Optimization of protocol for in vitro generation of HPV-specific T-cell lines

Abstract

Studies on several target factors like specific antigens, biomarkers and cellular signals involved in High-risk Human papilloma virus (HR-HPV) infection and its mediated immune response aid in narrowing the gap to generate effective therapeutic solutions for oropharyngeal carcinomas. Thus, establishing cytotoxic T-lymphocytes (CTL) lines will aid in investigating and carrying out functional studies in vitro of such responsible target factors. Optimization of various parameters involved in generating CTL lines specific for HPV16 was the core focus of this study. CD8+ T-cells were isolated from peripheral blood of healthy donors and HPV+ cancer patients. The cells were stimulated with HPV16 type E6 and E7 peptide-loaded monocyte derived dendritic cell (moDCs) and peripheral blood mononuclear cells (PBMC) by considering the human leukocyte antigen (HLA)-haplotype of the target (CD8+ T-cells) and stimulator cells (moDCs and PBMC). These stimulated CTL’s were expanded for 2 weeks by the addition of cytokines IL-2 and IL-15 to the co-culture. The viability and expansion of these CTL’s were assessed by trypan blue and cell trace violet stains. Activation of CTLs after stimulation with E6 and E7 peptides was evaluated by analysing the expression of lysosomal associated membrane protein 1 (CD107a) functional biomarker in a time-dependent manner. The specificity of expanded CTLs towards E6 and E7 peptides were analysed using suitable tetramer assays. In conclusion these optimized parameters will aid in quantitative, qualitative, and specificity evaluation of antigens, biomarkers and cellular signals involved in HR-HPV16 infection.

Popular Abstract

Human Papilloma Virus (HPV) is a sexually transmitted virus that is associated to the development of a number of cancers affecting e.g., mouth, throat, and genitals. There are several types of this virus which are associated with cancer, of which HPV type 16 (HPV16) is responsible for almost 80% of all the cancer caused in the oropharyngeal region located in the back of the throat. Two HPV oncoproteins, namely E6 and E7, are involved in tumor progression and they both act by interfering with the activity of proteins that have a suppressive function on the tumour cells.
The objective of this project was to set up and optimize an assay that could be used to assess the activation and specificity of immune cells, so-called Cytotoxic T-cells (CTL) isolated from blood from cancer patients and healthy individuals. CTLs in the tumor microenvironment are involved in killing the virus-infected tumor cells. Generation of T cell lines specific towards the proteins E6 and E7 is an important step towards understanding the interaction of CTLs and HPV E6 and E7 infected cells. The encounter of T cells with the protein parts occurs via helping cells, known as antigen-presenting cells, which also provide signals for activation. T-cell lines specific towards the HPV E6 and E7 proteins thus also allows further studies on the function of subpopulations of antigen-presenting cells.
During this thesis, each step involved in the setting up of the assay was carefully tested and monitored to be able to provide a lab- controlled environment. Immune cells were isolated from blood of healthy patients, with unknown HPV status, as well as from cancer patients with HPV-infections. Regular monitoring of the growth of the T cells was conducted using a technique known as flow cytometry and a stain known as Cell Trace violet. This helps in visualising the cells at a single cell level using lasers and fluorescence stains, and to monitor their growth. If T cells divide in response to protein stimulation, a disruption of the stain among the two daughter cells will occur and stain intensity is thus lowered. This loss of intensity can be seen in the flow cytometer and indicate whether the T cells are growing to act against the oncoproteins present in their environment. In addition to cell division, the regulation of various cell surface receptors was investigated to monitor activation and function. This project has led to the setup of an assay for assessment of proliferation using primary cells from patient samples, however, due to limited number of cells available from some blood samples, the experiments have to be repeated on a larger cohort of samples with known HPV status in order to generate conclusive results.

• author: Cornelius Singarayar, Ruban
• supervisor: Malin Lindstedt; Aastha Sobti
• organization: Department of Immunotechnology; Educational programmes, LTH
• course: KIMM01 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: immunology, flow cytometry, Oropharyngeal cancer, HPV16, PBMC
• language: English
• id: 9065316
• date added to LUP: 2021-09-13 12:21:51
• date last changed: 2021-09-13 12:21:51