Dynamics of human T cell memory in blood and secondary lymphoid tissue in shaping vaccine induced immunity in ageing

Vaccines protect by training the immune system to retain memory of an infection. This is made up of different kinds of cells which can retain information, like bits in a computer software system. This is called immune memory. It is this feature which allows us to be protected after vaccination if we encounter an infection. We do not fully understand the processes involved. One very important type of T cell interacts with B cells to help them make antibodies. These interactions happen in specialised tissues called lymph nodes.

These are small kidney-shaped organs found together in clusters in certain areas such as the underarm. It is here that T cells and B cells meet the vaccine or parts of the vaccine and as a result, the cells respond, change and multiply. In older people, we know that the processes of immune priming, maintaining immune memory and recalling it may all happen differently, perhaps more slowly, but we don't know precisely why. One possibility is that T cells respond differently in older people. Vaccines for older people often need a high dose or an extra activating component.

Although lymph nodes are important, they have not been well studied in humans. This is because taking a lymph node sample requires different skills from taking blood and these have not previously been invested in. A clinician who is skilled in ultrasound-guided needle biopsy, is needed. This means that we are missing key information in understanding how vaccines work, and how we might design them better in different circumstances; for older people for example, or to tackle highly complex infections that rapidly change such as HIV, flu and SARS-CoV-2.

This Fellowship aims to change that. Not just for the duration of this project but for the future of vaccine science.

Firstly, I will develop bedside imaging to determine which lymph nodes are responding to the vaccine and which are not. At present this is sometimes possible with greyscale ultrasound but the results are not always reliable, or with PET/CT but this involves a dose of radiation. I believe we can do this more frequently and in more people using harmless ultrasound. Volunteers will be vaccinated with a vaccine against influenza and attend for an imaging appointment on multiple days before and after vaccination. Each time they attend they will have a scan to see the size and blood flow in the lymph nodes near to the site of vaccination. This will determine the best site and best day on which to take a sample using these types of vaccines.

Secondly, I will use the ultrasound to guide a fine needle aspiration (USFNA) biopsy to take a sample of the lymph node before and after vaccination with influenza vaccine. I will also ask volunteers to give a sample of the blood at the same time. Samples from lymph nodes on the same side and opposite (control) side can be taken. The cells will be stained and measured using a machine called a flow cytometer so that I can determine which are T cells, what type of T cells, what they are responding to and whether they are growing in number (in cell cycle). I will compare this in the blood and in the lymph node.

Thirdly, I will measure how individual T cells are responding to vaccination by testing the ribonucleic acid (RNA) that they are producing. This will indicate in detail how different types of T cells respond to the vaccine.

To understand how age might affect the immune response, I will ask people of different ages to participate. My vision is to generate a picture of how T cells respond to vaccines in lymph nodes, and to demonstrate how, and why, this changes with age. This will provide new insight for the future of vaccine design.

Aims To compare T cell memory in secondary lymphoid tissue and blood in younger and older adults after intramuscular immunisation with a model (influenza) vaccine

Objectives To determine
a) peak reactivity in secondary lymphoid tissue
b) frequency, phenotype, activation and cells in cycle amongst cTfh/Tfh
c) the evolution of vaccine-specific T cell clones amongst cTfh/Tfh

Methodology
Participants aged 18-45 and 65+ years will receive adjuvanted influenza vaccine and donate samples at baseline and at Day 3-28. Lymph node cells will be processed alongside peripheral blood mononuclear cells and viably cryopreserved or used immediately.

Aim 1: USS will establish the peak time point of reactivity in secondary lymphoid tissue proximal to the site of vaccination in 5 healthy volunteers per group.
Aim 2: will measure the frequency, phenotype, activation status (CD38+) and proliferation status (Ki67+) of cTfh and Tfh amongst PBMCs and LNCs using multiparameter flow cytometry from 16 healthy volunteers per age group and cohort. Antigen specificity will be determined using an activation induced marker (AIM) assay or fluorochrome-conjugated tetramers. Specificity will be determined in PBMCs and the same TCR signature analysed amongst LNCs.
Aim 3: will use simultaneous scRNA-seq, antibody-dependent tagging (CITE-seq) and scTCR/Ig seq to comprehensively profile T and B cell subtypes and activation states in lymph node and blood before and after vaccination from 16 healthy volunteers in each age group. The well-established 10x Chromium platform will be employed using isolated CD45+ cells, and the single-cell TCR/Ig repertoire analysis will provide T/B clonality characterisation. Internal controls will come from sampling ipsilateral and contralateral nodes.
Scientific and medical opportunities: Discovery of how key vaccine T cell subsets respond in ageing will identify pathways for manipulation and an in vivo model in which to test them.