Dendritic Cells are Both Targets and Initiators of Peripheral Immune Tolerance to Self (original) (raw)
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The Journal of Immunology, 2013
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.
The Journal of Immunology, 2013
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-kB, 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.
Cancer Research
Dendritic cells (DCs) are professional antigen-presenting cells, well equipped to initiate an immune response. Currently, tumor antigenderived peptide loaded DCs are used in clinical vaccination in cancer patients. However, the optimal dose and route of administration of a DC vaccine still remain to be determined. Using indium-111-labeled DCs, we investigated whether the route of administration does affect the biodistribution of DCs in lymphoid organs and whether it influences the outcome of DC vaccination in the B16 mouse melanoma tumor model. The results demonstrate that i.v. injected DCs mainly accumulate in the spleen, whereas s.c. injected DCs preferentially home to the T-cell areas of the draining lymph nodes. Using tyrosinase-related protein-2-derived peptide-loaded DC vaccination in a fully autologous B16 melanoma tumor model, we observed a delay in tumor growth, improved survival as well as increased antitumor cytotoxic T-cell reactivity after s.c. vaccination as compared to i.v. vaccination. These data demonstrate that optimal induction of antitumor reactivity against the autologous melanocyte differentiation antigen tyrosinase-related protein-2-derived peptitde occurs after s.c. vaccination and correlates with the preferential accumulation of DCs in the T-cell areas of lymph nodes.
Dendritic Cells in Cancer Immunotherapy
Annual Review of Immunology, 2000
Dendritic cells (DCs) play a central role in the initiation and regulation of innate and adaptive immune responses and have increasingly been applied as vaccines for cancer patients. Ex vivo generation and antigen loading of monocyte-derived DCs allows a controlled maturation, with the aim of imprinting different DC functions that are essential for their subsequent induction of a T cell-mediated anti-tumor response. A better understanding of how DCs control T cell immunity is important for the design of novel DC-based cancer vaccines with improved clinical efficiency. The aim of this thesis was to evaluate how different maturation conditions used for generation of clinical grade DC-based cancer vaccines affect their capacity to assist type-1 polarized immune responses, important for elimination of cancer.
Cancer Research and Treatment, 2014
Purpose Targeted immunotherapy using dendritic cells (DCs) has been employed in numerous investigations aiming at combating neoplasms. We previously showed that copulsing of an antigen with a helper protein could considerably enhance antigen presenting capacity of ex vivo-generated DCs. In this study, we attempted to administer an effective treatment in a murine model of colon cancer with DCs pulsed with the mixture of a tumor-specific gp70-derived peptide (AH1) and a helper protein, ovalbumin (OVA). Materials and Methods First, the presence of gp70 in CT26 tumor cells and tumor tissues was verified using immunofluorescence and Western blot analyses. Next, DCs were purified from normal mice, loaded ex vivo with AH1 and OVA (DC-Pep-OVA), and injected into tumor-bearing mice. Tumor volume, in vitro antigen (Ag)-specific proliferation of splenic cells, and survival rate were measured to determine the efficacy of DC-Pep-OVA. As the control groups, tumor-bearing mice were vaccinated with DC-Pep, unpulsed DC, and DCs loaded with a mixture of OVA and an irrelevant peptide (P15), or were not vaccinated at all. Results DC-Pep-OVA showed superior efficacy over other groups, as indicated by smaller tumor volume, higher Ag-specific proliferation rate of splenic cells, and prolonged survival. Conclusion Overall, in the present study we showed for the first time that DCs copulsed with AH1 (tumor Ag) and OVA (helper molecule) could be considered as potentially robust weapons for use in future antitumor immunotherapies.
Unpulsed dendritic cells induce broadly applicable anti-tumor immunity in mice
Cancer Biology & Therapy, 2005
The discovery of dendritic cells (DC) as professional antigen presenting cells has opened up new possibilities for their use in the development of cancer vaccines. Here we show that unpulsed BM-derived CD11c + CD8α-DC administered s.c. to mice enhanced their resistance to subsequent lethal tumor challenges. The tumor resistance-inducing activity of unpulsed DC was dependent on their maturation status, involved CD4 + and CD8 + T cell activities, and was abrogated by pulsing with an irrelevant class I MHC-restricted peptide. Although the BALB/c Meth A tumor system was employed extensively to demonstrate the tumor resistance inducing activity of unpulsed DC, the immunogenicity of this vaccine was evident in other inbred strains of mice against a variety of syngeneic tumors. The broad anti-tumor responses induced by unpulsed DC appears to be due to their capacity to present "self" tumor-associated antigens, such as the H2-K d-restricted, wild type sequence p53 232-240 peptide. These findings highlight the potential broad applicability of unpulsed DC as a tumor vaccine. MATERIALS AND METHODS Mice. BALB/cJ, C57BL/6J (B6) DBA/2J and (BALB/c x C57BL/6)F1 (CB6F1) female mice, 6-8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the Animal Facility of the University of Pittsburgh Cancer Institute and used in protocols approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Tumors and tumor cell lines. The chemically induced BALB/c Meth A sarcoma has been previously described. 18,19 The BALB/c C26 colon carcinoma was obtained from Dr. M. Colombo (Milan, Italy), the renal cell carcinoma (RENCA) from Dr. E.M. Jaffee (Johns Hopkins School of Medicine, Baltimore, MD), and the B6 MC38 colon carcinoma from Dr. A. Gambotto (Pittsburgh, PA). 20,21 The DBA/2 KL205 lung carcinoma was purchased from the American Type Culture Collection, Rockville, MD (ATCC). Culture medium and monoclonal antibodies (mAb). Complete medium (CM) consisted of RPMI 1640 supplemented with 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 100 µg/ml streptomycin, 100 units/ml penicillin, Cellgro (Mediatech, Inc., Herndon, VA), 50 µM 2-ME (Bio-Rad, Hercules, CA) and either 10% heat-inactivated FBS (HyClone, Logan, UT) or 1% normal normal mouse serum (NMS). Murine rGM-CSF (15 ng/ml) and rIL-4 (15 ng/ml) from R&D Systems (Minneapolis, MN) were added to CM. Hybridomas producing rat anti-mouse CD4 (clone GK1.5, TIB207;) anti-mouse CD8 (clone 2.43,TIB217) mAb were from ATCC. The anti-CD4 and-CD8 mAbs were immunoaffinity purified from spent medium using Protein G and Gentle pH buffer (Pierce Chemical Co., Rockford, IL). DC generation. DC fbs and DC nms were generated from mouse BM cells using CM supplemented with either FBS or NMS, respectively, as previously described. 19 To obtain highly mature populations of C fbs+LBS , the DC were generated from BM cells and replated at day five and seven, as previously described by Labeur et al. 17 On day seven, Escherichia coli-LPS (0.1 µg/ml; Sigma) was added for an additional two days of culture before harvesting. Cell surface antigenic phenotype of DC. The phenotype of DC was determined by flow cytometry analysis using FITC-conjugated anti-CD8α (clone 5H10; Caltantigen, Burlingame, CA),-anti-CD80 (clone 16-10A1), PE-conjugated anti-CD4 (clone GK1.5),-CD40 (clone 3/23),-CD45R/B220 (clone RA3-6B2),-CD54 (clone 3E2),-CD86 (clone GL1),-Gr-1 (Ly-6G) (clone RB6-8C5) and-I-A d (clone AMS-32.1) mAbs from BD PharMingen. San Diego, CA. Biotinylated anti-CD11c mAb (clone HL3) was used for indirect immunofluorescent staining with the Streptavidin-Cy-Chrome TM system (both from BD PharMingen). The appropriate FITC or PE-conjugated isotype IgG antibodies were used as controls. Samples were stained with the antibody concentrations recommended by the manufacturer for 30 min at 4˚C and washed two times with staining buffer (PBS 0.1% sodium azide, 0.1% BSA). After the second step of staining, cells were washed twice with staining buffer, fixed with 2% paraformaldehyde and analyzed. CD11c + /CD8α-/ Gr-1cells were considered DC and used to determine the expression of costimulatory molecules. Immunization of mice. Mice were injected s.c. in the inguinal lymph node areas twice within a ten-day interval with 0.1 ml of various concentrations of DC for total doses of 2 x 10 4 to 2 x 10 5 DC. The H2-K drestricted wt p53 232-240 and enhanced green fluorescence protein (EGFP 200-208) (HYLSTQSAL) peptides were used in the study. 18,19,22 DC or splenocytes were pulsed for 60 min at RT with 10 µg/ml peptide/PBS, washed and resuspended in PBS prior to their use. Control mice received PBS alone. In tumor rejection assays, 7 to 10 days after the second immunization, mice were challenged s.c. in the dorsal flank regions with lethal doses of Meth A ascites cells or an enzyme digested single cell suspension of an in vivo grown solid tumor. 23 Areas of tumors were determined by measuring two tumor diameters perpendicular to each other with calipers. Data are reported as the mean tumor area (MTA) ± SEM. Student's t tests were performed to determine significant differences between experimental groups.
The Journal of Immunology, 2005
The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DCs) has been well characterized. There are many fewer examples of similar interactions between DCs and self-molecules, especially the abnormal self-proteins such as many tumor Ags, and their effects on DC function and the immune response. We show that human epithelial cell Ag MUC1 mucin is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DCs as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). On encounter with MUC1, similar to the encounter with LPS, immature DCs increase cell surface expression of CD80, CD86, CD40, and CD83 molecules and the production of IL-6 and TNF-α cytokines but fail to make IL-12. When these DCs are cocultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our d...