Dr. Elena Shklovskaya

Senior Research Officer

T Cell Biology Group (group head, A/Prof B.Fazekas de St.Groth)

Centenary Institute of Cancer Medicine and Cell Biology

Email: e.shklovskaya@centenary.usyd.edu.au

Phone 9565 6198

Project overview

The immune system can deal with a wide range of pathogens. Antigens derived from pathogens are acquired and processed by antigen-presenting cells, then presented to T cells in the form of peptide:MHC molecule complexes. Each antigen is then recognised by T cells bearing appropriate T cell receptors (TCRs). While both CD8⁺ and CD4⁺ T cells can recognise antigens and participate in their elimination, long-lasting memory develops only in the presence of CD4⁺ T cells. Being in charge of the initiation and cessation of the initial response, and regulating the potential involvement of other cell types and the development of immunological memory, CD4⁺ T cells are the ultimate controllers of the immune system.

How best can we measure immunological memory? The answer relies on the following characteristics of memory T cells:

· they have the highest affinity for the antigen (in the course of the response cells expressing TCR of one specificity become dominant and out-compete all other T cells)

· they survive long term and can be easily activated in “recall responses” (to proliferate and produce cytokines in response to the initial antigen)

· they have a characteristic phenotype (which can be measured using monoclonal antibodies recognising particular cell surface molecules)

· they can be found not only in secondary lymphoid organs, but throughout the body

Memory CD8⁺ T cells circulate at a relatively high frequency (in a mouse, any given specificity can reach 1-5% of all CD8⁺ T cells), and can be measured using peptide:MHC class I tetramers which bind to the TCR of interest. Memory CD4⁺ T cells circulate at a very low frequency (ie 0.001-0.01% of all CD4⁺ T cells), and class II tetramers are not readily available.

The aim of this project is to establish and compare ways of measuring CD4⁺ T cell memory in a transgenic mouse model.

What is already established: CD4⁺ T cells specific for the dominant epitope of moth cytochrome c (MCC) will be obtained from lymph nodes of TCR transgenic mice, labelled with a vital dye CFSE and adoptively transferred into Ly5 congenic mice. Recipient mice will be than challenged subcutaneously with MCC peptide in complete Freund’s adjuvant, a treatment known to result in CD4 memory. After 10 weeks or later, you will look for memory cells which will be i) negative for the host Ly5 allele ii) positive for CD4 iii) positive for the transgenic TCRV and V and iv) displaying a characteristic memory phenotype v) proliferating and producing cytokines in response to challenge with MCC peptide. What we need you to establish: the best way to measure a recall response of memory T cells (in vivo and/or in vitro). Some of the aspects to be measured include: the best site to find memory cells, the best type of recall immunisation in vivo, and the optimal time after immunisation

List of techniques that will be used in conducting the research

Mouse in vivo techniques (iv, sc injections; isolation of mouse lymph nodes and spleens, labelling of cell suspensions with CFSE dye and monoclonal antibodies for flow cytometry)

Flow cytometry 7-9 colour – and we promise you’ll learn your flow properly!

In vitro assays (T cell proliferation, cytokine re-stimulation)

Monoclonal antibody purification and conjugation (if required)