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1.  T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones 
Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer.
•Methodology allows isolation and cloning of multiple T-cell clones in parallel•Simple and requires ~ 6 weeks to validated clones•No requirement for peptide–MHC multimers or single cell sorting•CD4 + and CD8 + T-cell clones grown and validated•Clones produced for viral, autoimmune and cancer epitopes
PMCID: PMC4783706  PMID: 26826277
APC, Antigen presenting cells; CDH3, cadherin-3; 51Cr, chromium-51; DC, dendritic cell; EBOV-Z, Zaire Ebola virus; EBV, Epstein–Barr virus; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunospot assay; EN2, Engrailed-2; flu, influenza A; FBS, foetal bovine serum; GAD65, glutamic acid decarboxylase; gp, glycoprotein; HA, haemagglutinin; HLA, human leukocyte antigen; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein; IMP-3, Insulin-like growth factor 2 mRNA binding protein 3; InsB, insulin β chain; MAGE, melanoma-associated antigen; MHC, major histocompatibility complex; MP, matrix protein; NP, nucleoprotein; PAP-3, prostatic acid phosphatase-3; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; pMHC, peptide–MHC; PPI, preproinsulin; SFC, spot forming cells; TCR, T-cell receptor; T1D, Type 1 diabetes; Ebola; Library; Peptide-specific; T-cell clone; Tumour; Type 1 diabetes
2.  Innate and adaptive immunity to human beta cell lines: implications for beta cell therapy 
Diabetologia  2015;59(1):170-175.
Genetically engineered human beta cell lines provide a novel source of human beta cells to study metabolism, pharmacology and beta cell replacement therapy. Since the immune system is essentially involved in beta cell destruction in type 1 diabetes and after beta cell transplantation, we investigated the interaction of human beta cell lines with the immune system to resolve their potential for immune intervention protocol studies.
Human pancreatic beta cell lines (EndoC-βH1 and ECi50) generated by targeted oncogenesis in fetal pancreas were assessed for viability after innate and adaptive immune challenges. Beta cell lines were pre-conditioned with T helper type 1 (Th1) cytokines or high glucose to mimic inflammatory and hyperglycaemia-stressed conditions. Beta cells were then co-cultured with auto- and alloreactive cytotoxic T cells (CTL), natural killer (NK) cells, supernatant fraction from activated autoreactive Th1 cells, or alloantibodies in the presence of complement or effector cells.
Low HLA expression protected human beta cell lines from adaptive immune destruction, but it was associated with direct killing by activated NK cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta cell apoptosis and upregulation of HLA, increasing beta cell vulnerability to killing by auto- and alloreactive CTL and alloreactive antibodies.
We demonstrate that genetically engineered human beta cell lines can be used in vitro to assess diverse immune responses that may be involved in the pathogenesis of type 1 diabetes in humans and beta cell transplantation, enabling preclinical evaluation of novel immune intervention strategies protecting beta cells from immune destruction.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-015-3779-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC4670455  PMID: 26489735
Adaptive immunity; Beta cell; Innate immunity; Transplantation
3.  Reduction in CD4 Central Memory T-Cell Subset in Costimulation Modulator Abatacept-Treated Patients With Recent-Onset Type 1 Diabetes Is Associated With Slower C-Peptide Decline 
Diabetes  2014;63(10):3449-3457.
We previously reported that continuous 24-month costimulation blockade by abatacept significantly slows the decline of β-cell function after diagnosis of type 1 diabetes. In a mechanistic extension of that study, we evaluated peripheral blood immune cell subsets (CD4, CD8-naive, memory and activated subsets, myeloid and plasmacytoid dendritic cells, monocytes, B lymphocytes, CD4+CD25high regulatory T cells, and invariant NK T cells) by flow cytometry at baseline and 3, 6, 12, 24, and 30 months after treatment initiation to discover biomarkers of therapeutic effect. Using multivariable analysis and lagging of longitudinally measured variables, we made the novel observation in the placebo group that an increase in central memory (CM) CD4 T cells (CD4+CD45R0+CD62L+) during a preceding visit was significantly associated with C-peptide decline at the subsequent visit. These changes were significantly affected by abatacept treatment, which drove the peripheral contraction of CM CD4 T cells and the expansion of naive (CD45R0−CD62L+) CD4 T cells in association with a significantly slower rate of C-peptide decline. The findings show that the quantification of CM CD4 T cells can provide a surrogate immune marker for C-peptide decline after the diagnosis of type 1 diabetes and that costimulation blockade may exert its beneficial therapeutic effect via modulation of this subset.
PMCID: PMC4171657  PMID: 24834977
4.  β-cell-specific CD8 T cell phenotype in type 1 diabetes reflects chronic autoantigen exposure 
Diabetes  2014;64(3):916-925.
Autoreactive CD8 T cells play a central role in the destruction of pancreatic islet β-cells that leads to type 1 diabetes, yet the key features of this immune-mediated process remain poorly defined. In this study, we combined high definition polychromatic flow cytometry with ultrasensitive peptide-human leukocyte antigen class I (pHLAI) tetramer staining to quantify and characterize β-cell-specific CD8 T cell populations in patients with recent onset type 1 diabetes and healthy controls. Remarkably, we found that β-cell-specific CD8 T cell frequencies in peripheral blood were similar between subject groups. In contrast to healthy controls, however, patients with newly diagnosed type 1 diabetes displayed hallmarks of antigen-driven expansion uniquely within the β-cell-specific CD8 T cell compartment. Molecular analysis of selected β-cell-specific CD8 T cell populations further revealed highly skewed oligoclonal T cell receptor (TCR) repertoires comprising exclusively private clonotypes. Collectively, these data identify novel and distinctive features of disease-relevant CD8 T cells that inform the immunopathogenesis of type 1 diabetes.
PMCID: PMC4557541  PMID: 25249579
5.  Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes 
Diabetes  2014;63(11):3835-3845.
Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multi-parameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly-diagnosed disease. Multi-dimensional cluster analysis showed two equal-sized patient agglomerations, characterized by pro-inflammatory (IFN-γ+, multi-autoantibody-positive) and partially-regulated (IL-10+, pauci-autoantibody-positive) responses. Multi-autoantibody-positive non-diabetic siblings at high-risk of disease progression showed similar clustering. Second, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently-diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesion, distinguishable by degree of cellular infiltrate and presence of B-lymphocytes, that we term “hyper-immune CD20Hi” and “pauci-immune CD20Lo”. Notably, subjects had only one infiltration phenotype and were partitioned by this into two equal-size groups that differed significantly by age of diabetes diagnosis, hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with, and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment/prevention strategies.
PMCID: PMC4207393  PMID: 24939426
6.  Costimulation Modulation With Abatacept in Patients With Recent-Onset Type 1 Diabetes: Follow-up 1 Year After Cessation of Treatment 
Diabetes Care  2014;37(4):1069-1075.
We previously reported that 2 years of costimulation modulation with abatacept slowed decline of β-cell function in recent-onset type 1 diabetes (T1D). Subsequently, abatacept was discontinued and subjects were followed to determine whether there was persistence of effect.
Of 112 subjects (ages 6–36 years) with T1D, 77 received abatacept and 35 received placebo infusions intravenously for 27 infusions over 2 years. The primary outcome—baseline-adjusted geometric mean 2-h area under the curve (AUC) serum C-peptide during a mixed-meal tolerance test (MMTT) at 2 years—showed higher C-peptide with abatacept versus placebo. Subjects were followed an additional year, off treatment, with MMTTs performed at 30 and 36 months.
C-peptide AUC means, adjusted for age and baseline C-peptide, at 36 months were 0.217 nmol/L (95% CI 0.168–0.268) and 0.141 nmol/L (95% CI 0.071–0.215) for abatacept and placebo groups, respectively (P = 0.046). The C-peptide decline from baseline remained parallel with an estimated 9.5 months’ delay with abatacept. Moreover, HbA1c levels remained lower in the abatacept group than in the placebo group. The slightly lower (nonsignificant) mean total insulin dose among the abatacept group reported at 2 years was the same as the placebo group by 3 years.
Costimulation modulation with abatacept slowed decline of β-cell function and improved HbA1c in recent-onset T1D. The beneficial effect was sustained for at least 1 year after cessation of abatacept infusions or 3 years from T1D diagnosis.
PMCID: PMC3964491  PMID: 24296850
7.  Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs 
Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR–pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.
PMCID: PMC4273996  PMID: 25452566
8.  Human β-Cell Killing by Autoreactive Preproinsulin-Specific CD8 T Cells Is Predominantly Granule-Mediated With the Potency Dependent Upon T-Cell Receptor Avidity 
Diabetes  2012;62(1):205-213.
The end-stage immunopathology of type 1 diabetes resulting in β-cell destruction appears to be strongly dominated by cytotoxic CD8 T lymphocytes (CD8 T cells). However, the mechanism of cytotoxicity used by autoreactive CD8 T cells in the human setting remains unknown. Using type 1 diabetes patient–derived preproinsulin-specific CD8 T-cell clones recognizing either an HLA-A2 (A*0201) or HLA-A24 (A*2402)-restricted epitope (peptide of preproinsulin [PPI]15–24, ALWGPDPAAA; or PPI3–11, LWMRLLPLL), we assessed the use of conventional mediators of cytotoxicity in the destruction of human β-cells in vitro compared with virus-specific cytotoxic CD8 T-cell clones. We show that PPI-specific CD8 T-cell clones are mainly reliant upon cytotoxic degranulation for inducing β-cell death. Furthermore, we find that in comparison with virus-specific CD8 T cells, there are differences in the killing potency of PPI-specific CD8 T cells that are not due to cell-intrinsic differences, but rather are mediated by differences in strength of signaling by peptide–HLA ligands. The study highlights the regulation of β-cell killing as a potential point for therapeutic control, including the possibility of blocking autoreactive CD8 T-cell function without impacting upon general immune competence.
PMCID: PMC3526019  PMID: 22936177
9.  Circulating Preproinsulin Signal Peptide–Specific CD8 T Cells Restricted by the Susceptibility Molecule HLA-A24 Are Expanded at Onset of Type 1 Diabetes and Kill β-Cells 
Diabetes  2012;61(7):1752-1759.
Type 1 diabetes results from T cell–mediated β-cell destruction. The HLA-A*24 class I gene confers significant risk of disease and early onset. We tested the hypothesis that HLA-A24 molecules on islet cells present preproinsulin (PPI) peptide epitopes to CD8 cytotoxic T cells (CTLs). Surrogate β-cell lines secreting proinsulin and expressing HLA-A24 were generated and their peptide ligandome examined by mass spectrometry to discover naturally processed and HLA-A24–presented PPI epitopes. A novel PPI epitope was identified and used to generate HLA-A24 tetramers and examine the frequency of PPI-specific T cells in new-onset HLA-A*24+ patients and control subjects. We identified a novel naturally processed and HLA-A24–presented PPI signal peptide epitope (PPI3–11; LWMRLLPLL). HLA-A24 tetramer analysis reveals a significant expansion of PPI3–11-specific CD8 T cells in the blood of HLA-A*24+ recent-onset patients compared with HLA-matched control subjects. Moreover, a patient-derived PPI3–11-specific CD8 T-cell clone shows a proinflammatory phenotype and kills surrogate β-cells and human HLA-A*24+ islet cells in vitro. These results indicate that the type 1 diabetes susceptibility molecule HLA-A24 presents a naturally processed PPI signal peptide epitope. PPI-specific, HLA-A24–restricted CD8 T cells are expanded in patients with recent-onset disease. Human islet cells process and present PPI3–11, rendering themselves targets for CTL-mediated killing.
PMCID: PMC3379678  PMID: 22522618
11.  Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation 
Human skin-resident IL-10+ regulatory dendritic cells induce T reg cells that suppress allogeneic skin graft inflammation.
Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141+ DDCs). CD141+ DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D3 (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141+ DDCs from human blood DCs. These CD141+ DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141+ DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141+ DDC-like cells have potential clinical use for their capacity to induce immune tolerance.
PMCID: PMC3348099  PMID: 22547651
12.  Type 1 Diabetes-associated IL2RAvariationlowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T-cell function 
Numerous reports have demonstrated that CD4+CD25+regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including Type 1 diabetes (T1D),are deficient in theirability to control autologous pro-inflammatory responses when compared to non-diseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development.Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with T1D on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished interleukin (IL)-2-responsiveness in antigen-experienced CD4+ T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FoxP3 expression by Tregs, and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene impact upon immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.
PMCID: PMC3378653  PMID: 22461703
13.  Can we vaccinate against Type 1 diabetes? 
Vaccination is the administration of antigenic material to stimulate the immune system to develop adaptive immunity to a disease. As the most successful prophylactic in medical history, there is now an emerging interest as to whether vaccination can be applied in autoimmune and inflammatory conditions. These are diseases of failed immune regulation; vaccination in this context aims to exploit the power of antigenic material to stimulate immune homeostasis in the form of active, adaptive, regulatory immune responses. Type 1 diabetes is an autoimmune disease that could benefit from the therapeutic potential of vaccination. The major conditions necessary to make prophylaxis feasible are in place; the self antigens are known, the failure of existing immune regulation has been demonstrated, early studies of vaccine approaches have proved safe, and the preclinical prodrome of the disease can be easily detected by simple blood tests. Challenges for future implementation include finding the best mode of delivery and the best blend of adjunctive therapies that create the favorable conditions required for a vaccine to be effective.
PMCID: PMC3463895  PMID: 23112807
14.  Variation of Peripheral Blood Mononuclear Cell RNA Quality in Archived Samples 
Biopreservation and Biobanking  2011;9(3):259-263.
The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust.
PMCID: PMC3178418  PMID: 21977241
15.  How Representative Are Research Tissue Biobanks of the Local Populations? Experience of the Infectious Diseases Biobank at King's College, London, UK 
Biopreservation and Biobanking  2011;9(3):287-288.
Biobanks have a primary responsibility to collect tissues that are a true reflection of their local population and thereby promote translational research, which is applicable to the community. The Infectious Diseases BioBank (IDB) at King's College London is located in the southeast of the city, an area that is ethnically diverse. Transplantation programs have frequently reported a low rate of donation among some ethnic minorities. To determine whether patients who volunteered peripheral venous blood samples to the IDB were representative of the local community, we compared local government demographic data to characteristics of patients who have donated to the IDB. There was a good match between these statistics, indicating that the IDB's volunteer population of human immunodeficiency virus patients was similar to local demographics.
PMCID: PMC3178420  PMID: 21977243
16.  Structural basis of human β-cell killing by CD8+ T cells in Type 1 diabetes 
Nature immunology  2012;13(3):283-289.
The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid ‘lock-and-key’ binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an ‘aromatic-cap’, over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity.
PMCID: PMC3378510  PMID: 22245737
β-cell; crystal structure; peptide-major histocompatibility complex (pMHC); preproinsulin; surface plasmon resonance (SPR); T cell; T cell receptor (TCR); type 1 diabetes (T1D); vaccine
17.  Peripheral and Islet Interleukin-17 Pathway Activation Characterizes Human Autoimmune Diabetes and Promotes Cytokine-Mediated β-Cell Death 
Diabetes  2011;60(8):2112-2119.
CD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells.
Peripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined.
A total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ–induced and tumor necrosis factor (TNF)-α/IFN-γ–induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB.
Circulating IL-17+ β-cell–specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.
PMCID: PMC3142078  PMID: 21659501
18.  Long-Term Outcome of Individuals Treated With Oral Insulin 
Diabetes Care  2011;34(7):1585-1590.
To evaluate the long-term intervention effects of oral insulin on the development of type 1 diabetes and to assess the rate of progression to type 1 diabetes before and after oral insulin treatment was stopped in the Diabetes Prevention Trial–Type 1 (DPT-1).
The follow-up included subjects who participated in the early intervention of oral insulin (1994–2003) to prevent or delay type 1 diabetes. A telephone survey was conducted in 2009 to determine whether diabetes had been diagnosed and, if not, an oral glucose tolerance test (OGTT), hemoglobin A1c (HbA1c), and autoantibody levels were obtained on all subjects who agreed to participate.
Of 372 subjects randomized, 97 developed type 1 diabetes before follow-up; 75% of the remaining 275 subjects were contacted. In the interim, 77 subjects had been diagnosed with type 1 diabetes and 54 of the remainder have had an OGTT; 10 of these were diagnosed with type 1 diabetes, subsequently. Among individuals meeting the original criteria for insulin autoantibodies (IAAs) (≥80 nU/mL), the overall benefit of oral insulin remained significant (P = 0.05). However, the hazard rate in this group increased (from 6.4% [95% CI 4.5–9.1] to 10.0% [7.1–14.1]) after cessation of therapy, which approximated the rate of individuals treated with placebo (10.2% [7.1–14.6]).
Overall, the oral insulin treatment effect in individuals with confirmed IAA ≥80 nU/mL appeared to be maintained with additional follow-up; however, once therapy stopped, the rate of developing diabetes in the oral insulin group increased to a rate similar to that in the placebo group.
PMCID: PMC3120180  PMID: 21610124
19.  Antigen Targets of Type 1 Diabetes Autoimmunity 
Type 1 diabetes is characterized by recognition of one or more β-cell proteins by the immune system. The list of target antigens in this disease is ever increasing and it is conceivable that additional islet autoantigens, possibly including pivotal β-cell targets, remain to be discovered. Many knowledge gaps remain with respect to the disorder’s pathogenesis, including the cause of loss of tolerance to islet autoantigens and an explanation as to why targeting of proteins with a distribution of expression beyond β cells may result in selective β-cell destruction and type 1 diabetes. Yet, our knowledge of β-cell autoantigens has already led to translation into tissue-specific immune intervention strategies that are currently being assessed in clinical trials for their efficacy to halt or delay disease progression to type 1 diabetes, as well as to reverse type 1 diabetes. Here we will discuss recently gained insights into the identity, biology, structure, and presentation of islet antigens in relation to disease heterogeneity and β-cell destruction.
A single, primary autoantigenic β-cell target in type 1 diabetes (T1D), if it exists, remains to be identified with certainty. However, islet autoantigens have been used to study T1D pathogenesis and to develop therapies.
PMCID: PMC3312399  PMID: 22474615
20.  A Single Autoimmune T Cell Receptor Recognizes More Than a Million Different Peptides* 
The Journal of Biological Chemistry  2011;287(2):1168-1177.
Background: How does a limited pool of <108 T cell receptors (TCRs) provide immunity to >1015 antigens?
Results: A single TCR can respond to >one million different decamer peptides.
Conclusion: This unprecedented level of receptor promiscuity explains how the naïve TCR repertoire achieves effective immunity.
Significance: TCR degeneracy has enormous potential to be the root cause of autoimmune disease.
The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <108 human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>1015 distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8+ T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type “index” preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >108 peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8+ T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.
PMCID: PMC3256900  PMID: 22102287
Antigen Presentation; Major Histocompatibility Complex (MHC); T Cell; T Cell Biology; T Cell Receptor; Autoimmunity; Cellular Immune Response; Diabetes; Insulin; Pancreatic Islets
21.  Antigen-Specific Immunotherapy for Type 1 Diabetes: Maximizing the Potential 
Diabetes  2010;59(9):2087-2093.
PMCID: PMC2927927  PMID: 20805382
22.  Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell–Associated Epitopes Using Combinatorial MHC Multimers 
Diabetes  2010;59(7):1721-1730.
Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8+ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8+ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.
Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10–18, prepro-insulin (PPI)15–24, islet antigen (IA)-2797–805, GAD65114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)265–273, and prepro islet amyloid polypeptide (ppIAPP)5–13–specific CD8+ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.
Using this kit, islet autoreactive CD8+ T-cells recognizing insulin B10–18, IA-2797–805, and IGRP265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.
A kit was developed that allows simultaneous detection of CD8+ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.
PMCID: PMC2889772  PMID: 20357361
23.  Naturally Arising Human CD4 T-Cells That Recognize Islet Autoantigens and Secrete Interleukin-10 Regulate Proinflammatory T-Cell Responses via Linked Suppression 
Diabetes  2010;59(6):1451-1460.
Regulatory T-cells (Tregs) recognizing islet autoantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in humans, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine interleukin-10 (IL-10) is significantly associated with late onset of type 1 diabetes and is constitutively present in a majority of nondiabetic individuals. Here, we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses.
We isolated and cloned islet-specific IL-10–secreting CD4+ T-cells from nondiabetic individuals after brief ex vivo exposure to islet autoantigens using cytokine capture technology and examined their phenotype and regulatory potential.
Islet-specific IL-10+ CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC). The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells.
This hitherto undescribed population of islet autoantigen–specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen β-cell–specific tolerance.
PMCID: PMC2874706  PMID: 20299476
24.  Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T-cell recognition 
Improving T-cell antigens by altering MHC anchor residues is a common strategy used to enhance peptide vaccines but there has been little assessment of how such modifications affect TCR binding and T-cell recognition. Here, we use surface plasmon resonance and peptide-MHC tetramer binding at the cell surface to demonstrate that changes in primary peptide anchor residues can substantially and unpredictably alter TCR binding. We also demonstrate that the ability of TCRs to differentiate between natural and anchor-modified heteroclitic peptides distinguishes T-cells that exhibit a strong preference for either type of antigen. Furthermore, we show that anchor-modified heteroclitic peptides prime T-cells with different TCRs compared to those primed with natural antigen. Thus, vaccination with heteroclitic peptides may elicit T-cells that exhibit suboptimal recognition of the intended natural antigen and, consequently, impaired functional attributes in vivo. Heteroclitic peptide-based immune interventions therefore require careful evaluation to ensure efficacy in the clinic.
PMCID: PMC3024538  PMID: 20639478
adoptive therapy; anchor residue-modified peptide; cancer; clonotype; heteroclitic peptide; Melan-A/MART-1; peptide-major histocompatibility complex (pMHC); surface plasmon resonance (SPR); T-cell; T-cell receptor (TCR); type 1 diabetes (T1D); vaccine
25.  Validity and Reproducibility of Measurement of Islet Autoreactivity by T-Cell Assays in Subjects With Early Type 1 Diabetes 
Diabetes  2009;58(11):2588-2595.
Type 1 diabetes results from an immunemediated destruction of β-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression.
We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections.
Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays.
The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood.
PMCID: PMC2768166  PMID: 19675135

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