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. 2006 Nov 1:3:76.
doi: 10.1186/1742-4690-3-76.

HIV-1 infection and CD4 T cell depletion in the humanized Rag2-/-gamma c-/- (RAG-hu) mouse model

Bradford K Berges  1 , William H Wheat, Brent E Palmer, Elizabeth Connick, Ramesh Akkina
Affiliations

HIV-1 infection and CD4 T cell depletion in the humanized Rag2-/-gamma c-/- (RAG-hu) mouse model

Bradford K Berges et al. Retrovirology. .

Abstract

Background: The currently well-established humanized mouse models, namely the hu-PBL-SCID and SCID-hu systems played an important role in HIV pathogenesis studies. However, despite many notable successes, several limitations still exist. They lack multi-lineage human hematopoiesis and a functional human immune system. These models primarily reflect an acute HIV infection with rapid CD4 T cell loss thus limiting pathogenesis studies to a short-term period. The new humanized Rag2-/-gamma c-/- mouse model (RAG-hu) created by intrahepatic injection of CD34 hematopoietic stem cells sustains long-term multi-lineage human hematopoiesis and is capable of mounting immune responses. Thus, this model shows considerable promise to study long-term in vivo HIV infection and pathogenesis.

Results: Here we demonstrate that RAG-hu mice produce human cell types permissive to HIV-1 infection and that they can be productively infected by HIV-1 ex vivo. To assess the capacity of these mice to sustain long-term infection in vivo, they were infected by either X4-tropic or R5-tropic HIV-1. Viral infection was assessed by PCR, co-culture, and in situ hybridization. Our results show that both X4 and R5 viruses are capable of infecting RAG-hu mice and that viremia lasts for at least 30 weeks. Moreover, HIV-1 infection leads to CD4 T cell depletion in peripheral blood and thymus, thus mimicking key aspects of HIV-1 pathogenesis. Additionally, a chimeric HIV-1 NL4-3 virus expressing a GFP reporter, although capable of causing viremia, failed to show CD4 T cell depletion possibly due to attenuation.

Conclusion: The humanized RAG-hu mouse model, characterized by its capacity for sustained multi-lineage human hematopoiesis and immune response, can support productive HIV-1 infection. Both T cell and macrophage tropic HIV-1 strains can cause persistent infection of RAG-hu mice resulting in CD4 T cell loss. Prolonged viremia in the context of CD4 T cell depletion seen in this model mirrors the main features of HIV infection in the human. Thus, the RAG-hu mouse model of HIV-1 infection shows great promise for future in vivo pathogenesis studies, evaluation of new drug treatments, vaccines and novel gene therapy strategies.

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Figures

Figure 1
Figure 1
Human cell engraftment in the peripheral blood of CD34 cell-reconstituted Rag2-/-γc-/- mice and duration of engraftment. Conditioned neonatal mice were injected with CD34 cells intrahepatically. At different times post-reconstitution, mice were bled to detect human cell engraftment. Peripheral blood cells were stained with different antibodies after RBC lysis and analyzed by FACS. (A) Cells stained with antibodies against the human panleukocyte marker CD45 at 12 weeks post-engraftment. (B) Cells stained with antibodies against the T cell markers CD3 and CD4. (C) Cells stained with antibodies against the monocyte markers CD14 and CCR5. To analyze the duration of engraftment, peripheral blood cells from an engrafted mouse were stained with antibodies against CD45 at 12 weeks (D) and 52 weeks (E) post-engraftment.
Figure 2
Figure 2
Human T cell engraftment in lymphoid organs. CD34 cell-reconstituted mice were sacrificed at 19 weeks post-engraftment, and thymus, spleen and lymph nodes were collected. Tissue sections were subjected to immuno-staining with different antibodies specific for human T cells as described in methods.
Figure 3
Figure 3
Ex vivo productive HIV-1 infection in human cells differentiated in reconstituted Rag2-/-γc-/- mice. Thymus, spleen and lymph node tissues were collected at 16 weeks post-engraftment. Single cell suspensions were made and stimulated for 3 days with PHA and IL-2, and later challenged with HIV-1 NL4-3 HSA reporter virus. To detect productive viral infection, culture supernatants were analyzed by p24 ELISA at different days post-infection.
Figure 4
Figure 4
PCR detection of HIV-1 in infected RAG-hu mice. Peripheral blood was collected from infected mice at different weeks post-infection. Cellular and plasma fractions were separated by centrifugation. DNA from the cellular fractions was subjected to DNA PCR to detect integrated virus (A), whereas the RNA extracted from the plasma fraction was subjected to RT-PCR to detect cell-free virus (B). Results from a representative HIV-1 infected RAG-hu mouse (#16) are shown.
Figure 5
Figure 5
Detection of HIV-1 in infected RAG-hu mouse tissues by in situ hybridization. Thymus and spleen were collected at 12 weeks post-infection and sections were made from the frozen tissues. In situ hybridization was performed using digoxigenin-labeled antisense probes to detect HIV-1 RNA as described in methods. Dark staining cells indicate the presence of HIV-1.
Figure 6
Figure 6
CD4 T cell depletion in peripheral blood of HIV-1 infected RAG-hu mice. Peripheral blood was collected at different weeks post-infection and cells were stained with CD3 and CD4 antibodies and FACS analyzed. To determine the levels of CD4 T cells in the whole T cell population (stained with the pan T cell marker CD3), CD4:CD3 ratios were determined as described in methods. To obtain a baseline CD4:CD3 level for each individual mouse prior to HIV-1 infection, mice were bled a minimum of two times before infection. CD4 T cell levels are depicted as a percent of individual mouse baseline levels recorded at 1 week pre-infection. Shown are mean uninfected mouse levels (A, n = 4), infection with HIV-1 NL4-3 + NLENG1-IRES (B), infection with HIV-1 NLENG1-IRES alone (C), and infection with HIV-1 BaL (D). Also shown (E) are representative FACS plots from mouse #16 from various time points post-infection indicating the CD3CD4+/+ and CD3CD4+/- populations used to calculate the values shown in A-D.
Figure 7
Figure 7
Evidence for CD4 T cell depletion in HIV-1 infected RAG-hu mouse thymus. Thymus was collected at 12 weeks post-infection (from mouse #64) and sections were made from frozen tissues. Tissue sections were subjected to immuno-staining with antibodies specific for human CD4 T cells as described in methods.
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