Caroline B. Madsen

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

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 in vivo 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.

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Elevated mRNA levels of CTLA-4, FoxP3, and granzyme B in BAL, but not in blood, during acute rejection of lung allografts.

INTRODUCTION Regulatory T cells (Tregs) have been related to acute rejection as have the cytotoxic T cells, their immunological counterpart. High expression of cytotoxic markers has been related to acute rejection incidents following both kidney and intestine transplantation, while the correlation between FoxP3 expression and acute rejection is still being debated. Some studies have been performed on blood samples from lung-transplanted patients, while others have investigated the local immune response in the lungs by analysing broncho-alveolar-lavage (BAL) fluids or biopsies. Biopsies are considered the gold standard in diagnosis of acute rejection. AIM The aim was to measure the expression of both Treg (FoxP3, CD25 and CTLA-4) and cytotoxic (granzyme B, granulysin, and perforin) markers in BAL and blood samples from lung-transplanted patients to investigate the possible relation of expression and acute rejection incidents in order to develop a non-invasive diagnostic method for acute rejection. MATERIALS AND METHODS 24 lung-transplanted patients were included in this 6-month cross-section study. BAL and blood samples were analysed for FoxP3, CD25, CTLA-4, granzyme B, granulysin, perforin, CD4 and CD8 mRNA by QRT-PCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as primary reference. RESULTS We demonstrate that the mRNA levels in BAL relative to GAPDH of nearly all markers are elevated during acute rejection; CTLA-4, FoxP3, and granzyme B significantly, while a strong tendency is seen among the others. No significant differences were detected in blood. CONCLUSION CTLA-4, FoxP3 and Granzyme B mRNA levels are elevated during acute rejection in BAL, but not in blood, following lung transplantation, indicating that evaluation of Treg and cytotoxic marker expression in BAL can be used in the assessment of allograft rejection state.

Glycan Elongation Beyond the Mucin Associated Tn Antigen Protects Tumor Cells from Immune-Mediated Killing

Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galβ1–3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galβ1–3GalNAc-Ser/Thr) antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection.

BACKGROUND The transcription factor Forkhead Box P3 (FoxP3) is a marker of regulatory T cells (Tregs) - a subset of T cells known to suppress a wide range of immune responses. These cells are considered to be pivotal for the induction of tolerance to donor antigens in human allografts. We aimed to correlate the number of lymphocytes expressing FoxP3 in transbronchial biopsies from lung allografts with the FoxP3 expression in bronchoalveolar lavage fluid (BALF). In addition, we aimed to correlate the number of FoxP3+ cells in transbronchial biopsies with the degree of acute cellular rejection in lung allografts. MATERIALS AND METHODS The expression of FoxP3 was evaluated using immunohistochemical staining in 40 lung allograft biopsies obtained from 23 patients. The number of Tregs was related to the FoxP3 mRNA levels as determined using qRT-PCR in corresponding BALF samples from the same patients. Furthermore, the number of Tregs was related to the degree of acute allograft rejection (according to ISHLT criteria, A0-A4). RESULTS Regression analysis showed a significant concordance between the number of Tregs in lung tissue and the level of FoxP3 mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels in BALF (n=40, p=0.0001). In addition, we found a significant increase in the number of Tregs during acute allograft rejections of grades A2 and higher (median: 32.6Tregs/mm(2)) when compared to those of grades A1 and A0 (median: 4.9Tregs/mm(2)) (p=0.0002). DISCUSSION AND CONCLUSION The association between the distribution of Tregs in transbronchial biopsies and the level of FoxP3 mRNA in BALF indicates that assessment of FoxP3 mRNA in BALF is a reliable non-invasive method for evaluating the number of Tregs in lung tissue. Furthermore, the association between the number of Tregs in lung tissue and the degree of acute cellular rejection shows that Tregs are recruited to the site of inflammation and may be involved in the regulation of acute rejection. Thus, Tregs may play a role in the cellular processes that affect lung allograft outcome.

Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy

Abstract MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen made. We recently demonstrated that a monoclonal antibody against the immunodominant Tn-MUC1 (GalNAc-α-MUC1) antigen induced ADCC in breast cancer cell lines, suggesting the feasibility of targeting combined glycopeptide epitopes in future passive cancer immunotherapy.

Glycan-mediated modification of the immune response

Aberrantly glycosylated tumor antigens represent promising targets for the development of anti-cancer vaccines, yet how glycans influence immune responses is poorly understood. Recent studies have demonstrated that GalNAc-glycosylation enhances antigen uptake by dendritic cells as well as CD4+ T-cell and humoral responses, but prevents CD8+ T-cell activation. Here, we briefly discuss the relevance of glycans as candidate targets for anti-cancer vaccines.

Induction of Bcl‐xL‐Specific Cytotoxic T Lymphocytes in Mice

The induction of active immunity against tumour‐associated antigens to prevent relapse of cancer is a promising approach but has so far shown only low efficacy. This low efficacy may in part be due to clonal escape of tumour cell variants by the downregulation of antigen expression or inflammation‐induced dedifferentiation. Identification of novel tumour‐associated antigens that at the same time are essential for continued tumour cell survival is thus critical for the development of active cancer vaccinations. At the same time, identification of novel endogenous murine tumour antigens will help improve preclinical development of cancer immunotherapy. The anti‐apoptotic protein Bcl‐xL has been suggested to be such an essential tumour antigen, but the lack of well‐defined murine epitopes have delayed preclinical studies of Bcl‐xL‐targeting cancer vaccines. Here, we report the identification of two novel murine tumour‐associated epitopes TAYQSFEQV and AFFSFGGAL derived from mouse Bcl‐xL. Dendritic cell (DC)‐based vaccination induced CD8+ T cells capable of producing IFN‐γ upon restimulation with these epitopes. Thus, our data may benefit the design of future immunotherapy strategies by providing a preclinical model for cancer vaccination with an endogenous tumour antigen that can be combined with other cancer treatments.

MUC4-specific CTLs

To the editors: Wu et al. reported cytotoxic T lymphocyte (CTL) recognition of LLGVGTFVV HLA-A*02 binding MUC4-derived peptide in healthy individuals, and MUC4 has been considered a target in cancer immunotherapy. MUC4 is a membrane-bound mucin that has been identified in various cancer forms, including pancreatic cancer, colon, esophageal and lung carcinoma but not in, for example, normal pancreatic tissue. Of interest is also the occurrence of novel tumor-associated antigenic epitopes due to aberrant glycosylation. The approach used by Wu et al. was to generate mature monocyte-derived dendritic cells (DCs) pulsed with MUC4 peptides predicted and validated for binding to HLA-A*02. DCs were then cocultured with autologous purified CD8 T cells obtained from healthy individuals. After 7 days, responder cells were collected and stimulated additional one to two times with peptide pulsed DCs and IL-2 for a total of 2–3 weeks. Expanded CTLs in coculture with peptide pulsed T2 cells where then used to test for IFN-γ secretion in an ELISPOT assay, or the CTLs were used in a cytotoxicity assay against relevant tumor cells. This procedure is indeed efficient for the identification of potential naive precursor cells in blood, but it carry the risk of DC-mediated priming of these rare cells in vitro. The experiments are informative, as they demonstrate that CTLs can be raised against the identified MUC4 epitopes from precursor cells in healthy individuals. However, for the description of frequencies of existing CTLs, for example in cancer patients it may overestimate the frequency of specific cells. In contrast to the long expansion period of Wu et al., we pulsed peripheral blood mononuclear cells (PBMCs) for only 8–10 days with peptide and IL-2 before transfer to the ELISPOT assay. We investigated reactivity against four peptides predicted to bind HLA-A*02 (Table 1). We confirmed binding to HLA-A*02:01 and tested induction of peptide specific CTL in an ELISPOT assay in PBMCs from HLA-A*02-positive patients with colorectal cancer. However, we found no patients with responses to the LLGVGTFVV and only one response against the similar LLLGVGTFV peptide out of 15 patients and importantly this response was also identified in 3 out of 10 healthy individuals. Thus, we confirm that MUC4-specific CTLs can be raised in healthy donors, but they do not seem to be a dominant repertoire in colorectal cancer patients. In addition, the expanded CTL’s were not able to lyse T2 cells pulsed with the relevant peptide in a standard chromium release assay. Given these findings, it may be premature to conclude that MUC4-specific CTLs are important in immune responses against tumors and in immunotherapy. Indeed, we have data from ongoing studies that question the processing of glycosylated epitopes for presentation on major histocompatibility complex (MHC) class I molecules. In addition, MUC4-specific CTLs may even be more difficult to generate in cancer patients as compared to healthy controls due to tolerance induction. Finally, the epitopes tested by Wu et al. and in our lab represents unmodified peptides. In contrast, the peptide presented by tumor cells is likely to include posttranslational Letter to the editor