Figure 1.
In vitro expression of human cytokines and HLA-A2.1 in MC57G cells.
(A) Maps of Zac2.1 plasmids modified to encode human cytokines and HLA-A2.1 (HHD) containing α1 and α2 domains of HLA-A2.1, α3, cytoplasmic and transmembrane domains of murine H-2Db, and hβ2m, are shown. These plasmids were used to construct AAV9 viral particles. (B) MC57G cells were infected in vitro with AAV9 encoding the respective cytokine, and the production of each cytokine was determined using ELISA. (C) MC57G cells were infected in vitro with different doses (1×109, 1×1010, or 1×1011 GC/mL) of AAV9-encoding HLA-A2.1/hβ2m (AAV9-A2). Expression of HLA-A2.1 and hβ2m was evaluated using flow cytometric analyses.
Figure 2.
In vivo expression of human cytokines and HLA-A2/hβ2-m in NSG mice upon AAV9-mediated gene delivery.
(A) NSG mice were inoculated with 5×109 GC/mouse of AAV9 encoding each cytokine, and 1, 2, 4, 8, 10, or 16 weeks later, sera were collected from the mice and cytokine production was determined using ELISA. (B) The level of human GM-CSF produced in the sera was determined after inoculation of NSG mice with a high (5×109 GC/mouse) or a low (1×109 GC/mouse) dose of AAV9-GM-CSF. (C) Luciferase expression 2 weeks after inoculation of NSG mice with 1×1010 GC of AAV9-GFP-Luc via intrathoracic or i.v. route is shown by injecting D-luciferin intraperitoneally, followed by whole body in vivo imaging analyses. (D) NSG mice were administered intrathoracically with 5×1010 GC of AAV9-A2 and 4 weeks later, the expression of HLA-A2 and hβ2m by CD326HIGH cells within the thymus of AAV9-A2-infected NSG mice, A2-Tg NSG mice, and naïve NSG mice was determined using flow cytometric analyses. (E) Immunohistochemical analyses show HLA-A2 (green) and CD326 (red) staining of thymic tissue from AAV9-A2-transduced NSG mice, A2-Tg NSG mice, and naïve NSG mice. Hoechst 33342 (blue) was used to counterstain nuclei.
Figure 3.
GC number specific for AAV9 and the transgenes present in selected organs of NSG mice at 6 weeks and 20 weeks after AAV9 injection.
Lung, liver, spleen, kidney and bone marrow, were collected 6 and 20 weeks after NSG mice were given recombinant AAV9 vectors (5×109 GC of AAV9-GM-CSF, AAV9-IL-3, and AAV9-IL-15 by i.v., or 1×1011 GC of AAV9-A2 by i.v. and intrathoracically), and DNA was isolated from the respective organs. (A) The number of AAV9 vector GC was determined by using a set of primers specific to AAV9 vector. (B) The number of GC specific for the transgenes - HLA-A2, hGM-CSF, hIL-3 and hIL-15 - was determined by using a set of primers to the respective transgene. There was a low number (10–103 GC; depending on the transgene) of GC detected in naïve NSG mice, as a non-specific background, and, therefore, this background GC number was subtracted from the GC number in experimental groups.
Figure 4.
Human leukocyte reconstitution in the peripheral blood of NSG mice transduced with AAV9-hucytokines or AAV9-A2.
(A) Schematic representation of the strategic methodology for engrafting human CD34+ cells in AAV9-transduced NSG mice. NSG mice were inoculated with AAV9-hucytokines or AAV9-A2 and 2 weeks later, mice were irradiated to myeloablate mouse immune cells. The next day, mice were transplanted i.v. with 1×105 human CD34+ cells previously isolated from human fetal liver. (B) The level of human CD45+ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34+ cells into NSG mice transduced with AAV9 encoding human IL-3, IL-4, IL-7, IL-15, or GM-CSF. (C) The level of human CD45+ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34+ cells into NSG mice transduced with individual or combination of AAV9 encoding selected human cytokines, IL-3 (5×109 GC), IL-15 (5×109 GC), and GM-CSF (1×109 GC). (D) The level of human CD45+ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34+ cells into NSG mice transduced with AAV9-A2.
Figure 5.
Reconstitution of a human immune system in NSG mice transduced with AAV9-A2/hucytokines.
(A) Flow cytometric analyses were performed to determine the level of human CD45+ cell reconstitution in the blood of various groups of mice: AAV9-A2/hucytokines-transduced NSG mice, AAV9-A2-transduced NSG mice, A2-Tg NSG mice, or AAV9-GFP-transduced NSG mice 6, 10, or 14 weeks after engraftment of human CD34+ cells. (B) The level of human CD45+ cell reconstitution in the blood of various groups of NSG mice was determined using flow cytometric analyses 6, 10, or 14 weeks after engrafting human CD34+ cells. (C) Percentages of human CD3+ T cells and CD19+ B cells within the human CD45+ cells in the blood of various groups of HIS mice were determined 6, 10, and 14 weeks after HSC engraftment. (D) Percentages of human CD8+ and CD4+ T cells within the human CD3+ T cells in the blood of various groups of HIS mice were determined 6, 10, and 14 weeks after HSC engraftment. (E) The pie charts show the percentage of various human lymphocyte subsets, including naïve, central memory (CM), effector memory (EM) and effector CD8+ T cells, within the human CD45+ cells in the blood of various groups of HIS mice determined 14 weeks after HSC engraftment, compared to those in PBMCs of a healthy human subject. * indicates p values <0.05, ** indicates p values <0.01, and *** indicates p values <0.001.
Table 1.
Figure 6.
Magnitude of CD8+ T cell response induced in AAV9-A2/hucytokines transduced HIS mice immunized with Ad-p24 and AdPfCS.
Two weeks after NSG mice were transduced with either AAV9-A2/hucytokines, AAV9-A2, or AAV9-GFP (mock control), these NSG mice, as well as age-matched A2-Tg NSG mice, were engrafted with HSCs. Sixteen weeks after HSCs engraftment, all groups of NSG mice were immunized with Ad-p24 and AdPfCS, and 2 weeks later, splenocytes and liver mononuclear cells were collected. In (A) and (B), ELISpot assays were performed using splenocytes and liver mononuclear cells derived from respective mice in the presence or absence of peptides corresponding to A2-restricted CD8+ T-cell epitopes of HIV-p24 (A) and PfCS (B). (C) Intracellular cytokine staining (ICCS) was performed on splenocytes using a pool the peptides corresponding to A2-restricted CD8+ T-cell epitopes of HIV-p24 and PfCS. IFN-γ expression was measured in both assays. The results are expressed as IFN-γ-secreting cells/million lymphocytes for ELISpot and as the percentage of IFN-γ+ CD8+ T cells for ICCS. * indicates p values <0.05. NS, not significant.
Figure 7.
In vitro and in vivo A2-restricted, PfCS-specific cytotoxic T cell responses induced in AA-A2/hucytokines-transduced HIS mice immunized with AdPfCS.
(A) Splenocytes were collected from AAV9-A2/hucytokines transduced HIS mice 2 weeks after AdPfCS immunization. After CD8+ T cell enrichment, cells were used as an effector cells. A2+ hepatocytes were isolated from AA-A2/hucytokines-transduced NSG mice, labeled with CFSE, and used as target cells. These two cell populations were co-cultured and in vitro CTL assays were performed by measuring the amount of caspase 3 within the hepatocytes in the presence or absence of the peptide corresponding to the A2-restricted, CD8+ epitope of PfCS antigen. (B) Two weeks after AdPfCS immunization to AAV9-A2/hucytokines-transduced HIS mice, immunized and naïve AAV9-A2/hucytokines-transduced HIS mice were injected with 50 µg of DNA-PfCS dissolved in 2 ml PBS by HTV delivery. Another group of immunized and naïve AAV9-A2/hucytokines-transduced HIS mice received DNA-PyCS via HTV delivery as a negative control. Three days after DNA-PfCS or DNA-PyCS challenge, liver was collected and the amount of PfCS or PyCS mRNA was determined using real-time qRT-PCR.