Glycosylation is a complex and highly variable post-translational modification occurring to proteins and lipids. The combinatorial possibility of glycan signatures amplifies and enhances complexity of biological information.
Lectins are the receptors devoted to decipher such biological signals; deregulation of the glycan-lectin circuits results in immune dysfunction.
In epithelial cancer, changes in glycosylation occur since early steps of tumor transformation. Truncated core 2 O-glycans are generated as such as T (Galß1-3GalNAc¿1-O-Ser/Thr), and Tn (GalNAc¿1-O-Ser/Thr) and their sialylated version STn and ST. Their combined expression with protein backbone generates O-glycopeptide motifs that are tumor specific and immunogenic. The possibility to activate a specific immune response towards these tumor glycopeptide structures is extremely challenging for the design of immune intervention strategies.
While glycopeptides epitopes have been identified as target for humoral immune response, there is lack of consensus on T cell recognition of glycopeptides in the context of MHC.
This research project proposes to investigate the impact of Tn-glycosylation on CD8+ T cell activation. The project exploits unpublished results regarding Tn-glycoproteins relevant for tumor-immune cell interactions. MGL C-type lectin, specific for the Tn glycan, has been employed to isolate tumor Tn-glycoproteins. The analysis of glycosites of tumor Tn-glycoproteins will be interpolated with the predicted epitope binder sequences for HLAI-A2.01 molecule obtained by in silico analysis exploiting immune databases. The HLAI-A2.01 binders that are glycosylated in our database will be tested for their immunogenicity in vitro analysis employing immune cells from cancer patients, HLAI-A2.01+.
The final goal of the project is to obtain indication for anti-cancer glycopeptide targeted T cell immunotherapy to be used for designing vaccines or guide CAR-T and ACT therapies.
It is crucial to design immunotherapeutic strategies, to identify the target epitopes of anti-tumor T cells, that mediate the long-lasting anti-tumor immune response in cancer patients.
Glycosylation is a main post-translational modification occurring to proteins and lipids during their passage through the Endoplasmic Reticulum (ER) and Golgi Apparatus. The combinatorial possibility of glycan presentation dramatically increases the diversity of biological information and adds to the diversity already introduced by the genome and proteome.
In epithelial cancer, glycosylation undergoes to aberrant changes and tumor glycoforms of a plethora of molecules are generated. Although glycopeptide epitopes have been recognized as target for specific humoral immune response, no clear evidences has been conclusive on the recognition of glycopeptide by T cell in the context of MHC class I or II. Regarding T cell epitopes prediction, the structural basis for peptides-MHC cleft interactions are quite complex and no clear and definitive conclusion on the impact of glycosylation on MHC-peptide complex can be drawn. This is most probably due a combination of hurdles. For instance, crystallography studies of protein-glycoprotein interactions are quite difficult to perform due to the high flexibility of the glycan structure. Furthermore, the difficulty to have glyco-reagents at high standard in quality and reproducibility has strongly impaired the glycoimmune investigations.
In addition, the ¿non template¿ nature of carbohydrate synthesis and the heterogeneity of glycosylation pattern make unpredictable the identification of glycosylated residues in vivo.
The precision mapping of the O-glycosites occurring in vivo is a compelling challenge also for its implication for immune studies.
We propose to investigate the effect of tumor Tn-glycosylation on CD8+ T cell epitope recognition and T cell functions.
The project is based on the knowledge of tumor glycoproteins identified in ovarian tumor samples by means of MGL lectin recognition. MALDI-ToF analysis has mapped glycosites and confirmed Tn-glycosylation.
Therefore, the identification of putative HLAI-A2 glycoepitope springs out from tumor glycoproteins that are really glycosylated in vivo, as recognized by the immune system, and not from a predicted in silico glycosylated proteins.
The characterization of the in vitro T cell response to the glycoepitopes selected in the project, will give important information on the impact of tumor glycan to activate (or block) CD8+ T cell responses. The results obtained will be significative also for studying the general effect of glycosylation on CD8 T cell response balance and for future targeting strategies for T cell based anti-tumor immunotherapies.
From this project the following deliverables are expected:
D 1.1 - List of HLA-A binder epitopes and their Tn-glycosylated version.
D 2.1 - Collection of HLAI typed cryopreserved blood cells from cancer patients (at least 30 patients);
D 2.2 - Characterization of glycoantigen expression in tumor samples.
D 3.1 - Identification of Glycopeptides, target for CD8+ T cell response