In this study, we compared the engraftment of human CD34+ HSC in NOD-scid/γc−/−, Balb/c-Rag1−/−γc−/−, and C.B-17-scid/bg mice. To allow direct comparisons in these 3 mouse strains, a number of critical parameters were held constant throughout the study, including an established conditioning regime for each mouse strain, an intrahepatic injection site, age at the time of engraftment, and an identical number of input cells from each HSC source in a given experimental cohort of mice. Furthermore, on many occasions, cells from identical aliquots of HFL, UCB, or G-CSF-AB HSC were injected on the same day into mice of the 3 different strains. We discovered that mice of the NOD-scid/γc−/− strain, followed by those of the Balb/c-Rag1−/−γc−/− strain, were the most receptive for all HSC sources analyzed. The engraftment kinetics and breadth of developing immune cell types were similar when NOD-scid/γc−/− mice were reconstituted with either HFL or UCB HSC. HFL and UCB HSC-engrafted NOD-scid/γc−/− mice harbored T cells, B cells, NK cells, and DCs in multiple compartments and demonstrated human immune cell viability and immunological competence based on 1) splenocyte proliferation in response to PHA; 2) splenic and secondary node lymphoid enlargement in association with human immune cell engraftment; 3) human IgM and low levels of IgG antibody production following immunization with T-dependent antigens; and 4) human macrophage and T cell recruitment in response to a DTH challenge.
Previous reports have described administration of human HSC to mouse neonates via several routes, including the facial vein (18
), intraperitoneally (27
), and directly to the liver (19
). We found that direct injection of between 1.5 – 5.0 × 104
HFL HSC and 1.0 × 105
UCB HSC into the neonatal liver gave consistent engraftment if injected into 1–2 day old neonates, and in our hands, was technically simpler than the facial vein injection method described by Ishikawa and colleagues (18
). The survival rate of injected mice was ~ 90% using these methods, such that 113 of a total of 123 injected mice reached 6–10 weeks of age (ie. age of first blood draw) through the course of our studies. Typically, engraftment success rates of greater than 60% were achieved in NOD-scid/
mice engrafted with HFL or UBC HSC. Engraftment has been previously shown to vary depending on the age of Balb/c-Rag1−/−
mice injected intraperitoneally with HFL HSC, with younger neonates (1 day of age) more supportive of engraftment than older neonates (1 and 2 weeks of age) (27
). Furthermore, it is likely that a higher proportion of HSC would take up residence in the liver following direct intrahepatic injection compared to intraperitoneal delivery, circumventing the need for cell homing to the liver where hematopoiesis primarily occurs during the first several weeks of mouse postnatal development. Accordingly, direct delivery of greater numbers of HSC to the liver would lower the cell dose required to achieve optimal engraftment. For these reasons, intrahepatic injection of HSC into young neonates was held constant within our experimental design.
Our direct comparisons between various lines of mice and those of Takenaka and colleagues (28
) reveal that HSC engraftment is dramatically affected by the background strain of mouse. A polymorphism in the Sirpa
gene in the NOD background, but not in the Balb/c or C.B-17 backgrounds, may contribute to the increased survival and subsequent engraftment of transplanted HSC in NOD-scid/
mice by rendering the SIRP-α receptor on mouse macrophages cross-reactive with human CD47 expressed on HSC (28
). Appropriate binding of CD47 to SIRP-α prevents HSC phagocytosis by macrophages. In contrast, minimal or nonexistent engagement of Balb/c SIRP-α with human CD47 may result in the targeted destruction of the HSC compartment in Balb/c-Rag1−/−
mice, yielding lower engraftment levels than observed in NOD-scid/
mice. Because T cell engraftment in NOD-scid/
mice was positively correlated with the number of HFL HSC transplanted, failure to generate a reproducible T cell compartment in Balb/c-Rag1−/−
mice may be due to limited HSC survival. Though the C.B-17 background of scid
/bg mice used in this report is largely genetically identical to the Balb/c background, reconstitution was not apparent in neonates in spite of defects in NK cell function conferred by the beige mutation. The γc mutation, in contrast, renders mice deficient in NK cells, as well as causing deficiencies in T and B cell development and function, providing an absolutely critical environment for human HSC development in Balb/c-Rag1−/−
In congruence with other reports, engraftment of NOD-scid/
mice with human UCB HSC supported T and B cell reconstitution (17
), while engraftment with purified human G-CSF-AB HSC supports only B cells (26
). In contrast, we found that Balb/c-Rag1−/−
mice supported B cell and DC development, but negligible T cell development, following engraftment with either HFL or UCB HSC. Similarly, Gimeno et al. showed that engraftment of 1 × 106
HFL HSC into Balb/c-Rag1−/−
mice resulted primarily in B cell development, although lower numbers of T cells were also detected (27
). It appears that engraftment of HFL HSC provides no obvious advantages over UCB in either the Balb/c-Rag1−/−
strain except for the possibility of co-engrafting autologous thymic tissue to mediate positive clonal selection of developing T cells on a human thymus. In vivo
T cell responses have been reported in the presence of autologous human fetal thymic tissue, likely due to the requirement of positive selection on autologous human HLA class I-expressing stromal cells (1
). Similarly, a recent paper by Tonomura et al (30
) showed that co-engraftment of fetal thymus and liver with HFL HSC produced chimeric NOD-scid
mice capable of demonstrating T-dependent antibody responses to KLH. Very recently, Giassi and colleagues reported human immunoglobulin responses to T-dependent antigens following of UCB-reconstitution of TNF-α-treated NOD-scid/
mice, which were enhanced in the presence of the B-cell cytokine BLyS (31
). Though subclass typing was not done to rule out the possibility that our detected IgG is of subclass IgG3, which develops independently of T cell help, IgG was detected only in mice harboring T cells following immunization with KLH, suggesting that appropriate antigen-specific T cell-B cell interactions are likely occurring. Furthermore, low levels of human immunoglobulin were detected in both pre- and post-immunization plasma in those HFL HSC-engrafted NOD-scid/
mice immunized with influenza antigen. This latter group of mice were 5 months of age at the time of pre-immunization bleed and subsequent primary vaccination, compared to only 3 months of age for the KLH-immunized HFL HSC-engrafted mice, suggesting that successful class switching to IgG occurred as human immune responses matured and/or mice were exposed to more antigens throughout life. Reconstituted mice in our study do not likely possess a human thymic stromal microenvironment, however positive selection may be occurring on other human cells transferred in the inoculum, including DCs or other T cells (32
). Co-engraftment of autologous thymic tissue or bone marrow stromal cells with HFL HSC may enable more robust antigen-specific T-dependent responses than our current system supports.
Approximately half of human B cells in the peripheral blood and tissues of HSC reconstituted mice expressed the cell surface protein CD5, possibly suggesting they are B-1 cells that respond mainly to bacterial lipopolysaccharide and phosphorylcholine (33
) rather than conventional B-2 cells that mediate antigen-specific adaptive immunity. It is well established that CD5+ B cells represent B-1 cells in mice (36
) and are produced mainly in the fetal liver during development (37
). It is currently uncertain whether the similar B-1 and B-2 cell lineages are present in humans (37
). However if indeed human B-1 cells develop as described in mice, reconstitution of the neonatal mouse liver with HSC may account for the large proportion of B cells detected in HFL HSC-engrafted mice being of this subset. In mice, CD5+ B-1 cells (33
) produce mainly natural IgM antibodies without the requirement for T cell help. The relatively high levels of this B cell subset may explain why IgM, rather than IgG, is the predominant immunoglobulin class in our reconstituted mice. Alternatively, or perhaps in addition, class-switching is dysfunctional due to inadequate interactions between T cells and conventional B-2 cells. B-1 cells in naïve normal (immunocompetent) mice are known to produce natural IgM antibodies that react with influenza A and B strains (38
), and perhaps the influenza hemagglutinin molecule in particular (40
), providing a possible explanation for the presence of influenza-reactive antibodies in our mice prior to immunization. Higher levels of IgM antibody were apparent in pre-immunization plasma compared to post-immunization plasma, harvested at 5 and 12 months of age, respectively, in influenza-immunized NOD-scid/
mice, mimicking the typical decline of natural IgM throughout life. Analysis of cytokines within the B cell microenvironment may provide additional insight to explain the suboptimal immunoglobulin diversity observed in our mice.
Interestingly, mice from all 3 strains analyzed occasionally became ill during adult life and required premature euthanasia due to humane reasons if they failed to reconstitute or had very low engraftment levels (<1%). Infections (typically dermatitis) or a hunched and dehydrated appearance became predictive indicators of poor engraftment efficiency. This observation may be a consequence of a small (or steadily declining) human immune cell compartment that cannot (or can no longer) support an irradiation-compromised host innate immune system in the defense of ubiquitous pathogens. Alternatively, mice could have become clinically ill due to irradiation-induced anemia or thrombocytopenia. These mice were not used in the functional analysis experiments, rather were euthanized on humane grounds when clinical disease became severe.
We noted white pulp-like regions in the spleens of all engrafted NOD-scid/γc−/− mice and KLH-vaccinated Balb/c-Rag1−/−γc−/− mice at 15 weeks post-engraftment. In KLH-immunized NOD-scid/γc−/− mice, these follicle-like collections were remarkably larger. The presence of white pulp-like regions in non-immunized mice may arise spontaneously or be attributed to environmental antigen exposure. Within observed white pulp-like regions, distinct T and B cell zones were observed, but not well-organized. This lack of appropriate B and T cell compartmentalization may provide another explanation for the inadequate adaptive immune responses noted in HSC-engrafted mice.
HFL and UCB could be highly enriched for CD34+ cells using commercial anti-human CD34 columns in a single step; in contrast, adult G-CSF-mobilized blood preparations contained many mature leukocytes when processed using the same method, likely reflecting their predominance in a developmentally more mature sample prior to enrichment. These lin+ cells, and notably xenoreactive memory T cells, were presumably responsible for the graft-versus-host syndrome evident in many mice by 6–8 weeks when engrafted as irradiated neonates, as previously reported following engraftment of PBMC in adult scid
). Interestingly, column-enriched G-CSF-AB HSC did not cause disease when transplanted into irradiated, adult C.B-17-scid
/bg and NOD-scid
mice. Instead, mice remained healthy and could be used to investigate macrophage expansion, homing, and tissue injury to allogeneic tissues (44
). This suggests that it is imperative that HSC preparations be CD3+ T cell-deficient to prevent graft-versus-host symptoms following neonatal mouse engraftment.
The individual contributions of CD34-lin- and CD34+lin- progenitors, as well as more mature CD34-lin+ cells, to the multi-lineage hematolymphoid reconstitution of either NOD-scid/γc−/− and Balb/c-Rag1−/−γc−/− mice is unknown. However, the phenotype of specific leukocyte subsets observed in the peripheral blood of engrafted mice frequently correlated with the specific phenotypes of inocula mature contaminants. For example, both strains consistently harbored B cells, and all HSC preparations contained CD34-CD19+ population. Therefore, B cells detected in the chimeric mice could have originated from the de novo differentiation of true CD34+ hematopoietic stem cells or the expansion and homing of pre-existing mature CD19+ B cells. Interestingly, neonatal NOD-scid/γc−/− mice injected with Isolex-purified adult HSC, which lacked CD3+ T cells, never harbored T cells. In contrast, engraftment of HFL or UCB HSC preparations, which usually contained low levels of T cells, routinely produced mice harboring T cells, suggesting that a naïve T cells may be required for successful T cell development in our system. Consistent with mice inoculated with Isolex-purified, mice receiving HFL HSC from a preparation which lacked T cells, harbored B cells and DCs, but never showed any T cell engraftment. Lineage depletion prior to engraftment would allow further characterization of the developmental origin of each immune cell identified in vivo. Furthermore, studies citing HSC differentiation as the source for hematolymphoid development must be carefully evaluated.
Optimizing mouse models of human hematopoietic stem cell engraftment to further support a more complete and functional human immune system is paramount to a comprehensive understanding of human disease pathogenesis and progression. To our knowledge, our findings offer the first reported systematic comparison of neonatal hematopoietic reconstitution using four types of HSC preparations in 3 commonly-used strains of immunodeficient mice. We demonstrate successful multi-lineage hematolymphoid reconstitution of NOD-scid/γc−/− by intrahepatic injection of HFL and UCB HSC and demonstrate that this strain is more receptive to engraftment than age-matched Balb/c-Rag1−/−γc−/− mice. Immunologic function was confirmed based on proliferation of splenocytes in response to PHA and the presence of multiple human immunoglobulin isotypes following immunization with T-dependent antigens. In all cases, responses were less robust in Balb/c-Rag1−/−γc−/− mice likely owing to the limited T cell development observed in this strain.