Diabetes
Novel human regulatory T cells are co-generated in response to self
JA Dromey, BH Lee, HE Young, SI Mannering, KP Jensen, DJ Thearle, S Fourlanos, S Reinwald, ME Bandala, LC Harrison in collaboration with L Belov (Medsaic Pty Ltd)
Regulatory T cells (Tregs) are subsets of T cells that suppress pro-inflammatory immune responses and maintain immune homeostasis. Impaired generation of Tregs could be a forerunner of autoimmune disease, but Tregs have not yet been well characterised in terms of disease or autoantigen specificity. Type 1 diabetes (T1D) is an autoimmune disease in which destruction of insulin-producing β cells in the islets of the pancreas is associated with immunity to the autoantigens proinsulin and glutamic acid decarboxylase 65 (GAD65). We isolated CD4+ regulatory and non-regulatory clones specific for proinsulin and GAD65 from human blood. We determined that the regulatory clones have higher CD52 expression than those that lack regulatory function.
Populations of blood CD4+ T cells that divided in response to proinsulin or GAD65 could be separated into Tregs and non-Tregs on the basis of CD52 expression. CD4+ CD52hi cells suppressed proliferation and production of pro-inflammatory cytokines by CD52lo cells. Depletion of CD52hi cells enhanced the proliferation of T cells to antigen. The relative frequency of CD52hi cells (up to 15% of divided cells) was reciprocally related to autoantigen concentration; the CD52hi response to an infectious antigen (tetanus toxoid) was 5-8 fold less and not clearly dose-responsive. Generation of CD52hi cells to GAD65 was significantly impaired in individuals with, or at risk of T1D. We propose that the co-generation of CD4+CD52hi Tregs is an important proximal mechanism of self-tolerance, the impairment of which contributes to the pathogenesis of autoimmune disease.
CD4+CD52hi Tregs are co-generated in response to T-cell activation by autoantigen. (A) A minor proportion of CD4+ T cells dividing in response to proinsulin is CD52hi (healthy donor). (B) Proliferation of CD4+ T cells specific for tetanus was suppressed by CD52hi cells in a proinsulin-dependent manner. Sorted cells were co-cultured at a 1:1 ratio. Suppression was not observed when CD52lo cells were co-cultured with the tetanus-specific CD4+ T cells. (C) IFN-γ production by CD4+CD52hi and CD4+CD52lo T cells, sorted 7 days after incubating CFSE-labelled blood mononuclear cells with GAD65, measured by ELISpot in the absence (light blue) or presence (dark blue) of GAD65. Co-cultured at a 1:1 ratio, CD52hi cells suppressed IFN-γ production by CD52lo cells in response to GAD65. (D) Proliferative response of blood mononuclear cells to GAD65 following depletion of either CD4+CD52hi cells (upper 10% of CD4+CD52+ population) or CD4+CD52lo cells (bottom 10% of CD4+CD52+ population).
Adult pancreas stem cells
LJ Gonez, I Banakh, LC Harrison in collaboration with AJ Hussey, KR Knight, WA Morrison (Bernard O’Brien Institute of Microsurgery)
The existence of putative stem cells in the adult pancreas remains controversial. The problem in defining these cells is their rarity and the absence of specific markers. Tissue-specific stem cells can be isolated with FACS as a side population (SP) based on their ability to efflux the DNA binding dye Hoechst 33342 via membrane ABC transporters. We have isolated adult mouse pancreas SP and characterised its expression of putative stem cell markers including CD133, CD24 and aldehyde dehydrogenase. Their potential for differentiation into hormone-producing cells was demonstrated in vitro using cell cultures and in vivo using tissue engineered chambers.
Vitamin D in diabetes
S Elkassaby, JA Dromey, LC Harrison in collaboration with S Fourlanos (Department of Diabetes and Endocrinology, Royal Melbourne Hospital) Pub ref: 181, 190
Vitamin D has been shown to improve pancreatic β cell function in animal models of diabetes and in humans. Vitamin D has also been demonstrated to be an immunomodulatory agent, shifting the balance from pro-inflammatory to anti-inflammatory immune responses. We are investigating the effects of vitamin D on human immune function in vitro and in vivo, including randomised trials to determine the effects on immune and metabolic function in humans with autoimmune and non-autoimmune diabetes.
The proximal insulin A-chain contains two distinct T cell epitopes
SI Mannering, SH Pang, D Thearle, LC Harrison in collaboration with A Purcell, N Williamson (Bio21 Institute, University of Melbourne)
The targets of the autoimmune response that causes type 1 diabetes (T1D) need to be identified before antigen-specific therapies to prevent T1D can be developed. In 2005, we identified a new epitope in the A-chain of human insulin. Recently, we identified another epitope that overlaps the original A-chain epitope. The new epitope is present in proinsulin, but not insulin, indicating that residues from the C-peptide are required. This work reveals that the A-chain/ C-peptide region of proinsulin contains at least two related epitopes recognised by T cells from subjects with, or at-risk of, T1D.
Optimising the expansion of human islet antigen-specific regulatory T cell clones in vitro
H Yu, JA Dromey, BH Lee, SI Mannering, LC Harrison
Antigen-specific regulatory T cells (Tregs) have an important role in preventing autoimmune disease. However, owing to their anergic property, their therapeutic potential has been limited. We generated CD4+ Treg clones specific for the pancreatic islet autoantigen glutamic acid decarboxylase 65 (GAD65) from the blood of healthy and diabetic subjects. We found exogenous IL-2 maximally reversed anergy, compared to IL-4, IL-7 and IL-15. A higher concentration of IL-2 together with the immunosuppressive drug, rapamycin, did not promote expansion of GAD65-specific Treg clones. Compared with PHA, stimulation with anti-CD3 and anti-CD28 antibodies resulted in less expansion, but expanded clones consistently retained suppressor function in vitro.
Do adipose tissue macrophages cause type 2 diabetes?
JM Wentworth, G Naselli, LC Harrison in collaboration with PE O’Brien (Centre for Obesity Research and Education, Monash University)
Obesity is associated with increased numbers of macrophages in adipose tissue, which may promote insulin resistance via local inflammatory actions. We characterised adipose macrophages (AMs) in obese non-diabetic women and identified two AM subsets that differentially express a particular surface marker. Obese women with severe insulin resistance, an early marker of type 2 diabetes, have increased numbers of a subset of AMs compared to comparably obese women who are not insulin resistant, suggesting AMs with higher expression of the surface marker may cause diabetes. Ongoing work aims to determine the functional differences between the AM subsets, with the goal of developing anti-inflammatory treatments for type 2 diabetes.