Skeletal Muscle Is an Antigen Reservoir in Integrase-Defective Lentiviral Vector-Induced Long-Term Immunity

Abstract

We previously developed integrase-defective lentiviral vectors (IDLVs) as an antigen delivery system for inducing strong and prolonged immunity in animal models. Here, we examined the association between persistence of antigen expression and durability of immune response. Following a single intramuscular (i.m.) or subcutaneous (s.c.) injection of IDLV delivering GFP in mice, we evaluated antigen expression and inflammation at the site of injection and persistence of antigen-specific T cells at early and late time points. Durable antigen expression was detected up to 90 days only after i.m. immunization. Mononuclear inflammation was evident soon after IDLV injection in both i.m. and s.c. immunized mice, but remained detectable up to 30 days postinjection only in i.m. immunized mice. Similarly, GFP-specific T cells were more persistent in the i.m. immunized mice. Interestingly, GFP⁺ muscle fibers were co-expressing major histocompatibility complex (MHC) class I, suggesting that muscle cells are competent for presenting antigens to T cells in vivo. In in vitro experiments, we demonstrated that although both primary myoblasts and myocytes present the antigen to GFP-specific T cells through MHC class I, myoblasts are more resistant to Fas-dependent cytotoxic T lymphocyte (CTL) killing activity. Overall, these data indicate that muscle cells may serve as an antigen reservoir that contributes to the long-term immunity induced by IDLV vaccination.

Keywords: CTL; Fas; GFP; MHC I; integrase defective lentiviral vectors; intramuscular immunization; muscle; myoblasts; myocytes; vaccine.

Graphical abstract

Figure 1

Persistent GFP Expression at the Injection Site after Intramuscular Immunization with IDLV-GFP BALB/c mice injected with IDLV-GFP or PBS using either the intramuscular (i.m.) or subcutaneous (s.c.) route were sacrificed at different time points. Skeletal muscle and skin sections were stained for GFP (green) and DAPI (blue). (A and B) Representative confocal microscopy images of muscle after i.m. immunization (A) and skin after s.c. immunization (B) are shown at 3, 30, and 90 days (D3, D30, and D90). Longitudinal (a, d, and g) and cross-sectional images (b, e, and h) of skeletal muscle are shown. (C) Representative confocal microscopy images of dLNs stained for GFP and DAPI from mice injected either i.m. (n and q) or s.c. (p and s) with IDLV-GFP or PBS (o and r). (D) Quantitative analysis of GFP expression evaluated using the ImageJ software, shown as % GFP⁺ area on DAPI⁺ area in dLNs from IDLV-GFP-immunized mice (n = 3 for each group).

Figure 2

IDLV Immunization Induces Mononuclear Cellular Infiltration (A) H&E staining of skeletal muscle and skin sections from mice injected i.m. or s.c., respectively, with IDLV-GFP, PBS, or left untreated (naive) at 3 days (D3) postinjection. (B) H&E staining of muscle and skin tissues at 30 days (D30) after i.m. or s.c. injection, respectively, with IDLV-GFP or PBS. Arrows indicate foci of inflammatory cells. (C) F4/80 and CD3 as macrophage and T cell markers, respectively, were used to stain muscle sections from mice injected with IDLV-GFP or PBS at 30 days after injection. Arrows indicate macrophages and T cells within the cellular infiltration.

Figure 3

GFP-Specific T Cell Response Is More Persistent after i.m. Immunization GFP-specific T cells from mice immunized with IDLV-GFP were evaluated in splenocytes by IFN-γ ELISpot assay at 30 and 90 days after i.m. or s.c. injection. Cells were stimulated overnight with H-2d-restricted GFP 9-mer peptide HYLSTQSAL (GFP), concanavalin A (ConA), or left untreated. Data are expressed as numbers of specific spot-forming cells (SFCs) per million cells (left panel) and as % of GFP-specific SFCs normalized per ConA-induced SFCs (right panel). Data from three different experiments (n = 12) are shown. Black lines represent the mean and SEM. Asterisk indicates a statistically significant difference between the indicated groups (∗p < 0.05).

Figure 4

Baseline MHC Class I Expression in Muscle Is Not Modified by IDLV Injection (A) Muscle from naive and IDLV-GFP-immunized mice 3 days (D3) and 30 days (D30) postinjection was stained with the cell membrane marker beta-spectrin (green), MHC class I (red), and DAPI (blue) as nuclear staining. The right panels show MHC class I-positive area (white arrowheads) at higher magnification (far right panels). (B) MHC class I expression was quantified using ImageJ software (n = 3 for each group). Error bars represent the SD of three different samples. (C) Representative confocal images of muscle from IDLV-GFP-immunized mice at 3 and 30 days postinjection stained for GFP (green), MHC class I (red), and DAPI (blue). Arrowheads indicate MHC class I and GFP double-positive cells.

Figure 5

Primary Myoblasts and Myocytes Present the Antigen to Specific Effector T Cells through MHC Class I (A) Light microscopy images of primary myoblasts purified from 3-week-old BALB/c mice and after differentiation into myocytes. (B) Confocal images of myoblasts and myocytes transduced with LV-GFP or non-transduced (NT), stained for MHC class I (red), GFP (green), and DAPI (blue). White arrowheads indicate MHC class I expression on GFP-positive cells. (C) Layout of the experiment performed to demonstrate the ability of primary muscle cells to stimulate specific T cells. Primary myoblasts and myocytes were either pulsed with H-2d-restricted GFP 9-mer peptide HYLSTQSAL or transduced with LV-GFP and treated with anti-H-2d antibody or an unrelated IgG and then co-cultured with splenocytes from IDLV-GFP- or PBS-injected mice in the ELISpot plate. (D) The stimulation of GFP-specific T cells was assessed by IFN-γ ELISpot assay. Splenocytes pulsed with GFP peptide were used as a positive control of the assay. Results from a representative experiment are shown. Data are expressed as specific spot-forming cells (SFCs) per million splenocytes. Error bars represent the SD of duplicates.

Figure 6

Myoblasts, but Not Myocytes, Are Resistant to CTL-Induced Cytotoxicity (A) LDH assay was performed to assess the cytotoxicity mediated by specific CTLs. Splenocytes from IDLV-GFP-immunized mice were activated in vitro for 3 days with GFP peptide and then co-cultured for 8 h with myoblasts and myocytes pulsed with GFP peptide or left untreated. P815 cell line pulsed or not with GFP peptide was used as a positive control of the assay. Data are expressed as mean % specific cytotoxicity ± SD of three different experiments assessed at E:T ratio of 80:1. (B) Fas expression evaluated by confocal microscopy on myoblasts and myocytes before (NT) and after transduction with LV-GFP. Anti-Fas (red) and anti-GFP (green) antibodies were used. (C) Fas expression on myoblasts and myocytes by flow cytometry. Data in (C) are expressed as specific geomean fluorescence intensity (gMFI ± SD) from three different experiments after subtraction of the isotype-treated samples gMFI. A representative experiment of analysis by flow cytometry is shown. (D) FasL-His recombinant protein (1 and 2 μg/mL) and anti-His Ab were used to induce cytotoxicity on splenocytes, myoblasts, and myocytes using either complete medium (left) or medium without serum (right). FasL-induced cell death was measured by trypan blue staining and expressed as mean % cytotoxicity ± SD of three different experiments.

Figure 7

Overexpression of Fas in Myoblasts Resulted in Increased FasL-Mediated Cytotoxicity (A) Fas expression on myoblasts transduced with LV-GFP, LV-Fas/GFP, or NT was evaluated by flow cytometry. Data are expressed as gMFI. (B) FasL-His recombinant protein (2 μg/mL) and anti-His Ab were used to induce cytotoxicity in myoblasts transduced with LV-GFP or LV-Fas/GFP. FasL-induced cell death, quantified using a live/dead (L/D) stain by flow cytometry, is expressed as mean % cytotoxicity ± SD of triplicates. Flow cytometry analyses of a representative experiment are shown.

Figure 8

Fas Expression in Muscle Tissue Is Independent of Immunization Muscle tissue harvested at 3 and 30 days after i.m. injection with IDLV-GFP or PBS was stained with anti-Fas antibody (red) and DAPI (blue). White arrowheads indicate Fas-expressing cells.