Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy

Immunodeficient mice reconstituted with human immune tissues and cells (humanized mice) are relevant and robust models for the study of HIV-1 infection, immunopathogenesis, and therapy. In this study, we performed a comprehensive comparison of human immune reconstitution and HIV-1 infection, immunopathogenesis and therapy between immunodeficient NOD/Rag2−/−/γc−/− (NRG) mice transplanted with human HSCs (NRG-hu HSC) and mice transplanted with HSCs and thymus fragments (NRG-hu Thy/HSC) from the same donors. We found that similar human lymphoid and myeloid lineages were reconstituted in NRG-hu HSC and NRG-hu Thy/HSC mice, with human T cells more predominantly reconstituted in NRG-hu Thy/HSC mice, while NRG-hu HSC mice supported more human B cells and myeloid cells reconstitution. HIV-1 replicated similarly and induced similar T cell depletion, immune activation, and dysfunction in NRG-hu HSC and NRG-hu Thy/HSC mice. Moreover, combined antiretroviral therapy (cART) inhibited HIV-1 replication efficiently with similar persistent HIV-1 reservoirs in both models. Finally, we found that blocking type-I interferon signaling under cART treatment transiently activated HIV-1 reservoirs, enhanced T cell recovery and reduced HIV-1 reservoirs in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice. In summary, we report that NRG-hu Thy/HSC and NRG-hu HSC mice support similar HIV-1 infection and similar HIV-1 immunopathology; and HIV-1 replication responds similarly to cART and IFNAR blockade therapies. The NRG-hu HSC mouse model reconstituted with human HSC only is sufficient for the study of HIV-1 infection, pathogenesis, and therapy.

Immunodeficient mice reconstituted with human immune tissues and cells (humanized mice) are relevant and robust models for the study of HIV-1 infection, immunopathogenesis, and therapy. In this study, we performed a comprehensive comparison of human immune reconstitution and HIV-1 infection, immunopathogenesis and therapy between immunodeficient NOD/Rag2 −/− /γ −/− c (NRG) mice transplanted with human HSCs (NRG-hu HSC) and mice transplanted with HSCs and thymus fragments (NRG-hu Thy/HSC) from the same donors. We found that similar human lymphoid and myeloid lineages were reconstituted in NRG-hu HSC and NRG-hu Thy/HSC mice, with human T cells more predominantly reconstituted in NRG-hu Thy/HSC mice, while NRG-hu HSC mice supported more human B cells and myeloid cells reconstitution. HIV-1 replicated similarly and induced similar T cell depletion, immune activation, and dysfunction in NRG-hu HSC and NRG-hu Thy/HSC mice. Moreover, combined antiretroviral therapy (cART) inhibited HIV-1 replication efficiently with similar persistent HIV-1 reservoirs in both models. Finally, we found that blocking type-I interferon signaling under cART treatment transiently activated HIV-1 reservoirs, enhanced T cell recovery and reduced HIV-1 reservoirs in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice. In summary, we report that NRG-hu Thy/HSC and NRG-hu HSC mice support similar HIV-1 infection and similar HIV-1 immunopathology; and HIV-1 replication responds similarly to cART and IFNAR blockade therapies. The NRG-hu HSC mouse model reconstituted with human HSC only is sufficient for the study of HIV-1 infection, pathogenesis, and therapy.
Keywords: humanized mice, nrg-hu hsc, nrg-hu Thy/hsc, hiV-1 replication, hiV-1 immunopathology, combined antiretroviral therapy, hiV-1 immune therapy inTrODUcTiOn Human immunodeficiency virus type 1 (HIV-1) infects and progressively depletes CD4 + T cells, causing acquired immune deficiency syndrome (AIDS). Approximately 70 million people have been infected with HIV-1, and half of them have died of HIV/AIDS-related causes (1). The development of combined antiretroviral therapy (cART), which can efficiently suppress viral replication, has Humanized Hu-HSC and Hu-Thy/HSC Mice Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 817 significantly improved survival and life quality of HIV-1-infected patients who can both access and tolerate cART (2). However, cART is not curative and must be continued for life (3,4). Moreover, lifelong treatment is associated with significant side effects and non-AIDS-related "end-organ disease" (5). Thus, there is a great need for the development of novel therapies that can both control the epidemic and cure those individuals who have already been infected with HIV-1.
Understanding how HIV-1 infection leads to immunodeficiency is key for the development of new treatments. After more than 30 years of research, the precise mechanism by which HIV-1 infection causes AIDS development is still poorly understood, mainly due to the lack of robust small animal models. The recent development of humanized mice with functional human immune systems offer a relevant and robust model for the study of HIV-1 infection, replication, pathogenesis, and therapies (6)(7)(8). Humanized mice were constructed by transplantation of human CD34 + hematopoietic stem cells and/or implantation of human thymus tissue into immunodeficient mice, such as the NODscid γc −/− (NSG) mice and NOD-Rag2 −/− γc −/− (NRG) mice (9). Two major humanized mouse models, the NRG-hu HSC model and the hu-BLT model, are widely used for HIV-1 studies. The NRG-hu HSC model involves preconditioning neonate immunodeficient mice with radiation and then injecting them with human CD34 + HSCs (10)(11)(12)(13). In the hu-BLT model, implantation of human thymus tissue under the kidney capsule is combined with HSCs infusion into irradiated adult immunodeficient mice (14,15). We and others have reported that all the major human lymphoid lineage including T cell, B cell, and innate immune cells including NK cell, monocytes, myeloid dendritic cells (mDC), and plasmacytoid dendritic cells (pDC) are developed in both NRG-hu HSC mice and hu-BLT mice (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).
The advantage of the NRG-hu HSC model is that the procedure to construct the mice is simple, only involving pre-irradiating the neonate immunodeficient mice followed by injecting human CD34 + HSCs (10,11,13). To generate hu-BLT mice, a time consuming and technically difficult surgery procedure is needed to implant the human thymus tissue into the kidney capsule of the mice (14,15). Another major difference between these two models is that in NRG-hu HSC mouse, the human T cells are produced in the mouse thymus and presumed to be educated in the context of mouse major histocompatibility complex (MHC) (10)(11)(12)(13). In hu-BLT mice, human T cells can develop in the presence of human thymic epithelium, resulting in human MCH-restricted T cells (14,15,40). Thus, although both models are versatile tools for HIV-1 study, parallelly study to compare the human immune reconstitution, HIV-1 replication, immunopathology, and responses to therapy in both models will help to guide researchers how to balance and decide which system to use. In this study, we performed a comprehensive parallel comparison of human immune reconstitution and HIV-1 replication, immunopathogenesis and therapy between newborn immunodeficient mice transplanted with HSCs (NRG-hu HSC) and 6-to 8-week-old adult mice transplanted with HSCs and thymus (NRG-hu Thy/HSC) from same human donors into same background of immunodeficient mice. We report that both NRG-hu HSC and NRG-hu Thy/HSC mice support significant levels of human immune reconstitution and comparable levels of HIV-1 replication, immunopathology, and responses to cART and immune therapy.

construction of humanized Mice
NRG (NOD-Rag2 −/− γc −/− ) mice were obtained from the Jackson Laboratory. Human fetal liver and thymus (gestational age of 16-20 weeks) were obtained from medically or elective indicated termination of pregnancy through a non-profit intermediary working with outpatient clinics (Advanced Bioscience Resources, Alameda, CA, USA). Written informed consent of the maternal donors is obtained in all cases, under regulation governing the clinic. NRG-hu HSC mice were generated by intrahepatic injection of new born (1-5 days old) NRG mice (irradiated at 200 cGy from a 137Cs gamma radiation source) with 3 × 10 5 human fetal liver derived CD34 + HSCs as previously reported (41). To generate NRG-hu Thy/HSC mice, 6-to 8-week-old NRG mice were sub-lethally irradiated (250 cGy) and anesthetized, and ~1-mm 3 fragments of human fetal thymus fragments were implanted under the kidney capsule. 5 × 10 5 CD34 + HSCs purified from fetal liver of the same donor were injected i.v. within 3 h. All mice were housed and bred in a specific pathogen-free environment. All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee. Total lymphocytes were isolated prepared from peripheral blood, spleen, bone marrow (BM), and mesenteric lymph nodes (mLNs) according to standard protocols; red blood cells were lysed with ACK buffer. Intrahepatic lymphocytes were prepared Humanized Hu-HSC and Hu-Thy/HSC Mice Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 817 as described (42). Total cell number was quantified by Guava Easycytes with Guava Express software (Guava). For surface staining, single cell suspension was stained with surface markers and analyzed on a CyAn ADP flow cytometer (Dako). For intracellular staining, cells were first stained with surface markers and then fixed and permeabilized with cytofix/cytoperm buffer (BD Bioscience), followed by intracellular staining. Data were analyzed using Summit4.3 software (Dako).

T cell stimulation and intracellular cytokine staining
Splenocytes from humanized mice were stimulated ex vivo with PMA (phorbol 12-myristate 13-acetate) (

hiV-1 genomic rna Detection in Plasma
HIV-1 RNA was purified from the plasma with the QIAamp ® Viral RNA Mini Kit. The RNA was then reverse transcribed and quantitatively detected by real-time PCR using the TaqMan ® Fast Virus 1-Step PCR kit (ThermoFisher Scientific). The primers used for detecting the HIV Gag gene were (5′-GGTGCGAGA GCGTCAGTATTAAG-3′ and 5′-AGCTCCCTGCTTGCCCAT A-3′). The probe (FAM-AAAATTCGGTTAAGGCCAGGGGGA AAGAA-QSY7) used for detection was ordered from Applied Biosystems, and the reactions were set up following the manufacturer's guidelines and were run on the QuantStudio 6 Flex PCR system (Applied Biosystems). The detection limit of the real-time PCR reaction is four copies per reaction. Accordingly, the limit of detection of the assay with 50 µl of blood is 400 copies/ml in humanized mice.

In Vivo iFnar1 Blocking antibody Treatments
The α-IFNAR1 monoclonal antibody (mAb) was generated as previous reported (29). To block type-I interferon (IFN-I) signaling during chronic HIV-1 infection, humanized mice were treated i.p. with IFNAR1 blocking antibodies twice a week with the dose 400 µg/mouse at the first injection and 200 μg/mouse for the following treatments. Cohorts of mice were randomized into different treatment groups by level of HIV-1 RNA in plasma.

cell-associated hiV-1 Dna Detection
To measure total cell-associated HIV-1 DNA, nucleic acid was extracted from spleen and BM cells using the DNeasy Blood & Tissue Kit (Qiagen). HIV-1 DNA was quantified by real-time PCR. DNA from serial dilutions of ACH2 cells, which contain one copy of HIV genome in each cell, was used to generate a standard curve.

Viral Outgrowth assay
Viral outgrowth assay was performed as reported (43). Serial dilutions of human cells from splenocytes of humanized mice (1 × 10 6 , 2 × 10 5 , and 4 × 10 4 human cells) were stimulated with PHA (2 µg/ml) and IL-2 (100 U/ml) for 24 h. MOLT4/CCR5 cells were added on day 2 to enhance the survival of the cultured cells as well as to support and facilitate further HIV-1 replication. Culture medium containing IL-2 (NIH AIDS reagents program) and T cell growth factor (homemade as describe in the standard protocol) was replaced on days 5 and 9. After 7 and 14 days of culture, supernatant from each well was harvested, and HIV-1 RT-qPCR was performed to score viral outgrowth. Estimated frequencies of cells with replication-competent HIV-1 were determined by maximum likelihood statistics (43).

statistical analysis
In all other experiments, significance levels of data were determined by using Prism5 (GraphPad Software). Experiments were analyzed by two-tailed Student's t-test, or by one-way analysis of variance (ANOVA) and Bonferroni's post hoc test according to the assumptions of the test, as indicated for each experiment. A P value less than 0.05 was considered significant. The number of animals and replicates is specified in each figure legend.   (14,15), human thymic organoid was well developed and showed long-term sustained thymopoiesis in NRG-hu Thy/HSC mice ( Figure S1 in Supplementary Material). Human immune cell reconstitution in the peripheral blood was detected by flow cytometry 12 weeks after transplantation. All major human CD45 + leukocyte subsets were detected in peripheral blood of both NRG-hu HSC and NRG-hu Thy/HSC mice (Figure 1; Figure S1 in Supplementary Material). Similar level of human CD45 + cells was found in NRG-hu HSC (65.3 ± 5.3%) and NRG-hu Thy/HSC (68.8 ± 3.1%) mice ( Figure 1A). The percentage of human T cells within human CD45 + leukocytes was significantly higher in NRG-hu Thy/HSC mice (61.1 ± 3.2%) compared with the level in NRG-hu HSC mice (20.1 ± 3.4%) ( Figure 1B). The result indicated that fetal liver/thymus "sandwich" structure (14,15) is not essential for the long-term functioning human thymus development if human CD34 + HSCs were transplanted immediately after thymus transplantation. Progenitor cells derived from CD34 + HSCs can serve as the source of thymocyte progenitors. Both CD4 and CD8 T cells were developed in NRG-hu HSC and NRG-hu Thy/HSC mice (Figures 1C,D). The ratio of CD4 T cells to CD8 T cells was slightly higher in NRG-hu Thy/HSC mice compare to NRG-hu HSC mice (Figures 1C,D). The percentage of human B cells was 65.8 ± 3.3% in NRG-hu HSC mice and 33 ± 4.9% in NRG-hu Thy/HSC mice ( Figure 1E). The percentages of human NK cells, pDCs, and monocytes were also lower in NRG-hu Thy/HSC mice compared with NRG-hu HSC mice (Figures 1F-H).
human leukocytes are equally reconstituted in lymphoid Organs in Both nrg-hu hsc and nrg-hu Thy/hsc Mice We also detected human immune reconstitution in lymphoid organs including spleen, mLNs, liver, and BM of NRG-hu HSC and NRG-hu Thy/HSC mice. The percentage of human CD45 + cell and total number of human CD45 + cells were comparable in spleen, mLN, and BM between NRG-hu HSC and NRG-hu Thy/ HSC mice (Figures 2A,B). The level of human immune cells in the liver was slightly higher in NRG-hu Thy/HSC mice (95.7 ± 0.8%) compared with the NRG-hu HSC mice (85.6 ± 2.2%), consistent with the higher number of human CD45 + cells in the liver in NRG-hu Thy/HSC mice (Figures 2A,B).

similar Phenotype and Function of human T and B cells Developed in nrg-hu hsc and nrg-hu Thy/hsc Mice
We next determined the phenotype and function of human T cells and B cells from spleen of NRG-hu HSC and NRG-hu Thy/HSC mice. As in the peripheral blood, both the percentage and number of CD3 + T cells were higher in the spleen of NRG-hu Thy/HSC mice (Figures 3A,B). The percentages of CD4 and CD8 T cells in total T cells did not show difference in the spleen between these two models ( Figure 3A). Most of the T cells from both NRG-hu HSC (64.4 ± 3.1%) and NRG-hu Thy/HSC (64.3 ± 5.8%) showed naïve phenotype at 20 weeks posttransplantation ( Figure 3C). The function of T cells from both humanized mouse models were equal as they produced  (Figure 3D). No spontaneous IFN-γ and IL-2 production by T cells was detected from either the NRG-hu HSC or NRG-hu Thy/HSC mice. As in the peripheral blood, the percentage and number of B cells in the spleen were lower in NRG-hu Thy/HSC mice compared with the NRG-hu HSC mice ( Figure 3E). It has been reported that human B cells developed in humanized mice were immature and cannot produce significant level of antigen-specific IgG by vaccination (44)(45)(46). We also compared the phenotype of B cells from both NRG-hu HSC and NRG-hu Thy/HSC mice and found that they both express high level of immature marker CD10 ( Figure 3F). The expression of CD10 on B cells from NRG-hu Thy/HSC mice was slightly higher (Figure 3F), indicating that co-transplantation of thymus had minor effect on the maturation of B cells in NRG-hu Thy/HSC mice.
innate immune cells Were Developed in spleen of Both nrg-hu hsc and nrg-hu Thy/hsc Mice and responded similarly to Tlr-ls stimulation We compared the reconstitution of human innate immune cells including pDCs, mDCs and monocytes/macrophages in the spleen of NRG-hu HSC and NRG-hu Thy/HSC mice. The percentage and number of pDC ( Figure 4A) and monocytes/ macrophage ( Figure 4B) were comparable, while there were more mDCs in the spleen of NRG-hu HSC mice compared with NRG-hu Thy/HSC mice (Figure 4C).
To detect the function of innate immune cells developed in NRG-hu HSC and NRG-hu Thy/HSC mice, we treated the mice in vivo with the TLR9-ligands CpG-B, the TLR7/8-L R848 and the TLR3-L poly I:C and detected cytokine production in the serum. We found that all the three TLR-Ls induced significant levels of IFN-α and IL-6 in both NRG-hu HSC and NRG-hu Thy/HSC mice (Figure 4D). The induction of IFN-α by poly(I:C) and R848 stimulation is slightly lower in NRG-hu Thy/HSC mice compared with NRG-hu HSC mice ( Figure 4D) which probably due to the lower number of mDC developed in NRG-hu Thy/HSC mice compared with NRG-hu HSC mice ( Figure 4C).
In summary, most human lymphoid and myeloid lineage cells are reconstituted in both NRG-hu HSC and NRG-hu Thy/HSC mice, with human T cells predominantly developed in NRG-hu Thy/HSC mice, while NRG-hu HSC mice support better human B cell and myeloid cell development. The phenotype and function of human immune cells developed in NRG-hu HSC mice and NRG-hu Thy/HSC mice are similar.

nrg-hu hsc and nrg-hu Thy/hsc Mice support similar level of hiV-1 replication In Vivo
We and others have reported that both NRG-hu HSC and NRG-hu Thy/HSC mice supported HIV-1 replication in vivo (7,8,47). Here we compared the HIV-1 replication kinetics in NRG-hu HSC mice and NRG-hu Thy/HSC mice transplanted with HSCs ± thymus from the same donor tissue. We found the viremia reached to peak level at 2 weeks postinfection (wpi) in NRG-hu HSC mice (Figure 5A), while in NRG-hu Thy/HSC mice, the viremia reached the peak level at 4 wpi (Figure 5B). At 2 wpi, NRG-hu HSC mice supported efficient HIV-1 replication in nearly all mice (98%) but NRG-hu Thy/HSC mice supported HIV-1 replication in about 73% of infected mice ( Figure 5B). The results suggest that the immune cells in NRG-hu Thy/HSC mice may control/delay HIV-1 replication at the early stage of HIV-1 infection. At 4 wpi, all the infected BLT mice showed similar viremia as detected in NRG-hu HSC mice and sustained through 10 wpi when we terminated the mice (Figures 5A-C). HIV-1 p24 levels in CD4 T cells from the spleen were similar in NRG-hu HSC and NRG-hu Thy/HSC mice at 10 wpi (Figures 5D,E). In summary, the results indicated that both the NRG-hu HSC and NRG-hu Thy/HSC mice support similar levels of HIV-1 replication, although there was a 2 weeks delay reaching the peak viremia in NRG-hu Thy/HSC mice.

hiV-1 infection induces similar levels of T cell Depletion, activation, and exhaustion in nrg-hu hsc and nrg-hu Thy/hsc Mice
We next detected HIV-1 induced immunopathology including T cell depletion, activation and dysfunction in both NRG-hu Shown (e,F) are data from n = 15 (NRG-hu HSC) and n = 12 (NRG-hu Thy/HSC) hu-mice per group reconstituted with HSCs ± thymus from the same donor. Each dot represents one individual mouse; bars indicate mean (*P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, two-tailed Student's t-test). HSC and NRG-hu Thy/HSC mice. We found that HIV-1 induced similar level of total human CD45 + cells and human T cells depletion in both NRG-hu HSC and NRG-hu Thy/HSC mice (Figures 6A,B). HIV-1 infection also induced similar level of CD38 and HLA-DR expression on CD8 T cells in both NRG-hu HSC and NRG-hu Thy/HSC mice (Figures 6C,D). In addition, we detected the T cells exhaustion marker PD-1 expression and found that both CD8 T cell from NRG-hu HSC and NRG-hu Thy/ HSC mice expressed higher level of PD-1 than mock-infected mice, which indicated HIV-1 induced human T cell exhaustion in both humanized mouse models (Figures 6E,F). The results indicate that HIV-1 infection induced T cell depletion, activation and exhaustion to similar levels in both NRG-hu HSC and NRG-hu Thy/HSC mice.
carT efficiently inhibits hiV-1 replication in Both nrg-hu hsc and nrg-hu Thy/hsc Mice We and others have shown before that as in human patients, cART can efficiently inhibit HIV-1 replication in hu-mice (29,33,34,38). We compared the efficacy of cART to inhibit HIV-1 replication in NRG-hu HSC mice and NRG-hu Thy/ HSC mice. The results indicate that plasma viremia decreased to undetectable levels (<400 genome copies/ml) in all HIV-infected NRG-hu HSC and NRG-hu Thy/HSC mice within 3 weeks after cART treatment (Figures 7A,B). Similar to cART-treated patients, HIV-1 reservoirs persisted stably in both NRG-hu HSC and NRG-hu Thy/HSC mice and virus rebounded rapidly after cART cessation (Figures 7C,D). HIV-1 rebounded in 60% NRG-hu HSC mice at 1-week post cART cessation and in 100% NRG-hu HSC mice at 2 weeks post cART cessation (Figure 7E). Similarly, HIV-1 rebounded in 50% NRG-hu Thy/HSC mice at 1-week post cART cessation and in 100% NRG-hu Thy/HSC mice at 2 weeks post cART cessation ( Figure 7F).
iFnar Blockade During carT-suppressed hiV-1 infection reverses aberrant immune activation and exhaustion Phenotype of human T cells We next determined whether IFNAR blockade can reverses aberrant immune activation and exhaustion phenotype of human T cells in both NRG-hu HSC and NRG-hu Thy/HSC mice. We found that in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/ HSC mice, cART alone significantly rescued the number of human CD4 and CD8 T cells (Figures 8A,B), however, it only slightly decreased the expression level of CD38/HLA-DR (Figures 8C,D) and PD-1 on CD8 T cells (Figures 8E,F). CD8 T cells from both cART-treated NRG-hu HSC and NRG-hu Thy/HSC mice still expressed significantly higher levels of activation marker (Figures 8C,D) and exhaustion marker PD-1 (Figures 8E,F) compared with uninfected hu-mice. Interestingly, IFNAR blockade significantly reversed aberrant CD8 T-cell activation and exhaustion in the presence of cART in both NRG-hu HSC and NRG-hu Thy/HSC mice (Figures 8C-F).
iFnar Blockade reduce hiV-1 reservoirs in Both hiV-1 infected nrg-hu hsc and nrg-hu Thy/hsc Mice Under carT Combined antiretroviral therapy is able to suppress HIV-1 replication but does not eradicate HIV reservoir, which cause virus rebound after cART interruption. We have reported before that during chronic phase of HIV-1 infection in humanized mice, blockade of IFN-I signaling using a mAb targeting to IFN-I receptor (IFNAR) reduces the level of T cell activation, reverses T cell exhaustion, and improves HIV-specific CD8 + T cells (29). Most strikingly, we found that IFNAR blockade during cART administration markedly reduced HIV-1 reservoirs (29). Here we compared the effect of IFNAR blockade in HIV-1 reservoir reduction in NRG-hu HSC and NRG-hu Thy/HSC mice. We treated HIV-1-infected NRG-hu HSC and NRG-hu Thy/HSC mice that were fully cART-suppressed with α-IFNAR1 mAb for 3 weeks during 7-10 wpi (Figures 9A,B). Interestingly, IFNAR blockade led to low blips of HIV-1 replication, which returned to undetectable levels after stopping α-IFNAR1 mAb treatment, in the presence of cART in both NRG-hu HSC and NRG-hu Thy/ HSC mice (Figures 9A,B). We next analyzed the HIV-1 reservoir size in lymphoid organs 2 weeks after IFNAR blockade in both NRG-hu HSC and NRG-hu Thy/HSC mice. We measured cell-associated HIV-1 DNA by PCR, and replication-competent HIV-1 by the quantitative virus outgrowth assay. We found that IFNAR blockade reduced cell-associated HIV-1 DNA by 10.8-fold in the spleen of NRG-hu HSC mice ( Figure 9C) and by 7.9-fold  in NRG-hu Thy/HSC mice ( Figure 9D). More importantly and consistently, IFNAR blockade significantly reduced the size of replication-competent HIV-1 reservoirs measured by quantitative virus outgrowth assay in both NRG-hu HSC and NRG-hu Thy/HSC mice (Figures 9E,F). Taken together, we conclude that both NRG-hu HSC and NRG-hu Thy/HSC mouse models are valuable tools for the study of HIV-1 replication, pathogenesis and therapeutics.

DiscUssiOn
Humanized mice with human immune cells are highly relevant and robust models for HIV-1 study (6)(7)(8). The models are generated via transplantation of CD34 + HSCs and/or implantation of human tissue into immunodeficient mice. There are different humanized mouse models available as well as different means to prepare them (6,7,9). The degree of human immune system reconstitution can vary between different models, and between different batches of HSCs and/or tissue donors, and non-standardized operating procedure between laboratories. Also, HIV-1 infection, replication and HIV-1 induced pathology can vary between different models and dependant on which HIV-1 virus strain is used. These factors make researchers, especially those who have limited experiences on humanized mouse models difficult to decide which model to choose for their studies. Here we performed a comprehensive parallel comparison of systemic immune reconstitution and HIV-1 replication, HIV-1 induced pathology and their response to cART and immunotherapy between two humanized mouse models, the NRG-hu HSC and NRG-hu Thy/HSC models. We used NRG-hu HSC and NRG-hu Thy/HSC mice transplanted with HSCs without or with thymus fragment from same donors into same background of immunodeficient mice in our experiment to minimize the variation factors. Our results indicate that both NRG-hu HSC and NRG-hu Thy/HSC mice support significant level of human immune reconstitution and comparable level of HIV-1 replication, immunopathology and responses to ART and immune therapy.
We and others have reported that all the major human lymphoid and myeloid lineage cells are developed in both NRG-hu HSC mice and hu-BLT mice (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, no study has performed to parallelly compare the human immune reconstitution in these two models which transplanted with HSCs ± thymus from same donor into the same background of immunodeficient mice. In the NRG-hu Thy/HSC model, we co-transplanted CD34 + HSCs by intravenous injection within 3 h after thymus fragment (without fetal liver fragment) transplantation. Human thymic organoid developed under the kidney capsule in our NRG-hu Thy/HSC as well as reported in hu-BLT mice which indicated  that the fetal liver/thymus "sandwich" are not essential for thymic organoid development. Progenitor cells derived from CD34 + HSCs can serve as the source of thymocyte progenitors. It should be noted that the fetal liver fragments co-transplanted with thymus fragments in the BLT or SCID-hu Thy/Liv (48) mice also provide mostly human HSC/progenitors and no other liverrelated functions. Our results indicate that similar level of total human CD45 + cells were developed in peripheral blood, spleen, mLNs, and BM of both NRG-hu HSC and NRG-hu Thy/HSC mice transplanted with HSCs ± thymus from same donor. We also found that all the major human lymphoid and myeloid lineage including T, B, NK cells, monocytes/macrophages, mDC, and pDC were developed in both NRG-hu HSC mice and NRG-hu Thy/HSC mice. The major difference between these two models is that human T cells are predominantly developed in NRG-hu Thy/HSC mice due to more efficient T cell development in human thymus tissue (or xeno-reactive T cells) in NRG-hu HSC-Thy mice. This may lead to preferential reconstitution of T cells and reduced NK/monocyte/pDC engraftment in NRG-hu HSC-Thy mice (Figures 1 and 3).
Our results indicated that majority of human T cells from both NRG-hu HSC and NRG-hu Thy/HSC mice were with naïve phenotype and they responded similarly to mitogen stimulation. However, it is important to point out that human T cells can develop in the presence of human thymic epithelium, resulting in human HLA class I and class II restriction in NRG-hu Thy/HSC mice (14,15,40). While in NRG-hu HSC mice, human T cells are produced in the mouse thymus and presumed to be educated in the context of mouse MHC (10)(11)(12)(13). To study human HLArestricted immune response in NRG-hu HSC mice, an immunecompromised non-obese diabetic/SCID/IL2rg −/− strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD) was generated (49). Human CTLs developing in the NSG-HLA-A2/HHD mice recognized EBV-derived peptides in an HLA-restricted manner and showed HLA-restricted cytotoxicity against EBV-infected human B cells (49). We also reported that HIV-1 infection can induce HIV-1 antigen-specific, HLA-A2-restricted CD8 T cell responds in humanized NSG-HLA-A2/HHD mice (31).
Both NRG-hu HSC and NRG-hu Thy/HSC mice support HIV-1 replication in vivo. Our results show that plasma HIV-1 viremia reached to peak levels at 2 wpi in NRG-hu HSC mice, while the peak viremia appeared at 4 wpi in NRG-hu Thy/HSC mice. The results suggest that anti-HIV-1 immunity at the early Combined antiretroviral therapy is able to suppress HIV-1 replication but does not eradicate HIV-1 reservoir, which cause virus rebound after cART interruption. This lack of in vivo models of HIV-1 infection has hindered progress in finding a cure for HIV-1/AIDS. The use of both NRG-hu HSC and NRG-hu Thy/ HSC models of HIV-1 infection have made significant contribution to the field of HIV cure research (6,7). We found here that both the HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice responded similarly to cART. Importantly, we found that type-I IFN signaling contributed to HIV-1 induced immune activation, dysfunction and fostered viral persistence in both NRG-hu HSC and NRG-hu Thy/HSC mice. Blockade of IFNAR reduced the level of T cell activation, reversed T cell exhaustion, and reduced HIV-1 reservoirs in both models. Multiple mechanisms may lead to the reduction of HIV-1 reservoir size after IFNAR blockade. The rescued human T cells could target the HIV-1 reservoirs with elevated gene expression and clear the reservoir cells as we have reported (29,30). Other factors, including HIV-1 induced death of reservoir cells, reduced general T cell activation after IFNAR blockade, may also contribute to the reduction of HIV-1 reservoir size (29).
Taken together, we conclude that both NRG-hu HSC and NRG-hu Thy/HSC mouse models are relevant and robust for the study of HIV-1 replication, pathogenesis and therapeutics. Each model has its own advantage and disadvantages. Compared with the NRG-hu Thy/HSC or Hu-BLT models, the advantages of the NRG-hu HSC model are as follows: (1) the procedure to construct NRG-hu HSC mice is simple, which only involving pre-irradiating the neonate immunodeficient mice followed by injecting human CD34 + HSCs (10,11,13). To generate NRG-hu Thy/HSC or Hu-BLT mice, a time consuming and technically difficult surgery procedure is needed to implant the human thymus