Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice

Abstract

TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Ralpha, which contributed to high avidity, and decreased expression of programmed death-ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses.

Figure 1. TLR ligands in a triple combination in a peptide vaccine can markedly reduce viral load after virus challenge.

(A) BALB/c mice were primed and boosted 3 weeks apart with an HIV Env peptide vaccine together with MALP2+poly(I:C)+CpG by the i.c.r. route (see details in Methods). Four months later, immunized animals were challenged i.c.r. with vPE16, and after 6 days, paired ovaries were recovered for virus titration with a plaque forming assay. One of 2 independent experiments with similar results is shown. Asterisk indicates the difference between groups (*P < 0.05; **P < 0.01; ***P < 0.001; n = 5). TLR ligand combination group is significantly different from all other groups. (B) Induction of antigen-specific (tetramer⁺) T cells in the draining popliteal LNs after s.c. immunization in the footpad with PCLUS3-18IIIB and TLR ligands. LN cells were recovered at 5 days and enumerated by staining for tetramer and intracellular IFN-γ. Results represent 1 of 2 independent experiments. tet, tetramer. Results are shown as mean ± SEM.

Figure 2. The triple-TLR ligands induce high–functional avidity CD8⁺ T cells.

(A and B) Mice were immunized s.c. in the footpad with PCLUS3-IIIB and TLR ligands, and the LN cells were recovered at 5 days. Cells were stained with CD107a (B) upon restimulation with P18-I10 at various concentrations and stained for intracellular IFN-γ 5 hours after restimulation (A). (C) Mice were immunized s.c. in the back flank with BM-DCs pretreated with TLR ligands and pulsed with P18-I10. Spleen cells were recovered at 1 month and restimulated with P18-I10 at 10^–2 μM. IFN-γ and CD107a were measured 5 hours after restimulation. Values represent the percentage of tetramer⁺CD8⁺ T cells with the indicated function. Results represent 1 of 2 independent experiments with similar results. (n = 3). (D) s.c.-immunized mice received naive splenocytes as targets pulsed with difference concentrations of P18-I10 at 1 month after immunization. Amounts used in histograms are as follows: left, 0 μM; middle, 10^–5 μM; right, 10^–2 μM. In vivo–specific lysis of transferred targets in the spleen was assayed at 5 hours. (E) Splenocytes isolated from the immune mice were restimulated in vitro with 10^–2 μM of P18-I10 for 5 days and examined for their ex vivo 4-hour killing activity on vPE-16-infected P815 cells. Results represent 1 of 2 independent experiments and are shown as mean ± SEM. ** P < 0.01; *** P < 0.001.

Figure 3. Production of IL-15 by DCs is enhanced by TLR ligands in the triple combination.

Mice were immunized s.c. in the footpad as in Figure 2. At 2 days, popliteal LN cells were recovered for surface staining of IL-15 (surf IL-15) (A) and IL-15Rα expression (B) and analyzed based on MHC II^hi and CD11c⁺ (top panels). Numbers in the top panel (A) indicate percentage of double-positive DCs (from the gate excluding lymphocytes) out of total recovered LN cells. Results represent 1 of 3 independent experiments with similar results.

Figure 4. Enhanced IL-15 expression of the triple-TLR combination is associated with TIRAP and upregulated IL-15Rα.

BM-DCs were treated with the TLR ligands in different combinations for 20 hours and measured for IL-15 and IL-15Rα production by MHC class II^hi and CD11c⁺ DCs. (A and B) Surface staining of IL-15 (A) and IL-15Rα (B) on the DCs from Tirap^–/– mice in comparison with WT. Numbers indicate percentages of DCs positive for surface IL-15. (C) Surface staining of IL-15 and IL-15α on Ifnar1^–/– DCs versus WT DCs. (D) Staining of intracellular IL-15 (iIL-15) on WT DCs. *P < 0.05; **P < 0.01; ***P < 0.001. Results are shown as mean ± SEM.

Figure 5. DCs activated with the triple-TLR ligands have prolonged survival and reduced caspase-3.

(A) BM-DCs were treated in triplicate with TLR ligands for various times as indicated. Cells were enumerated with trypan blue exclusion. Asterisks indicate a significant difference in the number of remaining DCs between day 5 and day 3 or day 2. **P < 0.01. One representative result out of 3 similar experiments is shown. (B) After 2 days of treatment with TLR ligands, caspase-3 was stained in BM-DCs. Numbers indicate percentage of DCs positive for caspase-3. **P < 0.02. Results represent 1 of 2 independent experiments with similar results and are shown as mean ± SEM.

Figure 6. The triple–TLR ligand–treated DCs prevent expansion of Treg cells and exhibit minimal upregulation of PD-L1.

BM-DCs were treated with TLR ligands for 20 hours, and excess TLR ligands were removed. (A and B) Freshly isolated syngeneic splenic T cells were cocultured with TLR ligand–pretreated DCs. Foxp3⁺CD4⁺ cells were evaluated at 24 hours and 48 hours, respectively. Numbers indicate percentage of CD4⁺ T cells positive for Foxp3. (B) BM-DCs were treated with TLR ligands for 20 hours and stained with either anti-CD86 or anti–PD-L1 mAbs to measure levels of the costimulatory molecules. The experiments were repeated twice with similar results. *P < 0.05; **P < 0.01. Results are shown as mean ± SEM.