Careers & Students

Dissecting the antigen specificity of responding CD4+ T cells in a CFSE-based proliferation assays

Masters/Honours project

Type 1 diabetes (T1D) is an autoimmune disease caused by the CD4+ and CD8+ T-cell response against the insulin-secreting beta cells found in the islets of Langerhans in the pancreas (referred to here as islets)1. The CFSE-based proliferation assay has proven to be very sensitive assay for detecting CD4+ T-cell proliferation in responses to antigens, particularly autoantigens in T1D2,3. This assay works by ‘counting’ the number of cells that proliferate, identified by CFSE dilution, in response to an antigen compared to the number of cells that proliferated in absence of antigen. This assay has proven to be very powerful for detecting weak T-cell responses, particularly against autoantigens where the frequency of the responding cells is very low in the peripheral blood4-9. When an antigen specific T-cell proliferates in response to antigen there also some ‘bystander’ T-cell proliferation. Currently, it is not clear how many antigen-specific and antigen non-specific T cells proliferate in a CFSE assay. We know from cloning experiments3 that not all antigen responsive T cells are antigen specific.  Intriguingly, our preliminary sequencing data suggests that there may be a high proportion of antigen non-specific T-cell proliferation in assays measuring responses to beta-cell antigens, such as full-length C-peptide10-11. This information is important because it will help us interpret changes in the number and function of beta-cell antigen specific T-cells responses in the peripheral blood of people with T1D who have received immune therapies.

The aim of this project is to use single-cell sequencing to analyse the TCR genes expressed by human CD4+ T cells that respond to: allo-, auto-, or microbial antigens. The TCR genes will be expressed in a T-cell line and the specificity of the TCRs will be determined in a functional assay. This project builds on methods that have only very recently been established in the lab and will give a student expertise in cutting-edge single cell sequencing technologies, T-cell immunology and autoimmunity.

Primary Supervisor

Associate Professor Stuart Mannering:                      [email protected]

Co Supervisors

Dr Pushpak Bhattacharjee:                                         [email protected]


1.         Mannering SI, Bhattacharjee P. Insulin's other life: an autoantigen in type 1 diabetes. Immunology and cell biology. 2021;99(5):448-60.

2.         Mannering SI, Morris JS, Jensen KP, Purcell AW, Honeyman MC, van Endert PM, et al. A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. J Immunol Methods. 2003;283(1-2):173-83.

3.         Mannering SI, Dromey JA, Morris JS, Thearle DJ, Jensen KP, Harrison LC. An efficient method for cloning human autoantigen-specific T cells. J Immunol Methods. 2005;298(1-2):83-92.

4.         Mannering SI, Morris JS, Stone NL, Jensen KP, PM VANE, Harrison LC. CD4+ T cell proliferation in response to GAD and proinsulin in healthy, pre-diabetic, and diabetic donors. Annals of the New York Academy of Sciences. 2004;1037:16-21.

5.         Mannering SI, Harrison LC, Williamson NA, Morris JS, Thearle DJ, Jensen KP, et al. The insulin A-chain epitope recognized by human T cells is posttranslationally modified. The Journal of experimental medicine. 2005;202(9):1191-7.

6.         Mannering SI, Pang SH, Williamson NA, Naselli G, Reynolds EC, O'Brien-Simpson NM, et al. The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes. Clinical and experimental immunology. 2009;156(2):226-31.

7.         Dromey JA, Lee BH, Yu H, Young HE, Thearle DJ, Jensen KP, et al. Generation and expansion of regulatory human CD4(+) T-cell clones specific for pancreatic islet autoantigens. J Autoimmun. 2011;36(1):47-55.

8.         Prickett SR, Voskamp AL, Phan T, Dacumos-Hill A, Mannering SI, Rolland JM, et al. Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology. 2013;43(6):684-97.

9.         Di Carluccio AR, Tresoldi E, So M, Mannering SI. Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester. J Vis Exp. 2019(148).

10.       Pathiraja V, Kuehlich JP, Campbell PD, Krishnamurthy B, Loudovaris T, Coates PT, et al. Proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer-restricted CD4+ T cells infiltrate islets in type 1 diabetes. Diabetes. 2015;64(1):172-82.

11.       So M, Elso CM, Tresoldi E, Pakusch M, Pathiraja V, Wentworth JM, et al. Proinsulin C-peptide is an autoantigen in people with type 1 diabetes. Proceedings of the National Academy of Sciences of the United States of America. 2018;115(42):10732-7.

Supervised by:

  • A/Prof Stuart Mannering
  • Dr Pushpak Bhattacharjee
  • Disease Focus:

  • Type 1 diabetes
  • Research Unit:

  • Human immunology
  • For further information about this project, contact: [email protected]