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Human T cell

The goal of the Human T-cell Laboratory, within the Immunology of Diabetes Unit, is twofold. First, to understand why the insulin-producing cells are attacked by the immune system’s T cells and second, to use this information to develop new assays to measure these autoimmune responses and ways to stop them developing.

Research overview

The major current focus of the group is isolating and analyzing human islet infiltrating T cells. Through the Tom Mandel Islet Transplant Program we have received pancreata from deceased organ donors who suffered from type 1 diabetes. Using methods developed within the Human T-cell laboratory we have achieved a world first by isolating and cloning human islet-infiltrating T cells.  Our approach of analyzing islet-infiltrating T cells allows us to examine T cells strongly implicated in autoimmune beta-cell destruction because of their location within the pancreatic islets of people who had T1D. We have isolated a panel of >490 CD4+ T-cell clones from the residual pancreatic islets of six deceased organ donors who suffered from T1D.

Research Themes

What antigens do human islet infiltrating CD4+ T cells see?

Group Leader: Dr Stuart Mannering
Team Members: Vimukthi Pathiraja, Eleanora Tresoldi, Prerak Trivedi
Collaborators: Prof Tom Kay and A/Prof Helen Thomas
Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Drs Maki Nakayama and Aaron Michels, Barbara Davis center for Childhood diseases, Colorado, USA.
Prof Jamie Rossjohn and Dr Hugh Reid, Monash University, Melbourne.
Dr Eddie James, Benaroya Research Institute, Seattle, USA.

Genome wide association studies (GWAS) have confirmed that the strongest genetic risk for T1D is mediated by HLA alleles, specifically HLA DQ8 and DQ2. The second strongest association maps to the promoter region of the insulin gene. While these associations are well accepted in the field it remains unclear how HLA DQ2/8 and insulin lead to type 1 diabetes. From our analysis of human islet-infiltrating CD4+ T-cell clones we have identified several HLA DQ8-restricted CD4+ T-cell epitopes derived from the C-peptide of proinsulin. This project will build upon this success and identified epitopes from other antigens (including GAD-65, IGRP, IAPP, ZnT8). The long-term goal is to identify common features of epitopes seen by islet infiltrating CD4+ cells. This project will examine the epitope’s affinity for the restricting HLA allele, the avidity of TCR binding and TCR usage.

Do islet infiltrating CD4+ T-cells recognize epitopes formed by posttranslational modification?

Group Leader: Dr Stuart Mannering
Team Members: Vimukthi Pathiraja, Eleanora Tresoldi, Prerak Trivedi
Collaborators: Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Drs Maki Nakayama and Aaron Michels, Barbara Davis center for Childhood diseases, Colorado, USA.
Prof Jamie Rossjohn and Dr Hugh Reid, Monash University, Melbourne.
Dr Eddie James, Benaroya Research Institute, Seattle, USA.

In 2005 we published the first description of an epitope formed by posttranslational modification to be implicated in T1D (Mannering et al JEM 2005). While the concept of autoimmune responses against self-antigens that have undergone posttranslational modification remains popular this area remains largely unexplored in human studies. Our panel of islet-infiltrating CD4+ T-cell clones are invaluable tools that allow us to determine if these cells recognize epitopes formed by posttranslational modification. Our approach is to test panels of synthetic peptides that incorporate modified amino acids and determine if any of these are recognized by islet infiltrating CD4+ T cells. To date we have identified one HLA-DQ8 restricted epitope that is formed by posttranslational modification. Future work will screen a larger panel of peptides from other beta-cell antigens to find new epitopes formed by posttranslational modification. Using our very sensitive CFSE-based proliferation assays (Mannering JIM 2003) we will measure responses to modified epitopes in PBMC from individuals with and without T1D.  Our biochemical/functional analysis will also include structural analysis of TCR–peptide-HLA complex to gain insights into how human T cells ‘see’ autoantigens.

Identifying the antigens and epitopes seen by human islet-infiltrating CD8+ T cells

Group Leader: Dr Stuart Mannering
Team Members: Prerak Trivedi & Eleanora Tresoldi,
Collaborators: Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Prof Tom Kay, A/Prof Helen Thomas and Dr Bala Krishnamurthy.

CD8+ T cells that recognize beta-cell antigen derived epitopes presented by HLA class I molecules are believed to the ‘killers’ that destroy the insulin-secreting beta cells. The goal of this project is to identify the antigens and epitopes ‘seen’ by human islet-infiltrating CD8+ T cells. We have developed a Cos cell transfection system which allows us to screen CD8+ T-cell clones for responses to individual antigen/HLA combinations. This system has been validated using CD8+ T-cell clones specific for viral epitopes restricted by relevant HLA alleles. Antigens that stimulate the clones will be dissected to identify the epitope(s) and HLA restriction. This information will be used to generate HLA tetramers for characterizing beta cell antigen specific CD8+ T cells in human peripheral blood. 

Identifying the antigens and epitopes seen by human islet-infiltrating CD8+ T cells

Group Leader: Dr Stuart Mannering
Team Members: Eleanora Tresoldi
Collaborators: Dr Nadine Dudek, Monash University.
Dr Bala Krishnamurthy, SVI

Identification of antigens recognized by human T cells has been technically challenging. This is because relevant T-cell clones have been difficult to isolate and preparing antigen in a suitable format has been difficult. Hence the aim of this project is to develop improved techniques for identifying antigens recognized by human islet infiltrating T cells. We propose to genetically fuse the antigens to the Class II invariant chain that directs the protein to the lysosomes for loading into HLA Class II molecules. This project will investigate different antigen presenting cells, such as EBV transformed B cells and HLA transfected Cos cells. The capacity of the constructs to stimulate islet infiltrating CD4+ T-cell clones specific for proinsulin will be used to test the system. This project would be suitable for an Honors student. Successfully completing this project would be an excellent preparation for a PhD project using this system to characterize human islet-infiltrating T cells.

Honours and PhD Projects

Staff

Publication Highlights

  1. Pathiraja, V, Kuehlich JP, Campbell PD, Krishnamurthy B, Loudovaris T, Coates PT, Brodnicki TC, O'Connell PJ, Kedzierska K, Rodda C, Bergman P, Hill E, Purcell AW, Dudek NL, Thomas HE, Kay TW and Mannering SI (2015). "Proinsulin-Specific, HLA-DQ8, and HLA-DQ8-Transdimer-Restricted CD4+ T Cells Infiltrate Islets in Type 1 Diabetes." Diabetes 64(1): 172-182.
  2. Prickett, SR, Voskamp AL, Phan T, Dacumos-Hill A, Mannering SI, Rolland JM and O'Hehir RE (2013). "Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic." Clin Exp Allergy 43(6): 684-697.
  3. Lopez, JA, Jenkins MR, Rudd-Schmidt JA, Brennan AJ, Danne JC, Mannering SI, Trapani JA and Voskoboinik I (2013). "Rapid and unidirectional perforin pore delivery at the cytotoxic immune synapse." J Immunol 191(5): 2328-2334.
  4. Necula, A, Chand R, Albatat B and Mannering SI (2012). "Extraction of tissue antigens for functional assays." J Vis Exp(67).
  5. Joffe, M, Necula AS, Chand R, McWhinney BC, Krishnamurthy B, Loudovaris T, Goodman D, Thomas HE, Kay TW and Mannering SI (2012). "Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors." Int Immunol 24(7): 447-453.
  6. Broughton, SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, van Bergen J, Kooy-Winkelaar Y, Henderson KN, Beddoe T, Tye-Din JA, Mannering SI, Purcell AW, McCluskey J, Anderson RP, Koning F, Reid HH and Rossjohn J (2012). "Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease." Immunity 37(4): 611-621.
  7. Ciantar, JP and Mannering SI (2011). "An improved method for growing and analysing human antigen-specific CD4(+) T-cell clones." Diabetes Metab Res Rev 27(8): 906-912.
  8. Tye-Din, JA, Stewart JA, Dromey JA, Beissbarth T, van Heel DA, Tatham A, Henderson K, Mannering SI, Gianfrani C, Jewell DP, Hill AV, McCluskey J, Rossjohn J and Anderson RP (2010). "Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease." Sci Transl Med 2(41): 41ra51.
  9. Mannering, SI, Wong FS, Durinovic-Bello I, Brooks-Worrell B, Tree TI, Cilio CM, Schloot NC and Mallone R (2010). "Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes." Clin Exp Immunol 162(2): 197-209.
  10. Mannering SI, Pang SH, Williamson NA, Naselli G, Reynolds EC, O'Brien-Simpson NM, Purcell AW, Harrison LC, 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: 226-31.
  11. Campbell PD, Estella E, Dudek NL, Jhala G, Thomas HE, Kay TW, Mannering SI. Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro. Human Immunology 2008; 69: 543-551.
  12. Dudek NL, Maier S, Chen ZJ, Mudd PA, Mannering SI, Jackson DC, Zeng W, Keech CL, Hamlin K, Pan ZJ, Davis-Schwarz K, Workman-Azbill J, Bachmann M, McCluskey J, Farris AD. T cell epitopes of the La/SSB autoantigen in humanized transgenic mice expressing the HLA class II haplotype DRB1*0301/DQB1*0201. Arthritis & Rheumatism 2007; 56:3387-98.
  13. Henderson KN, Tye-Din JA, Reid HH, Chen Z, Borg NA, Beissbarth T, Tatham A, Mannering SI, Purcell AW, Dudek NL, van Heel DA, McCluskey J, Rossjohn J, Anderson RP. A Structural and Immunological Basis for the Role of Human Leukocyte Antigen DQ8 in Celiac Disease. Immunity 2007; 27:23-34.
  14. Mannering SI, Harrison LC, Williamson NA, Morris JS, Thearle DJ, Jensen KP, Kay TW, Rossjohn J, Falk BA, Nepom GT, Purcell AW. The insulin A-chain epitope recognized by human T cells is posttranslationally modified. Journal of Experimental Medicine 2005; 202: 1191-7.
  15. Mannering SI, Dromey JA, Morris JS, Thearle DJ, Jensen KP, Harrison LC. An efficient method for cloning human autoantigen-specific T cells. Journal of Immunological Methods 2005; 298:83-92.
  16. Mannering SI, Morris JS, Stone NL, Jensen KP, van Endert PM, 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.
  17. Mannering SI, Morris JS, Jensen KP, Purcell AW, Honeyman MC, van Endert PM, Harrison LC. A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. Journal of Immunological Methods 2003; 283:173-83.