Fig 1.
Diagram of MUC1 Domains and Peptides Chosen for Study.
The domains of MUC1 are shown on left side of the diagram with the sequences studied listed below the domains and the peptide name on the right side. Immunizing peptides and serines (S) and threonines (T) that are O-glycosylated with N-Acetylgalactosamine (Tn) are shaded.
Fig 2.
Diverse MUC1 Antigen Preparations Generate Specific Immune Responses in Tolerant MUC1.Tg and Non-Tolerant WT Mice.
MUC1.Tg mice were given three immunizations with vaccines containing 9mers, 22mers or rotating tumor lysates (Fig 1). Lymph node-derived T-cells were culture expanded for 7–14 days with DCs pulsed with the immunizing antigens. Antigen-specific T-cells were enumerated for intracellular IFN-γ production when re-stimulated with DCs pulsed with short peptides (A) and long peptides (B). Data are shown after subtracting background from unpulsed DCs to facilitate visual comparisons. See Fig 2D for examples of representative unsubtracted backgrounds. A representative of 2 experiments is shown; pools of 7 mice were used. (C) Wide specificity of the lysate-sensitized T-cells: Lysate sensitized T-cells from MUC1.Tg mice or WT mice showed specificity against 19 out of 19 MUC1 peptides from both TR and non-TR regions. (D) MUC1.Tg mice were immunized twice with vaccines containing either long peptides from TR, APG 22mer (APGSTAPPAHGVTSAPDTRPAP) or the CT peptide (SLSYTNPAVAATSANL). After in vitro stimulation with DCs pulsed with immunizing peptides, antigen specific T-cells were analyzed for intracellular IFN-γ against dendritic cells pulsed with peptides (APG 22mer or CT) or no peptide (UP). One representative of three experiments is shown; pools of 7 mice were used in each experiment.
Fig 3.
Aspects of Antigen Presentation Relevant to Tumor Recognition are Shown.
(A) Tumor cell expression of MUC1 was stable in vitro (C57mg.MUC1: 86%; KCM: 73%; EL4.MUC1: 79%; B16.MUC1 and Panc02.MUC1: 95%; MC38.MUC1: 99%) (B) Irradiated tumor digests passed in syngeneic mice prior to processing were used to assess T-cell recognition of tumor-associated MUC1. Representative flow data for Panc02.MUC1 is shown. MUC1 was predominantly expressed on the tumor cell population, while both MHC class I and II molecules were expressed by CD45+ host cells as well as tumor cells. (C) Glycosylation alters DC processing of MUC1. DCs were pulsed overnight with non-glycosylated APG 22mer or one of three glycoforms of APG 22mer (5-Tn; 4, 5-Tn or 18-Tn). The DCs were then analyzed for presentation of the C-terminal peptide SAPDTRPAP (PDTR) by flow cytometry with anti-MUC1 (BC2-Alexa488) specific for PDTR. Cells staining positively for PDTR also co-stained for CD11c+, Kb (Fig 3C) and I-Ab (not shown). Detection of PDTR on DCs was only observed if APG 22mer was glycosylated at 4-Tn or 4, 5-Tn (Fig 3C). (D) Glycosylation promotes co-localization of SAPDTRPAP with class I molecules. DCs in chamber slides were stimulated overnight with either non-glycosylated (APG 22mer) or glycosylated (APG 22mer 5-Tn) peptides and stained with anti-MUC1 (BC2-Alexa488; green) and anti-H-2Kb followed by secondary goat anti-mouse IgG-labeled-Alexa633 (red). Representative confocal images showed stronger co-localization (yellow) of epitope SAPDTRPAP with H-2Kb on the DCs pulsed with the glycopeptide. The experiment was repeated two times. (E) Individual forms of antigen during vaccination elicited differential recognition of MUC1-expressing tumors. Effector T-cells from MUC1.Tg mice immunized with vaccines containing either 9mers, 22mers or rotating tumor lysates were stimulated in vitro for 7–14 days with DCs pulsed with immunizing peptides or B16.MUC1-expressing tumor cell lysate. The stimulated T-cells were co-cultured with various MUC1-expressing or MUC1 non-expressing irradiated tumor digests (C57mg.MUC1, C57 WT; KCM, KCKO; EL4.MUC1, EL4.WT; B16.MUC1, B16.neo; Panc02.MUC1, Panc02.neo and MC38.MUC1, MC38.neo) and stained for intracellular IFN-γ. Data showed MUC1-specific responses that were determined by subtraction of background reactivity of the corresponding MUC1 non-expressing tumor digests. Representative data of two independent experiments are shown; pools of 7 mice were used.
Fig 4.
Lysate-Sensitized but not Peptide-Sensitized Mice are Protected Against Subsequent Challenge with MUC1-Expressing Tumor.
MUC1.Tg or WT mice were immunized with vaccines containing rotating MUC1-expressing tumor cell lysates or peptides (Fig 1) prior to challenge with MUC1-expressing cells (B16.MUC1, Panc02.MUC1). Tumor growth was monitored by palpation. (A) Peptide immunizations failed to protect B16.MUC1 tumor growth; (B) & (C) In both WT and MUC1.Tg mice, the vaccination with rotating lysates induced MUC1-specific immune responses that cured 4 out of 10 WT mice (B insert) (p = 0.047) and 5 out of 10 MUC1.Tg mice (C insert) (p = 0.0027). B16.MUC1 tumors that grew out in both (B) WT and (C) MUC1.Tg mice showed significantly delayed tumor growth (p<0.001). The same immunizations had no effect on the B16.neo tumor outgrowth (B) (p = n.s). (D) In Panc02.MUC1 tumor challenge, the onset of tumor was significantly delayed in the immunized mice as compared to the non-immunized controls (p = 0.02). Groups of 5 mice were tested and the experiment was repeated twice.
Fig 5.
Tumor Progression Following Immunization with Either Peptides or Rotating Lysates Prior to Tumor Challenge Consistently Resulted in Decreased Expression of Tumor MUC1 and MHC Proteins.
The resistant B16.MUC1 tumors that grew after peptide immunizations were excised, digested and analyzed by flow cytometry with anti-CD45 APC-Cy7, anti-MUC1 FITC (CD227), anti-H-2Kb PE or anti-I-Ab PE. Cell surface MUC1 and MHC expression were significantly decreased following peptide vaccination compared to non-immunized mice. (A) Each bar represents the average fluorescent index (FI) of surface MUC1 expression for each treatment group. A typical experiment with n = 3 mice per group is shown; experiments were repeated at least 3 times. (B) Representative histograms are shown. The peptide shown is SAP 22mer, which is representative of the results for 9mer peptides. (C) Similar results were seen for Panc02.MUC1 in those groups where prior lysate immunization failed to prevent tumor outgrowth. (D) The cell line generated from Panc02.MUC1 tumor in vitro in G418 maintained low MUC1 surface expression when expanded (right panel) as compared to the parent cell line (D, left panel).
Fig 6.
Peptide Vaccine is Rendered Therapeutically Effective if Repetitive Delivery is Continued Post Tumor Challenge.
MUC1.Tg mice were given two peptide immunizations before and two after tumor challenges. Three out of four peptide vaccinations significantly delayed the growth of B16.MUC1 tumors (APG+SAP 9mer, p = 0.001; APG+SAP Tn-9mer, p = 0.001; SAP 22mer, p = 0.001) and (B) showed complete protection in 4 out of 10 mice (SAP 22mer) and 3 out of 10 mice (9mers and Tn-9mers), if immunizations were continued post tumor challenges. Experiment was repeated 2 times, n = 5 mice/group.
Fig 7.
Lysate Sensitized T-Cells from MUC1.Tg Mice Conferred Protection in Adoptive Transfer Experiments (Winn Assay).
(A) The sorted spleen-derived effector T-cells from MUC1.Tg mice immunized with rotating tumor cell lysates were co-injected with B16.MUC1 tumor cells (T-cell to tumor cell ratio of 10:1) either directly or after stimulation in vitro with DCs pulsed with B16.MUC1 tumor cell lysates. T-cells from non-immunized MUC1.Tg mice were co-injected with B16.MUC1 tumor cells as controls. The mice that received T-cells after in vitro sensitization showed complete protection from tumor growth, (p<0.001). (B) The B16.MUC1 tumors resistant to T-cells from immunized MUC1.Tg mice showed low MUC1 expression vs non-immunized mice (p = 0.02). (C) Corresponding histograms of MUC1 expression are shown. Experiment was repeated two times, n = 4 mice/group.
Fig 8.
MUC1.Tg Mice Exhibited Incomplete Immunological Memory Following Tumor Rechallenge.
(A) Mice that rejected the primary tumor challenge (previously immunized with SAP 22mer or rotating cell lysates) were re-challenged with B16.MUC1 from 75 to 200 days later. There was no further vaccination following the re-challenge. WT mice consistently rejected the tumor whereas MUC1.Tg mice displayed tumor outgrowth that (B) down regulated MUC1 and class I expression. n = 2 for WT mice/group or 5 MUC1.Tg mice/group.