In type 1 diabetes, insulin-producing beta cells (arranged in clusters called islets) are destroyed by immune mechanisms. The major immune cell type involved is the CD8+ cytotoxic T lymphocyte (CTL) that directly recognises short peptides derived from proteins like insulin presented by major histocompatibility complex class I proteins on the surface of beta cells. We study the precise mechanisms by which T cells destroy beta cells, and test ways to prevent this from happening. Much of our work is based on the NOD mouse model that develops diabetes in a similar manner to humans. We also have several transgenic mice that express particular T-cell receptors that are able to cause rapid diabetes. We use these mice, and our expertise in flow cytometry, immunohistochemistry and molecular biology, to study the role that cytokines, death-receptor molecules and perforin/granzymes play in the development of diabetes. The major questions we are studying are the use of immune tolerance to insulin as a way of arresting diabetes and also how CTL differentiate into fully effective cytotoxic T cells.

Research Themes

The effect of immune tolerance to proinsulin at different stages of autoimmune diabetes.

Group Leaders: Dr Balasubramanian Krishnamurthy and Professor Tom Kay
Team Members: Mr Gaurang Jhala, Mr Jonathan Chee, Ms Claudia Selck, Mr Erwin Tanuwidjaya
Collaborators: Prof Len Harrison, Assoc Prof Andrew Lew, Dr Pere Santamaria, Dr Teresa DiLorenzo

Our long-term goal is to treat and prevent type 1 diabetes using antigen-specific therapy to restore immune tolerance without the risks of immunosuppression. We have been at the forefront of identifying pro-insulin as the best candidate auto-antigen for this. However, progress towards this goal in human diabetes has been slow, particularly because biomarkers that can indicate whether treatment has been effective are imperfect. We believe that mouse models continue to have an important role in guiding clinical trials although we recognise how critical it is to keep their applicability in perspective and to conduct properly powered and reproducible pre-clinical studies.

Our preliminary data have identified two major issues. First, after initiation of beta-cell autoimmunity specific for pro-insulin, which appears to be the driver antigen for the disease, the immune response spreads to other antigens such as IGRP. This means that therapy targeting the driver antigen may not be effective if given after spreading. A second issue is that auto-antigen-specific T cells have a memory phenotype once autoimmunity is established and may be harder to re-program to tolerance than naïve cells. Our current research is focused on exploring and solving these two questions, aiming to work out how antigen-specific therapy can be effective at a clinically relevant time after onset of beta cell autoimmunity. Alternatively it may be that this therapy must be given at a very early stage of life. We have assembled a powerful suite of resources in the NOD mouse model including numerous sophisticated transgenic strains and MHC class I and class II tetramers and are well placed to dissect the mechanisms of pro-insulin-specific immune tolerance by tracking, enumerating and phenotyping antigen-specific T cells.

Role of cytokines in generation of cytotoxic T lymphocytes capable of killing beta cells in type 1 diabetes

Group Leaders: Professor Tom Kay
Team Members: Dr Kate Graham, Dr Andrew Sutherland, Dr Balasubramanian Krishnamurthy, Mr Jonathan Chee, Ms Stacey Fynch
Collaborators: Dr Cecile King

During the onset of Type 1 diabetes the islet environment becomes enriched with cytotoxic T lymphocytes (CTLs) capable of killing beta cells. The overall objective of our project is to identify cytokines and cell interactions required within islets that result in the generation of fully armed CTLs capable of destroying beta cells. We have utilized a novel reporter mouse to demonstrate the CTLs within islets are responding to cytokines. We have also shown CTLs within islets have an enhanced ability to kill their target cells. We have now begun investigating if two particular cytokines IL-7 and IL-21 are stimulating CTLs within islets. We have preliminary data that demonstrates IL-21 but not IL-7 promotes the maturation of CTLs within the islet environment.

Development of effector memory T cells in type 1 diabetes

Group Leaders: Professor Tom Kay and Dr Balasubramanian Krishnamurthy
Team Members:, Dr Kate Graham, Mr Jonathan Chee, Ms Stacey Fynch
Collaborators: A/Prof Andrew Lew, Prof Mark Peakman

We are interested in understanding where effector memory T cells develop. We have developed a sensitive tetramer enrichment method for counting these T cells in the blood of NOD mice. Using this method, we identified that the number of effector memory T cells in the blood increases as NOD mice age and that the numbers of these memory T cells in the blood correlates with insulitis progression. These data demonstrate that T cells within islets expand and differentiate into effector-memory T-cells, and then migrate to the blood, where they can report progression of islet pathology. This is an important finding as blood is often the only sample we can access in humans.

Manipulating apoptosis to generate beta cell antigen-specific regulatory T cells

Group leader: Dr Bala Krishnamurthy
Team members: Prof Tom Kay, A/Prof Helen Thomas, Ms Eva Orlowski
Collaborators: Dr Daniel Gray, Prof Andreas Strasser

Immunological tolerance is maintained in normal individuals by a delicate balance between effector and Treg cells. We, and others, have found that activation of pro-apoptotic BIM is needed for deletion of autoreactive T cells in the thymus, a robust mechanism of T cell tolerance. In the absence of BIM, autoreactive T cells escape deletion and survive in the periphery. Paradoxically, this block in thymic deletion protects mice from type 1 diabetes (our unpublished data). This effect appears to be because many of the CD4+ T cells that escape deletion in the thymus develop into Treg cells capable of suppressing autoimmune diabetes. Therefore, regulation of apoptotic pathways in T cells can alter the balance between effector and Treg cells and we wish to explore this theme. Making Treg cells that are reactive to auto-antigens can direct their immune-suppressive activity to the insulitic lesion to halt autoimmune diabetes and Treg cells that bear auto-reactive T cell receptors suppress autoimmune diabetes better than a polyclonal Treg cell population. A better understanding of the mechanism of Treg cell development is necessary to generate auto-reactive Treg cells suitable as cellular therapy for autoimmunity and transplantation. We will examine the dynamics and function of antigen-specific Foxp3+ Treg cells in NOD mice. In our model, Treg cells respond to self-antigen, pro-insulin, within a polyclonal T cell repertoire and in the context of a pro-insulin-specific CD4+ T cell-initiated autoimmune disease.

Identification of pathways of beta cell death in diabetes.

Group leaders: A/Prof Helen Thomas, Prof Tom Kay
Team members: Dr Yuxing Zhao, Mr Nicholas Scott, Ms Stacey Fynch
Collaborators: Prof Andreas Strasser, Dr John Silke

Type 1 diabetes arises as a consequence of autoreactive T cell-mediated destruction of pancreatic beta cells. The mechanism of beta cell killing in vivo, however, remains unclear. Using an in vivo reconstitution assay, we are studying the mechanisms of beta cell death in a mouse model of autoimmune diabetes. Mouse islets with altered expression of critical cell death proteins were tested for their ability to survive the attack by diabetogenic T cells.  Our data suggest that apoptosis is not essential for beta cell killing. Using electron microscopy, we observed that dying beta cells showed features of necrosis. Collectively, our data indicate that necrosis is an important mechanism of beta cell death in T1D. It is not clear what effector mechanism induces the necrosis of beta cells, and the death signal is transduced through a death receptor-independent mechanism. We are addressing the hypothesis that T cells recruit other cells, such as macrophages, to kill beta cells. Macrophages are known to release cytokines and reactive oxygen species to kill target cells.  We are studying the role of macrophages in beta cell death in vitro and in vivo.

Role of cytotoxic T cells in human type 1 diabetes

Group Leaders: Professor Tom Kay, Dr Stuart Mannering and A/Prof Helen Thomas
Team Members: Dr Bala Krishnamurthy, Mr Prerak Trivedi, Dr Vimukthi Pathiraja
Collaborators: Prof Phil O’Connell, A/Prof Toby Coates, Prof Mark Peakman, Prof Matthias von Herrath, A/Prof Stephen Alexander

While a lot is known about the pathogenesis of type 1 diabetes in NOD mice, much less is known of human type 1 diabetes. The Tom Mandel Islet Transplant Program occasionally receives pancreata from organ donors with type 1 diabetes and it is of enormous value to study these. In 2008, we were fortunate to isolate islets from a donor who had type 1 diabetes for only three years. Pancreas sections from this donor reveal infiltrated islets. We expanded and sorted CD4+ and CD8+ T cell clones from the islets, and have these as well as islet RNA frozen for future characterization. We will use this and future specimens of this nature to study beta cell biology and immune phenotype of type 1 diabetes in humans.

Student Projects


Publication Highlights

  1. Krishnamurthy B, Dudek NL, McKenzie MD, Purcell AW, Brooks AG, Gellert S, Colman PG, Harrison LC, Lew AM, Thomas HE, Kay TWH (2006) Responses against islet antigens in NOD mice are prevented by tolerance to proinsulin but not IGRP. J Clin Invest 116:3258-65.
  2. Chong MM, Chen Y, Darwiche R, Dudek NL, Irawaty W, Santamaria P, Allison J, Kay TWH, Thomas HE (2004) Suppressor of cytokine signaling-1 overexpression protects pancreatic beta cells from CD8+ T cell-mediated autoimmune destruction. J Immunol 172:5714-21.
  3. O’Connell PJ, Holmes-Walker JD, Goodman D, Hawthorne WJ, Loudovaris T, Gunton JE, Thomas HE, Grey ST, Drogemuller CJ, Ward GM, Torpy DJ, Coates PT, Kay TW, On behalf of the Australian Islet Transplant Consortium. Multicenter Australian Trial of Islet Transplantation: Improving Accessibility and Outcomes Am. J. Transplant. 13:1850-8 (2013)
  4. Chee J, Ko H-J, Skowera A, Jhala G, Catterall T, Graham KL, Sutherland RM, Thomas HE, Lew AM, Peakman M,  Kay TWH, Krishnamurthy B Effector memory T cells develop in islets and report islet pathology in type 1 diabetes. J. Immunol 192:572-80 (2014)
  5. Pathiraja V, Kuechlich JP, Campbell PD, Krishnamurthy B, Loudovaris T, Coates PTH, Brodnicki TC, O'Connell PJ, Kedzierska K, Rodda C, Bergman P, Hill E, Purcell AW, Dudek NL, Thomas HE, Kay TWH, Mannering SI Proinsulin specific, HLA-DQ8 and HLA-DQ8 transdimer restricted, CD4+ T cells infiltrate the islets in type 1 diabetes. Diabetes 64:172-82 (2015)
  6. Jhala G, Chee J, Trivedi PM, Selck C, Gurzov EN, Graham KL, Thomas HE, Kay TW, Krishnamurthy B Perinatal tolerance to proinsulin is sufficient to prevent autoimmune diabetes. JCI Insight 1:e86065 (2016)
  7. Trivedi PM, Graham KL, Scott NA, Jenkins MR, Majaw S, Sutherland RM, Fynch S, Lew AM, Burns CJ, Krishnamurthy B, Brodnicki TC, Mannering SI, Kay TW, Thomas HE. Repurposed JAK1/JAK2 inhibitor reverses established autoimmune insulitis in non-obese diabetic mice. Diabetes 66:1650-60 (2017)
  8. Mollah, ZU, Quah HS, Graham KL, Jhala G, Krishnamurthy B, Francisca M Dharma J, Chee J, Trivedi PM, Pappas EG, Mackin L, Chu EP, Akazawa S, Fynch S, Hodson C, Deans AJ, Trapani JA, Chong MM, Bird PI, Brodnicki TC, Thomas HE, Kay TW. Granzyme A-deficiency breaks immune tolerance and promotes autoimmune diabetes through a type I interferon-dependent pathway. Diabetes, 66:3041-50 (2017)
  9. Sutherland RM, Londrigan SL, Brady JL, Carrington EM, Marchingo JM, Heinzel S, Hodgkin PD, Graham KL, Kay TW, Zhan Y, Lew AM Cognate antigen engagement on parenchymal cells stimulates CD8+ T cell proliferation in situ. Nat Commun 8:14809 (2017)
  10. Rickels MR, Stock PG, de Koning EJP, Piemonti L, Pratschke J, Alejandro R, Bellin MD, Berney T, Choudhary P, Johnson PR, Kandaswamy R, Kay TWH, Keymeulen B, Kudva YC, Latres E, Langer RM, Lehmann R, Ludwig B, Markmann JF, Marinac M, Odorico JS, Pattou F, Senior PA, Shaw JAM, Vantyghem MC, White S Defining the outcomes for beta-cell replacement therapy in the treatment of diabetes: a consensus report on the Igls criteria from the IPITA/EPITA opinion leaders workshop. Transplantation 102:1479-86 (2018)
  11. So M, Elso CM, Tresoldi E, Pakusch M, Pathiraja V, Wentworth JM, Harrison LC, Krishnamurthy B, Thomas HE, Rodda C, Cameron FJ, McMahon J, Kay TWH, Mannering SI Proinsulin C-peptide is an autoantigen in people with type 1 diabetes. Proc Natl Acad Sci USA 115:10732-7 (2018)