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. 2014 Jan 15;192(2):658-65.
doi: 10.4049/jimmunol.1301904. Epub 2013 Dec 11.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen

Adam M Farkas  1 , Olivera J Finn
Affiliations

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen

Adam M Farkas et al. J Immunol. .

Abstract

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

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Figures

Figure 1
Figure 1. DC isolated from MUC1p-immunized MUC1.Tg mice are less inflammatory than DC from immunized WT mice
(A) WT and MUC1.Tg mice (n=3/group) were immunized (i.v.) with DC loaded with MUC1p (1×106 cells). 24 hours later isolated splenic DC were cultured overnight in the presence of 500ng/mL LPS and brefeldin A. DC lysates were then incubated on a mouse cytokine blot array that were developed according to the manufacturer’s instructions. Blots show global expression of all 40 chemokines and cytokines queried in with a global preponderance of decreased expression in DC from immunized MUC1.Tg mice (B) Densitometry of the blots from A quantified with ImageJ. (C) qPCR performed for CCR2 on DC 24 hours post vaccination. Data are normalized to MUC1p-immunized WT mice, with bars representing the mean ± SEM. Data are representative of 2 (A) and 3 (C) independent experiments.
Figure 2
Figure 2. SOCS1 and HDAC11 do not contribute to the tolerized state, while sustained expression of Zbtb46 and CCR2 maintains an immature DC phenotype
WT and MUC1.Tg mice (n=3/group) were immunized (i.v.) with DC:MUC1p (MUC1p) (1×106 cells) or unloaded DC (Ctrl). 1d later, isolated DC were pooled for qRT-PCR for SOCS1 (A) or HDAC11 (B). Bars represent mean ± SEM after normalization to respective control vaccinations and are representative of 3 independent experiments. (C and D) Mice were immunized with MUC1p as in (A), splenic DC isolated at 24h and were left untreated (No Tx) or treated with 30ug/mL of Poly:ICLC (Poly:ICLC) for 4h before performing qPCR for zDC (C) or CCR2 (D).
Figure 3
Figure 3. Deficient NFκΒ activation and enhanced STAT3 signaling in tolerized DC
WT and MUC1.Tg mice (n=3/group) were immunized (i.v.) with DC:MUC1p (MUC1p) (1×106 cells) or unloaded DC (Ctrl). 24 hours later, isolated splenic DC were pooled and whole cell lysates were analyzed by Western blots for phospho-STAT3 (A), phospho-p65, (B) or total IκΒα (C). B-Actin is shown as a loading control for each blot. Data are representative of 2–3 independent experiments for each molecule.
Figure 4
Figure 4. Production of aldehyde dehydrogenase by tolerized DC
WT and MUC1.Tg mice (n=3/group) were immunized (i.v.) with DC:MUC1p (MUC1p) (1×106 cells) or unloaded DC (Ctrl). 24 hours later, isolated DC were pooled and qRT-PCR performed for Aldh1 (A). Concurrently, whole cell lysates from recovered DC were Western blotted for Aldh1/2 (B) and bands quantified via densitometry (C). (D) MUC1.Tg mice were immunized as described above immediately after injection of the aldehyde dehydrogenase inhibitor diethylaminobenzaldehyde (MUC1p+DEAB). 24 hours later, isolated splenic DC were pooled and qRT-PCR performed for Trypsin and CPB1. Bars represent mean ± SEM after normalization to respective control vaccinations (A and D). Data are representative of 2 (D) and 3 (A–C) independent experiments.
Figure 5
Figure 5. Activity of the metallopeptidase CPB1 in DC is required for optimal proliferation of MUC1p-specific CD4+ T cells
(A) BMDC were cultured overnight in the presence of 30ug/mL MUC1p and 30ug/mL Poly:ICLC with or without protease inhibitors (no inhibition, 10uM or100uM orthophenanthroline and 100ug/mL trypsin inhibitor, respectively). The following day, naïve CFSE-labeled MUC1p-specific CD4+ T cells were added at a 1:5 DC:T cell ratio and cultured for 3d. Cells were then harvested and percent proliferation determined by CFSE dilution. Histograms are from one experiment and are representative of 2 independent experiments. (B) Quantification of pooled data from 2 independent experiments. Bars represent mean ± SEM.
Figure 6
Figure 6. Trypsin activity in DC enhances degradation of the extracellular matrix
(A) Splenic DC were isolated from naïve WT mice and cultured for 40h in the presence or absence of 30ug/mL Poly:ICLC. Cell-free supernatants were collected and Western blotted for the presence of trypsin and CPB1. Data are representative of 3 independent experiments. (B) WT and MUC1.Tg mice (n=2/group) were immunized (i.v.) with DC:MUC1p (MUC1p) (1×106 cells) or unloaded DC (Ctrl). 2d later, splenic DC were isolated, pooled, and plated onto chamber slides coated with a FITC-Gelatin matrix in the presence or absence of 100ug/mL of trypsin inhibitor. Cells were fixed after 4h, and images collected on an epifluorescent microscope and quantified with Image J (C). Bars represent mean ± SEM. Data are representative of 2 independent experiments.
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