A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells

Abstract

Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8(+) T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8(+) T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8(+) T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8(+) T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.

Keywords: APC, antigen presenting cell; B cells; BCR, B cell receptor; CMV, cytomegalovirus; CTL, cytotoxic t lymphocyte; DC, dendritic cell; HCMV, human CMV; KO, knock out; LN, lymph node; MHC, major histocompatibility complex; OVA, avalbumin; PBMC, peripheral blood mononuclear cell; SMIP, Small Molecule Immune Potentiator; TLR, toll like receptor; cross presentation/priming; cytotoxic T cells; dendritic cells; vaccination.

Figure 1.

Activity of a new synthetic compound on human and mouse TLR2. (A), Structure of SMIP2.1 (IUPAC Name=(4R)-4-[(2S)-2-[(2R)-3-{[(2R)-2,3bis(dodecanoyloxy)propyl]sulfanyl}-2-hexadecanamidopropanamido]butanamido]-4-carbamoylbutanoic acid). (B) Luciferase expression in HEK293-cells stable transfected with FLAG-tagged human TLR2 (dotted lines) or with HA-tagged mouse TLR2 (solid lines) and a NF-κB-luciferase reporter gene after stimulation with different doses of SMIP2.1. Pam3CSK4 was used as positive control for activation of TLR2 HEK293 transfectants. (C) Human MoDCs were stimulated for 24 hours with different concentration of SMIP2.1 in the absence or in the presence of a blocking anti-TLR2 antibody. Secretion of TNFα in the supernatants was measured as indicator of the lipopeptide activity. (D) BM-DCs from the indicated mice were stimulated for 24 hours with different concentrations of SMIP2.1. Secretion of IL-6 in the supernatants was measured as indicator of the lipopeptide activity. Group of 6 BALB/c mice were immunized intramuscularly at 0, 21 and 35 d with vaccines containing 1 μg of TT antigen (E) or with 0.2 μg of H1N1 Solomon Flu subunit antigen (F) with the addition of the indicated concentration of SMIP2.1. 2 weeks after the third immunization sera were pooled and antigen specific antibody titers were measured by ELISA.

Figure 2.

SMIP2.1 induces cross-presentation in vivo. (A), Congenic Ly5 CD45.1⁺ mice, injected in the tail vein with CFSE-labeled OT-I CD8⁺ T cells, were immunized with PBS or OVA (10 μg/mouse) alone or adjuvanted with SMIP2.1 (10 μg/mouse). Proliferation of adoptively transferred cells was assessed after 3 d in draining LNs by flow cytometry, gating on viable CD3⁺, CD8⁺, CD45.2⁺, CFSE^low CD8⁺ T cells. Upper panels show flow cytometry analysis for one mouse in each group. The percentage of proliferating CD8⁺ T cells (mean ± SD) in each group is reported in the lower graph. Data are representative of 3 independent experiments (B-D), C57Bl/6 mice were immunized 3 times with PBS or OVA (25 μg/mouse) alone or adjuvanted with SMIP2.1 (10 μg/mouse). (B) 7 d after the first immunization OVA specific frequency of CD8⁺ T cells was measured in the blood by K^b/OVA_257–264 tetramer staining. Upper panels show flow cytometry analysis for one mouse in each group. The percentage of tetramer⁺ CD8⁺ T cells (mean ± SD) in each group is reported in the lower graph. Data shown are representative of 2 independent experiments. (C) 7 d after the third immunization splenocytes were stimulated for 6 h with OVA_SIINFEKL peptide (3 μg/ml) or PBS, as negative control, then fixed and stained for intracellular IFNγ and TNFα. (D) Total anti-OVA IgG serum titers were measured by ELISA 2 weeks after the third immunization. **P < 0.01; *** P < 0.001.

Figure 3.

SMIP2.1 induces antigen-specific CTL activity in vivo. (A and B), C57Bl/6 mice were immunized with PBS or OVA (25 μg/mouse) alone or adjuvanted with SMIP2.1 (10 μg/mouse). (A), After 7 days, syngeneic splenocytes loaded with 2 different concentrations of CFSE and pulsed with either OVA_SIINFEKL peptide (CFSE^high) or an irrelevant control peptide (CFSE^low) were injected i.v. into recipient mice at a ratio of 1:1. Twenty-four hours later, the CTL response was assessed in draining LNs by measuring the presence of CFSE^high target cells using flow cytometry. Upper panels show flow cytometry analysis while the graph at the bottom shows the percentage of specific lysis of fluorescent target cells in the different groups calculated as described in the Material and Methods section. (B), A peripheral blood sample was obtained from mice prior to infusion of cells. Cells were stained with K^b/OVA_257–264 tetramer to measure the frequency of OVA specific CD8⁺ T cells. The percentage of K^b/OVA_257–264 tetramer⁺ CD8⁺ T cells in mice immunized with PBS was subtracted from the other groups. (C), C57Bl/6 mice were immunized twice with PBS or OVA (25 μg/mouse) alone or adjuvanted with SMIP2.1 at the indicated concentration. Seven days after the second immunization, mice were implanted s.c. with OVA-expressing E.G7 tumor cells and mice were monitored for tumor growth. The graph shows the percentage of tumor-free mice along 47 d after tumor cells implantation. Mice were euthanized when moribund. Representative data of 3 independent experiments are shown. Statistical analysis was performed vs OVA immunized group: *P ≤ 0.05; *** P < 0.001.

Figure 4.

SMIP2.1 increases antigen deposition in the draining LN. (A), C57Bl/6 mice were immunized with PBS or OVA A555 (25 μg/mouse) alone or adjuvanted with SMIP2.1 (100 μg/mouse) and draining LNs were collected 24 h later. 8 μm thick cryosections of draining LNs were stained for CD169 (green) and CD45R (blue) and observed under a confocal microscope. The picture (63x magnification) shows the OVA antigen deposition (red) only in mice immunized with OVA and SMIP2.1. Bar represents 20 μm. (B), Groups of 3 mice were immunized with PBS or OVA A647 (25 μg/mouse) alone or adjuvanted with SMIP2.1 (100 μg/mouse). Draining LNs were collected 24 h later and analyzed in pool by FACS to identify specific cell types and antigen-content. The graph shows the number of OVA A647⁺ cells per 1 × 10⁶ total cells. The data shown are representative of 2 independent experiments.

Figure 5.

SMIP2.1 increases cross-priming of OT-(I)cells in vitro. CD8α⁺ CD11c⁺ and CD8α⁻ CD11c⁺ DCs were purified by cell sorting from the spleen of C57Bl/6 mice and incubated with medium or OVA (10 μg/ml) alone or in the presence of SMIP2.1 (10 μM) for 4 hours. After washing, DCs were co-cultured with purified OT-I CD8⁺ T cells for 60 hours and proliferation of CD8⁺ OTI T cells was measured by ³H thymidine incorporation. Data in the graph indicate counts per min (CPM) and are expressed as mean ± SD of triplicate wells. Values of CPM from each cell population incubated with medium alone were subtracted from values of CPM in all the other conditions.

Figure 6.

SMIP2.1 increases cross-presentation by OBI B cells in vitro. Isolated OBI B cells were incubated for 20 h with medium (Unstimulated) or SMIP2.1 (10 μg/ml). After washing, B cells were loaded with OVA (100 μg/ml) for 4 h and then co-cultured with purified CFSE labeled CD8⁺ OT-I T cells. (A), Surface expression of CD69 by OT-I cells was analyzed by flow cytometry after 20 hours. Histograms of flow cytometry data are shown in the upper panel while mean values for the percentage of CD69 positive cells from 3 samples in each condition are reported in the lower panel. (B), After 52 hours, proliferation of OT-I cells was assessed by flow cytometry detection of CFSE. Histograms of flow cytometry data are shown in the upper panel while mean values for the percentage of CFSE^low cells from 3 samples in each condition are reported in the lower panel. Error bars indicate SD. **P < 0.01; *** P < 0.001.

Figure 7.

SMIP2.1 induces cross-presentation in human cells in vitro. PBMCs from a HCMV seropositive donor were pulsed in vitro as indicated for 2 hours, washed and co-cultured for 4 hours with an expanded HCMV specific CD8⁺ T cell population from the same donor. Production of IFNγ by CD8⁺ T cells was quantified by intracellular staining and flow cytometry. Dot plot analysis (A) and the percent of CD3⁺, CD8⁺, pp65 HCMV tetramer⁺ IFNγ⁺ T cells (B) are shown.