Regulation of SIV antigen-specific CD4+ T cellular immunity via autophagosome-mediated MHC II molecule-targeting antigen presentation in mice

Abstract

CD4+ T cell-mediated immunity has increasingly received attention due to its contribution in the control of HIV viral replication; therefore, it is of great significance to improve CD4+ T cell responses to enhance the efficacy of HIV vaccines. Recent studies have suggested that macroautophagy plays a crucial role in modulating adaptive immune responses toward CD4+ T cells or CD8+ T cells. In the present study, a new strategy based on a macroautophagy degradation mechanism is investigated to enhance CD4+ T cell responses against the HIV/SIV gag antigen. Our results showed that when fused to the autophagosome-associated LC3b protein, SIVgag protein can be functionally targeted to autophagosomes, processed by autophagy-mediated degradation in autolysosomes/lysosomes, presented to MHC II compartments and elicit effective potential CD4 T cell responses in vitro. Importantly, compared with the SIVgag protein alone, SIVgag-LC3b fusion antigen can induce a stronger antigen-specific CD4+ T cell response in mice, which is characterized by an enhanced magnitude and polyfunctionality. This study provides insight for the immunological modulation between viral and mammalian cells via autophagy, and it also presents an alternative strategy for the design of new antigens in the development of effective HIV vaccines.

Figure 1. Construction of DNA vectors and Ad5-based vectors carrying the SIVgag-LC3b gene.

A, Schematic representation of constructs carrying various combinations of mouse LC3b gene or SIVgag under the CMV promoter, denoted as LC3b, SIVgag and SIVgag-LC3b, respectively. HeLa cells were transfected with plasmid-based or Ad5-based constructs, and expression of the protein of interest was detected using western blotting analyses. The GAPDH blot demonstrates equal protein loading. B, Expression of the fusion protein using recombinant pVAX plasmid DNA constructs. C, Expression of fusion protein using recombinant Ad5-based constructs. The left blots show proteins detected using anti-SIV Gag and the right blots show proteins detected using the anti-LC3b antibody, respectively.

Figure 2. Functionally targeted SIV Gag protein to autophagosomes using confocal microscopy.

HeLa-GFP-LC3 cell line stably expressing GFP-LC3 protein (green fluorescence) were transfected with pVAX-SIVgag plasmid (top) or pVAX-SIVgag-LC3 plasmid (bottom) with or without chloroquine (CQ) treatment, and then stained with anti-SIVgag and Cy3-labeled goat anti-mouse IgG as secondary antibodies (red fluorescence); the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. Representative cells are shown from one experiment out of three total experiments.

Figure 3. Functionally targeted SIV Gag protein to lysosomes and MHC II compartments using confocal microscopy.

A, HeLa cells were transfected with pVAX-SIVgag plasmid (top) or pVAX-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and then stained with mouse anti-SIVgag IgG antibody and rabbit anti-LAMP-2 IgG antibody. Subsequently, Cy3-labeled goat anti-mouse IgG (red fluorescence) and Alexa Fluor 488-labeled goat anti-rabbit IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. B, RAW 264.7 cells were stimulated with lipopolysaccharide(LPS) and infected with Ad5-SIVgag (top) or Ad5-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and subsequently stained with mouse anti-SIVgag IgG antibody and rat anti-MHC- II IgG antibody. Next, Cy3-labeled goat anti-rat IgG (red fluorescence) and 488-labeled goat anti-mouse IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. C, HeLa cells were transfected with pVAX, pVAX-SIVgag or pVAX-SIVgag-LC3b for 24 h, and then treated with 75 μM CQ or 200 nM RAPA for 24 h. LC3b-I, LC3b-II and SIVgag were visualized using anti-LC3b and anti-SIVgag immunoblotting, respectively. GAPDH blots demonstrate that CQ or RAPA treatment did not affect the overall protein level. D, SIVgag antigen-specific CD4+ T cells were obtained and isolated using anti-CD4 microbeads, and purified CD4+ T cells (effector cells) were then co-cultured with BMDC cells (target cells). The E:T ratios were 3∶1 or 9∶1. After 48 h, the culture supernatants were measured using the IFN-γ ELISA kit. As a positive control, the BMDC cells stimulated with LPS (1 μg/ml) were used as a positive control, and untreated DMDC cells were used as a negative control. These data were expressed as the mean±SEM from four mice samples.

Figure 4. Stronger antigen-specific IFN-γ-secreting CD4 T cell responses elicited by SIVgag-LC3b fusion protein compared to SIVgag antigen alone in mice.

A, Immunization schedule to evaluate the immunogenicity of the SIVgag-LC3b fusion antigen. C57BL/6 female mice were divided into four groups with 8–10 mice per group. Each mouse was intramuscularly injected 50 μg of the appropriate DNA plasmids at weeks 0 and 2, then boosted intramuscularly with 1×10⁹ vp of corresponding adenoviral vectors at weeks 4 and 6. To assess the immune responses, mice were sacrificed at weeks 4, 6 and 8 after the first immunization to collect splenocytes and serum for analysis of cellular and humoral immune responses. The symbol “↓” represents the time-point of injection; the symbol “Δ” represents the time-point of sacrifice and sample collection. The SIVgag-specific cellular immune responses, as assessed using the IFN-γ ELISPOT assay following stimulation with SIVgag peptide, were shown after DNA-based constructs immunization at week 4 (B) and after adenoviral-based constructs immunization at week 6 (C). The SIVgag-specific cellular immune responses, as assessed using the intracellular IFN-γ cytokine staining assay, were shown after DNA-based constructs immunization at week 4 (D) and after adenoviral-based constructs immunization at week 8 (E). Data were analyzed using the Student’s t-test, and a two-tailed p-value of less than 0.05 was considered statistically significant. These data were expressed as the mean±SEM from four mice samples (*: p<0.05;**: p<0.01; ***: p<0.001). Two independent experiments for the animal immunization were repeated.

Figure 5. Assessment of polyfunctional SIVgag-specific CD4+ T cellular immunity elicited by the SIVgag-LC3b fusion antigen.

Mice were immunized and splenocytes were collected as described in the Materials and Methods and Figure 4A. Splenocytes from four mice in each group were mixed together and 500,000 cells were acquired and analyzed by the FACSAria instrument using the FlowJo software. The ability of functional CD4+ T cell populations from immunized mice to secrete IFN-γ, TNF-α, and IL-2 cytokines in response to SIVgag peptide pool stimulation was assessed. (A) Gating strategy for flow cytometric scatter plots to analyze the frequency of cytokine(s)-positive CD4+ T cells positive in this study. Column graphs depicting subpopulations of single-, double-, or triple-positive CD4+ T cells secreting the cytokines IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (B) or adenoviral-based immunization at week 8 (C). Pie chart analysis was performed to represent subpopulations of cytokine-secreting CD4+ T cells positive for the combination of IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (D) or adenoviral -based immunization at week 8 (E), respectively. The representative data shown here were obtained from two independent experiments from 8–10 mice for each group.

Figure 6. Assessment of polyfunctional SIVgag-specific CD8+ T cellular immunity elicited by the SIVgag-LC3b fusion antigen.

Mice were immunized and splenocytes were collected as described in the Materials and Methods and Figure 4A. Splenocytes from four mice in each group were mixed together and 500,000 cells were acquired and analyzed by the FACSAria instrument using the FlowJo software. The ability of functional CD8+ T cell populations from immunized mice to secrete IFN-γ, TNF-α, and IL-2 cytokines in response to the SIVgag peptide pool stimulation was assessed. (A) Gating strategy for flow cytometric scatter plots to analyze the frequency of cytokine(s)-positive CD8+ T cells positive in this study. Column graphs depicting subpopulations of single-, double-, or triple-positive CD8+ T cells secreting the cytokines IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (B) or adenoviral -based immunization at week 8 (C). Pie chart analysis was performed to represent subpopulations of cytokine-secreting CD8+ T cells positive for combinations of IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (D) or adenoviral -based immunization at week 8(E). The representative data shown here were obtained from two independent experiments from 8–10 mice for each group.

Figure 7. Stronger SIVgag-specific antibodies elicited in mice using the SIVgag-LC3b fusion protein compared to SIVgag antigen alone.

Mice were immunized and serums were collected at week 8 as shown in the Materials and Methods. The SIV-binding antibody was assessed using ELISA. Data were analyzed using the Student’s t-test, and a two-tailed p-value of less than 0.05 was considered statistically significant (*: p<0.05). Each data point represents the antibody titer from an individual mouse (n = 4). The representative data were obtained from two independent experiments.