Philippe Blancou

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

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. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of β-cell autoimmunity. Taking advantage of a panel of HLA-A2–restricted β-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-γ enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying β-cell–reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention.

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

OBJECTIVE—Islet-reactive CD8+ 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 CD8+ T-cell γ-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+ adult type 1 diabetic patients close to diagnosis and at a second time point 7–16 months later. RESULTS—CD8+ 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 = 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 < 0.02). The previously subdominant IA-2206–214 and IGRP265–273 peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS—Shifts both in frequency and in immunodominance of CD8+ 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.

Immunization of HLA Class I Transgenic Mice Identifies Autoantigenic Epitopes Eliciting Dominant Responses in Type 1 Diabetes Patients

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic β cells. CD8+ T cells have recently been assigned a major role in β cell injury. Consequently, the identification of autoreactive CD8+ T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8+ T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8+ reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8+ T cells from newly diagnosed T1D patients (n = 19) but not from CD8+ T cells of healthy controls (n = 20). Among these, GAD114–123, GAD536–545 and IA-2805–813 were recognized by 53%, 25%, and 42% of T1D patients, respectively.

Equivalent Specificity of Peripheral Blood and Islet-Infiltrating CD8+ T Lymphocytes in Spontaneously Diabetic HLA-A2 Transgenic NOD Mice

CD8+ T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic β cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of β cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-γ ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8+ PBMC in human type 1 diabetes.

β2-Adrenoreceptor Agonist Inhibits Antigen Cross-Presentation by Dendritic Cells

Despite widespread usage of β-adrenergic receptor (AR) agonists and antagonists in current clinical practice, our understanding of their interactions with the immune system is surprisingly sparse. Among the AR expressed by dendritic cells (DC), β2-AR can modify in vitro cytokine release upon stimulation. Because DC play a pivotal role in CD8+ T cell immune responses, we examined the effects of β2-AR stimulation on MHC class I exogenous peptide presentation and cross-presentation capacities. We demonstrate that β2-AR agonist-exposed mature DC display a reduced ability to cross-present protein Ags while retaining their exogenous peptide presentation capability. This effect is mediated through the nonclassical inhibitory G (Gαi/0) protein. Moreover, inhibition of cross-presentation is neither due to reduced costimulatory molecule expression nor Ag uptake, but rather to impaired phagosomal Ag degradation. We observed a crosstalk between the TLR4 and β2-AR transduction pathways at the NF-κB level. In vivo, β2-AR agonist treatment of mice inhibits Ag protein cross-presentation to CD8+ T cells but preserves their exogenous MHC class I peptide presentation capability. These findings may explain some side effects on the immune system associated with stress or β-agonist treatment and pave the way for the development of new immunomodulatory strategies.

Immunoregulatory Properties of Heme Oxygenase-1

Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. HO-1 is induced by its substrate and by other stimuli, including agents involved in oxidative stress and proinflammatory cytokines as well as several anti-inflammatory stimuli. A growing body of evidence points toward the capacity of this molecule to inhibit immune reactions and the pivotal role of HO-1 in inflammatory diseases. We will first review the physiological role of HO-1 as determined by the analysis of HO-1-deficient individuals. This will be followed by an examination of the effect of HO-1 within immunopathological contexts such as immune disorders (autoimmunity and allergy) or infections. A section will be devoted to the use of an HO-1 inducer as an immunosuppressive molecule in transplantation. Finally, we will review the molecular basis of HO-1 actions on different immune cells.

HLA Class I Epitope Discovery in Type 1 Diabetes

Abstract:  Type 1 diabetes mellitus (T1DM) results from the destruction of β cells by autoantigen‐specific T cells. In the non‐obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I‐restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B‐chain and C‐peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA‐A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA‐A2.1‐transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA‐transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA‐2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot‐ and MHC tetramer‐based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.

Editorial: Heme oxygenase-1 and dendritic cells: what else?

HO are the rate-limiting intracellular enzymes that degrade heme to biliverdin and free divalent iron and CO. Three distinct HO enzymes have been identified: HO-1, HO-2, and HO-3 [1]. HO-1 is a stress-responsive gene whose expression is induced by a variety of stimuli including heme, heavy metals, inflammatory cytokines, and NO [1]. Demonstrating that HO-1 plays a role in immune regulation has been the objective of a small number of laboratories around the world. This is based on observations that induction of HO-1 expression by pharmacological activators or gene transfer, as well as delivery of heme degradation products, produces immunosuppressive effects in a variety of conditions or disorders involving activation of the immune system. As in vivo studies are the gold standard, data from HO-1 KO mice have established that HO-1 is crucial in the long run to inhibit not only spontaneous [1] but also induced inflammation [2], although to our knowledge, autoimmune disorders have not yet been described in HO-1deficient mice (probably as a result of their short life-spans). This situation forges two possibilities: HO-1 can provide immunosuppression through its expression by Tregs or by its expression by APC (mostly DCs but also macrophages). The initial idea that HO-1 expression in Tregs could be the key step for providing immune suppression came from Chung and co-workers [3], who observed that HO-1 was expressed by CD4 CD25 Tregs. His group subsequently showed that FoxP3 induces HO-1 expression in human T lymphocytes and most importantly, that the regulatory function of Tregs is lost when HO-1 activity is blunted by a pharmacological inhibitor [4]. The major setback of these experiments was that they were not designed to evaluate the effect of pharmacological inhibitors of HO-1 on CD4 Tregs or CD4 effector cells, thus leaving room for doubt. This gap is now widening, as in this issue of the Journal of Leukocyte Biology, Tiegs’ group [5] reports on the use of a well-designed assay, where effectors from Tregs can be distinguished in an in vitro proliferation assay, and shows that HO-1 induction in a CD4 CD25 T effector cell population does not confer an immunoregulatory function to these cells. Another important feature revealed by Biburger et al. [5] is the capacity of the CD4 CD25 Treg population to proliferate upon HO-1 inhibition by a pharmacological inhibitor. This result could be of interest for the amplification of the Treg population in clinical protocols dealing with suppression of graft rejection, graft-versus-host disease, or autoimmune or allergic diseases. The fact that HO-1 inhibition increases CD4 CD25 Treg proliferation casts doubt on the results of Choi et al.[4], where their suppressive assay could not distinguish between proliferation of CD4 CD25 Tregs or CD4 effector cells, as it used thymidine incorporation and not different fluorescence probes for each cell population (as described in Biburger’s manuscript [5]). KO mice for HO-1 provide a clear and definitive answer about the role of HO-1 in CD4 CD25 Treg function showing HO-1 is not required [6]. However, an indirect action of HO-1 on Treg immunosuppression activity by means of APCs and more specifically, by DCs, has been suggested in different pathological rodent models such as transplantation [7], experimental autoimmune encephalomyelitis [8], or diabetes [9]. HO-1 induction in matured DC leads to the loss of DC immunogenicity in humans, rats, and mice [10, 11]. In a recent and conclusive paper, George et al. [12] showed that expression of HO-1 by DCs is necessary for CD4 CD25 Tregs to exert their immunoregulatory activity (Fig. 1). Recently published data by our group [15] also showed that HO-1 is necessary for tolerogenic DC to be able to delay cardiac allograft rejection in the rat. However, one recent paper unexpectedly described induced anti-HO-1 CD8 Treg cells in cancer patients and suggested a processing of the

Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed that PBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

P2090 Rôle tolérogène de la stimulation b2-adrénergique des cellules dendritiques

Introduction Bien que les agonistes et antagonistes des recepteurs adrenergiques soient largement utilises en pratique, peu de donnees sont disponibles concernant les consequences potentielles de leur utilisation sur le systeme immunitaire en general et les maladies auto-immunes en particulier. Pourtant, les cellules immunitaires, dont les cellules dendritiques (DC), expriment les adrenorecepteurs. Dans ce contexte, nous avons etudie les effets de la stimulation β2-adrenergique des DC sur leur dialogue avec les lymphocytes T CD8+. Materiels et methodes En utilisant des approches pharmacologiques et genetiques, nous avons analyse les effets de la stimulation β2-adrenergique des DC sur leur phenotype, leur capacite a prendre en charge et a processer des antigenes particulaires et solubles afin de les presenter sur les molecules de CMH-I et sur leur efficacite a induire une reponse T cytotoxique in vitro et in vivo Resultats Nous avons demontre que la stimulation β2-adrenergique des DC leur confere un potentiel tolerogene caracterise par une inhibition de leur capacite a cross-presenter des antigenes aux lymphocytes T CD8+. Cet effet majeur est medie par l’activation d’une voie de signalisation dependante de la proteine Gαi/0 aboutissant a l’inhibition de la translocation nucleaire du complexe NFkB. De plus, cette inhibition de cross-presentation n’est due ni a une diminution de l’expression des molecules de co-stimulation ni a un defaut de captation des antigenes, mais a une diminution de la degradation des antigenes et de leur chargement sur les molecules de CMH-I. In vivo, l’administration de β2-agonistes a des souris inhibe la cross-presentation des antigenes et l’activation des lymphocytes T CD8+. Conclusion Ainsi, la stimulation β2-adrenergique des DC inhibe leur capacite a induire des reponses immunitaires specifiques d’antigene en interferant avec la voie de la cross-presentation. Ces resultats pourraient permettre d’identifier de nouvelles cibles therapeutiques du diabete auto-immun.