CD8+ T-Cell Responses Identify β-Cell Autoimmunity in Human Type 1 Diabetes

Mallone, Roberto; Martinuzzi, Emanuela; Blancou, Philippe; Novelli, Giulia; Afonso, Georgia; Dolz, Manuel; Bruno, Graziella; Chaillous, Lucy; Chatenoud, Lucienne; Bach, Jean‐Marie; Endert, Peter Van

doi:10.2337/db06-1419

Cited by 167 publications

(231 citation statements)

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“…Furthermore, more recent studies in type 1 diabetes (T1D) (Amrani et al, 2000;Toma et al, 2005;Mallone et al, 2007) and multiple sclerosis (Crawford et al, 2004) have highlighted the fundamental contribution of CD8 + T cells in autoimmunity (Liblau et al, 2002;Walter and Santamaria, 2005), thus challenging the paradigm depicting these diseases as predominantly CD4 + T helper 1-mediated.…”

Section: Introductionmentioning

confidence: 99%

Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Martinuzzi

Scotto

Énée

et al. 2008

Journal of Immunological Methods

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Abbreviations: ANOVA, analysis of variance; CMV, cytomegalovirus; EBV, Epstein-Barr virus; GAD, glutamic acid decarboxylase; HS, human serum; IA-2, insulinoma-associated protein 2; IGRP, islet glucose-6-phosphatase catalytic subunit related protein; MP, matrix protein; PI, proinsulin; PPI, preproinsulin; SFC, spot-forming cells; T1D, type 1 diabetes. ABSTRACTThe identification of parameters maximizing detection sensitivity in ELISpot assays is important to transfer this technology into the clinical setting for identifying rare Ag-specific CD8 + T cells. We have therefore considered human IFN-Ȗ CD8 + T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specific stimulation period. Two parameters were found to greatly enhance detection sensitivity (i.e., to specifically increase epitope-driven signal while keeping background noise to a minimum):use of human serum-free vs. serum-supplemented culture medium (2.4-fold median increase) and addition of low dose IL-7 (1.5-fold increase). Incorporating both of these parameters into the ELISpot procedure proved capable of greatly amplifying (35.1-fold increase) the low grade CD8 + T cell responses directed against ȕ-cell epitopes of type 1 diabetes patients, as compared to a previously optimized procedure using human serum-supplemented medium and low dose IL-2. Implementation of this ELISpot procedure should expedite development of "immune staging" protocols for autoimmune as well as tumor and infectious diseases.

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Section: Introductionmentioning

confidence: 99%

Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Martinuzzi

Scotto

Énée

et al. 2008

Journal of Immunological Methods

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“…Several β-cell Ags have been identified, among which insulin and its precursor (pre)proinsulin ((pre)PI) are major targets of islet-reactive T cells, both in humans [1] and in NOD mice [2]. This led to the Correspondence: Dr. Roberto Mallone e-mail: roberto.mallone@inserm.fr hypothesis that subcutaneous insulin administration could prevent disease, the rationale of which is twofold.…”

Section: Introductionmentioning

confidence: 99%

Short‐term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice

2012

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Despite encouraging results in the NOD mouse, type 1 diabetes prevention trials using subcutaneous insulin have been unsuccessful. To explain these discrepancies, 3-weekold NOD mice were treated for 7 weeks with subcutaneous insulin at two different doses: a high dose (0.5 U/mouse) used in previous mouse studies; and a low dose (0.005 U/mouse) equivalent to that used in human trials. Effects on insulitis and diabetes were monitored along with immune and metabolic modifications. Low-dose insulin did not have any effect on disease incidence. High-dose treatment delayed but did not prevent diabetes, with reduced insulitis reappearing once insulin discontinued. This effect was not associated with significant immune changes in islet infiltrates, either in terms of cell composition or frequency and IFN-γ secretion of islet-reactive CD8 + T cells recognizing the immunodominant epitopes insulin B 15-23 and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) [206][207][208][209][210][211][212][213][214] . Delayed diabetes and insulitis were associated with lower blood glucose and endogenous C-peptide levels, which rapidly returned to normal upon treatment discontinuation. In conclusion, high-but not low-dose prophylactic insulin treatment delays diabetes onset and is associated with metabolic changes suggestive of β-cell "rest" which do not persist beyond treatment. These findings have important implications for designing insulin-based prevention trials.Keywords: Insulin r NOD mouse r Prevention r T cells r Type 1 diabetes IntroductionType 1 diabetes (T1D) is an autoimmune disease in which failures in central and peripheral tolerance mechanisms lead to T-cellmediated destruction of insulin-producing β cells. Several β-cell Ags have been identified, among which insulin and its precursor (pre)proinsulin ((pre)PI) are major targets of islet-reactive T cells, both in humans [1] and in NOD mice [2]. This led to the Correspondence: Dr. Roberto Mallone e-mail: roberto.mallone@inserm.fr hypothesis that subcutaneous insulin administration could prevent disease, the rationale of which is twofold. First, insulin administration could induce a state of "β-cell rest" that would make β cells less vulnerable to metabolic stress, apoptosis [3], and possibly to immune-mediated destruction [4]. Second, repeated subcutaneous insulin injections could act as a vaccination protocol potentially restoring immune tolerance [5]. The relative role of these two putative mechanisms remains unsettled.Studies in the NOD mouse showed that subcutaneously administered insulin, started prior to disease development, is capable of preventing insulitis and diabetes [6]. This observation prompted the Diabetes Prevention Trial-Type 1 (DPT-1) [7] Eur. J. Immunol. 2012. 42: 1553-1561 developing disease [8]. However, both trials were unsuccessful, as no difference in T1D incidence was observed between insulinand placebo-treated individuals [7,8].It is still not clear why this translation from animal studies to human clinical trials fail...

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“…ϩ T-cells, we have recently proposed an isletspecific CD8 ϩ T-cell ␥-interferon (IFN-␥) ELISpot (ISL8Spot) assay (18,29). The ISL8Spot employs HLA-A2-restricted ␤-cell epitopes derived from preproinsulin (PPI) (30), GAD, insulinoma-associated protein 2 (IA-2) (17), and IGRP (31), which have been mostly identified by proteasome digestion and DNA immunization strategies (32).…”

Section: For Cd8mentioning

confidence: 99%

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

et al. 2008

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OBJECTIVE-Islet-reactive CD8ϩ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS-We took advantage of a recently validated islet-specific CD8ϩ T-cell ␥-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2 ϩ adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. RESULTS-CD8ϩ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P ϭ 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60 -67 to 20% (P Ͻ 0.02). The previously subdominant IA-2 206 -214 and IGRP [265][266][267][268][269][270][271][272][273] peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS-Shifts both in frequency and in immunodominance of CD8ϩ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements. Diabetes 57:1312-1320, 2008

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CD8+ T-Cell Responses Identify β-Cell Autoimmunity in Human Type 1 Diabetes

Abstract: Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials.

Cited by 167 publications

References 59 publications

Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Short‐term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

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