described>>16<<-18,27,28. Briefly, human fetal liver samples were cut in small fragments, treated for 45 min at 37°C with Collagenase D (Roche, 100 ng/mL) and a cell suspension was prepared.
Recipient mice were engrafted with human hematopoietic stem and progenitor cells as previously described16-18,>>27<<,28. Briefly, human fetal liver samples were cut in small fragments, treated for 45 min at 37°C with Collagenase D (Roche, 100 ng/mL) and a cell suspension was prepared.
Recipient mice were engrafted with human hematopoietic stem and progenitor cells as previously described16-18,27,>>28<<. Briefly, human fetal liver samples were cut in small fragments, treated for 45 min at 37°C with Collagenase D (Roche, 100 ng/mL) and a cell suspension was prepared.
Phagocytic cells were depleted by intravenous retro-orbital injection of 100 μl of clodronate-loaded liposomes>>50<<. Clodronate-liposomes were injected 3 times daily and human NK cells in mouse liver were analyzed 24h after the last injection.
Human NK cell cytotoxicity in vivo was determined following a previously reported protocol>>8<<. LCL721.221 (HLA class I negative) and LCL721.45 (HLA class I positive) cells were mixed in a 1:
discussion
permit the study of low-proliferative, primary human hematopoietic malignancies, such as myelodysplastic syndromes or myeloproliferative neoplasia, that thus far have proven difficult to efficiently engraft [AR: OK] immunodeficient mice>>41<<,42.
the study of low-proliferative, primary human hematopoietic malignancies, such as myelodysplastic syndromes or myeloproliferative neoplasia, that thus far have proven difficult to efficiently engraft [AR: OK] immunodeficient mice41,>>42<<.
For example, CD16+ monocytes, which develop in the MISTRG model but not in other humanized mouse models, are expanded in humans in inflammatory settings including those caused by HIV infection and atherosclerosis>>43<<. However, the functional significance of CD16+ monocytes in these settings is unknown; MI(S)TRG mice may enable the investigation of causal relationships between CD16+ monocytes and human inflammatory disease. Macrophages are also
monocyte-derived macrophages>>45<<, the trafficking of these cells in response to inflammation or infection, and the mechanisms of differentiation and effector function of M1 vs.
monocyte-derived macrophages45, the trafficking of these cells in response to inflammation or infection, and the mechanisms of differentiation and effector function of M1 vs. M2 macrophages>>46<<.
immunity might be improved by a number of approaches, including expression of human MHC elements in mice or use of the bone marrow-liver-thymus (BLT) approach where human fetal tissues are transplanted along with human CD34+ cells>>48<<,49. The BLT approach results in more robust adaptive immune responses because T cells develop and are selected in a human thymic tissue.
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