Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice

Abstract

Here we report that a new nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/interleukin 2 receptor [IL2r] gamma(null)) efficiently supports development of functional human hemato-lymphopoiesis. Purified human (h) CD34(+) or hCD34(+)hCD38(-) cord blood (CB) cells were transplanted into NOD/SCID/IL2rgamma(null) newborns via a facial vein. In all recipients injected with 10(5) hCD34(+) or 2 x 10(4) hCD34(+)hCD38(-) CB cells, human hematopoietic cells were reconstituted at approximately 70% of chimerisms. A high percentage of the human hematopoietic cell chimerism persisted for more than 24 weeks after transplantation, and hCD34(+) bone marrow grafts of primary recipients could reconstitute hematopoiesis in secondary NOD/SCID/IL2rgamma(null) recipients, suggesting that this system can support self-renewal of human hematopoietic stem cells. hCD34(+)hCD38(-) CB cells differentiated into mature blood cells, including myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes. Differentiation into each lineage occurred via developmental intermediates such as common lymphoid progenitors and common myeloid progenitors, recapitulating the steady-state human hematopoiesis. B cells underwent normal class switching, and produced antigen-specific immunoglobulins (Igs). T cells displayed the human leukocyte antigen (HLA)-dependent cytotoxic function. Furthermore, human IgA-secreting B cells were found in the intestinal mucosa, suggesting reconstitution of human mucosal immunity. Thus, the NOD/SCID/IL2rgamma(null) newborn system might be an important experimental model to study the human hemato-lymphoid system.

Figure 1.

Analysis of human hematopoietic cells in NOD/SCID/IL2rγ^null recipients. (A) In the scatter gates for nucleated cells, anti-hCD45 and anti-mCD45 antibodies (Abs) reacted exclusively with human and murine leukocytes, respectively. In the recipient blood, the majority of nucleated cells were human leukocytes (top row). High levels of engraftment by hCD33⁺ myelomonocytic cells, hCD19⁺ B cells, and hCD3⁺ T cells were achieved in peripheral blood of recipient mice given transplants of Lin^–hCD34⁺ CB cells (bottom row). (B) Analysis of circulating erythrocytes (top row) or platelets (bottom row) in a NOD/SCID/IL2rγ^null recipient. In the blood, Ter119⁺ murine erythrocytes as well as hGPA⁺ human erythrocytes were detected. mCD41a⁺ murine platelets were also reconstituted. (C) Multilineage engraftment of human cells in the NOD/SCID/IL2rγ^null murine bone marrow. hCD33⁺ myelomonocytic cells, hCD19⁺ B cells, and hCD3⁺ T cells were present. hGPA⁺ erythroid cells and hCD41a⁺ megakaryocytes were also seen in the nucleated cell gate of the bone marrow. (D, left) HLA-DR⁺hCD11c⁺ dendritic cells were detected in the spleen by a flow cytometric analysis. (Right) Immunohistochemical staining of CD11c in the spleen. CD11c⁺ cells displayed dendritic cell morphology.

Figure 2.

Purified Lin^–hCD34⁺hCD38^– CB cells reconstitute hematopoiesis via physiological intermediates, and display long-term reconstitution in the NOD/SCID/IL2rγ^null newborn system. (A) Serial evaluation of chimerism of human cells in peripheral blood of recipient mice injected with 2 × 10⁴ Lin^–hCD34⁺hCD38^– CB cells. White, gray, and black dots represent 3 individual recipients. (B) hCD34⁺ cells purified from a primary recipient marrow (left) were successfully engrafted into the secondary newborn recipients. hCD19⁺ B cells (middle) and hCD3⁺ T cells (right) in a representative secondary recipient is shown. (C) The Lin^– bone marrow cells contained hCD34⁺hCD38⁺hCD10⁺hCD127 (IL-7Rα)⁺ CLPs (top row). In the Lin^–hCD10^–fraction, hCD34⁺hCD38⁺hCD45RA^–hCD123 (IL-3Rα)^lo CMPs, hCD34⁺hCD38⁺hCD45RA⁺hCD123^lo GMPs, hCD34⁺hCD38⁺hCD45RA^–hCD123^– MEPs were present. Each number for progenitors indicates percentages of hCD45⁺ cells. SSC indicates side scatter. (D) Colony-forming activity of purified myeloid progenitor population in the methylcellulose assay. Representative data from 1 of 3 recipients are shown. Error bars represent standard deviation.

Figure 3.

Histology of lymphoid organs in engrafted NOD/SCID/IL2rγ^null recipients. (A) The thymus showed an increased cellularity after reconstitution. (B) The thymus stained with anti-hCD3 (green) and anti-hCD19 (red) antibodies. (C) The thymus stained with anti-hCD4 (green) and anti-hCD8 (red) antibodies. The majority of thymocytes are doubly positive for hCD4 and hCD8. (D-E) Lymphoid follicle-like structures in the spleen of a recipient. (F) The lymphoid follicles mainly contained hCD19⁺ B cells (red) that were surrounded by scattered hCD3⁺ T cells (green). (G) Histology of the intestine in an engrafted NOD/SCID/IL2rγ^null recipient (left). (H) In the intestine, DAPI⁺ (4′,6-diamidino-2-phenylindole)–nucleated cells (blue) contained both scattered hCD3⁺ T cells (green) and human IgA⁺ cells (red). (I) The DIC image of the same section shows that IgA⁺ B cells were mainly found in the interstitial region of the intestinal mucosal layer. White bars inside panels represent 80 μm (C), 100μm (F), and 20μm (I).

Figure 4.

Development of lymphocytes in NOD/SCID/IL2rγ^null recipients. (A) The flow cytometric analysis of human T cells in recipients. The majority of cells in the thymus were hCD4⁺hCD8⁺ double-positive thymocytes (top). The CD3⁺ spleen cells contained hCD4⁺ or hCD8⁺ single-positive mature T cells (bottom). (B) hCD34⁺hCD19⁺ pro-B, hCD10⁺hCD19⁺ pre-B, and hCD19⁺hCD20^hi mature B cells were seen in different proportions in the bone marrow and the spleen of recipient mice. Numbers represent percentages within total nucleated cells. (C) B cells expressing each class of human immunoglobulin heavy chain were seen in the bone marrow, the peripheral blood (PB), or the spleen of engrafted NOD/SCID/IL2rγ^null mice. Numbers represent percentages out of nucleated cells.

Figure 5.

Functional analysis of human T and B cells developed in NOD/SCID/IL2rγ^null recipients. (A) Production of OVA-specific human immunogloblins. Two weeks after immunization with OVA, sera of 5 independent recipients were sampled, and were evaluated for the concentration of OVA-specific human IgM (□) and IgG () by ELISA. Sera of 3 nonimmunized NOD/SCID/IL2rγ^null recipients were used as controls. O.D. indicates optical density. (B) Cytotoxic activity of human T cells generated in NOD/SCID/IL2rg-null mice. hCD4⁺ and hCD8⁺ T-cell clones derived from the recipient spleen were cocultured with allogeneic target cells (TAK-LCLs). KIN-LCLs that do not share any HLA type with effector cells or TAK-LCLs (X) were used as negative controls. Both hCD4⁺ and hCD8⁺ T-cell lines displayed cytotoxic activity against TAK-LCL in a dose-dependent manner. In hCD4⁺ T-cell clones, this effect was blocked by anti–HLA-DR antibodies (▴), whereas in hCD8⁺ T-cell clones, the effect was blocked by anti–HLA class I antibodies (▪). ♦ indicates cytotoxic response to TAK-LCLs without addition of antibodies.