2nd International Meeting on Precision Oncology and Personalized Medicine for Head and Neck Cancer

Prof. Dr.

Therapeutic vaccination for HPV-positive cancers

Keynote lecture

Marginal Zone B cells act as professional antigen-presenting cells in head and neck squamous cell carcinoma

Immunotherapy, cellular therapy and vaccination

Molecular landscape, multiomics and artificial intelligence

Background:Current data assign B lymphocytes a significant contribution to tumor development, including head and neck squamous cell carcinoma (HNSCC). Due to their high developmental diversity and different regulatory and functional roles, B cell subpopulations can promote or inhibit tumor growth. Therefore, the investigation of different B cell subpopulation within the microenvironment is of particular importance.Methods:Using flow cytometry, we discovered the presence of marginal zone B cells (MZB) both within tumor tissue and peripheral blood in a cohort of HNSCC patients. To confirm these data and to get more insight into the possible function of tumor-associated MZB, we analyzed four publicly available single-cell RNA sequencing (scRNA-seq) datasets.Results: Unsupervised clustering and reference-based cell annotation revealed two distinct MZB populations, characterized by their robust intercellular communication and anti-viral pathways. Cell communication analysis revealed abundant MHC-I and MHC-II antigen presentation between MZB clusters and various CD4+ and CD8+ T cell subpopulations. MZB-2 exhibit a higher degree of functional maturity, engage in more pronounced interactions with CD4+ T cells and antigen-presenting cells, and are of prognostic significance for HNSCC patients. A geographical proximity of MZB clusters and T cells was detected by spatial transcriptomics.Conclusion:Our study unveiled the existence of two distinct subsets of MZB within the tumor and peripheral blood of HNSCC patients, with a pivotal role in antigen presentation and T cell activation. Harnessing the potential of these immunogenic B cells represents a promising therapeutic avenue of which HNSCC patients could benefit in the future.

Abstract talk

Prof.

Glutamine synthetase expression enhances T cell effector functions in a glutamine-restricted environment

New technologies and preclinical models

Objective: T cells are highly dependent on glutamine to sustain metabolic activity and effector functions. Low glutamine levels within the tumour microenvironment have been associated with a diminished immune response, potentially affecting also the efficacy of adoptive T cell transfer therapies. We hypothesized, that expression of glutamine synthetase (GS), catalyzing the de-novo synthesis of glutamine from glutamate, could mitigate the effects of glutamine restriction on T cells.Methods: Retroviral transduction was used to express the GS in human CD4+ T cells. GS-expressing cells were analysed for metabolic activity, proliferation and cytokine production under glutamine-restricted conditions. Furthermore, the anti-tumour activity of GS-expressing T cells was evaluated in co-cultures with HNSCC 3D tumour spheroids. Results: Retroviral transduction resulted in strong and robust GS expression in human T cells. GS-expressing T cells displayed a restored respiratory and proliferative activity under glutamine-restricted conditions, in particular in the presence of glutamate. IFNγ secretion under glutamine limitation was elevated by GS expression. Of significant importance, upon co-cultivation with HNSCC spheroids GS-expressing T cells showed increased effector functions in the presence of low glutamine concentrations.Conclusion: The results of our study indicate that GS expression represents a promising strategy to enhance anti-tumour effector functions, suggesting that metabolic engineering of T cells could be a potential strategy in cell therapy approaches.

The effect of HNSCC-derived exosomes on the immunological function of neutrophil granulocytes

ObjectiveNeutrophil granulocytes are the most abundant immune cell type in the blood acting as a first line of immune defense. Recent findings show that neutrophils play an important role in the tumor microenvironment (TME), displaying an anti- or pro-tumor phenotype (N1-like / N2-like). This study aims to explore the effect of HNSCC-derived exosomes on neutrophil granulocytes to explore whether they are able to create an N2-like pro-tumor neutrophil phenotype.MethodsExosomes were isolated from the supernatant of two HNSCC cell lines (UD5, SAS) using size exclusion chromatography. Diameter and amount of exosomes were verified using a nanoparticle tracking video microscope. Venous blood was collected from healthy donors and neutrophils were isolated (n=4). The neutrophils were then co-incubated with cell-line-derived exosomes or two cytokine treatments (IL-8/TNF-α, TGF-β1) as a control. The stimulated neutrophils were harvested after a maximum of 24h co-culture and stained for cell surface proteins to investigate phenotypic changes using FACS.ResultsNeutrophils stimulated with IL-8 and TNF-α did show a CD66bhigh, CD62Llow, CD11bhigh, CD54high expression pattern, congruent with an N1-like phenotype. Neutrophils stimulated with TGF-β1 show a slight but not significant decrease in CD66b and no activation or degranulation. Neutrophils co-incubated with SAS- or UD5-derived exosomes show an insignificant decrease in CD66b and CD11b, the expression of PD-L1 was not increased.ConclusionNeutrophil granulocytes can be shifted into an N1-like phenotype using a combination of IL-8 and TNF-α in vitro. When stimulating neutrophils with TGF-β1, no clear N2-like phenotype could be established yet, raising the question if neutrophils can be shifted into an N2-like state by using this experimental setup. The co-incubation of neutrophils with HNSCC-derived exosomes doesn"t show significant marker shifts. To confirm whether these exosomes have an immunological effect on neutrophils, further experiments need to be conducted.

Session 2
Immunotherapy, Cellular Therapy and Vaccination

Appointment

Chairs

Programme

Session 2Immunotherapy, Cellular Therapy and Vaccination

Appointment

Chairs

Programme

Session 2
Immunotherapy, Cellular Therapy and Vaccination