Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/gammac-/-, Balb/c-Rag1-/-gammac-/-, and C.B-17-scid/bg immunodeficient mice

Abstract

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

Figure 1. Kinetics of human CD45+ cell development in NOD-scid/γc^−/− and Balb/c-Rag1^−/−γc^−/−mice injected as neonates

Prior to 2 days of age, NOD-scid/γc^−/− or Balb/c-Rag1^−/−γc^−/− mice were irradiated and then administered HFL or UCB HSC preparations by intrahepatic injection. The percentage of human CD45+ cells of the total leukocyte population in peripheral blood was determined in successive post-engraftment dates by flow cytometry in various cohorts of mice. A: 3 month comparative analysis of individual NOD-scid/γc^−/− or Balb/c-Rag1^−/−γc^−/− receiving 1.0 × 10⁵ UCB HSC; B: 6 month comparative analysis of individual NOD-scid/γc^−/− mice receiving either 1.5 × 10⁴, 3.0 × 10⁴ or 4.0 × 10⁴ HFL HSC; C: 12 month comparative analysis of individual NOD-scid/γc^−/− mice receiving 5.0 × 10⁴ HFL HSC.

Figure 2. Development of human immune cell subsets in NOD-scid/γc^−/− and Balb/c-Rag1^−/−γc^−/− mice receiving HFL HSC or UCB HSC

At 1 day of age, NOD-scid/γc^−/− or Balb/c-Rag1^−/−γc^−/− mice were irradiated and then injected intrahepatically with 4.0 × 10⁴ HFL HSC (A) or 1.0 × 10⁵ UCB HSC (B). The phenotype of human CD45+ cells expressing CD3, CD19, CD11c, or CD56 (at 8 or 13 weeks of age in A or B, respectively) as well as CD41a+ platelets (at 15 weeks or 28 weeks of age in A or B, respectively), in peripheral blood from HFL HSC- or UCB HSC-engrafted mice was determined by flow cytometry. Representative sets of plots from a NOD-scid/γc^−/− and a Balb/c-Rag1^−/−γc^−/− mouse are shown in the top and bottom row of panel A (HFL HSC-engrafted mice) and panel B (UCB HSC-engrafted mice), respectively. NT = not tested.

Figure 3. Dependence of human T and B lymphocyte development on HFL HSC inocula number in NOD-scid/γc^−/− mice

At 1 day of age, NOD-scid/γc^−/− mice were irradiated and then injected intrahepatically with 1.5 – 5.0 × 10⁴ HFL HSC. A: Flow cytometric plots showing human CD45+CD3+ cells in peripheral blood at 10 weeks of age from individual mice injected with either 1.5 × 10⁴ or 3.0 × 10⁴ HFL HSC. B: Percentages of CD45+CD3+ and CD45+CD19+ cells of total lymphocytes determined by flow cytometric analysis of peripheral blood at 10 – 12 weeks of age from individual mice injected with increasing numbers of HFL HSC.

Figure 4. Analysis of human immune cell subsets in spleens of representative NOD-scid/γc^−/− and Balb/c-Rag1^−/−γc^−/− mice receiving HFL or UCB HSC

At 1 day of age, NOD-scid/γc^−/− or Balb/c-Rag1^−/−γc^−/− mice were irradiated and then injected intrahepatically with 4.0 × 10⁴ HFL HSC or 1.0 × 10⁵ UCB HSC or left as non-irradiated, non-transplanted controls. The phenotype of human CD45+ splenocytes (CD3, CD19, or CD11c) from representative HFL HSC- or UCB HSC-engrafted mice was determined by flow cytometry following euthanasia at 15 or 28 weeks of life, respectively (A). Paraffin-embedded sections originating from a human spleen (B, H, N), or spleens from a KLH-immunized, HFL HSC-engrafted NOD-scid/γc^−/− mouse (C, I, O), KLH-immunized, HFL HSC-engrafted Balb/c-Rag1^−/− γc^−/− mouse (D, J, P), non-immunized, HFL HSC-engrafted NOD-scid/γc^−/− mouse (E, K, Q), KLH-immunized, UCB HSC-engrafted NOD-scid/γc^−/− mouse (F, L, R), and KLH-immunized, non-engrafted NOD-scid/γc^−/− mouse (G, M, S) were stained with H&E, CD20, or CD3, respectively (H&E: 4x magnification, CD20 and CD3: 10x magnification). White pulp in human splenic section and white pulp-like regions in engrafted mice are shown in panel B (black arrow) and panel C – F (white arrows), respectively.

Figure 5. Analysis of thymi and lymph nodes in NOD-scid/γc^−/− and Balb/c-Rag1^−/−γc^−/− mice receiving HFL or UCB HSC

A – C: Paraffin-embedded, H&E-stained thymic tissue sections from an immunocompetent NOD mouse (A, 4x); NOD-scid/γc^−/− (B, 4x) and Balb/c-Rag1^−/−γc^−/− (C, 10x) mice engrafted 28 weeks earlier with 4.0 × 10⁴ HFL HSC. White and red arrows show medullary-like and cortical-like regions, respectively. D – F: H&E-stained thymic tissue section (D, 4x) and flow cytometric plots of thymic leukocytes showing human CD45+CD3+ cells (E) and CD4+, CD8+ and CD4+CD8+ cells (F) from representative NOD-scid/γc^−/− mice injected with 1.0 × 10⁵ UCB HSC and analyzed at 28 weeks of age. G – I: Paraffin-embedded mesenteric (G, H; 4x and 20x magnification, respectively) and popliteal (I, 40x magnification) lymph node sections were harvested at 12 months of age from influenza-immunized NOD-scid/γc^−/− mice that received 5.0 × 10⁴ HFL HSC as neonates and stained with either H&E or anti-human CD45 (insets).

Figure 6. Splenocyte blastogenesis and human-specific immunoglobulin responses in HSC-engrafted, KLH- or influenza-immunized NOD-scid/γc^−/− and Balb/c-Rag1^−/− γc^−/− mice

At 1 day of age, NOD-scid/γc^−/− and Balb/c-Rag1^−/−γc^−/− mice were irradiated and then injected intrahepatically with 4.0 × 10⁴ HFL HSC (KLH-immunized group), 5.0 × 10⁴ HFL HSC (influenza-immunized group), 1.0 × 10⁵ UCB HSC (KLH-immunized group), or left as non-irradiated, non-transplanted controls. A: At 15 weeks of age, splenocytes were harvested and cultured in the presence or absence of 2 μg/ml phytohemagglutinin (PHA). An 18 hour ³H-thymidine incorporation pulse was determined in triplicate beginning at 48 hours of culture (+/− standard deviation). * indicates HSC-engrafted mice with <1% peripheral blood chimerism. B-D: Mice began a series of 2 intradermal immunization doses with either KLH at 12 weeks (HFL HSC-engrafted) or 25 weeks (UCB HSC-engrafted) or influenza at 21 weeks. Total human IgM, total human IgG, and influenza-specific human (IgM+IgG+IgA) immunoglobulin (B-D, respectively), present in the chimeric mouse plasma, both immediately prior to the first immunization dose (pre) and 2 weeks following the second dose (post), was determined by ELISA. E – F: Flow cytometric plots showing human CD19+ and CD5+ co-expression (F) in the gated human CD45+ population (E) in peripheral blood leukocytes from a representative Balb/c-Rag^−/−γc^−/− mouse injected at 2 days of age with 1.0 × 10⁵ UCB HSC and analyzed at 11 weeks of age.

Figure 7. Delayed type hypersensitivity responses in HFL HSC-engrafted, KLH-immunized NOD-scid/γc^−/− mice

At 1 day of age, NOD-scid/γc^−/− mice were irradiated and then injected intrahepatically with 4.0 × 10⁴ HFL HSC or left as an non-irradiated, non-transplanted controls. Mice were immunized twice intradermally with KLH then challenged in the footpad and the ear 18 days after their initial KLH exposure. Paraffin-embedded footpad sections originating from a challenged, non-engrafted (A, D, G) or engrafted (B, E, H) KLH-immunized NOD-scid/γc^−/− mouse, harvested at 15 weeks of age, or from relevant human sections were stained for human CD45 (A – C; 10x magnification), human CD3 (D-F; 40x magnification), or human CD68 (G-I; 20x magnification). Insets in B and H are 40x magnifications of the respective white-boxed regions.