Mice engrafted with human immune cells (mature peripheral blood lymphocytes or fetal thymus/liver transplant under kidney capsule) have emerged as a powerful model for studying the pathogeneses of species-specific virus pathogens, including HIV-1, human T-lymphotropic virus type 1, and human herpesviruses such as varicella-zoster virus and human herpesvirus 6 (8
). The ability to engraft human HSCs in small animals is extremely valuable, since it offers a new model system for studies on the ontogeny of the human immune system and for the analysis of the interplay of species-specific pathogens. Recent progress in the generation of humanized mice with long-term reconstitution of human functional lymphoid tissues by transplantation of HSCs was reviewed by Macchiarini et al. (17
) and Legrand et al. (16
). The best results so far, in terms of the longevity of engraftment and the functional properties of a HIS in a murine environment, have been achieved in mice with a deletion or truncation of IL-2Rγc
, and NOD/LtSz-scid
mice. Such animals can be engrafted with HSCs derived from a range of different sources, including umbilical cord blood, mobilized HSCs from peripheral blood, and fetal liver. However, to date, none of the published protocols has resulted in reproducible, efficient, and uniform engraftment of human immune cells in these mice. The first part of the present work was therefore dedicated to the development of an improved method for the reconstitution of mice with HSCs.
We used BALB/c-Rag2−/−
newborn pups in our reconstitution experiments (27
) but were unsuccessful in creating stable chimerism using the published irradiation dose of 375 cGy. The irradiation needed to create a niche for the engraftment of human cells was between 550 and 700 cGy. Even a high dose of 650 cGy did not guarantee higher levels of engraftment. Moreover, such doses induced significant health problems and reduced the survival of animals. We therefore combined lower-dose irradiation with busulfan-mediated myeloablation. We based our busulfan regimen on a protocol that was previously used for successful allotransplantation of bone marrow in C57BL/6 mice (7
Busulfan, a common component of pretransplant conditioning regimens in human bone marrow transplantation (9
), selectively destroys quiescent stem cells (2
). When combined with a lower (400-cGy) dose of irradiation, the intrapartum busulfan injection yielded efficient, stable, and reproducible engraftment of HSCs. The reduced irradiation dose in this combination regimen resulted in improved outcomes in terms of survival and neurologic damage (seizures or balance abnormalities).
In order to assess the kinetics of maturation of HIS in BALB/c-Rag2−/−
mice, we monitored human cells and immunoglobulin levels in peripheral blood. During the early postengraftment period, a significant expansion of CD19+
cells was observed, as previously noted in NOD/SCID/γcnull
). This may be related, in part, to the presence of immature B-cell precursors in cord blood and to ongoing stimulation of these cells by the murine environment. However, we observed that the expansion of these cells gradually tapered off, and the number of human B cells in circulation began to decline by 22 to 28 weeks after engraftment. At a slightly earlier time point (16 to 20 weeks), the frequency of human T cells in peripheral blood reached a stable peak, and lymph nodes and spleen were populated. Thus, all components of an HIS were present by 16 weeks of age. However, full functional maturation of the HIS was not complete until 5 to 6 months of age, as reflected by the ability to stably produce IgG and to mount a humoral immune response to ActHIB vaccination.
The second aspect of this study focused on an evaluation of HIV-1 infection in HIS mice. Researchers interested in the development of a mouse model for HIV-1 infection previously have used either (i) immune-deficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL mice) (21
) or (ii) immune-deficient mice engrafted with fetal human thymus/liver under the kidney capsule (Thy/Liv SCID-hu mice) (1
). There was considerable initial enthusiasm for the use of these models to study HIV-1 pathobiology. However, important technical limitations have precluded their widespread use and broad applicability. These limitations include reduced survival of the engrafted human immune cells, graft-versus-host reactions, an incomplete human T-cell receptor repertoire, deficiency of human antigen-presenting cells, and incomplete peripheral lymphocyte reconstitution. Perhaps most importantly, the functional properties of a human immune system were compromised, and HIV-1 infection led to very rapid depletion of human CD4+
T lymphocytes without most of the other hallmarks of HIV-1 infection in human subjects.
We show here that engrafted human immune cells in BALB/c-Rag2−/−γc−/− mice are susceptible to HIV-1 infection but that the dynamics of virus infection are substantially different in this model than in previously described models such as hu-PBL mice. The HIS mice are less susceptible to low-dose infection with HIV-1 and undergo a slower, more protracted course of CD4+ T-cell depletion compared to hu-PBL mice. This may be because HIS mice contain a larger fraction of naive human T cells than hu-PBL mice. Importantly, the large number of naive T cells in HIS mice is consistent with what is known about the T-cell population in normal human subjects, and this highlights the biological relevance of this new model.
In HIS mice the cytotoxicity of tested viral isolates did not correspond to the virus behavior in hu-PBL mice, where a rapid depletion of CD4+ cells occurs after administration of HIV-1C1157, as well as NL4-3 and UG 029A. In contrast, the same low doses of HIV-1 did not induce rapid viremia and depletion of CD4+ cells in HIS mice. Indeed, we were able to demonstrate the stable infection of HIS mice with HIV-1C1157, for at least 8 to 10 weeks following virus inoculation. This may provide a model for analysis of the early stages of natural HIV-1 infection in humans.
Infection of HIS mice with a high dose of HIV-1ADA
induced significant activation of the engrafted human immune system, with expansion of CD8+
cells, activation of B cells, and pathomorphologic changes in lymph nodes. These changes were similar to those observed in advanced HIV-1 infection in human subjects and again serve to underscore the relevance of the HIS mouse model for HIV-1 infection. At the same time, it is important to note that we were not able to detect humoral responses against HIV-1 in the HIS mice. This was unexpected, since the animals were capable of mounting an effective humoral immune response against a different antigen. It is possible that reconstitution of mice with HSCs may fail to provide key cells required for the efficient generation of HIV-1-specific humoral immune responses (such as human follicular dendritic cells) (27
). It is also possible that induction of humoral immune responses to HIV-1 in HIS mice may require a higher level of virus replication (30
) or that virus replication in HIS mice may interfere with B-cell maturation and preclude the generation of a HIV-1-specific antibody response, or it may simply be the case that seroconversion and development of HIV-1-specific humoral immune responses may require more than 10 weeks (3
). Further studies will be needed to distinguish these possibilities.
While our studies were in progress, Watanabe and colleagues reported on the outcome of HIV-1 infection in human HSC-transplanted NOD/SCID-IL-2Rγnull
mice, following exposure to both low and high doses of HIV-1JRCSF
, or SHIV-C/1 (30
). These studies showed that virus replication persisted for up to 40 days after inoculation and that it resulted in inversion of CD4/CD8 T-cell ratios in the spleen, at least in some cases. The present work corroborates these findings but also extends them considerably by (i) developing an improved and more reproducible engraftment procedure, (ii) demonstrating stable HIV-1 infection of HIS mice for up to 10 weeks following virus inoculation, and (iii) showing changes in lymphoid architecture that resemble those found in HIV-1-infected humans. These findings establish unequivocally the utility of the HIS model. Overall, we conclude that HIS mice (BALB/c-Rag2−/−
mice engrafted with CD34+
human hematopoietic stem cells) are a unique and valuable resource to study early stages of HIV-1 infection in its human host.