Transgenic expression of human signal regulatory protein alpha in Rag2-/-gamma(c)-/- mice improves engraftment of human hematopoietic cells in humanized mice

Abstract

Transplantation of human hematopoietic stem cells into severely immunocompromised newborn mice allows the development of a human hematopoietic and immune system in vivo. NOD/scid/γ(c)(-/-) (NSG) and BALB/c Rag2(-/-)γ(c)(-/-) mice are the most commonly used mouse strains for this purpose and a number of studies have demonstrated the high value of these model systems in areas spanning from basic to translational research. However, limited cross-reactivity of many murine cytokines on human cells and residual host immune function against the xenogeneic grafts results in defective development and maintenance of human cells in vivo. Whereas NSG mice have higher levels of absolute human engraftment than similar mice on a BALB/c background, they have a shorter lifespan and NOD ES cells are unsuitable for the complex genetic engineering that is required to improve human hematopoiesis and immune responses by transgenesis or knockin of human genes. We have generated mice that faithfully express a transgene of human signal regulatory protein alpha (SIRPa), a receptor that negatively regulates phagocytosis, in Rag2(-/-)γ(c)(-/-) mice on a mixed 129/BALB/c background, which can easily be genetically engineered. These mice allow significantly increased engraftment and maintenance of human hematopoietic cells reaching levels comparable to NSG mice. Furthermore, we found improved functionality of the human immune system in these mice. In summary, hSIRPa-transgenic Rag2(-/-)γ(c)(-/-) mice represent a unique mouse strain supporting high levels of human cell engraftment, which can easily be genetically manipulated.

Fig. 1.

hSIRP is faithfully expressed and is functional in BAC-transgenic mice. (A) Expression of mouse and human SIRPa in the blood was analyzed by FACS staining in hSIRPa-transgenic (gray solid line) and control (black solid line) mice. Isotype staining for hSIRP is shown as dashed line. (B and C) Human erythrocytes were transferred in Rag2^−/−γ_c^−/− (−hSIRPa), hSIRPa⁺ Rag2^−/−γ_c^−/− mice (+hSIRPa), and NSG mice. Their clearance was monitored by FACS staining for hGPA-expressing cells in the peripheral blood of mice. Data are a summary of two independent experiments with a total of 10 mice per group. *P < 0.01 by one-way ANOVA test.

Fig. 2.

hSIRPa-transgenic mice have improved hematopoiesis upon CD34⁺ cell engraftment. Irradiated newborn mice were transplanted with CD34⁺ cells and engraftment was monitored after 10 to 12 wk by FACS. (A) Representative staining pattern in the peripheral blood of a hSIRP⁺ Rag2^−/−γ_c^−/− mouse. (B–E) Frequencies of hCD45⁺, hCD3⁺, CD19⁺, and CD33⁺SSC^low cells in the blood were compared after engraftment between different immunodeficient strains. Data are a summary of at least four experiments with a total of 60 DKO mice, 63 hSIRPa-DKO mice, 26 NSG mice, and 29 NRG mice. (F) Representative staining pattern in the bone marrow of a hSIRPa⁺ Rag2^−/−γ_c^−/− mouse. (G and H) Frequency and number of human CD45⁺ cells was compared after 12–14 wk between different immunodeficient strains. (I and J) Numbers of human CD34⁺ progenitor cells and CD34⁺CD38⁻ cells were calculated on the basis of total cell number and frequencies determined by FACS. Data are a summary of three experiments with a total of 22 DKO mice, 24 hSIRPa-DKO mice, and 9 NSG/NRG mice. Data were analyzed by one-way ANOVA test and individual P values for posttest are displayed. *P < 0.05, **P < 0.01.

Fig. 3.

Elevated numbers of human cells in the periphery of hSIRPa-transgenic mice. (A) Representative staining pattern in the spleen of a hSIRPa⁺ Rag2^−/−γ_c^−/− mouse. (B and C) Frequencies and numbers of total human CD45⁺ cells in the spleen were determined after 12–14 wk. (D and E) At the same time, frequencies and numbers of CD3⁺ T cells and CD19⁺ B cells in the spleen were determined. (F) Representative staining pattern in the thymus of a hSIRPa⁺ Rag2^−/−γ_c^−/− mouse. (G) Enumeration of the number of human thymocytes and of (H) CD4⁺CD8⁺ thymocytes after 12–14 wk by combination of FACS staining and total cell count. Data are a summary of three experiments with a total of 22 DKO mice, 24 hSIRPa-DKO mice, and 9 NSG/NRG mice. Data were analyzed by one-way ANOVA test and individual P values for posttest are displayed. *P < 0.05, **P < 0.01.

Fig. 4.

Improved humoral antigen-specific immune responses in hSIRPa-transgenic mice. Characterization of humoral immune responses before (A–C) and after (D–I) immunization. (A and B) Total serum levels of human IgM (A) and IgG (B) were determined by ELISA in Rag2^−/−γ_c^−/− (−hSIRPa, n = 28) and hSIRPa-transgenic Rag2^−/−γ_c^−/− (+hSIRPa, n = 30) mice. (C) The frequencies of human IgG-producing cells in the spleen were measured using ELISPOT without immunization. (D–I) Mice were immunized with OVA/CFA and boosted 14 d later with OVA/IFA. (D–G) Anti-OVA IgM (D and E) and IgG (G and H) were assayed by ELISA and OD₄₅₀ nm readings are displayed for serial dilution of serum from individual DKO (D and F) or hSIRPa-DKO (G and H) mice. (F and I) OD₄₅₀ nm readings for a serum dilution of 1:33 are shown; each dot represents a mouse from one experiment. Data were analyzed using Mann-Whitney test, *P < 0.05.