Interleukin-12p70 expression by dendritic cells of HIV-1-infected patients fails to stimulate gag-specific immune responses

Abstract

A variety of immune-based therapies has been developed in order to boost or induce protective CD8(+) T cell responses in order to control HIV replication. Since dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique capability to stimulate naïve T cells into effector T cells, their use for the induction of HIV-specific immune responses has been studied intensively. In the present study we investigated whether modulation of the activation state of DCs electroporated with consensus codon-optimized HxB2 gag mRNA enhances their capacity to induce HIV gag-specific T cell responses. To this end, mature DCs were (i) co-electroporated with mRNA encoding interleukin (IL)-12p70 mRNA, or (ii) activated with a cytokine cocktail consisting of R848 and interferon (IFN)-γ. Our results confirm the ability of HxB2 gag-expressing DCs to expand functional HIV-specific CD8(+) T cells. However, although most of the patients had detectable gag-specific CD8(+) T cell responses, no significant differences in the level of expansion of functional CD8(+) T cells could be demonstrated when comparing conventional or immune-modulated DCs expressing IL-12p70. This result which goes against expectation may lead to a re-evaluation of the need for IL-12 expression by DCs in order to improve T-cell responses in HIV-1-infected individuals.

Figure 1

Production of pro- and anti-inflammatory cytokines by DCs modulated to express IL-12p70. (a) IL-12 secretion by DCs electroporated with IL-12p70-encoding mRNA. DCs were examined after incubation with a proinflammatory cytokine cocktail consisting of TNF-α and PGE₂ and electroporated with mRNA encoding IL-12p70 or with IL-12p70-encoding mRNA in combination with hHxB-2 gag-encoding mRNA. As a control mock-electroporated mature DCs were examined. DC culture supernatant was collected 24 h, 48 h, and 96 h after electroporation. Results are shown as mean ± standard deviation (n = 5). (b) IL-12 secretion by immune-modulated DCs. DCs were examined after incubation with a proinflammatory cytokine cocktail consisting of TNF-α and PGE₂ or activated under the same conditions supplemented with R848 and IFN-γ. As a control untreated, that is, immature DCs (iDCs) were examined. DC culture supernatant was collected 24 h after activation with proinflammatory stimuli, as well as 24 h and 72 h after a washout experiment. Results are shown as mean ± standard deviation (n = 5). (c), (d), and (e) Cytokine secretion by DCs activated under different experimental conditions and after a 24 h washout period. Values are shown for (c) IL-10, (d) IL-6, and (e) TNF-α. Results are shown as mean ± SD (n = 5).

Figure 2

Effect of coelectroporation of DCs with IL-12p70 mRNA on HIV gag-specific T cell responses after 1 week of stimulation. Freshly thawed PBLs were directly incubated in ELISpot with HIV gag-derived peptides (Day 0) or PBLs were stimulated for one week with autologous HIV gag mRNA-electroporated DCs. For this, DCs were either matured with a simplified cytokine cocktail (TNF-α and PGE₂) (a) and (b) or DCs were matured with this simplified cytokine cocktail in combination with IL-12p70 mRNA electroporation (c) and (d). Thereafter, cocultured PBLs were restimulated with HIV gag-derived peptides and evaluated in ELISpot for detection of antigen-specific IFN-γ (a), (c) and (e) or IL-2 (b), (d), and (f) secretion. Results are shown as mean of triplicate analyses for each donor displaying a positive response (n = 6), as defined in the Materials and Methods section.

Figure 3

Effect of addition of TLR7/8 ligand, R848, and IFN-γ for activation of DCs on HIV gag-specific T cell responses after 1 week of stimulation. Freshly thawed PBLs were directly incubated in ELISpot with HIV gag-derived peptides (Day 0) or PBLs were stimulated for one week with autologous HIV gag mRNA-electroporated DCs. For this, DCs were either matured with a simplified cytokine cocktail (TNF-α and PGE₂) (a) and (b) or DCs were matured with a simplified cytokine cocktail in combination with TLR7/8 ligand resiquimod (R848) and IFN-γ (c) and (d). Thereafter, cocultured PBLs were restimulated with HIV gag-derived peptides and evaluated in ELISpot for detection of antigen-specific IFN-γ (a), (c), and (e) or IL-2 (b, d, and f) secretion. Results are shown as mean of triplicate analyses for each donor displaying a positive response (n = 3), as defined in the Materials and Methods section.

Figure 4

Stimulatory capacity of DCs from HIV-seropositive individuals after a second stimulation round. Freshly thawed PBLs were directly incubated in ELISpot with gag-derived peptides (Day 0), or PBLs were stimulated for one week with autologous gag mRNA-electroporated DCs (day 7), or PBLs were stimulated for a second week with thawed autologous electroporated DCs and reevaluated in ELISpot (day 14). For this, DCs were matured with a simplified cytokine cocktail (TNF-α and PGE₂) or with a simplified cytokine cocktail in combination with IL-12p70 mRNA electroporation (a) and (b), or with a simplified cytokine cocktail in combination with TLR7/8 ligand resiquimod (R848), and IFN-γ (c) and (d). Cocultured PBLs were evaluated for antigen-specific secretion of the following cytokines: IFN-γ (a) and (c) and IL-2 (b) and (d). Results are shown as mean of triplicate analyses for each donor displaying a positive response (n = 2), as defined in the Materials and Methods section.