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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: J Immunol. 2013 Jul 5;191(4):1753–1764. doi: 10.4049/jimmunol.1201518

Characterization of human antiviral adaptive immune responses during hepatotropic virus infection in HLA-transgenic human immune system mice

Eva Billerbeck *, Joshua A Horwitz *, Rachael N Labitt *, Bridget M Donovan *, Kevin Vega *, William C Budell *, Gloria C Koo t, Charles M Rice *, Alexander Ploss *
PMCID: PMC3735836  NIHMSID: NIHMS493897  PMID: 23833235

Abstract

Humanized mice have emerged as a promising model to study human immunity in vivo. While they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in non-lymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL2RγNULL(NSG) mice to those of a novel NSG strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver-infiltration and activation of T cells, as well as the detection of antigen-specific humoral and cellular immune responses. When infected with an HCV NS3 expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201 restricted HCV NS3-specific CD8+ T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.

Introduction

Chronic human hepatotropic infections remain major medical problems. At least 500 million individuals are infected with hepatitis B (HBV) and C (HCV) viruses (1, 2) and an estimated 250 million cases of malaria result in nearly one million deaths each year (3). These pathogens exhibit an almost unique human tropism and the lack of amenable small animal models has slowed our understanding of pathogenesis, and stalled the search for drugs and vaccines. HCV, for example, establishes persistence in about 70% of infections, yet the immunological mechanisms that determine viral clearance versus persistence are not fully understood (4).

Over the past two decades, humanized mice, which are animals engrafted with human tissue and/or engineered to express human genes, have emerged as powerful systems to study species-restricted pathogens and human immunity in vivo (5, 6). Efficient immune system engraftment can be achieved by transplantation of human CD34+ hematopoietic stem cells (HSCs) into highly immuno-compromised xenorecipients, e.g. the non-obese diabetic (NOD), severe combined immunodeficiency (SCID) interleukin 2 receptor gamma deficient (IL2RγNULL, NSG) strain (7, 8). Such human immune system (HIS) mice can generate antigen-specific human immune responses when infected with lymphotropic viruses like Epstein-Barr virus (EBV) or HIV (6, 9). Similarly, the successful generation of human liver chimeric mouse models has been reported (10, 11). These animals can be infected with HCV and mount virus-specific immune responses when dually reconstituted with human liver and human immune system compartments (12).

Despite these advances, current HIS models still suffer from significant functional deficiencies (5). For example, development of human myeloid and NK cell lineages is impaired, possibly due to the limited cross reactivity of critical hematopoietic cytokines between mice and humans (13). In addition, the development of functional adaptive immune responses is limited by the lack of human leukocyte antigen (HLA) gene expression. It has recently been suggested that the expression of a human MHC class II molecule, HLA-DR4, partially improves the development of functional human T and B cells (14, 15). Furthermore, transgenic expression of a human MHC class I molecule, HLA-A2, has been shown to significantly increase antiviral HLA-restricted human T cell responses in HIS mice infected with the human lymphotropic pathogens EBV or dengue virus (16-18). It needs to be determined however whether these T cell responses can contribute to clearance of the virus infection in humanized mice. The lack of HLA expression also limits recognition of pathogen-derived antigen presented on infected non-hematopoietic tissue. Very little is known about immunity to pathogens of tissues such as the liver, however, since most studies in humanized mice to date have focused on lymphotropic pathogens. Here, we used hepatotropic adenoviruses expressing either firefly luciferase or HCV non-structural proteins to analyze the extent of functional intrahepatic immune responses in humanized mice, specifically focusing on the effect of HLA expression.

Material and Methods

Mice

NOD.Cg-Prkdcscid IL2rgtmlWjl/Sz (NSG) mice, BALB/c mice and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The NSG-DRB*0101 (NOD.Cg-Prkdcscid IL2rgtmlWjl/Sz Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz/GckJ) and NSG-A2*0201 (NOD.Cg-PrkdcscidIL2rgtmlWjl/Sz Tg(HLA-A2.1)1Eng/Sz) (17, 19-21) were originally crossed with support of Dr. Richard O’Reily (Memorial Sloan-Kettering Cancer Center, New York NY). Specifically, to obtain NSG-DRB*0101 mice, previously described NOD/scid-DRB*0101 mice (19, 20) were backcrossed to the NSG background exceeding a total of 10+ crosses. Microsatellite analysis was performed to confirm sufficient backcrossing on the NOD background. 128 microsatellites were tested and 127 were identical with NSG confirming the strain background. Subsequently, NSG-A*0201 and –DRB*0101 were intercrossed until homozygous expression of both transgenes on the NSG background was established. Genotyping was performed at Transnetyx (Cordova, TN) using a qPCR-based system. Genotyping data was further confirmed by FACS analysis of HLA-A2 and HLA-DR expression on murine tissue in transgenic mice. Homozygous NSG-A2/DR1 double transgenic mice were then bred with NSG mice and the hemizygous offspring was used for the generation of human immune system mice. An independent NSG breeding cohort was used to generate the control group of human immune system mice. Mice were bred and maintained with irradiated food supplemented with antibiotics and acidified water at the Comparative Bioscience Center of the Rockefeller University according to guidelines established by the Institutional Animal Committee.

Isolation of human HSC and generation of humanized mice

Human fetal livers were procured from Advanced Bioscience Resources (ABR), Inc. Human CD34+ HSC were isolated as described previously (22). 1-5 day old NSG and NSG-A2/DR1 mice were irradiated with 100 cGy and 1.5-2 × 105human CD34+ HSC were injected intrahepatically 6h after irradiation. NSG-A2/DR1 mice were transplanted with either HLA-A2.1+ or HLA-A2.1+/HLA-DR1.1+ human HSC. HLA-typing of HSC was performed by FACS analysis (HLA-A2) and PCR (HLA-A2 and HLA-DR1.1). Within a time period of 1.5 years we received approximately 80 human fetal livers. We identified 28 HLA-A2+ donors and 2 HLA-A2+HLA-DR1+ donors. Table I summarizes the number and HLA-type of human HSC donors used for this study and the number of mice transplanted with each respective donor.

Table I. Overview of human CD34+HSC donors used for transplantation of NSG and NSG-A2/DR1 mice.

HSC
donor		 HLA
type		 Number of
mice
transplanted		 Level of human cell
reconstitution:
% hCD45 (mean ±
SEM)		 Level of antiviral
immunity: photons/s at
day 10 post infection
(mean ± SEM)
1 A2+
DR1+		 NSG 0
NSG-
A2/DR1		 4 41.3 ± 4.6 2.3e7 ± 9.9e6
2 A2+
DR1−		 NSG 3 52.2 ± 6.2 5.6e7 ± 1.6e7
NSG-
A2/DR1		 5 53.8 ± 1.7 2.3e7 ± 5.7e6
3 A2−
DR1−		 NSG 6 61.2 ± 5.1e7 ± 1.5e7
NSG-
A2/DR1		 0
4 A2+
DR1 +		 NSG 0
NSG-
A2/DR1		 18 60.1 ± 5.3 2.4e7 ± 7.1e6
6 A2−
DR1−		 NSG 11 54.1 ± 4.4 8.6 ± 2.2e7
NSG-
A2/DR1		 0
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All experiments were performed with authorization from the Institutional Review Board and the IACUC at the Rockefeller University.

Generation of mouse immune system (MIS) NSG- chimeras

To obtain bone marrow, femur and tibia of 6-week-old C57BL/6 mice were flushed with PBS (Gibco, Invitrogen). Newborn conventional non-HLA transgenic NSG mice were irradiated with 100cGy and 1.5-2×105 C57BL/6 derived bone marrow cells were injected intrahepatically 6h after irradiation. 12 weeks after transplantation murine NK, T and B cell reconstitution was analyzed by flow cytometry.

Production of adenoviruses

Adenoviral constructs encoding firefly luciferase (Fluc) or HCV proteins were created using the AdEasy™ Adenoviral Vector System (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. Briefly, Fluc, HCV1-NS3 and –NS5B genes were PCR-amplified from Jc1FLAG2(p2Fluc2A) (23) or HCV1/SF (kindly provided by Dr. Robert Lanford) plasmids respectively and inserted into the pShuttle-CMV™ using KpnI/XhoI sites (SalI/EcoRV) for NS5B). Recombinant pShuttle-CMV plasmids were linearized with PmeI and ligated to pAdEasy™ by homologous recombination followed by electroporation into BJ5183 cells (Agilent). Recombinant pShuttle-pAdEasy constructs were identified by PacI restriction analysis. All plasmid constructs were verified by DNA sequencing. For the production of virus stocks adenoviral constructs were transfected into HEK293 cells using the calcium-phosphate method. Transfected cultures were maintained until cells exhibited full cytopathic effect (CPE), then harvested and freeze-thawed. Supernatants were serially passaged two more times with harvest at full CPE and freeze-thaw. For virus purification, cell pellets were resuspended in 0.01M sodium phosphate buffer pH7.2 and lysed in 5% sodium-deoxycholate, followed by DNAse I digestion. Lysates were centrifuged, supernatant were layered onto a 1.2-1.46 g/ml CsCl gradient and the virus was separated by ultracentrifugation. Adenovirus bands were isolated and further purified on a second CsCl gradient. Resulting purified adenoviral bands were isolated and twice-dialyzed against 4% sucrose. Adenovirus concentrations were measured at 1012 times the OD260. Adenovirus stocks were stored at −80°C. All mice were infected intravenously with 5×109 or 1010 particles of recombinant AdV5.

In vivo Imaging

For in vivo imaging mice were anaesthetized and injected intraperitoneally with 200μl (1.5 mg/ml) luciferin (Caliper Lifesciences). Bioluminescence was analyzed 5 min after luciferin injection for a period of 1 min using an IVIS Lumina II system (Caliper Lifesciences).

Cell isolation

Cells were isolated exactly as described previously (22). Briefly, blood-derived leukocytes were isolated by Ficoll-density gradient (Cellgro) centrifugation (20min, 2000rpm); spleen- and liver derived leukocytes were isolated by a collagenase IV digestion (HBSS, 0.1% collagenase IV, 40mM HEPES, 2M CaCl2 and 2U/ml DNAse I) (30min, 37°C) of the minced liver and spleen tissue followed by Ficoll-density gradient centrifugation. Isolated leukocytes were washed twice in PBS and directly analyzed. For the analysis of thymocytes and thymical epithelial cells the thymus was homogenized through a cell strainer (100 μm, BD) to obtain a single cell suspension. For the isolation of hepatocytes mice were anaesthetized (100 mg/kg ketamine/xylazine) and the liver was perfused through the vena cava by a collagenase solution (4.8 mM CaCl2, 100 U/ml collagenase type IV, 0.05 M HEPES pH 7.3 in Ca/Mg-free HBSS). The perfused liver tissue was subsequently passed through a cell strainer (100 μm, BD), washed twice in HBSS and fixed in 4% paraformaldehyde.

Peptides and Tetramers

The HLA-A2 restricted HCV1 peptides HCV-NS3 1073-1081 (CINGVCWTV), NS3 1406-1415 (KLVALGINAV) and HCV NS5B 2594-2602 (ALYDVVTKL) and the AdV5-hexon overlapping peptide library were generated at the Rockefeller University Proteomics Resource Center. HLA-A2 tetramers corresponding to the HCV peptides were obtained from the NIH Tetramer Core Facility (Emory University, Atlanta, GA).

Antibodies

The following anti-human antibodies were used: CD45-Pacific-Orange, CD14-Alexa-Fluor-700, CD16-Pacific-Blue, CD3-PE-Texas-Red, CD38-PE-Texas-Red (Invitrogen Corporation); CD8-FITC, CD19-APC, CD4-PE, CD33-PerCP-Cy5.5, CCR7-PE, HLA-DR-APC, Granzyme-B-Alexa-Fluor-700, HLA-A2-FITC (BD Biosciences); CD1c-Pacific-Blue, CD3-APC-eFluor780, CD8-APC-eFluor780, CD45RO-PerCP-eFluor710, CD27-Pey7, CD68-PE, CD127-PeCy7, PD-1-PerCP-eFluor710, IFN-γ-PeCy7, TNF-α-FITC and IL-4-PE (eBioscience); CD4-Pacific-Blue, CD56-FITC, CD62L-Alexa-Fluor-700, IL-17A-Alexa647, Perforin-PerCP and FoxP3-Alexa647 (Biolegend). The following anti-mouse antibodies were used: CD45-PeCy7, epCAM-PeCy7, F4/80-PE, CD49b-Alexa-Fluor488, CD80-Pacific-Blue, CD86-PerCP-Cy5.5 (Biolegend); CD3-APC-Cy7, H2kD-PE, (BD Biosciences); B220-Alexa-Fluor700 (Invitrogen); CD11b-PeCy7 (eBioscience). Appropriate isotype controls were also purchased from each company.

Flow cytometry

Antibody staining, MHC tetramer staining and FACS analysis was performed exactly as described previously (22, 24) using a LSRII Flow Cytometer (BD Biosciences).

In vivo T cell depletion

Human T cells were depleted from humanized mice (12 weeks post HSC transplantation) by i.p. injection of 100 μg Okt-4 and Okt-8 (BioXCell) on three consecutive days. T cell depletion was confirmed by FACS analysis. To maintain T cell depletion for the duration of the experiment (20 days), Okt-4 and Okt-8 treatment was repeated 10 days later.

Generation of bone marrow derived macrophages

Bone marrow cells were isolated from femur and tibia of NSG and NSG-A2/DR1 mice. L929 cells were used as a source of granulocyte/macrophage colony stimulating factor. For the generation of bone marrow derived macrophages (BMDM), bone marrow cells were cultured in L929 cell conditioned medium (DMEM (Gibco, Invitrogen) with 20% FBS, 30% supernatant from confluent L929 cells and 1% penicillin/streptomycin). After 7-10 days of culture 90% of cells stained positive for the macrophage-marker F4/80 and CD11b.

Macrophage-T cell cocultures

NSG and NSG-A2/DR1 derived BMDM were infected with 5×103 particles AdV5-Fluc/cell. Infection of BMDM was verified by testing cell lysates for firefly luciferase activity 48h post infection (Luciferase assay systems, Promega). Human T cells were isolated from AdV5-infected (day 20 post infection) and control HIS and HIS-A2/DR1 mice by using a human CD3 T cell MACS selection kit (Miltenyi Biotech). T cell purity post selection as determined by FACS analysis was ≥97%. T cells were cocultured with AdV5-infected or control BMDM (5:1 T/BMDM ratio) for 5 days in complete medium (RPMI (Gibco, Invitrogen) with 10% FBS, 1.5% HEPES and 1% penicillin/streptomycin). Supernatants were collected and analyzed for concentration of human IFN-γ and TNF-α (cytometric bead array, BD Biosciences).

Peptide-specific T cell stimulation

Leukocytes were isolated from blood, spleen and liver of AdV5-Fluc, AdV5-HCV-NS3 or AdV5-HCV-NS5B infected HIS and HIS-A2/DR1 mice and control groups 14 or 20 days post infection. Pooled blood-, spleen- and liver-derived leukocytes were plated on a 96 well plate and stimulated with 10μg/ml HCV-specific peptides, 10μg/ml AdV5-hexon peptide pools or PMA/Ionomycin (positive control) in the presence of Golgiplug (BD Biosciences). After 5h of incubation at 37°C cells were analyzed for intracellular production of INF-γ and TNF-α by flow cytometry. Cells were gated on human CD45, CD3 and CD4 or CD8 expression.

Neutralization assays

Serum from AdV5 infected (day 20-30 post infection) and control Balb/c, HIS and HIS-A2/DR1 mice was diluted 1:2, 1:10, 1:100 and 1:1000 in DMEM and incubated with 5×105 particles AdV5 for 1h at 37°C. Serum-virus mixtures were subsequently added to 105 HeLa cells. After 30 min of incubation HeLa cells were washed twice with DMEM and cultured in fresh medium. 24h later firefly luciferase activity in cell lysates was analyzed (Luciferase assay systems, Promega). Percentage of neutralization was calculated using the following equation: 100-(average # of sample/average # of control ×100).

Statistics

Unpaired Student’s t test was used to evaluate statistically significant differences (GraphPad Prism).

Results

Human immune system reconstitution in HLA transgenic mice

The expression of HLA improves human adaptive immune responses during lymphotropic virus infection in humanized mice (16, 17). In this study, we aimed to analyze the extent of functional human immune responses during a hepatotropic virus infection and the role of HLA expression in this context. First, we performed a comparative analysis of human immune system reconstitution efficiency in conventional NSG mice and a novel NSG mouse strain transgenic for HLA-A*0201 (HLA-A2) and a chimeric human HLA-DR*0101 (HLA-DR1), termed NSG-A2/DR1. In these mice HLA-A2 is ubiquitously expressed while HLA-DR1 is under the control of a murine MHC class II promoter, ensuring physiological expression only on lymphoid tissue. We confirmed the tissue-specific expression of the human transgenes in thymus, spleen, and liver of NSG-A2/DR1 mice by flow cytometry (Fig. 1A). Transplantation of human fetal liver-derived CD34+ HSC into sublethally irradiated NSG and NSG-A2/DR1 neonates resulted in comparable reconstitution of a human immune system in both mouse strains 12 weeks later (Fig. 1). Numbers and frequencies of human leukocytes, T cells, B cells, myeloid cells and NK cells in blood, spleen and liver (Fig. 1B-D) reflected what has been described previously for the NSG strain (16, 22). We refer to NSG mice engrafted with a human immune system as “HIS” mice and reconstituted NSG-A2/DR1 mice as “HIS-A2/DR1” mice. All NSG-A2/DR1 mice received HLA-A2+ HSC but only some fully matched HLA-A2+/HLA-DR1+ HSC due to limited availability. Figure 1 and 2 show combined data of HIS-A2/DR1 mice reconstituted with either HLA-A2+ or HLA-A2+/HLA-DR1+ HSC. As shown in supplementary figure 1A-C we did not detect statistically significant differences in the reconstitution of human immune cells between mice that received HLA-A2+ or HLA-A2+/HLA-DR1+ HSC. Table I further shows the human CD45+ cell reconstitution of groups of mice reconstituted with individual human HSC donors.

Figure 1. Human immune system reconstitution in HLA class I and class II double-transgenic mice.

Figure 1

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The tissue-specific expression of HLA-A2 and HLA-DR1 on murine thymical epithelial cells (epCAM+), splenocytes (H2-Kd+) and hepatocytes (albumin+) derived from NSG and NSG-A2/DR1 mice was determined by flow cytometry. Representative FACS plots are shown in (A). HIS (human immune system) mice were generated by transplantation of human CD34+ HSC into newborn NSG mice and NSG-A2/DR1 mice. 12 weeks post transplantation blood, spleen and liver were analyzed for human immune system reconstitution. (B) Total human CD45+ leukocytes, CD3+ T cell and CD19+ B cell numbers in spleen and liver of HIS and HIS-A2/DR1 mice. (C) Representative FACS plots showing the peripheral frequency of human leukocytes (hCD45+) within the total leukocyte population (upper left) and the frequencies of CD3+ T cells, CD19+ B cells (upper right) CD56+CD16+/− NK cells (lower left) and CD33+CD14+/− myeloid cells (lower right) within the human CD45+ cell population of HIS-A2/DR1 mice. (D) Group data showing the frequencies of B cells, T cells, NK cells and myeloid cells in blood, liver and spleen of HIS versus HIS-A2/DR1 mice. Summarized data of n=8 HIS and n=10 HIS-A2/DR1 mice are shown. Data represent mean+/−SEM. Statistically significant differences were analyzed using unpaired student’s t test

Figure 2. Tissue distribution of human T cell subsets in HIS and HIS-A2/DR1 mice.

Figure 2

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12 weeks after human CD34+ HSC transplantation the phenotype and function of human T cells present in HIS and HIS-A2/DR1 mice was analyzed by flow cytometry. Frequencies of human CD4+ and CD8+ T cells in blood, spleen and liver of HIS and HLA-transgenic HIS mice are shown in (A). To determine the percentage of effector T cells, CD4+ and CD8+ T cells were stained for the expression of CD45RO, CCR7, CD62L and HLA-DR. (B) Frequencies of human CD45RO+CCR7-CD62L-HLA-DR+ effector CD4+ (left) and CD8+ (right) T cells. (C) Representative FACS plot showing CD45RO expression on CD4+ T cells (left) and CD62L and CD45RO expression on gated CD8+ T cells (right) from HIS-A2/DR1 mice. (D) Histograms comparing the expression of CCR7 (left) and HLA-DR (right) on CD45RO+CD8+ effector T cells (black line) and CD45RO-CD8+ naïve T cells (gray solid). To determine the ability of human CD4+ and CD8+ T cells to produce effector cytokines and molecules cells were stimulated with PMA/Ionomycin and analyzed for the intracellular production of IFN-γ, TNF-α, granzyme B, perforin, IL-17 and IL-4. E) Group data showing the frequency of cytokine-producing CD8+ T cells (upper graphs) and CD4+ T cells (lower graphs) in blood, spleen and liver of HIS and HIS-A2/DR1 mice. (F) FACS plots showing granzyme B and IFN-γ expression of TNF-α+ CD8+ T cells from blood and liver of HIS-A2/DR1 mice. Summarized data of n=8 HIS and n=10 HIS-A2/DR1 mice are shown. Data represent mean+/−SEM.

Tissue distribution of human T cell subsets in HIS and HIS-A2/DR1 mice

The presence of functional T cells at the site of infection is essential for viral clearance. Little is known about the tissue distribution of different T cell subsets in humanized mice. Thus, we characterized human T cells present in blood, spleen and liver of HIS and HIS-A2/DR1 mice 12 weeks after human CD34+ HSC transplantation. Within the CD3+ population, CD4+ T cells comprised about 60-70% and CD8+ T cells about 30-40%; there was no significant difference in this ratio between different tissue compartments and the two mouse strains (Fig. 2A). About 15-20% of CD4+ and CD8+ T cells displayed a CD45RO+CD62L-CCR7-HLADR+ effector T cell phenotype (Fig. 2B-D).

We further analyzed the ability of T cells to produce IFN-γ, TNF-α, IL-4 and IL-17, granzyme B and perforin. After nonspecific stimulation with PMA/ionomycin, an average of 10-20% of blood-, spleen-, and liver-derived CD8+ T cells produced IFN-γ and granzyme B, while perforin production was almost undetectable (Fig. 2E). The most abundant cytokine produced by CD8+ T cells was TNF-α, specifically in blood (mean HIS: 30%, mean HIS-A2/DR1: 47%, p=0.02) and spleen (mean HIS and HIS-A2/DR1: 30%) (Fig. 2E). A large fraction of TNF-α+ CD8+ T cells also produced IFN-γ and granzyme B, highlighting the polyfunctionality of CD8+ T cells (Fig. 2F). About 20% of CD4+ T cells in blood, spleen and liver produced IL-4, while <10% produced IFN-γ. IL-17 producing cells were detectable only at low frequencies (Fig. 2E). Similar to the CD8+ T cell population, TNF-α was the most abundant cytokine produced by blood- and spleen-derived CD4+ T cells (Fig. 2E). In summary, these data show that different functional human T cell subsets are detectable in the blood, spleen and liver of HIS and HIS-A2/DR1 mice.

Improved in vivo clearance of a hepatotropic adenovirus in HIS-A2/DR1 mice

To analyze the extent of functional intrahepatic human immune responses in HIS and HIS-A2/DR1 mice, we established a model of a hepatotropic virus infection based on a replication incompetent adenovirus serotype 5 expressing firefly luciferase (AdV5-Fluc). Adenoviral vectors efficiently target the liver and induce strong innate and adaptive immune responses in immunocompetent mice, which result in clearance of the virus and loss of transgene expression (25-27). Intrahepatic infection kinetics could be readily monitored longitudinally by bioluminescent imaging (Fig. 3A). The luciferase reporter signal, which was readily detectable in the liver following infection of immunocompetent Balb/c mice with 1010 AdV5-Fluc particles intravenously, peaked shortly after infection and dropped below the limit of detection (105 photons/s) within 10 days (Fig. 3A-B). When HIS and HIS-A2/DR1 mice were infected with AdV5-Fluc 12 weeks post CD34+ HSC transplantation, substantially higher levels of luciferase were expressed in the liver at day 10 and 20 post-infection as compared to the Balb/c cohort (Fig. 3A-B). Together, this suggests that the human immune system does not efficiently control adenoviral infection in the liver of humanized mice.

Figure 3. Improved in vivo clearance of a hepatotropic adenovirus in HIS-A2/DR1 mice.

Figure 3

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Balb/c, MIS-NSG, HIS and HIS-A2/DR1 mice (12 weeks post HSC transplantation) were infected intravenously with 1010 particles of a hepatotropic replication-deficient E1/E2-deleted adenovirus serotype 5 expressing firefly luciferase (AdV5-Fluc). Fluc-expression was longitudinally monitored using an in vivo imaging system (IVIS) and quantified in photons/s. (A) Representative images showing the hepatic Fluc expression in Balb/c, MIS-NSG, HIS and HIS-A2/DR1 mice at day 1, 10 and 20 post infection. (B) Intrahepatic AdV5-Fluc clearance in groups of Balb/c (n=6), MIS-NSG (n=4), HIS (n=10) and HIS-A2/DR1 (n=10) mice quantified in photons/s. C) Comparison of Fluc-expression in HIS, HIS-A/DR1 and non-reconstituted NSG mice at day 10 and 20 post infection. (D) Comparison of Fluc-expression in HIS, HIS-A/DR1 and non-reconstituted NSG mice at day 20 and 40 post infection with 5×109 AdV5-Fluc particles. (E) Fluc-expression in T cell depleted HIS (n=5) and HIS-A2/DR1 (n=5) mice compare to control HIS (n=5) and HIS-A2/DR1 (n=5) mice at day 10 post AdV5 infection. Data represent mean+/SEM. Unpaired student’s t test: * p ≤ 0.05

To test whether the humanization procedure itself (irradiation and intrahepatic injection of neonates) interferes with the ability of chimeric animals to clear virus-infected cells from the liver, we transplanted NSG mice with MHC-mismatched C57BL/6 bone marrow cells. Twelve weeks post-transplantation, NSG recipients were engrafted to high levels with a heterologous mouse immune system (MIS), including NK, B and T cells (data not shown). We found that MIS-NSG mice were able to clear AdV5-Fluc-infected cells in the liver, albeit with slightly slower kinetics than Balb/c mice (Fig. 3A-B). The delay may be attributable to the MHC mismatch.

Interestingly, the photon-flux in HIS-A2/DR1 mice decreased significantly between day 10 and day 20 while it remained stable in HIS mice (mean HIS day 20: 1×108, mean HIS-A2/DR1 day 20: 1.6×107) (Fig. 3B). Further, compared to HIS mice and the non-reconstituted NSG background strain, HIS-A2/DR1 mice showed significantly reduced photon-flux at day 10 and day 20 post-infection (Fig. 3C). In separate groups of mice we analyzed luciferase expression up to day 40 post-infection (infectious dose: 5×109 particles) and detected a further significant decrease in HIS-A2/DR1 mice (Fig. 3D). However, at this time point a reduction in luciferase expression even in non-reconstituted NSG mice was observed. Also not statistically significant in these mice (Fig. 3D), this observation might indicate a general loss of the viral vector at time points beyond day 40 hence we stopped the kinetic study at day 40.

In sum, these results suggest a beneficial effect of HLA expression on the ability to clear the adenovirus from the liver of humanized mice. To determine whether this effect is directly mediated by T cells we depleted human CD4+ and CD8+ T cells in HIS and HIS-A2/DR1 mice prior to AdV5-Fluc expression. In HIS-A2/DR1 mice T cell depletion abrogated the observed reduction of luciferase expression at day 10 (Fig. 3E) and day 20 (data not shown) post-infection while in HIS mice T cell depletion had no significant effect (Fig 3E). This data clearly indicate that the partial clearance of an adenovirus infection from the liver of HIS-A2/DRR1 mice is mediated mostly by human T cells.

Data presented in figure 3 summarize the combined results of HIS-A2/DR1 mice reconstituted with either HLA-A2+ or HLA-A2+/HLA-DR1+ HSC. As shown in supplementary figure 2A we could not detect statistically significant differences between mice that received HLA-A2+ or HLA-A2+/HLA-DR1+ HSC. Table I further shows the average photon-flux (day 10 post infection) of groups of mice reconstituted with individual human HSC donors. Together, these findings suggest that HLA-DR1 expression does not play an essential role in the partial in vivo adenovirus clearance in HLA transgenic mice. Unless stated otherwise all following figures also show combined data of HIS-A2/DR1 mice reconstituted with either HLA-A2+ or HLA-A2+/HLA-DR1+ HSC. Significant differences were not observed between both groups.

Induction of functional human humoral immune responses during adenovirus infection

To further characterize the antiviral human immune responses during adenovirus infection on a cellular level, we analyzed human leukocytes isolated from blood-, spleen- and liver in HIS and HIS-A2/DR1 mice at day 10 following infection. In accordance with previously published data (13), innate human immune responses were limited in humanized mice (Fig. 1D). Nevertheless, we observed a significant increase in intrahepatic human CD68+ macrophages (from about 5% to 11%) in both HIS and HIS-A2/DR1 mice during infection (Fig. 4A-B). NK cell frequencies in blood, spleen and liver, however, did not change upon infection in HIS (data not shown) or HIS-A2/DR1 mice (Fig. 4C).

Figure 4. Increase of intrahepatic human B cells and macrophages during adenovirus infection.

Figure 4

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10 days post infection of HIS and HIS-A2/DR1 mice with 1010 particles AdV5-Fluc, the infiltration of human B cells, macrophages and NK cells into the liver was examined by flow cytometry. (A) Percentages of human CD68+ macrophages in the liver of virus-infected HIS (n=5), HIS-A2/DR1 (n=7) mice and controls (n=3 each). (B) Representative FACS plots showing intrahepatic CD68+ macrophages of HIS-A2/DR1 mice. (C) Frequencies of human CD3+CD56+ NK cells in blood, spleen and liver of HIS-A2/DR1 mice. (D) Frequencies of human CD19+ B cells in blood, spleen and liver of AdV5-infected HIS (n=6) and HIS-A2/DR1 (n=7) mice as compared to the control groups (n=8 each). (E) Representative FACS plot showing the frequency of CD19+ B cells in the liver of AdV5-infected HIS-A2/D1 mice and control.

The frequency of B cells significantly increased in the liver of HIS-A2/DR1 mice (mean control: 47%, mean AdV5: 57%, p=0.003) at day 10 post-infection, but decreased in the blood (mean control: 43%, mean AdV5: 32%, p=0.01) (Fig. 4D-E). These findings suggest that human B cells are recruited into the infected liver of HIS-A2/DR1 mice. We also determined human IgM and IgG serum concentrations in infected mice and control groups at day 10 and 20 post-infection. Serum IgM levels remained constant (approximately 50 μg/ml) in both HIS and HIS-A2/DR1 mice during the course of infection (Fig. 5A). While overall human IgG levels were very low in both strains (Fig. 5B), they were significantly higher in uninfected HIS-A2/DR1 mice as compared to HIS mice (HIS mean: 0.07μg/ml, HIS-A2/DR1 mean: 0.7μg/ml, p=0.01). IgG concentrations further increased at day 20 post AdV5 infection (mean: 4μg/ml) (Fig. 5B), indicating improved production of human IgG in HIS-A2/DR1 mice. Thereby, reconstitution with HLA-A2+/HLA-DR1+ HSC resulted in slightly elevated IgG levels but this trend did not reach statistical significance (supplementary figure 2B).

Figure 5. Increase of total human IgG and generation of neutralizing antibodies during adenovirus infection.

Figure 5

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The concentration of human IgM (A) and IgG (B) in sera of AdV5-infected HIS (n=5) and HIS-A2/DR1 mice (n=8) (days 10 and 20 post infection) and control groups (n=5 each) was quantified by cytometric bead array. (C) The presence of AdV5-Fluc-specific neutralizing antibodies in the serum of AdV5-infected Balb/c (n=3), HIS (n=4) and HIS-A2/DR1 (n=4) mice (20-30 days post infection) and control mice was determined by incubation of virus with serum dilutions (1:2-1:1000) and subsequent infection of HeLa cells. 24 h after infection firefly luciferase activity in HeLa cell lysates was determined and neutralization efficiency was calculated. Data represent mean + SEM.

To test whether HIS and HIS-A2/DR1 mice produce virus-specific antibodies, we tested sera from AdV5-Fluc infected mice (20-30 days post infection) and control groups for the capacity to neutralize AdV5 infection in vitro. As expected, serum of AdV5-Fluc immune but not naive Balb/c mice efficiently neutralized the virus at dilution of 1:2-1:1000 (Fig. 5C). The serum of about 50% of HIS and HIS-A2/DR1 mice tested had a similar capacity to neutralize AdV5-Fluc at a 1:2 and 1:10 dilutions (Fig. 5C). These results indicate that both humanized mouse strains are able to generate antigen-specific human antibodies that partially neutralize AdV5-Fluc in an in vitro assay.

Adenovirus infection induces liver-infiltration and activation of human T cells

We next characterized the induction of human T cell responses during AdV5 infection. While frequencies and total numbers of intrahepatic CD3+ T cells were only slightly elevated HIS mice at 10 days post-infection (mean control: 30%, mean AdV5: 39%, p=ns), those values were significantly increased in HIS-A2/DR1 mice (mean control: 27%, mean AdV5: 45% p=0.0005) (Fig. 6A-B). The ratio CD4+ to CD8+ T cells remained unchanged (data not shown). Within the blood- and liver-derived CD4+ and the CD8+ populations from HIS-A2/DR1 mice, the percentage of CD45RO+ effector T cells was elevated at day 10 post-infection; this was most pronounced in the intrahepatic CD8+ T cell population (p=0.01) (Fig. 6C-D). In HIS mice, we also observed an increase in CD45RO+ T cells after AdV5 infection, although this did not reach statistical significance (data not shown). CD4+ and CD8+ effector T cells, particularly in the livers of infected HIS-A2/DR1 mice (Fig. 6E) were highly activated, as indicated by the expression CD38, HLA-DR or both on greater than 90% of these subsets. Significant increase in human CCL5 serum concentration at day 3 and 10 post-infection provides additional evidence for T cell activation (HIS-A2/DR1 mean control: 26 pg/ml, mean day 3: 96 pg/ml, mean day 10: 85 pg/ml, p=0.01) (Fig. 6F). CCL5 is expressed by activated T cells and plays an important role in the recruitment of leukocytes to the site of infection. Of note, we did not detect changes in the serum concentration of IFN-α, IFN-γ, IL-12, TNF-α, IL-10, IL-6, IL-1b, IL-8, CXCL9, CCL2 or CXCL10 (data not shown).

Figure 6. Adenovirus infection induces liver-infiltration and activation of human T cells.

Figure 6

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HIS and HIS-A2/DR1 mice were infected i.v. with 1010 particles of AdV5-Fluc. At day 10 post infection human T cell activation in blood, spleen and liver was analyzed by flow cytometry. (A) Group data showing the frequency of human CD3+ T cells in blood, spleen and liver of AdV5-infected HIS and HIS-A2/DR1 mice as compared to the control groups. (B) Total human T cell numbers in the liver of AdV5-infected HIS and HIS-A2/DR1 mice and control groups. (C) Percentages of human CD45RO+ effector CD4+ (left graph) and CD8+ (right graph) T cells in blood, spleen and liver of virus-infected HIS-A2/DR1 mice. (D) Representative FACS plots showing the expression of CD45RO and HLA-DR on intrahepatic CD8+ T cells in control and AdV5-infected HIS-A2/DR1 mice. (E) Activation status (HLA-DR and CD38 expression) of human CD45RO+CD4+ (left graph) and CD8+ (right graph) T cells derived from blood, spleen and liver of uninfected and virus-infected HIS-A2/DR1 mice. (F) Serum concentration of human Rantes/CCL5 at day 1, 3 and 10 after AdV5 infection in HIS and HIS-A2/DR1 mice. Summarized data of n=8 control HIS, n=6 AdV5 HIS, n=10 control HIS-A2/DR1 and n=7 AdV5 HIS-A2/DR1 are shown. Data represent mean+/SEM.

We then focused on the expression profiles of CD127 and programmed death-1 (PD-1) to phenotype human T cells in humanized mice during AdV infection. CD127, the IL-7 receptor alpha chain, is highly expressed on naïve T cells and long-lived memory T cells but down-regulated on effector cells after antigen recognition (28). PD-1 is an inhibitory receptor that is transiently expressed on activated effector T cells during acute infection and highly up-regulated on exhausted T cells during chronic infection (29). We detected significant changes in CD127 and PD-1 expression within the peripheral T cell populations of HIS and HIS-A2/DR1 mice (Fig. 7A,B,D). In naïve mice, the majority of CD8+ and CD4+ T cells displayed a CD127+PD-1-phenotype (about 75-80% in both mouse strains). During virus infection (day 10 and day 20), CD127 expression was down-regulated and large subsets of CD127+PD1+, CD127-PD1- and CD127-PD1+ T cells emerged (Fig. 7A-B). These phenotypic changes were most significant in the CD8+ T cell population of HIS-A2/DR1 mice. Similar to blood-derived T cells, we detected changes in CD127 and PD-1 expression patterns on intrahepatic T cells at day 20 post infection (HIS-A2/DR1: Fig. 7C-D, HIS: data not shown). Taken together, these data provide further evidence for T cell antigen recognition and development of activated effector cells during AdV5 infection.

Figure 7. Change of CD127 and PD-1 expression patterns on human T cells during adenovirus infection.

Figure 7

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The expression of CD127 and PD-1 on blood-derived human CD8+ T cells (A) and CD4+ T cells (B) was analyzed in naïve HIS (n=6) and HIS-A2/DR1 (n=7) mice and on day 10 and 20 post AdV5 infection (1010 particles i.v.). (C) CD127 and PD-1 expression on intrahepatic CD8+ (left graph) and CD4+ (right graph) of uninfected A2-DR1 mice (n=4) and at day 20 post infection (n=5). D) Representative FACS plots showing CD127 and PD1 expression on blood- and liver-derived CD8+ T cells of HIS-A2/DR1 mice 20 days post infection and of controls. Data represent mean+/SEM. Unpaired student’s t test: * p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0001.

Generation of antigen-specific and HLA-restricted T cells during adenovirus infection

Finally, we analyzed whether the humanized mice could mount antigen-specific T cell responses during adenovirus infection. To assess the ability of human T cells to acquire antigen-specific effector functions when exposed to a virally infected mouse cells, we co-cultured T cells isolated from HIS and HIS-A2/DR1 mice (20 days post-infection) or control mice with AdV5-infected or control autologous bone marrow derived macrophages (BMDM) of NSG or NSG-A2/DR1 mice. We detected elevated concentrations of human TNF-α, but not IFN-γ, in the supernatants of T cells derived from AdV5-infected HIS-A2/DR1 mice and co-cultured with AdV5 infected BMDM (Fig. 8A). Of note, TNF-α levels were not increased in co-cultures of T cells derived from control mice and AdV5 infected BMDM (Fig. 8A). These results suggest antigen-specific cytokine secretion by in vivo primed human T cells when encountering an infected mouse cell. Next, we determined whether humanized mice generate antigen-specific T cell responses directed against antigens of the adenoviral vector. Specifically, we stimulated isolated leukocytes of AdV5-infected HIS-A2/DR1 mice for 5h with peptide pools specific for the AdV5 hexon protein followed by intracellular cytokine staining. Hexon-specific T cells responses were readily detectable in immunocompetent AdV5 infected mice and also in adenovirus-exposed humans (30, 31). In HIS and HIS-A2/DR1 mice we detected TNF-α production by CD4+ T cells, but not by CD8+ T cells, in responses to several hexon peptide pools (supplementary fig. 3A-B and data not shown) indicating the priming of antigen-specific CD4+ T cells directed against viral vector proteins in both mouse strains.

Figure 8. Generation of HLA-restricted antigen-specific T cells during adenovirus infection.

Figure 8

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(A) To analyze antigen-specific IFN-γ and TNF-α production, human CD3+ T cells isolated from AdV5 infected HIS (n=3) and HIS-A2/DR1 (n=4) mice (day 20 post infection) and controls (n=3 each) were co-cultured with AdV5 infected or control NSG or NSG-A2/DR1 bone-marrow derived macrophages (BMDM). Cytokine concentrations in supernatants after 3 days of co-culture are shown. (B-C) To analyze the generation of HLA-A2 restricted CD8+ T cell responses we infected HIS (n=3) and HIS-A2/DR1 (n=6) mice with adenoviruses expressing HCV-NS3 and HCV-NS5B. 14 days post infection pooled blood-, spleen- and liver-derived leukocytes were stained ex vivo with HLA-A2 restricted NS31073-1081 and NS5B2594-2602 tetramers. Representative plots from two individual mice (M1 and M2) are shown (B). Leukocytes from the same mice were also stimulated ex vivo for 5h with the cognate antigen followed by intracellular TNF-α and IFN-γ staining (C). Summarized data of HLA-A2 restricted HCV-specific CD8+ T cells responses are shown in (D).

To analyze the presence of HLA-A2 restricted CD8+ T cells, we infected HIS and HIS-A2/DR1 mice with adenoviruses expressing the HCV-NS3 or HCV-NS5B protein. NS3 and NS5B contain several well-described immuno-dominant HLA-A2 restricted epitopes (32, 33) and CD8+ T cells specific for these epitopes are readily detectable in HCV infected patients. We stained isolated pooled blood-, spleen- and liver-derived leukocytes of uninfected and AdV5-infected HIS and HIS-A2/DR1 mice directly ex vivo with HLA-A2 tetramers specific for NS31073-1081, NS31406-1415 and NS5B2594-2602. CD8+ T cells specific for NS31073-1081, but not the two other epitopes, were detectable in about 50% of infected HIS-A2/DR1 mice (Fig. 8B and D). Tetramer+ CD8+T cells were not detectable in non-transgenic (Fig. 8D) and uninfected mice (data not shown). To verify the positive tetramer results we stimulated leukocytes from the same mice for 5h with the cognate antigen followed by intracellular cytokine staining. CD8+ T cells from mice positive for the NS31073-1081 tetramer also produced TNF-α in response to the same antigen (Fig. 8C and D). These results indicate that hepatotropic virus infection in HLA-transgenic humanized mice can induce HLA-restricted T cell responses towards epitopes that have been described to be immuno-dominant in humans.

Discussion

Mice engrafted with a human immune system are becoming more widely used to study human infectious diseases. However, due to significant shortcomings in the function of the engrafted human immune system, a major topic in current humanized mouse research is the improvement of the model systems (5, 13). Transgenic HLA expression, for example, has been shown to improve human antiviral HLA-restricted T cell responses during human lymphotropic virus infections, such as EBV or dengue virus infection (17, 18). However, little is known about human immune responses during hepatotropic virus infection. One recent study demonstrated the generation of HCV-specific T cell responses in HCV-permissive mice reconstituted with both, a human liver and a human immune system (12). However, in this study the functional capacity of the engrafted human immune system was not evaluated and it remains unclear whether the observed immune responses could contribute to clearance of HCV infection in humanized mice (12).

In our study, we analyzed the extent of functional antiviral intrahepatic human immune responses in humanized mice, specifically focusing on the impact of ectopic HLA expression. To simplify a functional readout, we devised a hepatotropic virus infection system based on a replication-incompetent recombinant adenovirus serotype 5 (AdV5) expressing firefly luciferase. AdV5 is an attractive model virus because it efficiently infects the murine liver, and infection elicits significant innate and adaptive immune responses without being lethal or causing severe disease (23, 26).

Under steady state conditions, we detected significant numbers of human immune cells in the liver, in particular macrophages, B cells and polyfunctional T cells, suggesting homing of these immune cell subsets to the mouse liver. Of note, NK cells, which are abundant in the normal mouse and human liver (34), were detectable only at only low frequencies in the liver of humanized mice. This finding is in line with previous reports about general defects in human NK cell development in these mice (13).

During virus infection, we observed an improved capacity of humanized HLA-transgenic mice to clear AdV5 from the liver as compared to non-transgenic humanized mice. Indeed, these mice showed a significant loss of in vivo firefly luciferase expression over time that correlated with an intrahepatic accumulation of human macrophages, B cells and T cells, the induction of human T cell activation and the generation of antigen-specific B and T cell responses. T cell depletion abrogated the loss of in vivo firefly luciferase expression in HLA-transgenic mice clearly demonstrating the direct role of human T cells in mediating virus clearance in these mice.

Although antiviral immune responses were also detectable in non-HLA transgenic humanized mice, they did not correlate with in vivo virus clearance. These findings indicate that HLA expression in humanized mice enables the generation of partially functional adaptive human immune responses during virus infection of the mouse liver. HLA expression also enabled the generation of HLA-A2 restricted virus-specific T cells that were detectable by tetramer staining ex vivo.

Despite partial virus clearance and the generation of antiviral adaptive immune responses during adenovirus infection, HLA transgenic humanized mice failed to completely eliminate the virus from the liver, as indicated by a detectable firefly luciferase signal even at day 20-40 post-infection after infection with a low infectious dose of 5×109 particles. Remaining deficits in humanized immune system function may be responsible for this observation: human NK cells are present only at very low frequency; possibly impaired homing and recruitment of human immune cells through murine tissue; and limited inter-species cross-reactivity of chemokines and cytokines produced during the infection. The firefly luciferase adenovirus model system should serve as a useful and simple platform to evaluate future improvement strategies, such as human cytokine or chemokine expression.

To analyze immune responses and pathogenesis of clinically relevant hepatotropic pathogens, it is necessary to dually engraft donor matched human liver cells and human immune cells. Proof-of-concept for this approach has recently been established (12), but dually reconstituted animals are difficult to generate and are limited in numbers. It is also important to note that the liver sinusoidal endothelium, even in human liver chimeric mice, is of mouse origin. Liver sinusoidal endothelial cells (LSEC) play an important role in shaping intrahepatic immune responses by mediating antigen-presentation and immune cell homing into the liver (35, 36). Thus, insights into human immune cell migration through the liver endothelium and possible modes of antigen-presentation of murine LSEC to human T cells will be of great importance to evaluate the utility of dually engrafted mice for the study of human hepatotropic pathogens.

In conclusion, our study provides a detailed characterization of the extent of functional human antiviral immune responses during a hepatotropic virus infection in humanized mice. We show that the transgenic expression of HLA improves human adaptive immune responses during virus infection of the mouse liver and enables the detection of HLA-A2-restricted virus-specific CD8+ T cells. The experimental system used in our study should help to evaluate future strategies for the improvement of intrahepatic immune responses in humanized mice. These observations might guide improvements in dually reconstituted HBV or HCV permissive mice harboring both, human liver and immune system, and contribute to the generation a functional and reliable model for the preclinical evaluation of drug and vaccine candidates for human hepatotropic pathogens.

Supplementary Material

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Acknowledgements

We thank Ellen Castillo, Brenna Flatley and Tamar Friling for technical assistance, Dr. Julia Sable for administrative assistance and Dr. John Schoggins for helpful advice.

Funding

This study was supported in part by a Center for Translational Science Award (CTSA) Pilot Grant CCL3001018 (to AP), a CTSA grant UL1 RR024143 (to Rockefeller University), the National Center for Research Resources (NCRR), a component of NIH, a U19 AI057266 subcontract with Emory University (to CMR and AP), the National Institutes of Health through the NIH Roadmap for Medical Research, Grant 1 R01 DK085713-01, the Greenberg Medical Research Institute and the Starr Foundation. Funded in part also by a Grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health initiative (to AP and CMR). EB was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. AP is a recipient of an Astella Young Investigator Award from the Infectious Disease Society of America and a Liver Scholar Award from the American Liver Foundation.

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Supplementary Materials

1
NIHMS493897-supplement-1.eps (5MB, eps)
2
NIHMS493897-supplement-2.eps (2.2MB, eps)
3
NIHMS493897-supplement-3.eps (2.4MB, eps)
4
NIHMS493897-supplement-4.docx (154KB, docx)

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