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. 2013 Apr 1;190(7):3319-27.
doi: 10.4049/jimmunol.1203321. Epub 2013 Feb 18.

Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic dendritic cells that are marked by differential expression of pancreatic enzymes

Adam M Farkas  1 , Douglas M Marvel, Olivera J Finn
Affiliations

Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic dendritic cells that are marked by differential expression of pancreatic enzymes

Adam M Farkas et al. J Immunol. .

Abstract

Dendritic cells (DC) elicit immunity to pathogens and tumors while simultaneously preserving tolerance to self. Efficacious cancer vaccines have been a challenge because they are based on tumor Ags, some of which are self-Ags and thus subject to self-tolerance. One such Ag is the tumor-associated mucin MUC1. Preclinical testing of MUC1 vaccines revealed existence of peripheral tolerance to MUC1 that compromises their efficacy. To identify mechanisms that act early postvaccination and might predict vaccine outcome, we immunized human MUC1 transgenic mice (MUC1.Tg) i.v. with a MUC1 peptide vaccine against which they generate weak immunity and wild-type (WT) mice that respond strongly to the same peptide. We analyzed differences in splenic DC phenotype and function between the two mouse strains at 24 and 72 h postvaccination and also performed unbiased total gene expression analysis of the spleen. Compared to WT, MUC1.Tg spleens had significantly fewer DC, and they exhibited significantly lower expression of costimulatory molecules, decreased motility, and preferential priming of Ag-specific Foxp3(+) regulatory T cells. This tolerogenic DC phenotype and function was marked by a new putative biomarker revealed by the microarray: a cohort of pancreatic enzymes (trypsin, carboxypeptidase, elastase, and others) not previously reported in DC. These enzymes were strongly upregulated in the splenic DC from vaccinated WT mice and suppressed in the splenic DC of vaccinated MUC1.Tg mice. Suppression of the enzymes was dependent on regulatory T cells and on signaling through the IL-10R and correlated with global downregulation of DC immunostimulatory phenotype and function.

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Figures

Figure 1
Figure 1. Immunization of MUC1.Tg mice with MUC1p results in decreased splenic DC number and costimulatory molecule expression, and preferential priming of Foxp3+ Treg
(A) WT and MUC1.Tg mice were immunized with unloaded DC (ctrl) or DC loaded with MUC1p. 24h post-immunization total splenic DC numbers were analyzed. Each symbol represents one mouse with bars showing mean ± SEM from three pooled independent experiments, with each experiment including 2–4 mice per group. (B–D) WT and MUC1.Tg mice were immunized as in (A). 48h post immunization bulk splenocytes were stained for FACS analysis. Data represent percentage of positive cells within the CD11c+ gate (B–C) or the MFI of cells within the CD11c+ gate (D). Symbols represent individual mice with bars showing mean ± SEM and are representative of 2 independent experiments. (E–H) WT and MUC1.Tg mice were immunized as in (A). 24h later, splenic DC were bead isolated, loaded with OVA and co-cultured with OT-II CD4 T cells for 7 days. On day 7, OT-II cells were treated with PMA/Ionomycin and analyzed by FACS. Each symbol represents an individual mouse with bars depicting mean ± SEM. Data are pooled from two independent experiments. (G) OT-II CD4+ T cells were labeled with CFSE and cultured as in (E–H). On day 7, CFSE dilution was assessed in CD4+Foxp3+ cells. Representative dot plots from MUC1p vaccinated WT and MUC1.Tg mice are shown (G). (H) Bars represent mean percentage proliferation ± SEM of OT-II CD4+Foxp3+ cells. Data are representative of two independent experiments.
Figure 2
Figure 2. Immunization of MUC1.Tg mice with MUC1p results in decreased DC motility
(A) WT and MUC1.Tg mice were vaccinated i.v. with DC loaded with MUC1p. RNA was extracted from pooled splenic DC 72h post vaccination for qRT-PCR. Bars represent mean ± SEM. Data are representative of three independent experiments. (B) and (C) WT and MUC1.Tg mice were vaccinated as in (A). 72h post immunization, splenic DC were bead isolated for live cell imaging. The track length (B) and displacement (C) were analyzed after 20h in culture. Each dot represents a single DC and bars depict mean ± SEM. Data are from two mice comparing 6×103 DC per group.
Figure 3
Figure 3. Immunization of WT but not MUC1.Tg mice with MUC1p results in up-regulation of pancreatic enzymes in splenic DC
(A) WT and MUC1.Tg mice were injected i.v with unloaded BMDC (ctrl) or BMDC loaded with MUC1 peptide (MUC1p). 24h later spleens were harvested, pooled according to group, and RNA extracted for qRT-PCR. Arbitrary Units were normalized to WT mice given the ctrl vaccine. Bars represent mean ± SEM. Data are representative of two independent experiments. (B) Splenic DC from unvaccinated mice were isolated with CD11c+ beads (n=3), total splenic T cells were isolated using negative selection via MACS depletion of CD3− cells, and BMDM (MΦ) were cultured for 8 days in the presence of L-cell supernatant as a source of M-CSF. RNA was isolated from all populations for qRT-PCR analysis. Units were normalized to expression levels in CD11c+ cells.Bars represent mean ± SEM. Data representative of two independent experiments. (C) WT and MUC1.Tg mice were immunized as in (A). At 24h, splenic DC were isolated using CD11c+ beads for analysis by qRT-PCR or Western blotting for trypsin and CPB1 (D). Bars represent mean ± SEM after normalization to control vaccination. Data are representative of two (C) and three (D) independent experiments. (E) Mice were immunized i.v. with PBS (ctrl), Poly-ICLC (adj), or soluble MUC1p admixed with Poly-ICLC (MUC1p + Adj). 24h later spleens were harvested for qRT-PCR analysis. Bars represent mean ± SEM normalized to PBS control and are representative of four independent experiments.
Figure 4
Figure 4. Failure of DC to up-regulate pancreatic enzymes following immunization with MUC1p as a self-antigen is recapitulated in the OVA model of self-tolerance
(A) MUC1.Tg mice were immunized i.v. with PBS (ctrl), soluble MUC1p or ovalbumin (OVA) admixed with Poly-ICLC. Spleens were harvested at 24h post immunization and pooled for qRT-PCR analysis. Bars represent mean ± SEM normalized to PBS control. Data are representative of three independent experiments. (B) MUC1.Tg mice were immunized i.v. with unloaded DC (ctrl) or DC loaded with OVA (OVA). 24h post-immunization splenic DC were MACS purified for qRT-PCR analysis. Bars represent mean ± SEM normalized to ctrl. Data are representative of three independent experiments. (C) RIP.OVA mice were immunized and processed as in (B). Bars represent mean ± SEM normalized to ctrl vaccination. Data are representative of two independent experiments.
Figure 5
Figure 5. Interactions between DC and CD4 T cells regulate expression levels of pancreatic enzymes in DC
(A) DC were cultured alone (ctrl), with LPS, or with polyclonally activated CD25-CD4+ T cells (Teff) and/or CD25+CD4+ T cells (Treg). After 24h of co-culture DC were separated and mRNA was extracted for qRT-PCR analysis. Units were standardized against levels pre-culture (baseline). Bars represent mean ± SEM. Data are representative of two independent experiments. (B) MUC1.Tg mice were treated with an antibody against CD25 to deplete regulatory CD4 T cells (Anti-CD25) or with an isotype control (ctrl). 2 days following depletion, mice were vaccinated with soluble MUC1p plus Poly-ICLC i.v. Splenic RNA was extracted 24h post vaccination for qRT-PCR analysis. Units were standardized against isotype control treated mice. Bars represent mean ± SEM respectively. Data are representative of 3 independent experiments.
Figure 6
Figure 6. IL-10 is required in vivo for regulation of pancreatic enzymes expression in DC
(A) MUC1.Tg mice were immunized with PBS (ctrl) or a soluble MUC1p admixed with Poly-LCIC (MUC1p). IL-10 expression was measured by qRT-PCR on total splenic mRNA 24h post vaccination. Bars represent mean ± SEM. Data are representative of at least 4 independent experiments. (B) WT and MUC1.tg mice were treated with an IL-10R blocking antibody followed by i.v. immunization with PBS (ctrl) or MUC1p as in (A). 24h post vaccination splenic RNA was extracted for qRT-PCR analysis. Units were normalized to WT ctrl. Bars represent mean ± SEM. Data are representative of two independent experiments.
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