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. 2013 Nov 15;191(10):5085-96.
doi: 10.4049/jimmunol.1301730. Epub 2013 Oct 2.

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity

Kylie M Quinn  1 , Ayako Yamamoto, Andreia Costa, Patricia A Darrah, Ross W B Lindsay, Sonia T Hegde, Teresa R Johnson, Barbara J Flynn, Karin Loré, Robert A Seder
Affiliations

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity

Kylie M Quinn et al. J Immunol. .

Abstract

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

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Figures

Figure 1
Figure 1
Characterisation of CD8 T cell responses after vaccination with Poly I:C and ISCOMs. (A) Experimental schema for Figures 1 and 2. Balb/c mice are vaccinated with one of the 5 indicated combinations of Gag (30 μg), Poly I:C (50 μg) and ISCOMs (12 μg) given as two doses, 21 days apart. Splenocytes were harvested 10 days later and used in MHCI tetramer- or ICS-based assays for T cell responses. (B) Frequency of CD3+CD8+ T cells that are Tetramer+. (C) Proportion of CD3+CD8+Tetramer+ T cells that express any combination of CD127+or KLRG1+. The black arc represents the proportion of cells that express CD127. (D) Histograms (left) and average MFI (right) of CD3+CD8+Tetramer+ T cells with each adjuvant as compared to total CD8+ T cells from a naïve animal. (E) Frequency of CD3+CD8+ T cells producing IFNγ, IL-2, or TNF (total cytokine+) by ICS. (F) Frequency of CD3+CD8+ T cells producing IFNγ, IL-2, or TNF individually. (G) Proportion of CD3+CD8+ T cells producing any combination of IFNγ, IL-2, or TNF; where 3+ cells produce IFNγ, IL-2 and TNF (black), 2+ cells produce any two of IFNγ, IL-2 and TNF (grey) and 1+ cells produce IFNγ, IL-2 or TNF alone (white). The black arc represents cells that do not produce IFNγ. (H) Frequency of CD3+CD8+ T cells producing IFNγ, IL-2, or TNF 6 weeks after the second dose. (I) Frequency of CD3+CD8+ T cells producing IFNγ, IL-2, or TNF 2 weeks after boosting with rAd5:Gag. Bars and error bars represent mean ± SEM. Statistical differences for bar graphs are represented as NS= no significant difference, * = p ≤ 0.05 and ** = p ≤ 0.01. Statistical differences for pie graphs are represented as # = p ≤ 0.05 compared to ISCOMs alone, $ = p ≤ 0.05 compared to Poly I:C alone. Each group is representative of at least two independent experiments and 4-8 Balb/c mice per group.
Figure 2
Figure 2
Characterisation of CD4 T cell responses after vaccination with Poly I:C and ISCOMs. (A) Frequency of CD3+CD4+ T cells producing IFNγ, IL-2, or TNF by ICS. (B) Frequency of CD3+CD4+ T cells producing IFNγ, IL-2, or TNF individually. (C) Proportion of CD3+CD4+ T cells producing any combination of IFNγ, IL-2, or TNF; 3+, 2+ or 1+ cells and the black arc represents cells that do not produce IFNγ. (D) Frequency of CD3+CD4+ T cells producing IFNγ, IL-2, or TNF 6 weeks after the second dose. Frequency of CD3+CD4+ T cells producing (E) IL-10 or (F) IL-17 at peak. Bars and error bars represent mean ±SEM. Statistical differences for bar graphs are represented as NS= no significant difference and * = p ≤ 0.05. Statistical differences for pie graphs are represented as # = p ≤ 0.05 compared to ISCOMs alone, $ = p ≤ 0.05 compared to Poly I:C alone. Each group is representative of at least two independent experiments and 4-8 Balb/c mice per group.
Figure 3
Figure 3
Protection conferred by vaccination with Poly I:C and ISCOMs against infectious challenge. (A) Bacterial load in the spleen (colony forming units, CFU) after Listeria:Gag challenge of mice vaccinated with formulations containing Gag, ISCOMs and Poly I:C as indicated. (B) Bacterial load in the spleen (CFU) after Listeria:Gag challenge of mice vaccinated with Gag, ISCOMs and Poly I:C (vaccine) and either left untreated or treated with a control antibody (Control Ab), a CD4-depleting antibody (Anti-CD4 Ab), a CD8-depleting Ab (Anti-CD8 Ab) or both of the latter (Anti-CD4/8 Abs). (C) Weight loss as % of original body weight at day 6 after rVACV:Gag challenge in mice vaccinated with formulations containing Gag, ISCOMs and Poly I:C as indicated. (D) Weight loss as % of original body weight at day 6 after rVACV:Gag challenge in mice vaccinated with Gag, ISCOMs and Poly I:C and left untreated or treated the indicated antibodies, as above. Bars and error bars represent geometric mean ±GEM. Statistical differences for bar graphs are represented as NS= no significant difference, * = p ≤ 0.05 and ** = p ≤ 0.01. Each group is representative of at least two independent experiments with 4-6 Balb/c mice per group.
Figure 4
Figure 4
Composition of DC populations and antigen uptake in dLN of Balb/c mice after vaccination with Gag:AF488, Poly I:C and ISCOMs. (A) Flow cytometric plots illustrating gating strategy with CD11c+ enriched samples for identification of DC subsets and evaluation of uptake of AF488 labeled antigen. (B) Number of total live CD11c+ DCs recovered per dLN at indicated times after vaccination. (C) Number of total leukocytes in dLN that stain with AquaBlue, a marker of non-viable cells, and (inset) the proportion of total leukocytes in dLN that are non-viable at 24 hours. (D) Number (bar graphs) and relative proportion (pie graphs) of total live CD11c+ DCs that distribute to each DC subset at indicated times after vaccination. (E) Number of live Gag:AF488+ CD11c+ DCs recovered per dLN and (inset) the number at 168 hours on an expanded y axis. (F) Number and relative proportion of live Gag:AF488+ CD11c+ DCs that distribute to each DC subset at 24 hours after vaccination. Results are representative of three independent experiments, with dLN from 10 mice pooled for each formulation.
Figure 5
Figure 5
Degradation of antigen by DC subsets in dLN of Balb/c mice after vaccination with DQ-OVA, Poly I:C and ISCOMs. (A) Flow cytometric plots illustrating gating strategy with CD11c+ enriched samples for identification of DC subsets and evaluation of degradation of DQ-OVA, indicated by fluorescence in the B515 channel on an LSRII. (B) Number of live DQ-OVA+ CD11c+ DCs recovered per dLN at indicated times after vaccination. (C) Dot plots with frequency of total live CD11c+ DCs that are DQ-OVA+. (D) Number and relative proportion of live DQOVA+ CD11c+ DCs that distribute to each DC subset at indicated times after vaccination. Results are representative of three independent experiments, with dLN from 4 mice pooled for each formulation.
Figure 6
Figure 6
Role of DC subsets for in vivo priming of T cells after vaccination with Poly I:C and/or ISCOMs. Frequency of (A) CD3+CD8+ T cells or (B) CD3+CD4+ T cells producing IFNγ, IL-2, or TNF by ICS after vaccination of WT Balb/c or Batf3 −/− mice with Gag and each adjuvant formulation. Bars and error bars represent mean ±SEM. Statistical differences for bar graphs are represented as NS= no significant difference and * = p ≤ 0.05. Each group is representative of at least two independent experiments and 4-5 mice per group.
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