HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model
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The major targets of HIV infection in humans are CD4+ T cells. CD4+ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages.
NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4+ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4+ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo.
NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.
KeywordsRaltegravir Humanize Mouse Human Thymus Systemic Reconstitution Humanize Mouse Model
SCID-hu thy/liv mice develop a bona-fide human thymic organ and have a marginal level of systemic reconstitution with human T cells [1, 2]. The human thymic organoid present in the SCID-hu model is susceptible to HIV infection . However, infection of these animals requires this tissue to be surgically exposed and virus administration via direct injection . HIV injection results in infection of the human thymocytes present but there is no viremia in these mice, thus analysis of virus replication and its effect on thymocytes requires surgical removal of a piece of tissue . Subsequent monitoring of infection over time also requires additional surgical collection of tissue for analysis. Although the use of this model is extremely labor intensive and requires large numbers of animals to make meaningful observations, the SCID thy/liv model has been extensively used to evaluate HIV pathogenesis of the thymus, the effect of HIV on thymocyte development, the establishment of HIV latency in thymocytes in vivo, the efficacy of antiviral drugs on thymocytes and the role of auxiliary genes of HIV in virus replication and CD4+ thymocyte destruction [5, 6, 7, 8].
Following the development of the SCID thy/liv model, several other novel strains of mice have been derived with a higher degree of immune suppression. These include the NOD/SCID and the NOD/SCID common gamma chain null (NSG) strains of immunodeficient mice . Both of these strains have been extensively and successfully used in the derivation of a variety of humanized mouse models . However, neither of these two strains has been extensively used to produce humanized thy/liv implanted mice .
Resting CD4+ T cells represent a well-characterized reservoir for latent HIV-1 infection, and this reservoir persists long-term despite treatment with highly active antiretroviral therapy (HAART) [11, 12, 13]. Incubating resting CD4+ T cells with CCL19, secreted by mature dendritic cells, ex vivo increases HIV-1 integration efficiency . Additionally, the chemokines CXCL9 and CXCL10, secreted by monocyte-derived cells and induced by IFN-γ production, seem to mediate similar effects in resting T cells [11, 14, 15, 16]. Secretion of IL-7 by dendritic cells may be important for the survival of memory T cells, and secretion of IL-15 by macrophages and other mononuclear phagocytes is important for the low level of proliferation necessary to maintain a resting memory pool over time . Thus while it is known that several myeloid-derived cell types secrete cytokines and chemokines that facilitate the development of latency and maintain the resting CD4+ T cell pool, whether or not these cells are necessary for the establishment of latency in vivo remains unknown .
With the long-term goal of obtaining a better understanding of HIV replication, CD4+ T cell depletion, HIV latency and persistence in vivo, we sought to study HIV-1 in a humanized mouse model that possesses human T cells but is devoid of human myeloid (and B) cells. To this effect, we implanted human thymus and liver into NOD/SCID and NSG mice. In this study we show that whereas NOD/SCID hu thy/liv mice do not develop high levels of systemic reconstitution with human cells, NSG hu thy/liv mice develop high levels of human T cells in the peripheral blood. Remarkably, flow cytometric analysis of blood and tissues demonstrate the complete absence of human B and myeloid cells in these mice. Interestingly, in contrast to mice reconstituted with human peripheral blood mononuclear cells (PBMC) and some other types of humanized mice [19, 20], these T cell-only mice (or TOM) do not develop signs of GVHD. In addition, TOM are readily susceptible to HIV infection after parenteral exposure and can sustain high levels of HIV replication. Virus replication can be efficiently suppressed by antiretroviral therapy and HIV latency is established in resting T cells.
Results and discussion
Although SCID-hu thy/liv animals have been used extensively to study thymopoiesis and HIV-1 infection of the thymus, additional applications of this model has been limited by the lack of peripheral access to the human cells [31, 32]. Specifically, in this model a lack of systemic reconstitution with human cells requires invasive surgery for infection and monitoring of virus replication . In one report, low levels of human cells in PB, spleen and lymph nodes of SCID-hu thy/liv implanted mice were noted . However, this required implantation of twenty pieces of human thy/liv tissue under both kidney capsules of each mouse. Using this more invasive implantation strategy combined with 20X more tissue, HIV-1 infection was achieved after IP or intra-implant injection. Using the original implantation strategy described for SCID-hu mice, the use of more immunodeficient mouse strains, like the NSG strain, has overcome the limited systemic reconstitution previously seen in SCID-hu mice. Interestingly, thy/liv implantation of NOD/SCID mice did not result in systemic reconstitution with T cells suggesting that the additional immunosuppression due to the lack of a functional common gamma chain observed in NSG mice resulting in a complete lack of natural killer cells  is likely contributing to the increased T cell levels in these mice.
TOM were systemically reconstituted with human T cells. This reconstitution is consistent with the continued production of human T cells from the implanted thy/liv organoid as it showed a substantial and robust population of CD3+/CD4+/CD8+ thymocytes for as long as the animals were examined (1.2 years). Consistent with the lack of cryptopatches in NSG mice  TOM showed essentially no significant accumulation of human T cells in the intestinal tract (data not shown). TOM show phenotypically normal CD4+ T cell development. However, we noted somewhat limited CD8+ T cell development in TOM, with few effector memory CD8+ cells. These differences in the formation of effector phenotypes in the CD4+ and CD8+ T cell populations may be due to the absence of cytokine signals from professional APCs as well as CD4+ helper T cells that limit CD8+ T cell activation/differentiation . The reduction in the percentage of CD4+ T cells with an effector memory phenotype pre- and post- HIV infection cannot be attributed to differences in the source of donor tissues since tissue from a total of 11 different donors were used to generate the mice used for these experiments.
One salient feature of TOM is the fact that despite robust levels of human T cells, they do not develop GVHD. GVHD has been observed in multiple humanized mouse models [19, 36]. Some investigators have reported a significance incidence of GVHD leading to death of some of the animals at 25-28 weeks post-humanization . In contrast, we did not notice any of these effects on TOMs at these or subsequent time points (up to 78 weeks longest time point analyzed). The longevity of TOM systemically reconstituted with high levels of human T cells in the absence of GVHD is an important feature of this model.
ART offers significant benefits to HIV infected patients. Our results show that combination ART is able to suppress viral replication in TOM validating this model for the evaluation of the effect of antivirals on HIV replication in vivo. As in humans, therapy interruption resulted in rapid viral rebound. Furthermore, we show that human T cells alone are sufficient for the establishment of HIV latency in resting CD4+ T cells. Additionally, latently infected cells in TOM can be induced ex vivo to produce virus. The frequency of latently infected resting human CD4+ T cells in ART suppressed TOM are within the range seen circulating in PB of ART suppressed patients, regardless of when therapy was initiated. T cells represent the major reservoir of latent HIV in humans. Therefore, TOM may represent a unique tool for studies of HIV eradication strategies, as they have latently infected resting CD4+ T cells in the complete absence of any myeloid cells.
In summary, TOM represent a significant advance over the original SCID-hu thy/liv model because they have substantial levels of T cells in both PB and tissues. TOM are systemically and exclusively reconstituted with human T cells enhancing their utility for the study of T cell development, repopulation, function and response to stimuli in vivo. The presence of human T cells in blood permit direct inoculations with HIV and direct monitoring of virus infection via blood plasma facilitating the longitudinal analysis of HIV infection and its effects on CD4+ T cells. ART efficiently inhibits HIV replication in TOM resulting in strong viral suppression. The ability to suppress HIV replication by ART in TOM allows the use of these mice to investigate latently infected resting human CD4+ cells in vivo. Because these mice do not develop GVHD and systemic reconstitution with human T cells is sustained at high levels for over a year, long-term experiments are greatly facilitated in this model.
Generation of humanized mice
Humanized TOM were prepared by implanting allogeneic thymus and liver tissue into, 6–8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG, The Jackson Laboratory) mice. NOD/SCID hu-thy/liv (N/S-hu) mice were prepared in the same manner by implanting thymus and liver tissue into NOD.CB17-Prkdcscid/J mice (NOD/SCID, The Jackson Laboratory). Seven different tissue sets were used to generate the humanized mice presented in this manuscript. The thymus and liver implants consisted of a 1-2 mm piece of liver tissue sandwiched between two pieces of autologous thymus that were placed under the left kidney capsule (Advanced Bioscience Resources, Alameda, CA). All mice were maintained in a specific pathogen-free facility with the Division of Laboratory Animal Medicine at the University of North Carolina at Chapel Hill (UNC-CH) according to protocols approved by the Institutional Animal Care and Use Committee. Human reconstitution of mice was monitored by flow cytometric analysis for human CD45+ cells in peripheral blood, as previously described [37, 38]. Peripheral blood samples were obtained via submandibular venipuncture and were collected in tubes containing EDTA. Whole peripheral blood was stained with antibodies, red blood cells were lysed, and the remaining cells were washed and fixed using a 1% paraformaldehyde solution. A total of 10,000-30,000 events were collected per animal at each time point as indicated below.
Tissue harvesting and flow cytometric analyses of humanized mice
Mononuclear cells (MNCs) were isolated from the bone marrow, spleen, lymph nodes, lung, liver, and thymic organoid as previously described . Tissues were minced and/or digested and filtered through a 70 μm strainer. The liver and lung were processed as previously described . For all latency determinations, mononuclear cells, with the exception of the lymph nodes, were isolated using a Percoll gradient. Red blood cells were lysed as needed (namely for the spleen, bone marrow and liver tissues). MNCs were washed, counted via trypan blue exclusion, and flow cytometric analyses were performed for the indicated markers [13, 24, 37, 38, 39]. Live cells were distinguished by their forward and side scatter profiles as previously described . Flow cytometry data was collected on either a BD FACSCanto or a BD LSRFortessa flow cytometer, and analyzed using BD FACSDiva software (v.5.0.2 or v.6.1.3).
Stocks of HIV-1JR-CSF were prepared and titered as previously described . Briefly, virus supernatants were prepared via transient transfection of 293 T cells, and were tittered using TZM-bl cells essentially as we have previously described . Parenteral exposures were performed using HIV-1JR-CSF (90,000 TCIU) administered either intravenously or intraperitoneally. A total of two intraperitoneal and six intravenous exposures were performed, yielding 2/2 and 6/6 systemically infected animals, respectively.
Analysis of HIV-1 infection
Peripheral blood was collected via retro-orbital bleed using EDTA coated capillary tubes (approximately 100 ul total). Infection of TOM with HIV-1 was determined with a one-step reverse transcriptase real-time PCR assay (ABI custom TaqMan Assays-by-design) according to the manufacturer’s instructions (with primers 5′-CATGTTTTCAGCATTATCAGAAGGA-3′ and 5′-TGCTTGATGTCCCCCCACT-3′; assay sensitivity of 400 RNA copies per mL). Additionally, the percent of human CD4+ T cells in the peripheral blood of TOM pre- and post-exposure to HIV-1 were monitored by flow cytometry (using 40-60 ul of blood). Changes in the percent of CD4+ T cells present in the tissues of infected and uninfected animals were compared by two-way ANOVA, and were not significantly different. Statistical analysis was performed in Prism version 5 (GraphPad Software, Inc., San Diego, CA).
Antiretroviral treatment of TOM
For HIV treatment we used a previously described triple combination of drugs that we have shown to be effective at suppressing viral load in humanized mice. Specifically, infected TOM were administered daily intraperitoneal injections of emtricitabine (FTC; 140-200 mg/kg), tenofovir disoproxil fumarate (TDF; 146-208 mg/kg) and raltegravir (RAL; 56-80 mg/kg) for six to nine weeks, as previously described . HIV-1 infection was monitored throughout ART as described above.
Resting cell isolation and latency determinations of TOM and patient samples
All MNCs from individual mice were pooled. Resting human CD4+ T cells were isolated from pooled tissues or from leukopheresis product of patient samples using negative magnetic selection (STEMCELL Technologies, Vancouver) as previously described [12, 13, 43]. Briefly, MNCs obtained from mouse tissues were incubated with a cocktail of antibodies composed of mouse anti CD45 and TER119, and anti-human CD8, CD14, CD16, CD19, CD56, CD41, CD25, CD31, CD105, HLA-DR, and glycophorin A. For the separation of cells from human samples, the mouse antibodies were not included in the isolation cocktail. Antibody-bound cells were removed using a column based-magnetic purification system and the purified resting cells were collected as flow through. This approach resulted in a >99% pure resting CD4+ T cell population. Resting CD4+ T cells were then cultured with 15 nM efavirenz and 1 μM raltegravir for 2 days prior to performing viral outgrowth assays to prevent any de-novo infection from unintegrated virus . Viral outgrowth was achieved by maximally stimulating resting cells in limiting dilution cultures containing 60 U/ml IL-2, 1ug/ml phytohemagglutinin (PHA) and irradiated allogeneic PBMC from an uninfected donor.al. [12, 13]. The culture media was replaced every 3-4 days with fresh media containing 5 U/ml IL-2. CD8 depleted PHA-stimulated PBMC from an uninfected donor were added twice during the experiment to facilitate virus spread/amplification in cultures. Cultures were scored positive if p24 was detectable at day 15 and confirmed on day 19. The number of infected resting cells was estimated by a maximum likelihood method and was expressed as the infectious units per million resting CD4+ T cells (IUPM) .
This work was supported in part by National Institutes of Health grants AI096113, AI073146, AI096138 (J.V.G.), the UNC Center for AIDS Research grant P30 AI50410, a National Institute of Allergy and Infectious Disease Institutional Training Grant (5T32AI007273-27) (A.W.) and 5R01DA030156 (D.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank I. Chen for providing pYK-JR-CSF (cat#2708) via the AIDS Research and Reference Reagent Program. We also thank former and current members of the Garcia laboratory and husbandry technicians at the UNC Division of Laboratory Animal Medicine for their assistance with aspects of this work.
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