MD Roberto Mallone
email: roberto.mallone@inserm.fr.

Clémentine Halliez

Jinxiu Rui

Kevan C Herold

The Beta Cell in Type 1 Diabetes Pathogenesis: a Victim of Circumstances or an Instigator of Tragic Events?

Keywords: antigens, apoptosis, autoimmunity, beta cells, senescence, T cells, type 1 diabetes

Recent reports have revived interest in the active role that beta cells may play in type 1 diabetes pathogenesis at different stages of disease. Some studies suggested an initiating role and proposed that type 1 diabetes may be primarily a disease of beta cells, and only secondarily a disease of autoimmunity. This scenario is possible, and invites the search for environmental triggers damaging beta cells. Another major contribution of beta cells may be to amplify autoimmune vulnerability, and to eventually drive it into an intrinsic, self-detrimental state that turns the T-cellmediated homicide into a beta-cell suicide. On the other hand, protective mechanisms are also mounted by beta cells and may provide novel therapeutic targets to combine immunomodulatory and beta-cell protective agents. This integrated view of autoimmunity as a disease of T-cell/betacell cross-talk will ultimately advance our understanding of type 1 diabetes pathogenesis and improve our chances to prevent or reverse disease progression.

Introduction

Beta-cell failure in type 1 diabetes creeps forward at a pace that is defined by a dynamic relationship between immune cells and beta-cell intrinsic responses [START_REF] Mallone | Presumption of innocence for beta cells: why are they vulnerable autoimmune targets in type 1 diabetes?[END_REF][START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF]. This relationship is affected by host factors such as genetics, age, and poorly defined environmental triggers. In some individuals, autoimmunity is initiated but does not progress further, notably in the presence of a single autoantibody (aAb) [START_REF] Insel | Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the Endocrine Society, and the American Diabetes Association[END_REF]. This may reflect an aborted immune process or responses of beta cells, or other cells, that prevent progression to the next stage of autoimmune amplification and beta-cell destruction (4). However, in others, particularly young individuals, beta-cell destruction is more rapid once autoimmunity (stage 1) or hyperglycemia (stage 3) appears [START_REF] Frohnert | Late-onset islet autoimmunity in childhood: the Diabetes Autoimmunity Study in the Young (DAISY)[END_REF][START_REF] Hao | Type 1 Diabetes TrialNet Study G: Fall in C-Peptide During First 4 Years From Diagnosis of Type 1 Diabetes: Variable Relation to Age, HbA1c, and Insulin Dose[END_REF], with little evidence of remission phases.

The precise molecular and cellular mechanisms that account for beta-cell death remain unclear.

Direct cytotoxic lysis by CD8 + T cells reactive to beta-cell antigens such as preproinsulin is postulated. However, immune infiltrates in the pancreas are sparse at the time of diagnosis. While the late timing of these histological studies, i.e. years after autoimmune initiation and even after beta-cell destruction and clinical onset, may account for these findings [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF], other mechanisms capable of mediating beta-cell killing at a distance have been suggested. Inflammatory molecules are produced by several cell types, including beta cells themselves [START_REF] Nakayasu | Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention[END_REF], and may cause dysfunction or killing. Findings of shrinkage of the exocrine pancreas before type 1 diabetes diagnosis suggest that inflammation of the whole organ may occur and that beta cells are perhaps killed by soluble mediators produced by cells that are not present in the islets. Indeed, a recent trial of anti-TNF-α antibody showed how neutralizing this cytokine may arrest progression in new-onset type 1 diabetic patients [START_REF] Quattrin | Investigators TGS: Golimumab and Beta-Cell Function in Youth with New-Onset Type 1 Diabetes[END_REF]. However, the way in which these acute inflammatory mediators affect the chronic progression remains undefined.

As part of this immune/beta-cell dynamics, a number of investigators have described how beta cells may respond to the autoimmune attack in ways that may promote their demise or protect them [START_REF] Atkinson | How does type 1 diabetes develop?: the notion of homicide or beta-cell suicide revisited[END_REF]. The question of whether beta cells die by homicide vs. suicide was originally proposed by G.F. Bottazzo [START_REF] Bottazzo | Death of a beta cell: homicide or suicide?[END_REF], but newer studies call for revisiting this question. Clearly, both T cells and beta cells are active disease players, but is one the initiator and the other the victim or do both accelerate beta-cell death? We here review this dynamic interplay in the light of recent studies.

"Absolving" T cells

The increasing evidence that T cells, together with other immune players, are not the sole culprits in type 1 diabetes pathogenesis has been recently reviewed [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF]. Briefly, arguments for their nonexclusive involvement include the following. First, experimental autoimmune diabetes cannot be induced in animal models by immunization with islet extracts or beta-cell antigens alone (2), as is the case for other autoimmune diseases such as experimental autoimmune encephalomyelitis (a model of multiple sclerosis). Second, human T cells adoptively transferred into immunodeficient mice can cause insulitis but are not diabetogenic in the absence of strong priming conditions, e.g. streptozotocin treatment to induce some beta-cell death and self-antigen release [START_REF] Tan | Type 1 diabetes induction in humanized mice[END_REF][START_REF] Milam | A Humanized Mouse Model of Autoimmune Insulitis[END_REF]. Third, histopathological, age-related endotypes [START_REF] Leete | The Effect of Age on the Progression and Severity of Type 1 Diabetes: Potential Effects on Disease Mechanisms[END_REF][START_REF] Leete | Studies of insulin and proinsulin in pancreas and serum support the existence of aetiopathological endotypes of type 1 diabetes associated with age at diagnosis[END_REF] mirroring clinical endotypes defined by age and aAb targets [START_REF] Frohnert | Late-onset islet autoimmunity in childhood: the Diabetes Autoimmunity Study in the Young (DAISY)[END_REF][START_REF] Hao | Type 1 Diabetes TrialNet Study G: Fall in C-Peptide During First 4 Years From Diagnosis of Type 1 Diabetes: Variable Relation to Age, HbA1c, and Insulin Dose[END_REF] indicate that a number of other factors may determine rates of progression.

Finally, a universal state of 'benign' autoimmunity is found in the circulation of all individuals, as witnessed by the presence of islet-reactive (and largely naïve) CD8 + T cells [START_REF] Culina | ImMaDiab Study G: Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors[END_REF][START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF][START_REF] Azoury | Peptides Derived From Insulin Granule Proteins are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice[END_REF] and isletreactive CD4 + T cells [START_REF] Viglietta | GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes[END_REF][START_REF] Danke | Autoreactive T cells in healthy individuals[END_REF]. T cells with the same antigen specificities are enriched in the pancreas of type 1 diabetic donors [START_REF] Culina | ImMaDiab Study G: Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors[END_REF][START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF], pointing to local factors that may affect the phenotypes of T cells and attract them into the target organ. Although two case reports of type 1 diabetes transfer upon grafting of bone marrow from a diabetic donor [START_REF] Lampeter | Transfer of insulin-dependent diabetes between HLA-identical siblings by bone marrow transplantation[END_REF] and grafting of islets into a diabetic recipient ( 21) argue for a central role of T cells, an alternative explanation contemplating the contribution of beta cells is possible, as explained below.

"Incriminating" beta cells

The active role of beta cells in type 1 diabetes pathogenesis has been linked to their specialized function of insulin secretion (1)a paradigm that may apply more generally to other endocrine cells. Namely, the high endoplasmic reticulum (ER) and oxidative stress imposed by the requirements for rapid and considerable insulin bio-synthesis, the rich islet vascularization -which exposes cells to inflammatory soluble mediators and cells, and the secretion of insulin and other antigenic granule contents directly in the bloodstream with high amplitude [START_REF] Wan | Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides[END_REF] are all features that may render beta cells more vulnerable to autoimmunity.

Importantly, new emerging data extend this picture to alterations of the exocrine pancreas [START_REF] Alexandre-Heymann | Structure and function of the exocrine pancreas in patients with type 1 diabetes[END_REF]. A reduced pancreas organ weight was reported in single-aAb + donors [START_REF] Campbell-Thompson | Pancreas organ weight in individuals with disease-associated autoantibodies at risk for type 1 diabetes[END_REF] and even in aAb-negative first-degree relatives [START_REF] Campbell-Thompson | Relative Pancreas Volume Is Reduced in First-Degree Relatives of Patients With Type 1 Diabetes[END_REF]. Other studies reported reduced serum trypsinogen and lipase levels starting at stage 1 (≥2 aAbs) type 1 diabetes [START_REF] Li | Serum Trypsinogen Levels in Type 1 Diabetes[END_REF][START_REF] Ross | Exocrine Pancreatic Enzymes Are a Serological Biomarker for Type 1 Diabetes Staging and Pancreas Size[END_REF], and enriched exocrine immune infiltration [START_REF] Rodriguez-Calvo | Increased immune cell infiltration of the exocrine pancreas: a possible contribution to the pathogenesis of type 1 diabetes[END_REF] and HLA Class II expression in ductal cells from type 1 diabetic donors [START_REF] Fasolino | Singlecell multi-omics analysis of human pancreatic islets reveals novel cellular states in type 1 diabetes[END_REF]. Another notable recent single-cell epigenomics study [START_REF] Chiou | Interpreting type 1 diabetes risk with genetics and single-cell epigenomics[END_REF] documented high expression of novel type 1 diabetes risk gene variants (e.g. chymotrypsinogens, lipase and cystic fibrosis transmembrane conductance regulator) in acinar and ductal cells, which is postulated to promote subclinical pancreas inflammation favoring later immune infiltration.

The pathogenic role of beta cells could theoretically be called into question at three sequential stages (Fig. 1):

1) Self-initiated homicide: are beta cells the original drivers of T-cell autoimmunity?

2) Self-amplified homicide: do beta cells enhance T-cell-mediated killing?

3) Self-finalized suicide: do beta cells under attack eventually die by suicide?

1) Self-initiated homicide

Studies by Larger and Boitard, nearly 30 years ago, showed that beta cells are needed to initiate autoimmune diabetes in NOD mice [START_REF] Larger | Pancreatic islet beta cells drive T cell-immune responses in the nonobese diabetic mouse model[END_REF]. These results support a primary role of beta cells in developing an autoimmune repertoire endowed with beta-cell specificity, since splenocytes from beta-cell-deficient mice could not transfer diabetes, but the mice were otherwise immune competent and developed other autoimmune manifestations, i.e. sialitis. This observation pointed to an early critical time window in which T cells are primed by beta-cell antigens and trigger diabetes. Of further note, this priming occurs primarily in pancreatic lymph nodes and is an early event, as pancreatic lymphadenectomy at 3 weeks, but not at 10 weeks of age, led to near-complete diabetes protection in NOD mice [START_REF] Gagnerault | Pancreatic lymph nodes are required for priming of beta cell reactive T cells in NOD mice[END_REF]. Pancreatic lymph nodes may also harbor stem-like CD8 + T cells that can proliferate and generate effector T cells that invade islets [START_REF] Gearty | An autoimmune stem-like CD8 T cell population drives type 1 diabetes[END_REF].

Several lines of experimental evidence suggest that beta-cell aberrancies, which may include increased antigen exposure and neo-antigen expression, may be the first drivers of islet autoimmunity. Islet autoimmunity does not develop if beta cells are not visible to the immune system due to reduced antigen expression, i.e. if immune ignorance is maintained. An emblematic example is provided by NOD mice carrying an insulin B16 mutation that abolishes the immunogenicity of the initiating epitope Ins B9-23, which are completely protected from diabetes [START_REF] Nakayama | Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice[END_REF]. Serreze et al found that initiation of autoimmune diabetes in NOD mice is dependent on MHC Class I [START_REF] Serreze | Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent[END_REF]. β2-microglobulin (β2m) -/-mice, which are MHC Class I-deficient, did not develop insulitis or diabetes, whereas splenocytes from diabetic NOD donors transferred diabetes into both wild-type and β2m -/-NOD/scid recipients. In contrast, splenocytes from young prediabetic NOD donors transferred diabetes only to wild-type NOD/scid recipients, while β2m -/-NOD/scid recipients required prior grafting with wild-type islets to become diabetic. The authors concluded that islet expression of MHC Class I (i.e. beta-cell visibility) was needed to prime T cells, which could then mediate beta-cell destruction independent of MHC Class I [START_REF] Serreze | Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent[END_REF]. Other adoptive T-cell transfer models suggest that, in addition to antigen presentation, beta-cell alterations such as those imprinted in the NOD background or induced by streptozotocin, are needed to enable antigendriven T-cell priming and beta-cell killing (2). On the same lines, T-cell receptor transgenic models in which T cells recognize a foreign antigen transgenically expressed in beta cells show that provision of costimulatory or inflammatory signals is required to activate these T cells [START_REF] Brezar | Beyond the hormone: insulin as an autoimmune target in type 1 diabetes[END_REF]. These mouse studies may provide an interpretation for some observations in patients. A case report of identical twin-to-twin transplantation of a non-diabetic pancreas into a type 1 diabetic recipient led to rapid autoimmune relapse [START_REF] Sutherland | Twin-to-twin pancreas transplantation: reversal and reenactment of the pathogenesis of type I diabetes[END_REF]; and conversely, bone marrow transplantation from a type 1 diabetic sibling into a HLA-identical non-diabetic recipient triggered type 1 diabetes [START_REF] Lampeter | Transfer of insulin-dependent diabetes between HLA-identical siblings by bone marrow transplantation[END_REF]. In both cases, the previous priming of the autoimmune T-cell repertoire prior to transplantation may have bypassed the need for a beta-cell trigger.

Recent elegant studies have revived interest in this hypothesis of self-initiated homicide by showing that the induction of an early and transient beta-cell dedifferentiation protects NOD mice from diabetes. A first model of NOD mice with high beta-cell proliferation rates and decreased antigen expression [START_REF] Dirice | Increased beta-cell proliferation before immune cell invasion prevents progression of type 1 diabetes[END_REF] was obtained either with a liver-specific insulin receptor genetic knock out (LIRKO) or by pharmacological treatment with an insulin receptor antagonist from 4 to 6 weeks of age. In a second model [START_REF] Lee | Beta Cell Dedifferentiation Induced by IRE1alpha Deletion Prevents Type 1 Diabetes[END_REF], a conditional knock-out of the inositol-requiring enzyme (IRE)1α, which activates the unfolded protein response (UPR) in beta cells, was induced by tamoxifen treatment during the neonatal period (1 to 5 days of life), resulting in a similar dedifferentiated beta-cell phenotype. Both NOD models are protected from diabetes.

Can all these extreme murine models that "blind" the immune system from beta cells be taken as evidence for an active role of beta cells at initiating autoimmunity? Other arguments from human genetic studies need to be considered. First, the contribution of type 1 diabetes-associated gene variants expressed in beta cells to genetic susceptibility is marginal when compared to HLA Class II polymorphisms (2)a feature that type 1 diabetes shares with several other autoimmune diseases. Second, the major influence of HLA Class II haplotypes is on the antigen specificity and rate of aAb seroconversion (i.e., autoimmune initiation) rather than on the rate of actual beta-cell destruction (i.e., clinical progression) [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF][START_REF] Valdes | Use of class I and class II HLA loci for predicting age at onset of type 1 diabetes in multiple populations[END_REF][START_REF] Dufort | Cell type-specific immune phenotypes predict loss of insulin secretion in new-onset type 1 diabetes[END_REF], suggesting a driving effect on pathology. This may further suggest that beta-cell-related non-HLA gene variants may act as modulators of this later clinical progression rather than drivers. Third, monogenic forms of neonatal diabetes causing beta-cell defects, including mutations (e.g. in INS and EIF2AK3 genes) that induce severe ER stress, show little evidence of islet autoimmunity, even in the context of high-risk HLA Class II haplotypes, with only 5.4% found positive for islet aAbs, mostly (92%) for a single aAb (41). This is observed in spite of a dysregulated expression of genes (and proteins) associated with monogenic forms of diabetes resulting from ER stress in the pancreas of type 1 diabetic and aAb + donors [START_REF] Hiller | Monogenic Diabetes and Integrated Stress Response Genes Display Altered Gene Expression in Type 1 Diabetes[END_REF], suggesting a role of primary beta-cell alterations as disease contributors rather than drivers.

However, an alternative explanation could be that the beta-cell contribution to type 1 diabetes pathogenesis may simply be less genetically imprinted and more environmentally driven than the immune contribution. This hypothesis may invite the search for environmental triggers that may compromise beta-cell health rather than immune tolerance per se, e.g. islet-tropic viral infections and/or xenotoxic agents.

Altogether, this literature suggests that beta-cell stress/dysfunction may play a role at initiating islet autoimmunity, and then significantly contribute to its amplification, as discussed in the next section.

2) Self-amplified homicide

A key observation suggesting the role of beta-cell-driven initiation and amplification mechanisms is the heterogeneity of insulitis across islets, which do not appear to be attacked and destroyed at the same time [START_REF] Campbell-Thompson | Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes[END_REF]. There are multiple mechanisms by which beta cells amplify the autoimmune attack (Fig. 2). The inflammatory environment of insulitis, particularly interferons, stimulate several active responses in beta cells that go beyond the passive effect of inducing ER stress, dysfunction and apoptosis. First, inflammation drives beta cells themselves to release chemokines (e.g. CCL5, CXCL2, CXCL9, CXCL10) and cytokines (e.g. IL-1β, IL-6) [START_REF] Nakayasu | Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention[END_REF], which further attract immune cells to the pancreas and contribute to their activation in situ.

The second major outcome of islet inflammation is HLA Class I upregulation, which results in the exposure of a larger and more diversified repertoire of antigenic peptides [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF], thus increasing the visibility and vulnerability of beta cells to infiltrating T cells. Not surprisingly, peptides derived from proteins of secretory granules (e.g. insulin, chromogranin A, secretogranin-5, urocortin-3, proconvertase-2) are highly represented across different human and murine MHC restrictions [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF][START_REF] Azoury | Peptides Derived From Insulin Granule Proteins are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice[END_REF] and are targeted by diabetogenic CD8+ T cells, which induce disease when transferred into NOD/scid mice [START_REF] Azoury | Peptides Derived From Insulin Granule Proteins are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice[END_REF]. This pathogenic role may rely on features that granule proteins share with (pro)insulin [START_REF] Carre | Making Insulin and Staying Out of Autoimmune Trouble: The Beta-Cell Conundrum[END_REF]. All these antigens are synthesized as pro-protein precursors, which are subsequently enzymatically cleaved, mostly by proconvertases, to generate their bio-active products. Hence, the impaired proinsulin processing by proconvertases described in beta cells from type 1 diabetic patients [START_REF] Carre | Making Insulin and Staying Out of Autoimmune Trouble: The Beta-Cell Conundrum[END_REF][START_REF] Sims | Elevations in the Fasting Serum Proinsulin-to-C-Peptide Ratio Precede the Onset of Type 1 Diabetes[END_REF] may extend to all these proteins, possibly diverting their degradation towards the MHC Class I presentation pathway. In addition, all of these proteins are released in the bloodstream with insulin exocytosis, both in intact form and as antigenic degradation products [START_REF] Wan | Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides[END_REF]. They can thus be taken up by extra-pancreatic antigen-presenting cells and prime T cells at distance upon uptake by extra-pancreatic antigen-presenting cells (1).

The MHC-peptide repertoire displayed by beta cells further includes neo-antigens, i.e. peptide sequences that are not templated in the genome and may therefore be regarded as non-self and trigger autoimmunity, since they have not participated in the physiological mechanisms of immune tolerance. These neo-antigens are produced through different mechanisms: alternative mRNA splicing [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF][START_REF] Azoury | Peptides Derived From Insulin Granule Proteins are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice[END_REF], out-of-frame protein translation due to defective ribosomal scanning under high insulin demand [START_REF] Kracht | Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes[END_REF], post-translational modifications [START_REF] Purcell | The Evolving Landscape of Autoantigen Discovery and Characterization in Type 1 Diabetes[END_REF] and transpeptidation [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF][START_REF] Delong | Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion[END_REF], i.e. the fusion of non-contiguous peptide fragments from the same protein or from two distinct partners.

Other amplification mechanisms may be related to beta-cell sensitivity to stress-induced senescence. A recent study [START_REF] Thompson | Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes[END_REF] documented the accumulation of senescent beta cells in NOD mice during a critical early time window that marks the transition from peri-insulitis to destructive insulitis. Although these senescent beta cells increase the expression of pro-survival factors such as Bcl2, they also have a key role in amplifying the autoimmune response because they produce a specific secretome (the so called SASPsenescence-associated secretory phenotype) enriched in factors (e.g. IGFBP3, serpin E1) that propagate this senescent phenotype to neighboring beta cells; and in cytokines/chemokines (e.g. IL-6, CXCL10), that as noted above, attract and activate immune cells in the pancreas. Senescence markers (CDKN1A, serpin E1, IL-6) were also observed in the pancreas of type 1 diabetic patients [START_REF] Thompson | Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes[END_REF]. Importantly, when senescence was inhibited by an anti-Bcl2 small molecule that drives apoptosis specifically in these cells, diabetes was prevented, thus documenting the pathogenic role of the senescent phenotype. This observation identifies a novel therapeutic target involving a rather counter-intuitive strategy of inducing apoptosis of selected beta-cell subsets. This also suggests that the physiologic process of clearing senescent beta cells, or hypersecreting mutants (adenomas) [START_REF] Kohanim | Endocrine Autoimmune Disease as a Fragility of Immune Surveillance against Hypersecreting Mutants[END_REF], is misdirected in autoimmunity. It will be important to clarify whether the relationship between beta-cell dysfunction, senescence and apoptosis is sequential, mutually exclusive and/or naturally reversible.

Besides inciting autoimmunity via all these immunogenic signals, beta cells may further amplify these mechanisms by ceasing to secrete protective anti-apoptotic factors. A notable example is growth/differentiation factor (GDF)15, a transforming growth factor-β superfamily member that was found downregulated in beta cells from type 1 diabetic individuals and upon in-vitro exposure to IL-1β and IFN-γ [START_REF] Nakayasu | Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention[END_REF]. Although its mechanisms of action remain unclear, GDF15 protects NOD mice from diabetes [START_REF] Nakayasu | Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention[END_REF], presumably through anti-inflammatory effects, and exerts additional effects in the hindbrain (reducing food intake and promoting weight loss) and in peripheral tissues (hepatocytes, myocytes and adipocytes), by increasing insulin sensitivity.

3) Self-finalized suicide vs protection

Further compensatory mechanisms mounted by beta cells may be self-detrimental ("suicidal") in the long term, acting through intrinsic mechanisms that do not require the participation of other cells (Fig. 2). Some cytokines (e.g. IL-1β, IL-6) that are secreted by beta cells themselves ( 7) can be proapoptotic in an autocrine/paracrine loop. Their secretion may be particularly important during the extended period of timeas long as 5 yearswhen insulin secretion may be impaired but clinically silent. The ability to compensate for metabolic demands becomes critical during this time for betacell survival. As metabolic demands increase, reactive oxygen species are needed for glucose-induced insulin secretion (51) and mass expansion [START_REF] Alfar | Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity[END_REF]. However, the low expression of antioxidant enzymes such as catalase and superoxide dismutase exposes beta cells to cell-intrinsic oxidative damage, particularly in the ER [START_REF] Tersey | Islet beta-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model[END_REF]. Even beta-cell identity may be affected by this oxidative stress, since exposure to hydrogen peroxide can induce loss of nuclear localization of MafA, a transcription factor associated with mature beta cells that may enhance the beta-cell antioxidant defenses, protecting them against deterioration during hyperglycemia (54-56). Increased metabolic demands also upregulate the UPR, which is constitutively active in beta cells due to their basal ER stress from high protein (mostly insulin) biosynthesis rates. This increased UPR can drive apoptosis when reaching a critical threshold (1). Indeed, UPR upregulation was observed in beta cells from two type 1 diabetes mouse models and from patients [START_REF] Engin | Restoration of the unfolded protein response in pancreatic beta cells protects mice against type 1 diabetes[END_REF], as identified by the expression of activating transcription factor-6 (ATF6) and spliced X-box binding protein-1 (sXBP1). The deleterious effect of what is initially a compensatory response is suggested by the observation that surviving beta cells display lower ATF6 and sXBP1 expression, and that relieving UPR with tauroursodeoxycholic acid (TUDCA) protects mice from diabetes [START_REF] Engin | Restoration of the unfolded protein response in pancreatic beta cells protects mice against type 1 diabetes[END_REF].

We recently showed that Tet2 is increased in beta cells from NOD mice and in human insulitis [START_REF] Rui | Tet2 Controls the Responses of beta cells to Inflammation in Autoimmune Diabetes[END_REF]. Its deleterious beta-cell-intrinsic outcomes were documented in several ways. In beta cells that survive autoimmune attack, Tet2 expression was reduced. Despite unchanged insulitis and Tcell diabetogenicity, Tet2 -/-NOD mice were protected from diabetes, even after grafting Tet2 +/+ bone marrow or transferring diabetogenic splenocytes. Tet2 affects the epigenetic control of signaling molecules such as STAT1, NFB and IRF2. While its loss was associated with CXCL10 downregulation by beta cells, there was also reduced expression of the CXCR3 homing receptor on beta-cell-reactive T cells in pancreatic lymph nodes.

Protective mechanisms are also mounted by beta cells (Fig. 2). During progression of autoimmunity in NOD mice, we described degranulation, loss of Ins1/Ins2 gene expression and acquisition of a more undifferentiated phenotype, with expression of other islet hormones (e.g. Gcg, Sst) and stem cell markers (Sox9, L-Myc, Oct-4) in residual beta cells [START_REF] Rui | Beta Cells that Resist Immunological Attack Develop during Progression of Autoimmune Diabetes in NOD Mice[END_REF]. In addition to insulin, there was reduced expression of other diabetes autoantigens (GAD, ZnT8, IA-2). In line with these results, Damond et al. compared beta cells from new-onset and long-standing type 1 diabetic patients to non-diabetic individuals by image cytometry pseudo-timing using nPOD specimens [START_REF] Damond | A Map of Human Type 1 Diabetes Progression by Imaging Mass Cytometry[END_REF]. Beta-cell destruction was preceded by downregulation of identity markers (INS, PIN, IAPP, PTPRN) with (pseudo)time, without changes in beta-cell transcription factors PDX1 and NKX6-1. Loss of lineage-specific markers may thus be an early event, before further disease progression driven by beta-cell death. In our study [START_REF] Rui | Beta Cells that Resist Immunological Attack Develop during Progression of Autoimmune Diabetes in NOD Mice[END_REF], dedifferentiated beta cells also expressed immunoregulatory ligands (PD-L1, Qa-2) and resisted immune killing by islet infiltrates or cytokines, unlike their mature counterparts. PD-L1 is induced on human and murine beta cells by IFN- and IFN-α [START_REF] Osum | Interferon-gamma drives programmed death-ligand 1 expression on islet beta cells to limit T cell function during autoimmune diabetes[END_REF]. Beta cells also secrete anti-microbial peptides (e.g., CRAMP), a process triggered by in-vitro exposure to IL-1β or LPS [START_REF] Sun | Cathelicidins positively regulate pancreatic β-cell functions[END_REF] that is impaired in NOD mice [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF]. Besides inducing tolerogenic antigen-presenting cells and Tregs [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF], these peptides reduce beta-cell apoptosis and production of inflammatory prostaglandin E2 [START_REF] Sun | Cathelicidins positively regulate pancreatic β-cell functions[END_REF]. These mechanisms, while protective against killing in specific experimental settings, clearly fail to arrest the autoimmune response in most clinical settings.

Altogether, the available literature suggests that, following autoimmune insult, beta cells may also contribute in a direct way to their own killing. This concept of self-detrimental vs protective beta-cell responses has clinical relevance. Successful immunotherapies (e.g. teplizumab, antithymocyte globulin, alefacept, rituximab) share a mechanism of modulation/deletion of inflammatory cells. While treatment is often followed by C-peptide stabilization, a decline subsequently occurs in many patients [START_REF] Greenbaum | Through the fog: recent clinical trials to preserve beta-cell function in type 1 diabetes[END_REF], suggesting either that the conditions that led to clinical onset have recurred or that beta-cell-intrinsic mechanisms may drive this decline. As an example, even continuous administration of CTLA-4-Ig at type 1 diabetes onset did not avoid a decline in beta-cell function after an initial improvement [START_REF] Orban | Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial[END_REF]. This suggests that maintaining beta-cellintrinsic protective mechanisms together with immunomodulation may limit this later decline.

Therapeutic strategies under development to boost beta-cell protective mechanisms

The concepts of beta-cell responsiveness to autoimmune attack also suggest ways in which autoimmunity might be arrested with a combination of agents that target immune and beta cells.

Evidence [START_REF] Gearty | An autoimmune stem-like CD8 T cell population drives type 1 diabetes[END_REF][START_REF] Abdelsamed | Beta cell-specific CD8(+) T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes[END_REF] for stem-like features of islet-reactive CD8 + T cells that can proliferate and are the source of short-lived effector cells that migrate to the pancreas highlights the challenge in purging the repertoire of effector T cells sufficiently to prevent disease [START_REF] Gearty | An autoimmune stem-like CD8 T cell population drives type 1 diabetes[END_REF]. Intriguingly, results from teplizumab trials [START_REF] Herold | Type 1 Diabetes TrialNet Study G: An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes[END_REF] suggest that immunotherapies may be most effective when beta-cell stress is occurring, i.e. at the stage 2 of rapid C-peptide decline, consistent with preclinical data in NOD mice. Although the beta-cell changes occurring during this period of rapid progression may expose new and more antigen targets, the T cells that recognize them may represent the more differentiated ones that are amenable to treatment.

Modulating the beta-cell and antigen targets of autoimmune T cells may thus be needed in addition to immunotherapies. Clinical studies are ongoing with agents that are postulated to limit beta-cell stress responses, such as verapamil (NCT04545151), a calcium channel blocker that inhibits TXNIP, a mediator of oxidative stress [START_REF] Ovalle | Verapamil and beta cell function in adults with recent-onset type 1 diabetes[END_REF], and TUDCA (NCT02218619), a chemical chaperone that mitigates ER stress [START_REF] Engin | Restoration of the unfolded protein response in pancreatic beta cells protects mice against type 1 diabetes[END_REF]. In a previous study of the tyrosine kinase inhibitor imatinib as a single agent, a significant effect on C-peptide responses was not sustained [START_REF] Gitelman | Imatinib therapy for patients with recent-onset type 1 diabetes: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial[END_REF], but ongoing studies are evaluating effects on markers of beta-cell stress. Other studies with agents such as JAK1/2 inhibitors, that can block critical signaling pathways in beta cells as well as blunting Tcell activation, are under development. Finally, in a randomized 4-arm placebo-controlled trial, anti-IL-21 antibodies and the GLP-1 receptor agonist liraglutide significantly reduced C-peptide decline at 54 weeks in patients with recently diagnosed type 1 diabetes compared to placebo [START_REF] Von Herrath | contributors: Anti-interleukin-21 antibody and liraglutide for the preservation of beta-cell function in adults with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled, phase 2 trial[END_REF].

The finding that this decline was lower than with either agent alone suggests that immune and beta-cell combination therapies may be synergistic in achieving clinical outcomes.

Conclusions and perspectives

These studies highlight the dynamic relationships between immune effector cells and their betacell targets. The findings have shown how both parties contribute to killing, but also suggest that therapies that are directed only at one arm may be insufficient to completely arrest the ongoing process. Much work has been done to characterize the autoimmune repertoire, including the targets and features of T and B cells that can mediate disease. Attention to the attributes of beta cells that can instill protection from or participation in autoimmunity are now needed.

Figure 1 .

 1 Figure 1. Pathogenic role of beta cells in type 1 diabetes. The three possible stages of initiation, amplification and finalization of the pathogenic cascade where beta cells can come into play are depicted.

Figure 2 . 2 ) 3 )

 223 Figure 2. Beta-cell mechanisms of self-amplified homicide/suicide (red) vs. protection (blue). See text for details.

Acknowledgements.

We apologize with colleagues whose publications could not be referenced due to space constraints. Figures 1 and2 were created with elements from Servier Medical Art (smart.servier.com).

Funding. Work performed in the Laboratory of R.M. is supported by The Leona M. and Harry B. Helmsley Charitable Trust (Helmsley #1901-03689), the Agence Nationale de la Recherche (ANR-19-CE15-0014-01) and the Fondation pour la Recherche Medicale (EQU20193007831); and by the Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 115797 and 945268 (INNODIA and INNODIA HARVEST), which receive support from the EU Horizon 2020 program, the European Federation of Pharmaceutical Industries and Associations, JDRF, and The Leona M. & Harry B. Helmsley Charitable Trust. C.H. was funded by an Année Recherche fellowship of the Paris Saclay University. Work performed in the Laboratory of K.C.H. is supported by NIH grant DK057846, DK101122, DK104205, DK045735, JDRF grant SRA2014-142-S-B, a Pilot Award from the Yale Diabetes Research Center P30DK116577 and a gift from the Howalt family. Duality of Interest. K.C.H. is co-inventor on a patent application for use of teplizumab. No other potential conflicts of interest relevant to this article were reported.

.


Fatoumata Samassa

MD Roberto Mallone
email: roberto.mallone@inserm.fr.

Self-antigens, benign autoimmunity and type 1 diabetes: a beta-cell and T-cell perspective

Keywords: epitope, exhaustion, immune synapse, MHC, stem-like T cells

Purpose of review: recent work using immunopeptidomics and deconvolution of the antigenic reactivity of islet-infiltrating CD8+ T cells has expanded our knowledge about the autoimmune target epitopes of type 1 diabetes. The stem-like properties of autoimmune CD8+ T cells have also been described. We here propose a possible link between these findings.

Recent findings: weak major histocompatibility complex (MHC)-binding epitopes list among the major targets of human islet-infiltrating CD8+ T cells, likely resulting in low peptide-MHC presentation that delivers weak T-cell receptor (TCR) signals, especially in the face of low-affinity autoimmune TCRs. These weak TCR signals may favor the maintenance of the partially differentiated stem-like phenotype recently described for islet-reactive CD8+ T cells in the blood and pancreatic lymph nodes. These weak TCR signals may also be physiological, reflecting the need for self-peptide-MHC contacts to maintain homeostatic T-cell survival and proliferation. These features may underlie the universal state of benign autoimmunity that we recently described, which is characterized by islet-reactive, naïve-like CD8+ T cells circulating in all individuals.

Summary: these observations provide novel challenges and opportunities to develop circulating T-cell biomarkers for autoimmune staging. Therapeutic halting of islet autoimmunity may require targeting of stem-like T cells to blunt their self-regeneration.

Introduction

Increasing efforts are being invested to provide a comprehensive catalog of the self-antigens exposed by pancreatic beta cells that become autoimmune targets in type 1 diabetes (T1D). This catalog would provide critical information for detecting the corresponding T cells and autoantibodies to improve disease staging, for developing antigen-specific immunotherapies, and for understanding T1D pathogenesis. Major gaps in knowledge exist, as there have been few systematic discovery studies to map this antigenic landscape. This is particularly true for T cells, which are the key actors in T1D pathogenesis and recognize peptides bound to and presented by HLA molecules (Class I for CD8+ T cells, Class II for CD4+ T cells). Typically, T-cell antigen discovery has been inherently biased by its reliance on prior knowledge of the same antigens being targeted by auto-antibodies, or by T cells found in islet infiltrates and pancreatic lymph nodes (PLNs) in the non-obese diabetic (NOD) mouse. Other efforts have been eminently hypothesisdriven, e.g. by looking at particular post-translational modifications (e.g. transglutamination, citrullination) or at processes such as defective ribosomal scanning that may generate neo-antigens.

A unifying picture is missing. We will here discuss recent advances and some common emerging themes, and highlight controversial or understudied aspects that deserve further scrutiny.

What makes an antigenic epitope a solid one?

A recent meta-analysis [*1] of the T-cell epitopes identified to date has proposed a ranking based on the degree of available evidence. This classification considers 3 key features (Fig. 1):

1) The source of the T cells found to recognize a given epitope: humanized mice, e.g. transgenically expressing human HLA molecules, have been extensively used [START_REF] Blancou | Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients[END_REF][START_REF] Jarchum | In vivo cytotoxicity of insulin-specific CD8+ T-cells in HLA-A*0201 transgenic NOD mice[END_REF][START_REF] Scotto | Zinc transporter (ZnT)8(186-194) is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients[END_REF][START_REF] Scotto | HLA-B7-restricted islet epitopes are differentially recognized in type 1 diabetic children and adults and form weak peptide-HLA complexes[END_REF]. Most work has however focused on human tissues, with two different levels of evidence depending on whether the studied T-cell source is peripheral (i.e. blood, spleen) or in the target organ (i.e. PLNs, pancreas). Arguably, the finding of T cells at the site of pathology adds an important argument for the relevance of the antigens identified.

2) The demonstration that the epitope is naturally processed and presented by target beta cells and/or antigen-presenting cells. This is important to avoid the pitfall of identifying antigens that are predicted in silico but may not exist in the real world, simply because they cannot be produced and exposed on the cell surface. Without this evidence, the odds of finding serendipitous, irrelevant T-cell reactivities are high, which reflect the broad cross-reactivity of T cells [START_REF] Sewell | Why must T cells be cross-reactive?[END_REF][START_REF] Quiniou | Human thymopoiesis selects unconventional CD8+ α/β T cells that respond to multiple viruses[END_REF]. A teaching example of this cross-reactivity is provided by our recent work analyzing the recognition of HLA-A2-restricted epitopes in donors that express or not the HLA-A2 restriction element [START_REF] Culina | Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors[END_REF]. When looking at the recognition of self or viral epitopes recognized by naïve CD8+ T cells (reflecting lack of autoimmune disease or lack of exposure to the source viruses), the frequency of these T cells was similar in HLA-A2+ and negative donors. This exemplifies the permissiveness of the natural T-cell repertoire for recognition of disparate peptide-HLA (pHLA) complexes, even those that HLA-A2-negative donors will never encounter.

3) The isolation of T cells recognizing the epitope provides another strong evidence, as it allows more in-depth investigations to confirm antigen specificity.

Our approach to antigen discovery: strengths and limitations

We recently launched a systematic epitope discovery effort by focusing on the antigenic peptides that are naturally processed and presented by target beta cells [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF]. With the exception of a similar study performed on murine beta-cell lines and primary islets from NOD mice [START_REF] Dudek | Constitutive and inflammatory immunopeptidome of pancreatic beta-cells[END_REF], this information was not available from previous literature. This strategy is based on immunopeptidomics techniques, which require the isolation of pHLA complexes from cell lysates by immunoaffinity purification, followed by peptide elution and identification by mass spectrometry. This work provided a first cartography of the peptides naturally processed and presented by beta cells, with some important messages. First, several known antigenic proteins and peptides were represented, particularly those derived from preproinsulin that have been more extensively studied. Second, some known antigens were marginally represented, with very few or no peptides identified, e.g. 1/each for GAD65 and ZnT8 and none for IGRP and IAPP. Third, secretory granules contributed one third of the total number of source proteins, in line with the functional specialization of beta cells to secrete insulin. Fourth, some novel granule antigens were identified, such as secretogranin-5, urocortin-3 and proconvertase-2. Fifth, some neo-antigens (Fig. 2A) derived from protein or mRNA splicing were found.

The exposure of these HLA-bound peptides by beta cells is a prerequisite, but does not warrant autoimmune recognition if the T-cell repertoire is not poised to do so. The second step of this work was therefore to test which of these peptides were recognized by circulating CD8+ T cells in T1D patients and age/sex-matched healthy controls, using peptide-loaded HLA Class I (HLA-A2 and -A3) multimers [9,*11]. Results were surprising: the frequency of these CD8+ T cells recognizing the peptides identified was similar irrespective of disease statusan observation that we previously reported for known islet antigens [START_REF] Culina | Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors[END_REF]. Moreover, a large fraction of these self-reactive CD8+ T cells displayed a naïve-like (CD45RA+CCR7+) phenotype. The overall frequency of these T cells was in line with this naïve status, as it fell in a predictable range (1-50/10 6 CD8+ T cells, i.e. 0.0001-0.005%) previously reported for viral-reactive naïve CD8+ T cells [12]. CD8+ T cells recognizing these same peptides were instead enriched in the pancreas of T1D vs. healthy donors and displayed CD45RO expression compatible with an effector/memory phenotype, suggesting their pathogenic relevance. Such relevance was directly demonstrated in the NOD mouse, where CD8+ T cells recognizing the murine orthologs of the novel granule antigens identified were found to infiltrate the islets of prediabetic animals, and to transfer disease in NOD/scid mice [*11].

Based on these results, we proposed the existence of a universal state of "benign" islet autoimmunity hardwired in all individuals [13,*14], which we and others traced back to a marginal deletion of autoreactive T cells in the thymus [START_REF] Culina | Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors[END_REF]15]. This benign autoimmune state is characterized by the circulation of naïve-like autoreactive CD8+ T cells, which become activated and sequestered in the pancreas only in T1D patients. The progression of this benign state to T1D may rely on two non-mutually exclusive mechanisms: related to beta cells, which become accessible, visible and vulnerable to CD8+ T cells under inflammatory conditions (i.e. loss of immune ignorance); and related to T cells, which escape from the control of immune regulatory mechanisms, both intrinsic (e.g. anergy, exhaustion) and extrinsic (e.g. Treg suppression). This approach to antigen discovery carries strengths and limitations. Strengths include robust evidence of natural processing and presentation by beta cells, with enhanced antigen exposure under inflammatory conditions [START_REF] Gonzalez-Duque | Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors[END_REF]; evidence of T-cell recognition in both blood and pancreas; and demonstration of diabetogenic properties in NOD mice for some of the novel antigens identified [ *11]. A first limitation relates to the lack of exploration of the natural processing and presentation by antigen-presenting cells, which are the major source of T-cell priming in PLNs. This may explain the absent of marginal detection of some known antigens. A second limitation is the difficulty in providing stringent evidence for neo-epitopes derived from post-translational modifications (PTMs), which requires to differentiate biological PTMs from artifactual ones. Indeed, peptides synthesized in their native form are often found to carry these same PTMs, suggesting that they may arise spontaneously (e.g. by oxidation) or may be introduced during sample preparation and mass spectrometry acquisition. Although this does not negate the existence of biological PTMs, it masks their actual prevalence. Other PTMs that may be particularly relevant in T1D (e.g. glycosylation/glycation) are challenging to study, still others have been elusive

altogether. Citrullination provides a revealing example: despite its detection in inflamed cartilages and the diagnostic importance of anti-citrullinated protein auto-antibodies in rheumatoid arthritis, direct evidence of HLA-eluted citrullinated peptides is missing. Moreover, our recent study on citrullinated GRP78 peptides [START_REF] Azoury | CD8+ T cells variably recognize native versus citrullinated GRP78 epitopes in type 1 diabetes[END_REF] documented that preferential T-cell recognition of posttranslationally modified vs. native epitopes is not always the rule (Fig. 2B). First, this rule has been proposed based on the assumption that citrullinated peptides are not presented in the thymus, thus leading to the escape of their cognate T cells. Contrary to this assumption, citrullinated proteins were detected in the thymus and medullary epithelial cells, as was the case for the peptidyl-arginine deiminases catalyzing this PTM. Second, T-cell preference for some native rather than citrullinated GRP78 epitopes was driven by cross-recognition of bacterial mimotopes/homotopes.

Other limitations have been complemented by the work of other investigators and are discussed in the next section.

Lessons from human CD8+ T cells isolated from islet infiltrates: possible over-reliance on HLA binding affinity and functional implications.

Another limitation of our work is that we did not isolate T cells recognizing our antigens from the pancreas. M. Nakayama developed an elegant approach overcoming this limitation, based on the isolation of islet-infiltrating T cells and single-cell sequencing of their T-cell receptors (TCRs).

Their antigen specificity is subsequently reconstructed by re-expressing these TCRs in a 5KC fluorescent reporter cell system, in which a ZsGreen protein is expressed under the control of an NFAT-driven promoter activated upon TCR triggering [START_REF] Mann | Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System[END_REF]. Using this system, several TCRs were assigned to preproinsulin-derived epitopes [**18], including an epitope derived from a defective ribosomal product (DRiP) generated by mis-initiated translation using an alternative open-reading frame [START_REF] Kracht | Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes[END_REF]. The large fraction of "orphan" TCRs with unassigned antigen specificities is a residual challenge that may yield additional information in the near future. This seminal work [**18] provided two additional key messages. First, it was noted that several preproinsulin-reactive TCRs recognize their cognate antigens with low affinity, with EC50 mostly in the 0.1-100 µM range. This is line with the lower affinity of self-reactive TCRs, and may reflect the outcome of thymic negative selection, which prunes high-affinity TCRs from the self-reactive T-cell repertoire [START_REF] Yu | Clonal deletion prunes but does not eliminate self-specific alphabeta CD8(+) T lymphocytes[END_REF].

A closer look reveals another striking feature (Fig. 3A): the predicted binding affinity for the identified HLA Class I restriction elements is extremely low for several of these peptides, with a percent rank in the NetMHCPan 4.1 algorithm >2%, which is classically regarded as evidence for lack of binding altogether. The implications of these observations are two-fold. First, these peptides could be missed by immunopeptidomics strategies relying on pHLA purification. Second, this means that the TCR signal that these autoimmune T cells receive upon antigen encounter is extremely weak, as it results from the interaction between pHLA complexes that are poorly presented on the surface of beta cells, often in the face of a low-affinity TCR. An increasing number of reports documented the existence of epitopes that are highly immunogenic despite poor or undetectable HLA binding [START_REF] Brennick | Prediction of cancer neoepitopes needs new rules[END_REF]. A recent elegant work [**22] described a mutated tumor neoantigen that mediated tumor rejection by CD8+ T cells in a mouse model, despite undetectable MHC Class I binding. While a strong HLA binding is a good predictor of immunogenicity for viral peptides, this seems less the case for tumor and self-epitopes [START_REF] Brennick | Prediction of cancer neoepitopes needs new rules[END_REF].

How can this poor MHC presentation translate into T-cell recognition, even more so if low-affinity

TCRs are involved? For CD4+ T cells, another elegant study demonstrated that both high-and low-affinity TCRs reactive to Ins B9-23 are diabetogenic [START_REF] Bettini | TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential[END_REF]. We can speculate that the immune synapse (IS) formed by the interaction of TCRs (often of low affinity) and weak pHLA complexes may harbor some specific features (Fig. 3B). First, the local peptide concentration achieved in the constrained IS space may be sufficiently high to favor binding to HLA. Although unstable, this binding may result in intermittent, repeated "on-off" TCR signals. Conversely, it has been shown that a single pHLA complex can serially engage up to ~200 TCRs [START_REF] Valitutti | Serial triggering of many T-cell receptors by a few peptide-MHC complexes[END_REF]. Second, the signal generated might be sufficient to trigger a transcriptional program that differs from the one triggered by high-affinity TCR/strong peptide-HLA complexes, but still lead to T-cell activation. In line with this, a self-reactive, low-affinity human CD4+ T-cell clone isolated from a T1D individual formed an IS where the usual central TCR cluster was replaced by micro-clusters, still able to trigger signaling, cytokine production and proliferation [START_REF] Schubert | Self-reactive human CD4 T cell clones form unusual immunological synapses[END_REF]. Third, compensatory interactions mediated by the CD4 and CD8 co-receptors, adhesion molecules and/or actin might increase the stability of the IS and subsequent strength of TCR signaling [START_REF] Laugel | Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties[END_REF].

What could be the functional consequences of this weak TCR signals on the biology of autoimmune CD8+ T cells? The stem-like properties of circulating [**27] and/or PLN-residing [**28] autoimmune CD8+ T cells are increasingly recognized. An enrichment in the stem-cell memory fraction of circulating autoimmune CD8+ T cells from T1D patients compared to healthy donors was originally reported by the group of P. Monti [START_REF] Vignali | Detection and Characterization of CD8(+) Autoreactive Memory Stem T Cells in Patients With Type 1 Diabetes[END_REF]. This stem-like phenotype (mainly driven by the transcription factor TCF1) [START_REF] Gonzalez | Schrodinger's T Cells: Molecular Insights Into Stemness and Exhaustion[END_REF] is strikingly different from that of CD8+ T cells recognizing viral epitopes in the context of chronic infection (Fig. 2C), which rather display an exhausted phenotype (mainly driven by the transcription factor TOX) [START_REF] Gonzalez | Schrodinger's T Cells: Molecular Insights Into Stemness and Exhaustion[END_REF], characterized by reduced effector functions due to prolonged antigen exposure [** [START_REF] Abdelsamed | Beta cell-specific CD8(+) T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes[END_REF][START_REF] Zajac | Viral immune evasion due to persistence of activated T cells without effector function[END_REF][START_REF] Gallimore | Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes[END_REF]. In contrast, the stemlike properties of CD8+ T cells in T1D may be driven by these weak TCR signals that maintain T cells alive without differentiating them into a full effector/memory phenotype. This is in line with the notion that immunization with subdominant or low-affinity CD8+ T-cell epitopes can achieve superior viral and tumor clearance by limiting exhaustion and sparing T-cell stemness [START_REF] Holst | Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection[END_REF][START_REF] Gross | High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy[END_REF]. We can speculate that these stem-like CD8+ T cells may transiently recirculate in the bloodstream [**27] prior to reaching the pancreas, where the re-encounter with their cognate antigen (possibly at higher density than in PLNs) drives their full differentiation into effector/memory cells with cytotoxic activity against beta cells. These so-called "autoimmune mediators" [**28] display higher levels of apoptosis markers, suggesting that the antigen re-encounter in the pancreas may drive activation-induced cell death rather than exhaustion. The continual replenishing of the shortlived autoimmune mediator pool by stem-like CD8+ T cells from PLNs may prevent the exhaustion seen at the high antigen loads of chronic viral infection. This may explain the challenge of durably blunting islet autoimmunity, which, without proper targeting of stem-like T cells, may self-regenerate and mediate autoimmune progression or relapse. Conversely, it may also explain the autoimmune manifestations induced by therapeutic PD-1 blockade in cancer patients, which has been shown to target PD-1-expressing stem-like CD8+ T cells rather than their terminally differentiated, exhausted counterparts, which are also PD-1+ [START_REF] Im | Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy[END_REF][START_REF] Miller | Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade[END_REF].

Benign islet autoimmunity: challenges and opportunities for translation into circulating Tcell biomarkers

The dynamics that may favor the maintenance of autoimmune T cells with stem-like properties may be physiological to a large extent. Prior to encounter with their cognate antigens, which is the most common situation outside some restricted time windows (e.g. outside viral infection episodes), tonic, low-grade TCR signals triggered by self-p/MHC complexes ensure homeostatic T-cell survival and proliferation [START_REF] Takeda | MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span[END_REF][START_REF] Tanchot | Differential requirements for survival and proliferation of CD8 naive or memory T cells[END_REF][START_REF] Dorfman | CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling[END_REF]. In the absence of MHC contact, the half-life of peripheral CD8+ and CD4+ T cells is reduced, even more so for CD8+ T cells [START_REF] Polic | How alpha beta T cells deal with induced TCR alpha ablation[END_REF][START_REF] Labrecque | How much TCR does a T cell need?[END_REF][START_REF] Seddon | TCR and IL-7 receptor signals can operate independently or synergize to promote lymphopenia-induced expansion of naive T cells[END_REF].

Moreover, most (~90%) of the energy required for pMHC/TCR interaction is peptide-independent and is given solely by the MHC molecule [START_REF] Wu | Two-step binding mechanism for T-cell receptor recognition of peptide MHC[END_REF]. The cognate peptide turns this initial association into stable binding, thus imparting specificity and eliciting T-cell activation by modulating the duration of the contact. These features may underlie the benign autoimmunity phenomenon that we recently described. It is also possible that the naïve-like status described in our work may overlap, to a certain extent, with the epigenetically imprinted stem-like phenotype, intermediate between naïve and effector/memory, described by B. Youngblood and co-workers [**27].

How could the benign CD8+ T-cell autoimmunity be missed by previous investigations? A first

key problem relates to the sampling size employed by several studies, in terms of number of peripheral blood mononuclear cells (PBMCs) processed. The frequency of these self-peptidereactive CD8+ T cells falls in a relatively narrow range, between 1 and 50/10 6 total CD8+ T cells [8,9,*11]. As CD8+ T cells account for ~20% of PBMCs, this means that at least 20x10 6 PBMCs (~20 ml of blood) are needed for appropriate sampling, i.e. to be able to count and phenotype 4-200 HLA multimer+ CD8+ T cells recognizing a given epitope. Several previous studies have analyzed a much more limited number of cells. A second problem may be a "streetlight effect" [START_REF] Battaglia | The streetlight effect in type 1 diabetes[END_REF], i.e. our bias of over-emphasizing experimental results in line with prior assumptions and deemphasizing discordant ones. Indeed, a closer scrutiny of previous works reveals that the frequency differences in circulating islet-reactive CD8+ T cells (detected by multimer staining) between T1D and healthy donors have been often marginal, and that major naïve fractions within this autoimmune repertoire have been neglected altogether [START_REF] Kracht | Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes[END_REF][START_REF] Luce | Single insulin-specific CD8+ T cells show characteristic gene expression profiles in human type 1 diabetes[END_REF][START_REF] Skowera | Beta-cell-specific CD8 T cell phenotype in type 1 diabetes reflects chronic autoantigen exposure[END_REF]. Does the universal presence of circulating islet-reactive CD8+ T cells put a gravestone on our ambition to develop circulating T-cell biomarkers for autoimmune staging? These recent observations pose additional challenges, but also offer unprecedented opportunities to better understand autoimmune pathology and ultimately improve our disease staging capabilities. Indeed, previous T-cell assays using functional readouts, e.g. interferon-γ ELISpot, have repeatedly documented the possibility to discriminate between T1D and healthy donors [START_REF] Blancou | Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients[END_REF][START_REF] Scotto | Zinc transporter (ZnT)8(186-194) is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients[END_REF][START_REF] Mallone | CD8+ T-cell responses identify beta-cell autoimmunity in human type 1 diabetes[END_REF][START_REF] Skowera | CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope[END_REF]. This may reflect the fact that these assays are performed using unfractionated PBMCs, which may maintain the immune regulatory networks at play under healthy conditions. On one hand, it will be important to broaden our knowledge of target islet antigens, to be able to measure the overall autoimmune T-cell burden of a given individual as comprehensively as possible. This is similar to staging strategies using islet auto-antibodies, where the presence of multiple autoantibodies rather than a single one marks a higher risk of disease progression [START_REF] Insel | Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the Endocrine Society, and the American Diabetes Association[END_REF]. Similarly, we may expect that analyzing autoreactive T cells recognizing more antigens rather than just a few may provide better prognostic stratification. On the other, it will be critically to "grade" the pathogenic potential of these autoreactive T cells by identifying their surface or epigenetic markers associated with disease progression.

Conclusion

Besides providing a more comprehensive description of the autoimmune T-cell repertoire, islet antigen discovery is advancing our understanding of T1D pathophysiology. The distinctive features of the self-peptides presented by HLA molecules and of the TCRs that recognize them may help explaining the peculiar stem-like properties of their cognate autoreactive T cells and their benign vs. pathogenic profile. The complementary perspectives of beta cells (HLA-driven) and T cells (TCR-driven) on the antigens that they eventually select for presentation and recognition (Fig. 4) trace the path to pursue these integrated investigations. Such investigations are embedded into a more holistic view of islet autoimmunity as a disease of T cells, beta cells and beyond [13,*14].

Key points

 A combined beta-cell and T-cell perspective is needed to maximize our understanding of T1D pathogenesis through islet antigen discovery.

 Recent data suggest that weak MHC-binding epitopes list among the major targets of human islet infiltrating CD8+ T cells, likely resulting in low peptide-MHC presentation that delivers weak TCR signals, especially in the face of low-affinity autoimmune TCRs.

 These weak TCR signals may favor the maintenance of the partially differentiated stem-like phenotype recently described for islet-reactive CD8+ T cells in the blood and PLNs.

 These weak TCR signals may also be physiological, reflecting the need for self-peptide MHC contacts to maintain homeostatic T-cell survival and proliferation.

 These features may underlie the universal state of benign autoimmunity that we recently described. peptides [START_REF] Azoury | CD8+ T cells variably recognize native versus citrullinated GRP78 epitopes in type 1 diabetes[END_REF] demonstrates that preferential T-cell recognition of post-translationally modified vs. native epitopes is not always the rule. On one hand, the citrullinating peptidyl-arginine deiminase enzymes are expressed in the thymus and citrullinated proteins are detected, suggesting that posttranslationally modified peptides do not necessarily lead to thymic escape of their cognate T cells.

On the other, the cross-reactivity of native GRP78 epitopes with bacterial mimotopes/homotopes shapes the T-cell preference for some native epitope versions. high antigen availability is usually present in chronic viral infections. In the lymph node (LN), APCs interact with naive T cells, triggering a strong TCR signal that leads to full effector/memory differentiation. These T cells subsequently recirculate toward the infection site, where antigen 20 availability may be even higher and eventually trigger exhaustion. Bottom: antigen availability may be lower in T1D, where several weak HLA-binding peptides are presented. Together with the frequently low affinity of autoimmune TCRs, this may trigger a weak TCR signal that leads to partial effector/memory differentiation, thus maintaining T-cell stemness that continuously replenish fully differentiated, cytotoxic T cells in the pancreas. These stem-like T cells transit through the blood to recirculate toward the pancreas to reach their beta-cell targets, and may provide circulating biomarkers of pathogenic islet autoimmunity. In the pancreas, activationinduced apoptosis rather than exhaustion seems to be triggered, possibly favored by a higher antigen availability.

Figure 1 .

 1 Figure 1. Levels of evidence for islet-derived T-cell epitopes. The epitope scoring criteria

Figure 2 .

 2 Figure 2. Generation and recognition of neo-antigens in beta cells. A. Mechanisms of neo-

Figure 3 .

 3 Figure 3. Weak HLA-binding peptides: possible mechanisms of T-cell recognition and

Figure 4 .Figure 4

 44 Figure 4. The complementary beta-cell and T-cell perspective on HLA-driven antigen

Acknowledgements. Figures 1, 2 and 3 were created with elements from Servier Medical Art (smart.servier.com). Financial support and sponsorship. Work performed in the Laboratory of R.M. is supported by The Leona M. and Harry B. Helmsley Charitable Trust (Helmsley #1901-03689), the Agence Nationale de la Recherche (ANR-19-CE15-0014-01) and the Fondation pour la Recherche Medicale (EQU20193007831); and by the Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 115797 and 945268 (INNODIA and INNODIA HARVEST), which receive support from the EU Horizon 2020 program, the European Federation of Pharmaceutical Industries and Associations, JDRF, and The Leona M. & Harry B. Helmsley Charitable Trust.

Conflicts of interest.

None.


Understanding the communicative value of ultrasonic calls in male and female laboratory mice: implications for mouse models of neurodevelopmental disorders

Beside the number of USVs, parameters like the mean duration of the calls or the calling time are sensitive to sex and genotype differences. The qualitative analysis of the call types is useful to reveal subtle differences in communication phenotypes. The proportion of complex calls seems particularly sensitive to sex and genotype differences, suggesting their distinctive communicative potential. Female mice may be more interesting than males to investigate adult communicative patterns and their potential alterations in NDDs.

BACKGROUND

  Ultrasonic vocalizations (USVs) are a major tool for assessing social communication in laboratory mice during their entire lifespan.  At adulthood, male mice preferentially emit USVs towards a female conspecific, while females mostly produce ultrasonic calls when facing an adult intruder of the same sex. Recent studies have largely developed several sophisticated tools to analyze adult mouse USVs, although the focus has been largely on males. Little is still known about the communicative value of the different types of ultrasonic calls emitted by adult male and female mice, an issue that is of particular relevance for the behavioral phenotyping of mouse models of Neurodevelopmental disorders (NDDs). METHODS  Female and male subjects (B6 background) encountered a CD1 female during 3 min  Video files were scored with Observer XT (Noldus)  USVs were recorded by Avisoft microphone and analyzed by Sonotrack (Metris). RESULTS: STUDY 1 (WT MICE)  WT Females (B6 background) tended to emit more and longer USVs than males  WT females displayed more affiliative behaviors than males  WT females emitted less short, more down, step-double and complex calls than males  These sex differences were mostly detectable on all testing sessions  No effect of estrous cycle Premoli, Petroni, Bonini, Bulthuis and Pietropaolo, Front Behav Neurosci, In press Call types Call types RESULTS: STUDY 2 (CD MICE, MODEL OF WBS)  CD Females (B6 background) emit less and shorter USVs than WT  CD females emitted more 1 component calls and less complex (+3 components) calls than WT  No genotype effect in males  No effect of estrous cycle  No difference in other social behaviors


Valeria Petroni

Enejda Subashi

Marika Premoli

Markus Wöhr

Wim E Crusio

Valerie Lemaire

Susanna Pietropaolo

AUTISTIC-LIKE BEHAVIORAL EFFECTS OF PRENATAL STRESS IN THE FMR1-KO MOUSE MODEL OF FRAGILE X SYNDROME

Stress effects on FXS-phenotype are present only in the cognitive domain and in males. These effects are stable (adult and old mice) -Stress behavioral effects on locomotion, SI and USVS appear only as long-term effects  Gene-environment interactions play a major role in modulating the behavioral FXS-like phenotype of Fmr1-KO mice

BACKGROUND

Fragile X Syndrome (FXS) is the most common heritable form of mental retardation and the main monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 human gene consisting in more than 200 CGG repetitions leading to the absence of FMRP protein and is characterized by motor, cognitive and social alterations, mostly overlapping with autism spectrum disorder behavioral phenotypes.

FXS behavioral phenotype can be critically modulated by environmental factors e.g., stress exposure, both in terms of its severity and of the timing of appearance AIMS Study 1: Evaluate the short-term effects of the prenatal exposure to unpredictable chronic stress on the behavioral phenotype of juveniles of both sexes in the Fmr1 knock-out (KO) mouse model of FXS, in order to asses whether the prenatal stress could advance and/or exacerbate the behavioral phenotype of Fmr1-KO offspring.

Study 2: Evaluate the long-term effects of prenatal exposure to unpredictable chronic stress during the last prenatal week on the FXS-and ASD-like behavioral phenotype of the Fmr1-KO model to asses the stability of the stress effects in Fmr1 mutant mice, extending their behavioral characterization during aging.

STUDY 1: SHORT-TERM EFFECTS -JUVENILES METHODS:

• Unpredictable mild stress  restrain stress (3 sessions 30-min day 1) and overnight housing with wet bedding; sawdust and cage changes (3 sessions day 2), overnight housing with novel glass black beads. • Behavioral tests (7-8 weeks): Open Field, Y-Maze, Social Interaction, Ultrasonic vocalizations

• 45 males (n=9-15 for stress condition) and 48 females (n=12 for stress condition).

RESULTS:

WT

  • Mutants: Number and duration of USVs in HET females • Stress: No selective effect We observed: • Mutants: HET females affiliation behavior • Stress: 1. Cognitive deficit in Y maze only in males 2. Affiliation behavior in WT males and females 3. Locomotion in females (HET and WT) STUDY 2: LONG-TERM EFFECTS -ADULTS AND OLD METHODS: • 38 males (n=8-10 for stress condition) tested at 3 months (Adults). • 31 surviving animals: n=8 for each stress condition) tested again at 18 months (Old). • Same stress procedure as in study 1  Unpredictable mild stress • Behavioral tests: Elevated Plus Maze, Open Field, Y-Maze, Social Interaction, Ultrasonic vocalizations RESULTS

•

  . 2022, Scientific Reports. Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions We observed: • Mutants: No effects • Stress: 1. Duration of ultrasonic calls in old mice We observed: • Mutants: Qualitative alterations of ultrasonic calls in adults KO( complex calls) • Stress: 1. The percentage of flat calls in old mice (KO and WT) 2. The percentage of complex calls in adults and old mice (KO and WT) Petroni et al., Frontiers -Under review. Long-term behavioral effects of prenatal stress in the Fmr1-KO mouse model for Fragile X syndrome Stress induced the cognitive deficits only in Fmr1-KO males (deficit in Y maze at juvenile age) • In females stress attenuated the Fmr1-KO phenotype because of its effects on locomotion, SI and USVs in WT mice • Stress exacerbates the cognitive deficits of Fmr1-KO mice at both ages • Stress affected anxiety, locomotion, social interaction and communication in mice of both genotypes, especially at aging We observed: • Mutants: Qualitative alterations of USVs ( complex calls) in HET females • Stress: No selective effect We observed: • Mutants: 1. Locomotion at both ages 2. Affiliative behavior at both ages • Stress: 1. Anxiety and locomotion at adulthood 2. Hyperactivity (KO and WT) at aging 3. Cognitive deficit at both ages KO ( old) 4. Affiliative behavior at adulthood

31% 12% 17% 9% 9% 11% 2% 8% Adults Old WT no stress WT stress KO no stress KO stress WT no stress WT stress KO no stress KO stress

									2%
	2%						2%		10%	Short
		11%	2%		14%		11%	19%	21%	Flat
			21%			18%			10%	Up
	10%					11%			Down
			6%	11%		6%			8%	6%	Chevron
									15%	Step down
	14%									Step up
				12%		14%		19%	22%	Step double
			23%			25%			12%	Complex tot
		11%
					10%		13%	2%
	2%
				2%			3%
			2%						Short
	12% 2%	14%	27%	12%	10,5%	29%	2% 12% 12%		29%	Flat Up Down Chevron
										Step down
			11%					1%	Step up
	12%	7%			10%	10%		8%	Step double
				8%					Complex tot
			14%		17%
		11%	0,5%			8%	18%
							1%
	1%
	Adults
	Old
	No Stress
	Stress

*


Ounissi, M J Arslan

RESPONSIBLE ARTIFICIAL INTELLIGENCE: A REVIEW OF CURRENT TRENDS

INTRODUCTION SOME CONCERNS AROUND AI WHAT SHOULD RAI ENCOMPASS?

• With the advent and rise of artificial intelligence (AI), its social, legal, and ethical implications in society have come into question.

• Chiefly amongst these concerns are the safety of AI, the treatment and handling of big data to train AI, particularly when dealing with personal data and privacy, and the ethics of AI, with concern over AI-related bias and inequity.

• High-risk and high-stake areas, such as security, finance, healthcare, medicine, and the criminal justice system, are of particular concern, as all these areas impact on human lives, interests, and rights [START_REF] Islam | Towards quantification of explainability in explainable artificial intelligence methods[END_REF].

• Responsible AI (RAI) has emerged as a new discipline of the AI domain to ensure AI are developed to produce equitable outcomes.

• The objective of RAI is to ensure that AI applications are developed with good intentions, that any applications are safely integrated, pose no bias (e.g., the use of AI predictions in insurance which may render some individuals ineligible) and are designed to serve humanity [START_REF] Islam | Towards quantification of explainability in explainable artificial intelligence methods[END_REF].

• RAI is still a hot topic in the social sciences. However, RAI concepts have not been fully embedded in the development of AIbased products. Additionally, terminologies surrounding RAI are often confusing, thus delaying its complete integration in reallife applications.

OBJECTIVE:

To understand the current trends in opinions surrounding AI, propose RAI definitions with the hope they may become universal, and briefly summarize some attempts made in quantifying RAI.

• While there is no consensus regarding definitions surrounding RAI, Figure 4 offers an alternative perspective to what RAI should include:

• Accountability: Determining whether the responsibility lies with the AI, AI developer, or data providers in the event the AI does not behave in an expected way.

• Authority: Which outcome should have more authority -the one determined by a human or the one determined by the AI?

• Fairness & equity: Ensuring the AI functions in an equitable manner and does not bias any group of individuals.

• Data Privacy & Governance: Prevention of adversarial attacks on AI-based systems by using mathematical systems, such as differential privacy.

• Social & Environmental Impact: Ensuring the AI is designed with good intentions and improves our everyday lives for the better.

• Mechanism & Translations: This includes explainable AI (xAI), intelligible AI (intellAI), interpretable AI (interAI), and transparent AI (tAI) and directly deals with understanding, translating, and improving AI systems uisng qualitative and quantitative measures.

• Safety & Security: Ensure the AI poses no real danger or threat.

• Ethics: Ensuring AI developed are in line with not only legal processes but human values.

• One of the primary concerns is AI bias (Figure 1)

• Prominent case of AI bias -Amazon's Hiring Algorithm: A computer program designed to assist with hiring future Amazon staff revealed to have a bias towards female candidates in 2015.

• Another prominent case of AI bias: the Correctional Offender Management Profiling for Alternative Sanctions (COMPAS). A tool designed to predict future crimes in courtrooms in the United States, in 2016 the COMPAS was revealed to be biased towards African American defendants.

• Search terms 'AI bias' started gaining interest in Google searches around 2016-2017 (Figure 1).

• In parallel, search terms "responsible AI" and "explainable AI" started gaining interest in Google searches around 2017, with interest continually increasing up until 2021 (Figure 2). • The world has begun a countdown of sorts to AI takeover, as is illustrated by the book "2062: The World that AI Made" by Prof. Toby Walsh (Figure 3). The 'deadline' year of "2062" is an average estimate, calculated based on the interviews conducted by Prof. Toby Walsh, with interviewees including prominent members of the AI community. • Ultimately, RAI development should include engineers, data scientists, regulators, legal experts, as well as sociologists.

• Burden should not be placed on the shoulders of data scientists and engineers alone. RAI requires a multi-disciplinary collaboration to ensure AI-based systems developed within an institution are not only responsible but are aligned with what the organization has defined for itself (i.e., organizational values and mission statements).

• In lieu of any quantifiable metrics specifically designed for use in RAI (i.e., detection and correction of any bias), some cautionary measures can be taken in the interim. For example, frontline developers can check available data for any inadvertent discrimination before delving into AI development. CONTACT AUTHOR CAN WE QUANTIFY RAI?

• At the moment, RAI and subdisciplines are predominantly assessed from a social sciences perspective. However, for RAI to truly be effective, quantification of RAI is necessary. Such quantification can be integrated into a logical AI framework with ease and will ensure that future AI machines and software developed will adhere to expected standards.

• • These methods are all statistical based. Future works in this space can include quantification of other aspects of RAI, including accountability, ethics, and authority. These concepts are discussed extensively, but no consideration has been given to their quantification.

SOME FINAL TAKE AWAY MESSAGES

•

  Fast and Horvitz's (2017) study assessed public response to AI trends over the course of 30 years [3]: • Initially, AI was celebrated by the general public • Over time, this initial optimism morphed into outright concern as examples of AI bias rose, and the public started wondering "When would AI takeover?"

Figure 1 .

 1 Figure 1. Google Trends Data for search terms "AI bias" from 2004-2021

Figure 2 .Figure 3 .

 23 Figure 2. Google Trends Data for search terms "Responsible AI" and "Explainable AI" from 2004-2021

Figure 4 .

 4 Figure 4. For a harmonious human and AI interaction, the incorporation of RAI is essential. RAI should consist of multiple components, including: accountability, fairness, safety, security, transparency, privacy and ethics.

  The only part of RAI currently most assessed in terms of quantification is fairness, and this is most likely attributable to the awareness built around AI bias in recent years. Some examples of fairness quantification include: • Individual and group fairness -statistical parity: For any two individuals 𝑥, 𝑦 that are at distance 𝑑(𝑥, 𝑦) ∈ [0, 1] map to distributions 𝑀(𝑥) and 𝑀(𝑦), respectively, such that the statistical distance between 𝑀(𝑥) and 𝑀(𝑦) is at most 𝑑(𝑥, 𝑦) [4]. In other words, the smaller the distance, the more likely fairness is achieved. • Predictive fairness and parity: This is to ensure same level of fairness is demonstrated, irrespective of group membership, i.e.. 𝑃(𝑌 = 1 | 𝑆 = 𝑠, 𝑅 = 𝑏) = 𝑃(𝑌 = 1 | 𝑆 = 𝑠, 𝑅 = 𝑤) [5].