Adult AIDS-Like Disease in a Novel Inducible Human Immunodeficiency Virus Type 1 Nef Transgenic Mouse Model: CD4+ T-Cell Activation Is Nef Dependent and Can Occur in the Absence of Lymphophenia

CD4C/HIV^nef transgenic (Tg) mice express Nef in CD4⁺ T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA × TRE/HIV^Nef) or (CD4C/rtTA2^S-M2 × TRE/HIV^Nef) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4⁺ T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4⁺ T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV^Nef Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.Small animal models able to express the entire human immunodeficiency virus (HIV) genome or selected HIV genes have provided useful information on the pathogenesis of AIDS and still represent important research tools toward this goal. Among these models, transgenic (Tg) mice containing intact copies of HIV DNA, defective provirus with the gag and pol genes deleted, or individual HIV-1 genes have been reported to develop various pathologies, some of which resemble those found in human AIDS (2, 3, 8, 9, 16, 17, 18, 24, 27, 29, 30, 38, 44, 45, 46, 49, 51, 52). The cell type context in which the HIV-1 transgene is expressed in these Tg mice appears to play an important role in determining the type of pathological lesions. Tg mice generated in our laboratory and expressing the entire coding sequence of HIV-1 (CD4C/HIV^WT) or HIV-1 Nef alone (CD4C/HIV^Nef) in the relevant target cells of HIV-1, namely, CD4⁺ T cells, macrophages, and dendritic cells, develop pathologies very similar to those in human AIDS (17, 18). The AIDS-like disease of CD4C/HIV^Nef Tg mice is characterized by immunodeficiency, loss of CD4⁺ T cells, thymic atrophy, activation of T cells and pathologies in heart, lungs, and kidneys (18, 53). Similarly, expression of simian immunodeficiency virus (SIV) Nef in Tg mice under the control of the same promoter sequences (CD4C) results in an AIDS-like disease (42). These studies demonstrated that Nef plays an important role in the development of the AIDS-like disease induced by HIV-1 or SIV in Tg mice.Among the AIDS-like phenotypes of these models, the T-cell activation observed by a number of groups in Tg mice expressing Nef (3, 33, 44, 53) may be of special interest for its resemblance to that of humans or macaques infected with HIV-1 or SIV, respectively. HIV infection results in a state of chronic immune activation which correlates very closely with disease progression in humans (11, 14, 23). Similarly, SIV-infected macaques which develop AIDS show aberrant immune activation (35), while SIV-infected sooty mangabey monkeys, natural hosts of SIV, do not develop immunopathologies and do not show immune activation either (41). Various factors may contribute to this immune activation, including increased plasma lipopolysaccharide levels due to microbial translocation from the gut (4), impaired regulatory T cell function (32), or the action of the HIV-1 gene products themselves, such as Env gp120 and Nef (10, 12, 43). Consistent with this latter scenario, we reported that in CD4C/HIV^Nef Tg mice the extent of T-cell activation correlates with levels of Nef expression in CD4⁺ T cells, thus suggesting a direct involvement of Nef in this activation (53). In contrast, Koenen and coworkers reported that T-cell activation in CD2/Nef Tg mice is induced indirectly by lymphophenia (26). In that study, chimeric mice, which were generated from a mixture of non-Tg and Nef Tg bone marrow (BM) cells, were not lymphopenic, and the donor-derived Nef-expressing Tg T cells did not show an activated phenotype. However, the donor Nef Tg T cells constituted only 1 to 2% of peripheral T cells of these chimeric mice (26). Clearly, alternative experimental approaches are needed to study this phenotype in a more physiological context.In the previously described CD4C/HIV^Nef Tg mice (18), Nef expression begins early in life and is constitutively expressed throughout the life of the animal. The AIDS-like disease caused by this early expression of Nef best represents a model for pediatric AIDS. However, in these Tg mice, Nef may interfere with normal developmental processes and these latter defects may contribute to some of the phenotypes observed. To assess the effects of Nef in fully mature adult animals, and thus develop a model of adult AIDS, temporal regulation of Nef expression in adult mice using an inducible system is required.In the present study, we chose the tet-On (rtTA and rtTA2^S-M2) system (13, 15, 25, 48) to induce expression of HIV-1 Nef in CD4⁺ T cells and cells of the macrophage/dendritic lineage of mice using the CD4C tissue-specific regulatory elements. These CD4C sequences were previously used to generate the constitutively Nef-expressing CD4C/HIV^Nef Tg mice (18). These inducible adult (TRE/HIV^Nef × CD4C/rtTA) and (TRE/HIV^Nef × CD4C/rtTA2^S-M2) double-Tg (DTg) mice express Nef when treated with doxycycline (DOX) and develop an AIDS-like disease very similar to that seen in constitutively Nef-expressing CD4C/HIV^Nef Tg mice. We took advantage of this novel biological system to reassess the role of Nef in T-cell activation. Using a mixed chimera made with BM cells from these inducible Nef Tg mice and from non-Tg mice, we could document CD4⁺ T-cell activation only in donor-derived Nef-expressing Tg cells, but not in non-Tg cells, in the absence of lymphopenia. This result strongly suggests that this CD4⁺ T-cell activation phenotype is most likely driven by expression of Nef in these cells.