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Defining parameters for multi-colour flow cytometry and molecular identification of human T cell subsets

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posted on 28.03.2022, 12:49 authored by Nicoleen Gasparillo
Flow cytometry is widely used to define cell populations in immunology. Here, we describe an optimised multi-parameter flow cytometry protocol that enables T cell subset identification in human blood and tissue, using a comprehensive panel of surface and intracellular markers. Antibody selection, cell membrane permeabilisation, T cell stimulation, controls and surrogate markers were considerations taken in establishing the methodology. With the optimised method, CD4 T helper cell subsets, Th1 (T-bet⁺, IFNγ⁺,) Th2 (GATA3⁺, IL-4⁺, IL-13⁺), Th17 (RORγt⁺, IL-17A⁺) and regulatory T cells (FoxP3⁺CD25⁺TNFR2⁺, IL-10⁺,TGFβ⁺), as well as CD8 T cell subsets Tc1 (IFNγ⁺) and Tc2 (IL-4⁺), are detectable at varying frequencies in healthy human blood and in blood and stromal vasculature fractions from lymphoedema patients. Suggested surrogate markers for Th1 (CD183 [CXCR3]), and Th2(CD194 [CCR4]), are evaluated as well. Furthermore, naïve and memory T cells are comprehensively assessed using a combination of antibodies to CD45RA/RO, CD27, CD62L,CCR7 and CD31. CCR7 expression further distinguishes the central and effector memory subsets, while CD31 expression differentiates the recent thymic emigrants from peripherally expanded CD4 naïve T cells. These two naïve T cell subsets can be further investigated for Tcell receptor diversity by way of next-gen sequencing, to confirm if clonality decreases due to homeostatic expansion, especially as thymic output is said to decrease with age. A thorough definition of these T cell subsets will be valuable to a wide range of human immunology studies where the T cells play a role in exacerbating or in alleviating a condition.

History

Table of Contents

1. Introduction -- 2. Material and methods -- 3.Results -- 4. Discussion.

Notes

Empirical thesis. Bibliography: pages 65-76

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Medicine and Health Sciences, Department of Clinical Medicine

Department, Centre or School

Department of Clinical Medicine

Year of Award

2015

Principal Supervisor

Lisa Sedger

Additional Supervisor 1

Seray Adams

Rights

Copyright Nicoleen Gasparillo 2015. Copyright disclaimer: http://www.copyright.mq.edu.au

Language

English

Extent

1 online resource (viii, 106 pages) colour illustrations

Former Identifiers

mq:44714 http://hdl.handle.net/1959.14/1071561