Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection
The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins’ chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known.
We mutated mouse CMAH in the NOD/scid-IL2Rγc−/− (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8.
NSG-cmah−/− mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.
KeywordsCMP-N-acetylneuraminic acid hydroxylase NOD/scid-IL2Rγc−/− mice Hematopoietic stem cells HIV-1
CMP-N-acetylneuraminic acid hydroxylase
Human immunodeficiency virus type 1
Hematopoietic stem/progenitor cells
Peripheral blood immune cells
Recombinant adeno-associated viral vector
Sialic acid–binding Ig-like lectins
- WT mice
All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids that have multiple functions due to their location and diverse modifications . The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom by CMP-Neu5Ac hydroxylase (Cmah). The pattern of proteins glycosylation affects the physiology of the cell, cell-to-cell communication, adhesion, migration, recognition by other cells and antibodies . In infectious diseases, sialylation patterns influence how humans interact with some pathogens or viral vectors including HIV-1, malaria, influenza, and streptococcus [3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. In xenotransplantation and stem cell biology, it is a key factor for graft acceptance and preservation of self-renewing properties . Of the two most common sialic acids forms Neu5Gc is widely expressed on most mammalian tissues but has limited accumulation in human cells . The human deficiency of Neu5Gc is explained by an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase (CMAH), the rate-limiting enzyme in generating Neu5Gc in cells of other mammals . This deficiency also results in an excess of the precursor Neu5Ac in humans. This mutation appears universal to modern humans and happens to be one of the first known human-great ape genetic differences with an obvious biochemical readout. In particular, it is important for interaction with Sialic acid–binding Ig-like lectins, or Siglecs. Expression of such lectins vary in their specificity for sialic acid–containing ligands and are mainly expressed by cells of the immune system. For example, humans, compared to mice and rats, express a much larger set of CD33rSiglecs . CD33rSiglecs have immune receptor, tyrosine-based inhibitory motifs, and signal negatively . Interaction with Siglec-7 has the potential to also affect monocyte migration and function  along with T-cell activation [8, 20, 21]. During B cells activation and germinal center formation (GC), Siglecs are important for appropriate activation of B cells and responses to T-cell-dependent and independent antigens . In B-cell antigen receptor (BCR) engagement, interaction of CD22 and Siglec-G has been shown to inhibit the BCR signal . Most importantly, exposure to exogenous Neu5Gc is known to cause rapid phosphorylation of beta-catenin in both CMAH-overexpressing cells and bone marrow-derived mesenchymal stem cells, thereby inactivating Wnt/β-catenin signaling and, as a consequence, possibly forcing stem cells to lose pluri- or multipotency .
Immunodeficient mice transplanted with human hematopoietic stem/progenitor cells (HSPC) are an established model to study human-specific infections like HIV-1 . However, if a particular sialic acid residue is missing in a donor species (Neu5Gc) and present in the recipient, biologic consequences can be difficult to delineate. Exclusion of mouse Neu5Gc has the potential to improve immune responses to pathogens with non-human patterns of glycosylation like HIV-1 and HCV (hepatitis C virus) and to study the pathogenicity of human-like sialylated pathogen surfaces [12, 17, 26, 27].
To distinguish the effect of the expression of CMP-N-acetylneuraminic acid hydroxylase in mice on human HSPC biology, improve the development of a human immune system in mice, and study responses to HIV-1 infection, we generated mutation in exon 6 of the gene on NSG strain using CRISP/Cas9 technology . We compared original NSG and NSG-cmah−/− strains for multiple parameters and observed changes in the human lymphocyte phenotype and repertoire. Human lymphocytes generated from HSPC in a human-like sialylation environment exhibit persistence of naïve non-activated T-cell phenotypes and are more sensitive to HIV-1 mediated depletion of CD4+ T-cells. Alternatively, mature human lymphocytes derived from human peripheral blood expand more efficiently in the NSG-cmah−/− mice with higher levels of activation.
This new strain expands the utility of the NSG standard strain used to study human hematopoiesis and immunity and allows comparison of new viral vectors for gene therapy and sensitivity to a wider variety of pathogens.
Generation of NSG-cmah −/− mice
NSG-cmah −/− phenotype
Comparison of human immune system development after CD34+ hematopoietic stem/progenitor cell transplantation
Analysis of T and B cell repertoires in NSG-cmah and wild type NSG mice
Gaussian-like distribution patterns of IgH CDR3 length in NSG-cmah−/− mice were similar to wt NSG (Fig. 4a, and b) mice. In contrast, the frequency of clonal B-cell expansion was more common in the wt NSG mice, and the difference was close to statistical significance. A similar size distribution pattern of IgH CDR3 length was observed in bone marrow samples evaluated for five NSG-cmah−/− mice. The NSG animals analyzed also had significant variability (Additional file 1: Figure S1b, two animals). The variability in NSG mice was also reflected by variable total number of templates (Additional file 1: Figure S1e). We observed lower diversity of B cell repertoires in NSG mice compared to NSG-cmah−/− strain according to higher clonality of IgH genes in bone marrow and spleen (Fig. 4c and Additional file 1: Figure S1c). The average IgH CDR3 length naturally generated by the rearrangement machinery was found to be reduced during B cell development, and we observed a slight shift to the left of CDR3 length in 4 of 5 analyzed NSG-cmah−/− mice by comparing bone marrow and spleen compartments (Additional file 2: Figure S2). The use of IgH genes families was similar in spleen and bone marrow (Additional file 3: Figure S3C) and correspond to the known human peripheral blood B cell data [38, 39]. We observed similar changes in IgH D and J gene family usage between bone marrow (pre- and immature B cells) and spleen compartment with mature B cells (Additional file 3: Figure S3C). The clonality of IgH gene loci were two folds higher in spleen compared to bone marrow, and in cmah−/− mice reached statistical significance (Fig. 4d).
Comparison of human immune system responses to HIV-1 infection in NSG-cmah −/− and wt NSG mice
Effects of NSG-cmah −/− phenotype on transplanted human peripheral blood mononuclear cells behavior
Host glycoproteins sialylation pattern affects the clearance of HIV-1 virus, rAAV2/DJ8 biodistribution but not half-life of infused human IgG
The clearance of sialylated glycoproteins going through the multiple types of receptors and changes of mouse sialylation could affect interaction with human immune globulins. We compared the time of circulation of human IgG in NSG-cmah−/− and wt mice (Fig. 8b). We were not able to determine the first minutes of injected IgG dose clearance, but overall clearance of human IgG was nearly identical in the two strains of mice. A slightly higher concentration remained in NSG-cmah−/− mice compared to NSG at the end of observation (1.1 ± 0.04 and 0.9 ± 0.03 μg/ml for NSG-cmah−/− and wt mice, respectively).
Biodistribution and expression levels of recombinant adeno-associated virus (rAAV) vector delivered genes could be affected by sialylation of host receptors [5, 10, 44]. We compared the luciferase expression delivered by rAAV2/DJ8. We were not able to detect statistically significant differences in luminescence in liver and spleen between the two strains at 7, 14, and 21 days post-inoculation (data not shown). However, at 32 days the expression of luciferase RNA was significantly higher in the spleen and brain of NSG-cmah−/− as determined by ddPCR (Fig. 8c). It was higher by 0.60 log10 copies in spleen (4.72 ± 0.12 vs 4.12. ± 0.07 log10) and 0.54 log10 in brain (1.40 ± 0.11 vs 0.85. ± 0.12 log10) of cmah−/− mice compared to NSG (P < 0.05).
We created NSG-cmah−/− mice with the intent to use this strain for different aspects of biomedical research. As initial phenotypic characterization, we compared several parameters involved in human immune system development in NSG-cmah−/− to the parent NSG strain. Glycoprotein-lectin interactions (for example, hematopoetic cells surface glycoporteins – bone marrow stromal cell lectins/selectins) are an important mechanism of human B-cell selection. The NSG mouse environment that contained Neu5Gc (non-human type of sialylation) exhibited a higher frequency of clonal B-cell outgrowth that may represent responses against the Neu5Gc moiety. In the absence of Neu5Gc, human B cells appear to remain less activated. This finding indicates that non-human sialylation has a negative effect on human B cell development, and the NSG-cmah−/−strain provides a more supportive environment with good repertoire development and less clonal outgrowth . The observations of lower IgD+ and CD22+ human B cells and sustained naïve CD45RA+CD3+ cells in the NSG-cmah−/− strain also support this conclusion.
We observed better bone marrow B-cell precursor engraftment in control NSG mice compared to NSG-cmah−/−, and better bone marrow T-cell engraftment in NSG-cmah−/−. The HIV-1 infected NSG-cmah−/− mice showed a significant reduction in bone marrow T-cells and a subsequent increase in bone marrow B-cell precursor engraftment. These findings may indicate T-cell suppression of B-cell engraftment is occurring in this mouse strain but will require further study to confirm. In contrast, the NSG strain showed similar levels of bone marrow T-cell and B-cell engraftment regardless of infection with HIV-1.
The T cell TCRβ chain repertoires in NSG-cmah−/− and wt NSG mice were not different in clonality metrics and remain polyclonal in bone marrow and spleen. However, compared to wt NSG mice, NSG-cmah−/− exhibited better evenness of repertoire between spleen and bone marrow. We did not observe statistically significant differences in clonal T-cell expansion based on TCRβ CDR3 length frequency in bone marrow versus spleen. This was previously reported for NOD/scid mice transplanted with cord blood derived hematopoietic CD34+ stem cells .
In the studies presented here, we are not showing effects of vaccination with the childhood vaccines DTaP, HiB, and MMRII, which were tested in comparison on both strains of humanized mice at 6 months of age. Only a few animals developed antigen-specific IgM. The inability of CD34-NSG humanized mice to efficiently respond to vaccination is related to multiple deficiencies recognized in the humanized animals. This includes the absence of supportive cytokines, human follicular dendritic cells responsible for accumulation of antigen and stimulation of germinal center B-cells, deficiency of follicular helper cells involved in T-cell dependent immune responses, deficiency of complement, and others (reviewed in ). Several approaches to improve human adaptive immune responses were explored by elimination of mouse tissue histocompatibility antigens and introducing human MHC class I and II, cytokines and co-transplantation of bone, spleen, liver, and thymus. These approaches improved adaptive responses to varying degrees. It is possible that the introduction of these additional factors on the NSG-cmah−/− background will further optimize the function of the human immune system and development of adaptive immune responses.
In addition to the comparison of human immune system cell development and phenotype, we assessed the behavior of human mature peripheral blood mononuclear cells in their ability to colonize mouse spleen and peripheral blood. In contrast to lymphocytes generated from human stem cells transplanted into NSG-cmah−/− mice, which showed reduced levels of activation, transplanted mature lymphocytes very quickly expand and lose the naïve CD45RA+ phenotype. This could be attributed to the selection of human T cells in mouse thymus and reduced responsiveness to the mouse MHC (major histocompatibility) antigens.
Sialic acid on cellular membrane molecules has been identified as an attachment receptor for several pathogens and toxins. The composition could influence the viral capture by different cell types and trans-infection . We observed increased HIV-1 mediated depletion of human CD4+ T-cells in the NSG-cmah−/− strain of mice compared to the parental NSG. We also found a reduction of circulating HIV-1 RNA in non-engrafted NSG-cmah−/− mice suggesting the viral particles may be more efficiently absorbed by cells with human-like sialylation patterns. We previously showed that mice transplanted with human hepatocytes also had enhanced clearance of virus from the circulation . The increased pathogenicity of HIV-1 in NSG-cmah−/− mice may be related to both the properties of human cells raised in the more human-like modified mouse environment as well as interaction of virus with the modified mouse stroma.
As humanized mice serve as a model to test biologic activity of human therapeutic antibodies [50, 51], we compared the clearance of human IgG. Human IgG contain oligosaccharides with N-acetylneuraminic acid (Neu5Ac, NANA); whereas, rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (Neu5Gc, NGNA). The asialoglycoprotein receptor on hepatocytes  and FcRn on endothelial cells and placenta  could potentially be affected by human-like sialylation of receptors. Human FcRn binds to human, rabbit, and guinea pig IgG, but not significantly to rat, bovine, sheep, or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG as previously analyzed . Our expectation was not confirmed, and the clearance of human Intravenous immunoglobulin (IVIG) injected intravenously in NSG-cmah−/− mice was not statistically different. It is possible that purified from blood donor serum IgG with the sialylated fraction contributing about 10% of the total IgGs was not the appropriate reagent for comparison. Future studies with known precise Neu5Ac content in the Fc region of IgG will help to understand applicability of the model . Another miscellaneous application of NSG-cmah−/− mice could be testing the transduction efficacy of viral vectors as well as viruses . We used a common vector of rAAV serotype 2 with luciferase and expected to see the differences of transduction efficacy between wt NSG and NSG-cmah−/− mice. We found the expression of luciferase was not different in liver (the major affected organ), but we did observe differences in less commonly affected organs, such as spleen and brain. These findings suggest that endothelial and splenic hematopoietic cells with human-like sialylation profiles could be more sensitive to viral infection.
Humanized mice are widely used to study the human immune system responses to pathogens and therapeutics. However, mouse specific glycosylation affects the development of the human immune system and responses to various agents, such as viruses or biological, human-specific products like antibodies. We demonstrated that human-specific sialylation established by mutation of the CMAH gene supports naïve B and T cell generation with polyclonal receptors repertoires. In contrast to NSG wild type mouse sialylation background, we found the NSG-cmah−/−background increased sensitivity to HIV-1 infection and influenced rAAV vector transduction patterns. As such, NSG-cmah−/− mice may accelerate translational research that target human infections and therapeutics development.
Material and methods
Strains obtained from the Jackson Laboratories C57Bl/6 (Stock No: 000664), CMAH knock-out (B6.129X1-Cmahtm1Avrk/J, Stock No: 017588), and NSG (Stock No:05557) were bred and housed in the pathogen-free animal facility at the University of Nebraska Medical Center (Omaha, NE, USA). Mice were housed in individually ventilated cages that are changed once every two weeks within a laminar flow work station using micro-isolator technique with ClidoxR-S sterilant. All caging, bedding (cob), and nesting materials were autoclaved prior to use. Water is R/O filtered, chlorinated, and supplied by the HydropacR system. Rodent diet used was autoclaved or irradiated prior to use.
Generation of B6.129X1-Cmahtm1Avrk/J referred hereafter as C57Bl/6-Cmah−/− was described previously . In this mouse, exon 6 was deleted by targeting a cassette containing LoxP flanked exon 6 with a neomycin cassette into 129/SvJ derived ES cells, followed by removal of the exon and the neomycin cassette through Cre enzyme and injection of deleted clone into blastcysts to generate chimera. The mice line was backcrossed to C57BL6/J strain for 10 generations.
CRISPR reagents, mice generation, and genotyping
We deleted exon 6 using CRISPR approach in NSG strain background. Two guide RNAs were identified to target part of exon 6 (Cmah gRNA 1 CTTTGTGCATTTAACGGACCTGG and Cmah gRNA 2 TGAAATATATCAACCCTCCAGGG; PAM sequences underlined and italicized). The sgRNAs were transcribed from DNA templates generated by annealing two primers using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA) following manufacturer’s instructions. Cas9 mRNA was prepared using the pBGK plasmid as described in . Injection mixture was prepared by dilution of the components into injection buffer (5 mM Tris, 0.1 mM EDTA, pH 7.5) to obtain the following concentrations: 10 ng/μl Cas9 mRNA, 10 ng/μl Cmah Left Guide and Right Guide RNA. Female NSG mice 3–4 weeks of age (JAX Laboratories, Bar Harbor, ME, USA) were superovulated by intraperitoneal injection with 2.5 IU pregnant mare serum gonadotropin (National Hormone & Peptide Program, NIDDK), followed 48 h later by injection of 2.5 IU human chorionic gonadotropin (hCG, National Hormone & Peptide Program, NIDDK). Mouse zygotes were obtained by mating NSG stud males with superovulated NSG females. The animals were sacrificed using CO2 inhalation followed by cervical dislocation 14 h following hcG administration, and oviducts were collected to isolate fertilized embryos. Injection mixture was introduced into the pronuclei and cytoplasm of fertilized NSG embryos by microinjection using a continuous flow injection mode. Surviving embryos were surgically implanted into the oviducts of pseudopregnant CD-1 recipient females. Genomic DNAs were extracted from tail biopsies and genotyping PCRs were performed as previously reported . Cmah Forward TCCCAGACCAGGAGGAGTTA and Cmah Reverse TCCACTCCGAGTTTCAGATCA primers were used for screening by PCR. The expected amplicon sizes were 455 bases and 428 bases respectively for wild type and mutant mice. The PCR products were column purified and were sequenced using the primers used for PCR amplification. NSG-cmah−/− mice were bred as homozygous.
Western blot and flow cytometry analyses of NeuGc expression
Tissue samples from NSG and NSG-cmah−/− mice were collected and homogenized in ice cold RIPA buffer with HALT protease inhibitor (cat#78430, ThermoFisher Scientific, Waltham, MA, USA). Twenty microgram of protein/sample was denatured in Laemmli sample buffer, then loaded and ran on a 12% SDS-polyacrylamide gel. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane, blocked in 0.5% cold fish gelatin in TRIS buffered saline with 0.05% Tween-20 (TBS-T) for 2 h at room temperature and then incubated in the primary antibody, Neu5GC (1:4000 in 0.5% cold fish gelatin TBS-T; chicken polyclonal IgY antibody, Biolegend Cat. No 146901) for overnight at 4 °C. After washes, the membrane was incubated in HRP-conjugated goat-anti-chicken IgY (GeneTex Cat. GTX26877, Lucerna-Chem AG, Switzerland) secondary antibody (1:4000) for 1 h at room temperature. The immunoblot was developed with chemiluminescence and imaged using FluorChem M imager (Proteinsimple, San Jose, CA, USA). GAPDH was used as housekeeping control (clone GA1R, cat #MA5–15738, Thermoscientific, Waltham, MA USA). For flow cytometry, whole blood was obtained by bleeding the mice from the facial vein. After spinning it down at 1800 rpm for 8 min and removing the excess serum, 50 μl of the 1:1 mixture of the whole blood and 0.5% gelatin from cold water fish was incubated in the Fc block for 15 min on ice. Next, the primary anti-Neu5Gc was added and incubated for 30 min on ice. Washing was done by re-suspending the samples in 1 ml of the 0.5% gelatin from cold water fish and spinning it at 1500 rpm for 5 min for 3 times. The secondary FITC-labeled anti-chicken reagent (ab46969) staining followed by red blood cells lysis using red blood cell lysis (Cat. #349202 from BD Bioscience, San Jose, CA) were performed. Further, acquisition was done on BD LSR II flow cytometer cells and analyzed on gated leukocytes using FLOWJO, analysis software (Tree Star, Ashland, OR, USA).
Organs collected from NSG-cmah−/−, NSG wt, and C57Bl/6 mice were fixed and paraffin embedded; 5 μ thick sections were stained with antibodies for Neu5Gc (anti-Neu5Gc antibody, Biolegend, San Diego, CA, USA, Cat. No: 146903, dilution 1:100) at 4 °C overnight. Secondary anti-chicken antibodies (Biolegend Cat. No. 146901) were used at dilution 1:100 and DAB as chromogen. Tissues counterstained with hematoxylin.
Human PBMC transplantation and engraftment analyses
NSG-cmah−/− and wild type NSG mice at 8 weeks of age were transplanted with single donor human PBMC intraperitoneally (20 × 106 cells/mouse). Blood samples were collected by facial vein bleeding for immunophenotyping at variable intervals (7, 14, 24, 31 days). Five-week post-injection, mice were sacrificed by overdose of isoflurane; blood, liver, and spleen tissues were collected for immunophenotyping and immunohistochemistry. Briefly, 50 μL of whole blood was stained in EDTA-coated tubes with two different monoclonal antibody panels (T cells and B cells) in a final volume of approximately 100 μl. Cells not stained with any of the antibody were initially used to define the gating strategy. Compensation beads (BD Biosciences, cat. #552843) were stained with each antibody separately and run at each acquisition to calculate the compensation matrix. Immunophenotyping was performed to determine the absolute count and frequency of blood cells: leukocytes (CD45, BD Biosciences, Cat. #555482); CD3 (#557943); CD4 (#560650); CD8 (#562428); CD19 (#562653) and CD14 (eBioscience, San Diego, CA, Cat. #17–0149-42) for blood and spleen, respectively. The gating strategy for identification of cell subsets is presented in Fig. 1a. Presence of activation markers CD45RA/CD45RO (BD Biosciences, #560674; #563215) and CD62L (#555544) was also checked for CD4+ and CD8+ T cells subpopulations, CD22 on B cells (#563940) in blood and spleen as absolute count and frequency of parent, respectively. After staining of cells, red blood cells were lysed with FACS Lysing solution (BD Biosciences), and cells were washed with phosphate buffered saline (PBS) and resuspended in 1% paraformaldehyde. For absolute counting of the blood samples, CountBrightTM absolute counting beads (Invitrogen, Carlsbad, CA, USA; catalog C36950) were added to each sample before acquisition. Acquisition of stained cells was performed on BD LSR II (BD Biosciences) using acquisition software FACS Diva (BD Biosciences), and data were analyzed using FLOWJO, analysis software (Tree Star). Event counts of each cell population were exported, and absolute count/μl of blood was calculated using the following formula: [(Number of cell events / number of bead events) × (assigned bead count of the lot (beads/50 μl) / Volume of sample)].
Human CD34+ cell transplantation and engraftment analyses
NSG-cmah−/− and wild type NSG mice reconstituted with the same cord blood sample derived CD34+ cells (5 × 104/mouse intrahepatically) were bled at three and again at 6 months of age. For both time points, ~ 100 μl of blood was collected into MiniCollect 0.5 mL EDTA tubes (Greiner Vacuette North America Inc., Monroe, NC, USA; Cat. #450475). Plasma was collected and stored at − 80 °C for future use. Remaining cell samples were diluted at a 1:1 ratio with 50 μl FACS buffer (2% fetal bovine serum in PBS) and mixed thoroughly. The samples were divided into two panels, B cell and T cell. The B cell panel consisted of mouse anti-human CD45-FITC (BD Pharmingen; Cat. #555482), CD19-BV605 (BD Biosciences; Cat. #562653), CD22 BV421(BD Pharmingen; #563940), IgG-PerCP/Cy5.5 (Biolegends, # 409312), IgD-PE (eBiosciences, #12–9868-42), IgM-APC (eBiosciences, #17–9998-42), and Brilliant Stain Buffer (BD Horizon, #563794) cocktails. The T cell panel consisted of: anti-human CD45-FITC (BD Pharmigen, #555482), CD3-Alexa Fluor 700 (BD Biosciences #557943), CD4-APC (BD Pharmingen #555349), CD8-BV421 (BD Horizons #562428), CD45RA-APC-H7 (BD Biosciences #560674), CD14-PE (BD Pharmingen #555398), and Brilliant Stain Buffer. After 30 min incubation at 4 °C, red blood cell lysis (BD Bioscience #349202) and two washes, samples were fixed with 2% paraformaldehyde and acquisition was performed on BD LSR II flow cytometer.
Animals with comparable peripheral blood repopulation with human leucocytes were infected with HIV-1ADA at 104 50% tissue culture infectious (TCID50) dose intraperitoneally. At four and 9 weeks post infection, animals were euthanized for the VL analysis (COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0, Roche Molecular Systems Inc., Pleasanton, CA, USA) and human cells phenotypes by FACS. Peripheral blood, spleen, and bone marrow cells were analyzed as described above.
Additional bone marrow human population analysis was performed by flow cytometry at the end of observation at 5–6 weeks post infection. Approximately 106 isolated bone marrow cells were aliquoted into three tubes for each mouse evaluated. The cells were washed once with 2 mL PBS and were then incubated with the 8 antibody cocktails for 30 min at room temperature in 200 uL of PBS with 10% fetal calf serum (PBS-FCS). The following antibodies were used in combination: T-NK cocktail consisting of APC-H7 conjugated anti-CD3 (clone SK7), PE-Cy7 conjugated anti-CD4 (clone SK3), PE conjugated anti-CD7 (clone 8G12), PerCP-CY5–5 conjugated anti-CD8 (clone SK1), FITC conjugated anti-CD14 (clone MϕP9), V450 conjugated anti-CD16 (clone 3G8), APC conjugated anti-CD56 (clone NCAM16.2), and V500c–conjugated CD45 (clone 2D1); MYELO cocktail consisting of APC-H7 conjugated anti-HLA-DR (clone L243), PE conjugated anti-CD10 (clone H10a), PE-Cy7 conjugated anti-CD13 (clone L138), APC conjugated anti-CD24 (clone 2G5), V450 conjugated anti-CD33 (clone WM53), FITC conjugated anti-CD34 (clone 8G12), PerCP-CY5–5 conjugated anti-CD117 (clone 95C3), and V500c–conjugated CD45 (clone 2D1); and the BCL tube consisting of FITC conjugated anti-kappa (clone TB28–2), PE conjugated anti-lambda (clone 1–155-2), PE-Cy7 conjugated anti-CD10, PerCP-Cy5.5 conjugated anti-CD19 (clone SJ25C1), APC-H7 conjugated anti-CD20 (clone L27), APC conjugated anti-CD24 (clone 2G5), V450 conjugated anti-CD38 (clone HB7), and V500c–conjugated CD45 (clone 2D1). All antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA) except CD24 and CD117, which were obtained from Beckman Coulter (Brea, CA, USA).
After incubation, the cells were washed twice with 1 mL PBS and were resuspended in 500 uL PBS containing stabilizer (BD Biosciences) to fix the cells. Fifty thousand to 100,000 cell events were collected for each sample on a Becton Dickinson FACS Canto II (Franklin Lakes, NJ, USA). Analysis of the FCS files was performed using Kaluza 1.3 analysis software (Beckman Coulter).
Gating schemes for the bone marrow analysis are shown in the Additional file 6: Figure S6.
For all bone marrow cell populations characterized, total cell events were derived based on gating and logical antigen (Boolean) profiles. Population frequencies were then derived by dividing the specific cell population events by the total human cell events after CD45 and singlet gating.
HIV-1 viral clearance
Viral clearance naïve non-humanized animals were injected with HIV-1 stock 3 × 103 TCID50/mouse), and blood was collected on days 1, 2, 5, and 7 post inoculation. Number of viral RNA copies were determined as described above.
Recombinant adeno-associated virus biodistribution
NSG-cmah−/− and wild type NSG mice were injected intravenously with rAAV2/DJ EF1a-luciferase 1.825E+ 11 GE/mouse (Viral Vector Core, University of Iowa, Iowa City, IA; Cat No. Uiowa-6161: Lot AAV3240; http://www.medicine.uiowa.edu/vectorcore/). D-Luciferin Bioluminescent Substrate (Cat 770,504, PerkinElmer, Waltham, MA, USA) was used for in vivo detection. The biodistribution of luminescence was analyzed by IVIS® Spectrum an in vivo imaging system at 1, 2, and 4 weeks after rAAV administration to validate and compare transduction efficiency. Liver, spleen, and brain tissues were collected at day 32 for detection of luciferase gene on droplet digital PCR (ddPCR) (QX200™ Droplet Digital™ PCR System, Bio-Rad, Hercules, CA, USA) with forward primer 5’CTTCGAGGAGGAGCTATTCTT-3′, reverse primer 5’-GTCGTACTTGATGAGAGTG-3′, and luciferase probe 5′−/56 FAM/TGCTGGTGC/ZEN/CCACACTATTTAGCT/3IABKFQ/− 3′ (Integrated DNA Technologies, Inc., Coralville, IA, USA). Briefly, isolation of RNA was performed using a RNeasy Plus Universal Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. The final PCR reaction was comprised of ddPCR supermix (Bio-Rad), 20 U/μl reverse transcriptase, 15 mM Dithiothreitol (DTT), 900 nM primers, 250 nM probe, and 100 ng of RNA template in a final volume of 20 μl and loaded into an eight-channel disposable droplet generator cartridge (Bio-Rad). Generated droplets were then transferred into a 96-well PCR plate, heat-sealed with foil and then amplified to endpoint using a BioRad C1000 Touch PCR cycler at 95 °C for 10 min then 40 cycles of 94 °C for 15 s and 60 °C for 1 min (2 °C/s ramp rate) with a final step at 98 °C for 10 min and 4 °C hold. Plates containing amplified droplets were loaded into a QX200 droplet reader (Bio-Rad). Discrimination between negative droplets (no luciferase copies) and positives (with luciferase copies) was used to estimate concentration of targets (luciferase copies/ul) using QuantaSoft analysis software (Bio-Rad). The resulted copies were normalized to input RNA and represented as luciferase copies in log scale.
Human IgG clearance
We compared the circulation time of human IgG (IVIG, PRIVIGEN, CSL Behring LLC). NSG-cmah−/− and control mice were injected with 100 μl of 10% IVIG in saline. Blood samples were collected at 30 min after IVIG injection as point day 0. The actual concentration of human IgG in peripheral blood was measured at days 1, 2, 3, 6, 7, and 14. Plasma human IgG concentration was determined by ELISA kit (Bethyl Laboratories, Inc. Montgomery, TX, USA, cat# E80–104).
We would like to thank Mr. Edward Makarov and Mr. Weimin Wang for technical assistance with animal work; the Elutriation Core Facility in the Department of Pharmacology and Experimental Neuroscience provided peripheral blood mononuclear cells; and the Flow Cytometry Research Facility, the DNA Sequencing Core Facility and the Comparative Medicine Department; Robin Taylor for editing assistance. All located at University of Nebraska Medical Center.
This work was supported by NIH grant 5R24OD018546–04 (LYP/SG). The funding source had no role in the study design, data collection or analyses, the decision to publish, or the preparation of this manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
SG and LYP conceived and designed the study. RSD, ABW, and YC performed in vivo studies on mice, flow cytometry. RSD, SM, and PSJ performed immunohistochemistry, qPCR, and Western blot procedures. CBG, RMQ, and DWH performed embryo isolation, microinjection, and generation of founder mice. ABW, SM, YC, and SMM performed cord blood collection, CD34+ cells isolation, FACS analysis of peripheral blood. SJP performed flow cytometry of bone marrow. RSD, SJP, CBG, SG, and LYP interpreted the results and wrote the manuscript. All authors have read and given approval of the final version of the manuscript.
All animal experiments were conducted following NIH guidelines for housing and care of laboratory animals and in accordance with the University of Nebraska Medical Center protocols approved by the institution’s Institutional Animal Care and Use Committee (protocol numbers 13–009, 14–100 and 06–071) in animal facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Human cord blood samples were obtained after receiving informed consent from parents and then approved by the Institutional Review Board (IRB) of the University of Nebraska Medical Center.
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- 20.Buchlis G, Mingozzi F, Soto PC, Pearce O, Hui DJ, Varki AP, et al. Intrinsically hyperactive and Hyperproliferative CD8<sup>+</sup> T cells in <em>Cmah</em>−/− mice as a model of human gene transfer responses. Blood. 2010;116(21):3773.Google Scholar
- 22.Naito Y, Takematsu H, Koyama S, Miyake S, Yamamoto H, Fujinawa R, et al. Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation. Mol Cell Biol. 2007;27(8):3008–22.CrossRefGoogle Scholar
- 28.Harms DW, Quadros RM, Seruggia D, Ohtsuka M, Takahashi G, Montoliu L, Gurumurthy CB. Mouse Genome Editing Using the CRISPR/Cas System. Curr Protoc Hum Genet. 2014;83:15 17 11–27.Google Scholar
- 32.Giltiay NV, Shu GL, Shock A, Clark EA. Targeting CD22 with the monoclonal antibody epratuzumab modulates human B-cell maturation and cytokine production in response to toll-like receptor 7 (TLR7) and B-cell receptor (BCR) signaling. Arthritis Res Ther. 2017;19:91. https://doi.org/10.1186/s13075-017-1284-2.
- 35.Thome JJ, Grinshpun B, Kumar BV, Kubota M, Ohmura Y, Lerner H, Sempowski GD, Shen Y, Farber DL. Longterm maintenance of human naive T cells through in situ homeostasis in lymphoid tissue sites. Sci Immunol. 2016;1(6).Google Scholar
- 38.Vasta GR, Feng C, Gonzalez-Montalban N, Mancini J, Yang L, Abernathy K, Frost G, Palm C. Functions of galectins as ‘self/non-self’-recognition and effector factors. Pathog Dis. 2017;75(5). https://doi.org/10.1093/femspd/ftx046.
- 39.Poluektova LY, Garcia-Martinez JV, Koyanagi Y, Manz MG, Tager AM. Humanized Mice for HIV Research, 1 ed. Switzerland: Springer International Publishing AG.; 2014.Google Scholar
- 42.Bishop JR, Gagneux P. Evolution of carbohydrate antigens--microbial forces shaping host glycomes? Glycobiology. 2007;17(5):23r-34r.Google Scholar
- 49.Dagur RS, Wang W, Cheng Y, Makarov E, Ganesan M, Suemizu H, Gebhart CL, Gorantla S, Osna N, Poluektova LY. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice. Biol Open. 2018;7(2). https://doi.org/10.1242/bio.029785.
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