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Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

<div><p>Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the <em>in vivo</em> response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.</p> </div

MUC16 expression is inversely correlated with sensitivity to CTL-mediated killing.

<p>Influenza peptide pulsed CFSE labeled Capan-1 cells stained for MUC16 expression and propidium iodide (PI) uptake after 4 hrs co-culture with influenza peptide specific CTLs as analyzed by flow cytometry. The % PI positive cells are quantified in the MUC16 high and low expressor populations within the CFSE positive Capan-1 WT cells. Experiments performed with four different PBMC donors. Data from one representative donor is shown.</p

Expression profile of Capan-1 and T47D WT and COSMC KO cells.

<p>Surface/non-permeabilized (Non-perm) glycoepitope and MUC1 expression of Capan-1 and T47D WT (pink) and COSMC KO (blue) cell lines as quantified by flow cytometry using HMFG-2 (MUC1), 5E10 (MUC1), 5F4 (Tn), 3F1 (STn) and 3C9 (T) Abs. In the last row, cells have been treated with neuraminidase (neu) prior to staining, thereby removing the sialic acid from ST and allowing 3C9 to stain both the existing T structure and the former ST structures after the sialic acid removal. Isotype control for WT (purple) and KO (green) cells was used as background control.</p

Capan-1 COSMC KO cells are more susceptible to CTL-mediated killing than Capan-1 WT.

<p>Chromium release assay with Capan-1 WT/Cosmc KO target cells either (GILGFVFTL ) influenza peptide loaded or not. Influenza peptide primed CD8+ T cells were used as effector cells in an E∶T ratio of 4∶1. The bars show mean +/− SD of the calculated specific killing from quadruplicates. Student's two-tailed t test was applied to test for significance; stars indicate level of significance. Results from two out of three donors are shown.</p

COSMC KO renders Capan-1 and T47D cells more susceptible to ADCC.

<p>A)–C) WT and COSMC KO target cells were labeled with chromium and co-cultured with PBMC effector cells in an E/T ratio of 50∶1 (black) and 25∶1 (white) either with or without Ab (Erbitux®). After 4 hrs the chromium release into the media was measured and the % specific killing was calculated. The bars show mean +/− SD from quadruplicates. Student's two-tailed unpaired t test was applied to test for significance, stars indicate level of significance. Experiments performed with two to six different PBMC donors. Data from one representative donor is shown. Experiments where cells were treated with +/− neuraminidase to remove sialic acid before ADCC is included in B) and COSMC KO cells transiently rescued with COSMC pcDNA3 transfection as well as mock transfected control are included in C). D) Capan-1 and T47D were treated with hydrogen peroxide for 20 hrs (0–0.2 mM range for Capan-1 and 0–0.5 mM range for T47D) prior to apoptosis staining using Annexin V and PI. Annexin V positive cells are considered early apoptotic whereas double positive cells are considered late apoptotic. The % positive is defined by the percent of cells above the MFI of an unstained background control in a flow cytometric analysis. One representative data set is shown out of two to three individual experiments performed.</p

Peptide sequences for DC T cell hybridoma co-culture.

<p>1 and 2 (H2-Kb restricted fusion), 3 and 4 (I-Ab restricted fusion). Underlining indicate sites of glycosylation. Internal (I), terminal (T), N-Acetylgalactosamine (GalNAc).</p

Peptide glycosylation inhibits activation of antigen specific CD8+ T cell hybridoma.

<p>IL-2 production from OVA specific CD8+ T cell hybridoma (RF 33.70) co-cultured with bone marrow derived DCs (A) or CD11c+ DCs purified from mouse spleen (B). DCs were pulsed with two peptide variants with and without 2 and 4 glycan residues (GalNAc). Full length OVA was used as a positive control. Individually cultured T cells and DCs had an OD value equal to the background. C, D) Surface expression by flow cytometry of SIINFEKL in the H2kb peptide binding groove on DCs after pulsing with the two peptide variants (non-glycosylated peptide (thin purple dashed line), peptide with 2 GalNAcs (thick green line), or 4 GalNAcs (pink line)). E) Surface expression of SIINFEKL in the H2kb peptide binding groove on DCs without pulsing (blue line) and after pulsing with OVA control (purple line). Gray histograms represent cells stained only with secondary antibody.</p

MUC1 and 16 expression is inversely correlated with sensitivity to ADCC-mediated killing.

<p>CFSE labeled Capan-1 cells stained for MUC1 or MUC16 expression and propidium iodide (PI) uptake after 4 hrs co-culture with Erbitux® and PBMCs as analyzed by flow cytometry. A) Gating strategy on CFSE positive Capan-1 cells and from that population the selection of high and low MUC16 expressing cells. The gating strategy is the same for the MUC1 stained cells. B) The % PI positive cells are quantified in the MUC1/16 high and low expressor populations within the CFSE positive Capan-1 WT or COSMC KO cell population. Experiments performed with six different PBMC donors. Data from one representative donor is shown.</p

<i>In vivo</i> response to degMUC1 in WT Balb/c and HLA-A2 transgenic mice.

<p>A) Serum reactivity to different mucin glycoforms from Balb/c mice immunized with degMUC1+/− GalNAc by ELISA. Data are representative of a minimum of 4 Balb/c mice immunized with each antigen. (B) T cell proliferation from mice Balb/c mice or (C) HLA-A2 mice immunized with GalNAc degMUC1 (white bars) and non-glycosylated degMUC1 (grey bars) for each peptide used for <i>in vitro</i> re-stimulation (100 ug/ml) as indicated on the x-axis. D) Lymphocytes from immunized HLA-A2 mice pulsed with the 9 mer degMUC1 peptide, ALGSTAPPV, with and without glycosylation. DegMUC1 specific CD8+ T cells were selected based on anti-CD8 Ab (x-axis) and anti-IFNγ Ab (y-axis) after 4 hrs of Golgi stop treatment. E) Spleen cells after 24 days of re-stimulation with the non-glycosylated ALGSTAPPV peptide. All T cell data is generated from spleen or lymph nodes from at least 4 mice, but in most cases 6 mice.</p