Using mass spectrometry to identify neoantigens in autoimmune diseases: The type 1 diabetes example

In autoimmune diseases, recognition of self-antigens presented by major histocompatibility complex (MHC) molecules elicits unexpected attack of tissue by autoantibodies and/or autoreactive T cells. Post-translational modification (PTM) may alter the MHC-binding motif or TCR contact residues in a peptide antigen, transforming the tolerance to self to autoreactivity. Mass spectrometry-based immunopeptidomics provides a valuable mechanism for identifying MHC ligands that contain PTMs and can thus provide valuable insights into pathogenesis and therapeutics of autoimmune diseases. A plethora of PTMs have been implicated in this process, and this review highlights their formation and identification.     

The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3+ regulatory T cells

Foxp3⁺ regulatory T (Treg) cells are required to prevent the immune system from spontaneously mounting a severe autoaggressive lymphoproliferative disease and can modulate immune responses in a variety of settings, including infections. In this review, we describe studies that use transgenic mice to determine how signals through the T-cell receptor (TCR) contribute to the development, differentiation, and activity of Treg cells in in vivo settings. By varying the amount and quality of the self-peptide recognized by an autoreactive TCR, we have shown that the interplay between autoreactive thymocyte deletion and Treg cell formation leads to a Treg cell repertoire that is biased toward low abundance agonist self-peptides. In an autoimmune disease setting, we have demonstrated that diverse TCR specificities can be required in order for Treg cells to prevent disease in a mouse model of autoimmune inflammatory arthritis. Lastly, we have shown that Treg cells initially selected based on specificity for a self-peptide can be activated by TCR recognition of a viral peptide, and that they can acquire a specialized phenotype and suppress antiviral effector cell activity at the site of infection. These studies provide insights into the pivotal role that TCR specificity plays in the formation and activity of Treg cells.     

Taming the TCR: Antigen-Specific Immunotherapeutic Agents for Autoimmune Diseases

Current treatments for autoimmune diseases are typically non-specific anti-inflammatory agents that affect not only the autoreactive cells but also the parts of the immune system that are required to maintain health. There is a need for the development of antigen-specific therapeutic agents that can effectively prevent the autoimmune attack while leaving the rest of the immune system functioning as normal. The simplest way to achieve this is using the autoantigen itself as a tolerizing agent; however, there is some risk involved with administering a potentially pathogenic antigen. In this review, we focus instead on the development and use of modified T cell receptor (TCR) ligands, in which the peptide ligand is modified to change the response by the T cell from a disease inducing to a protective response, and still retain the antigen-specificity necessary to target the autoreactive T cells. We review the use of modified TCR ligands as therapeutic agents in animal models of autoimmunity and in human autoimmune disease, and finally consider how they need to be improved in order to use them effectively in patients with autoimmune disease.     

Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue

Summary:  The epithelium of the human small intestine contains a large population of intraepithelial cytolytic αβ T-cell receptor (TCR) CD8αβ T lymphocytes (IE-CTLs), whose main role is to sustain epithelial integrity by rapidly eliminating infected and damaged cells. In mouse, the recognition of inducible/modified self-molecules, i.e. non-classical major histocompatibility complex (MHC) class I molecules, is mediated by the TCR and natural killer receptors (NKRs) co-expressed on the cell surface of a non-conventional autoreactive CD8αααβTCR cell subset. In contrast, in humans, the recognition of non-classical MHC class I molecules induced by stress and inflammation on intestinal epithelial cells (IECs) is principally mediated by NKRs expressed on conventional CD8αβαβTCR cells. By sensing microenvironmental signals of inflammation and stress through NKRs, IE-CTLs fine tune their TCR activation threshold. Furthermore, IE-CTLs under particular conditions, involving interleukin-15 upregulation, acquire the capacity to kill distressed intestinal epithelial cells in an antigen non-specific manner. Adaptive IE-CTLs appear hence to have autoreactive properties and modulate their immune response based on innate signals, reflecting the fitness of the tissue.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

T-cell receptor revision: friend or foe?

T‐cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post‐thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.     

Development and function of autospecific dual TCR+ T lymphocytes

Recent studies have challenged the long held concept that each T lymphocyte expresses on its surface only a single, unique alphabetaTCR. Dual TCR+ T cells have been recognized, however, their origin and potential to escape screening for self-reactivity remain obscure. We now report the thymic generation of dual alphabetaTCR+ T cells in the H-2Db/H-Y-specific TCR transgenic (Tg) mouse. Dual TCR+ thymocytes were positively selected less efficiently than single TCR+ thymocytes, although a subset attained maturity. Importantly, when TCR Tg mice were bred onto a negatively selecting background, auto-specific cells survived central deletion and matured as CD4+ dual TCR+ cells. These cells were autoreactive when CD8 expression was restored. The existence of autospecific, dual TCR+ T cells may have implications for the maintenance of self tolerance.     

Strength of TCR signal from self‐peptide modulates autoreactive thymocyte deletion and Foxp3+ Treg‐cell formation

Autoreactive CD4⁺CD8^? (CD4SP) thymocytes can be subjected to deletion when they encounter self‐peptide during their development, but they can also undergo selection to become CD4SPFoxp3⁺ Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP‐76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3⁺ thymocytes. CD4SP thymocytes expressing TCR Vβ‐chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3⁺ thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg‐cell formation in response to a defined self‐peptide, SLP‐76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3⁺ Treg‐cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg‐cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg‐cell formation.     

A novel form of self tolerance dictated in the thymus of transgenic mice with autoreactive TCR {alpha} and {beta} chain genes

Transgenic (TG) mice with TCR and ß chain genes froma CD4-dependent auto-l-A^k reactive T cell clone were generated.H-2^k TG mice had a large number of thymic and splenic CD4 Tcells expressing the autoreactive TCR without manifestationof autolmmunlty. The cells were not anergic, as they could respondto autologous antigen presenting cells and antl-TCR antibodiesin vitro to proliferate and to produce interleuklns. Variousdegrees of down-regulation of CD2 and CD44 was observed in TGmice, Indicating the presence of a defective co-stlmulatoryprocess in TG T cells. These features indicate that the selftolerance in autoreactive TCR TG mice is due not to clonal deletionand anergy but to a novel mechanism where T cells cannot sufficientlyrespond to normally existing self ligand in vivo. That suchan in vivo unresponsiveness of autoreactive T cells is dictatedin the thymus during CD4 T cell differentiation as an atypicalform of positive selection of autoreactive T cells was suggestedby the abnormal surface expression of CD69 and HSA.     

Apoptotic signal transduction and T cell tolerance

The healthy immune system makes use of a variety of surveillance mechanisms at different stages of lymphoid development to prevent the occurrence and expansion of potentially harmful autoreactive T cell clones. Disruption of these mechanisms may lead to inappropriate activation of T cells and the development of autoimmune and lymphoproliferative diseases [such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, diabetes and autoimmune lymphoproliferative syndrome (ALPS)]. Clonal deletion of T cells with high affinities for self-peptide-MHC via programmed cell death (apoptosis) is an essential mechanism leading to self-tolerance. Referred to as negative selection, central tolerance in the thymus serves as the first checkpoint for the developing T cell repertoire and involves the apoptotic elimination of potentially autoreactive T cells clones bearing high affinity T cell receptors (TCR) that recognize autoantigens presented by thymic epithelial cells. Autoreactive T cells that escape negative selection are held in check in the periphery by either functional inactivation ("anergy") or extrathymic clonal deletion, both of which are dependent on the strength and frequency of the TCR signal and the costimulatory context, or by regulatory T cells. This review provides an overview of the different molecular executioners of cell death programs that are vital to intrathymic or extrathymic clonal deletion of T cells. Further, the potential involvement of various apoptotic signaling paradigms are discussed with respect to the genesis and pathophysiology of autoimmune disease.     

Apoptotic signal transduction and T cell tolerance

The healthy immune system makes use of a variety of surveillance mechanisms at different stages of lymphoid development to prevent the occurrence and expansion of potentially harmful autoreactive T cell clones. Disruption of these mechanisms may lead to inappropriate activation of T cells and the development of autoimmune and lymphoproliferative diseases [such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, diabetes and autoimmune lymphoproliferative syndrome (ALPS)]. Clonal deletion of T cells with high affinities for self-peptide-MHC via programmed cell death (apoptosis) is an essential mechanism leading to self-tolerance. Referred to as negative selection, central tolerance in the thymus serves as the first checkpoint for the developing T cell repertoire and involves the apoptotic elimination of potentially autoreactive T cells clones bearing high affinity T cell receptors (TCR) that recognize autoantigens presented by thymic epithelial cells. Autoreactive T cells that escape negative selection are held in check in the periphery by either functional inactivation (“anergy”) or extrathymic clonal deletion, both of which are dependent on the strength and frequency of the TCR signal and the costimulatory context, or by regulatory T cells. This review provides an overview of the different molecular executioners of cell death programs that are vital to intrathymic or extrathymic clonal deletion of T cells. Further, the potential involvement of various apoptotic signaling paradigms are discussed with respect to the genesis and pathophysiology of autoimmune disease.     

Analysis of the relationship between viral infection and autoimmune disease

The clinical association between viral infection and onset or exacerbation of autoimmune disorders remains poorly understood. Here, we examine the relative roles of molecular mimicry and nonspecific inflammatory stimuli in progression from infection to autoimmune disease. Murine herpes virus 1 (HSV-1 KOS) infection triggers T cell-dependent autoimmune reactions to corneal tissue. We generated an HSV-1 KOS point mutant containing a single amino acid exchange within the putative mimicry epitope as well as mice expressing a TCR transgene specific for the self-peptide mimic to allow dissection of two pathogenic mechanisms in disease induction. These experiments indicate that viral mimicry is essential for disease induction after low-level viral infection of animals containing limited numbers of autoreactive T cells, while innate immune mechanisms become sufficient to provoke disease in animals containing relatively high numbers of autoreactive T cells.     

Molecules in medicine mini review: the αβ T cell receptor

As an integral part of the mammalian immune system, a distributed network of tissues, cells, and extracellular factors, T lymphocytes perform and control a multitude of activities that collectively contribute to the effective establishment, maintenance, and restoration of tissue and organismal integrity. Development and function of T cells is controlled by the T cell receptor (TCR), a heterodimeric cell surface protein uniquely expressed on T cells. During T cell development, the TCR undergoes extensive somatic diversification that generates a diverse T cell repertoire capable of recognizing an extraordinary range of protein and nonprotein antigens presented in the context of major histocompatibility complex molecules (MHC). In this review, we provide an introduction to the TCR, describing underlying principles that position this molecule as a central regulator of the adaptive immune system involved in responses ranging from tissue protection and preservation to pathology and autoimmunity.     

The central residues of a T cell receptor sequence motif are key determinants of autoantigen recognition in murine experimental autoimmune encephalomyelitis

The autoreactive response in murine experimental autoimmune encephalomyelitis (EAE) is dominated by an oligoclonal expansion of V beta 8(+) CD4(+) T cells. These T cells recognize the immunodominant N-terminal nonapeptide of myelin basic protein (MBP1-9) associated with the MHC class II molecule, I-A(u). Amongst the autoreactive cells, T cells bearing TCR containing the CDR3 beta motif Asp-Ala-Gly-Gly-Gly-Tyr (DAGGGY) play a dominant role in the disease process. Here we have investigated the molecular basis for antigen recognition by a representative TCR (172.10) that contains the DAGGGY motif. The roles of the three glycines in this motif in the corresponding TCR-peptide-MHC interactions have been analyzed using a combination of site-directed mutagenesis and surface plasmon resonance. Our data show that mutation of either of the first two glycines (G97, G98) to alanine results in soluble, recombinant TCR that do not bind to recombinant antigen at detectable levels. Mutation of the third glycine (G99) of the 172.10 TCR results in a substantial decrease in affinity. The importance of the triple glycines for antigen recognition provides an explanation at the molecular level for the recruitment of T cells bearing the DAGGGY motif into the responding repertoire during EAE induction.     

Targeting the TCR: T-cell receptor and peptide-specific tolerance-based strategies for restoring self-tolerance in CNS autoimmune disease

A principal theme in autoimmunity is the breakdown of central tolerance resulting in the persistence and eventual activation of autoreactive T cells. Because CD4(+) T cells are key contributors to the underlying pathogenic mechanisms responsible for the onset and progression of most autoimmune diseases, they are a logical target for therapeutic interventions. One technique for restoring self-tolerance is to exploit the endogenous regulatory mechanisms that govern CD4(+) T-cell activation. In this review, we discuss promising techniques with the common goal of inducing antigen (Ag)-specific tolerance. Emphasis is given to the use of non-mitogenic anti-CD3 and peptide-specific tolerance strategies that specifically target the T-cell receptor (TCR) in the absence of costimulatory signals. These approaches produce a TCR signal of insufficient strength to cause CD4(+) T-cell activation and instead induce functional T-cell anergy or deletion while avoiding generalized long-term immunosuppression.     

A molecular basis for NKT cell recognition of CD1d-self-antigen

The antigen receptor for natural killer T?cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3???resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.     

CD24 on thymic APCs regulates negative selection of myelin antigen-specific T lymphocytes

Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2(+) CD24(-/-) ), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if CD24 regulated negative selection, and if so, what cellular mechanisms were involved. Here, we show that elimination of MOG or Aire gene expression in 2D2(+) CD24(-/-) mice - through the creation of 2D2(+) CD24(-/-) MOG(-/-) or 2D2(+) CD24(/) ～Aire(-/-) mice - completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on DCs, but not on thymocytes also partially restores 2D2 T-cell generation in 2D2(+) CD24(-/-) mice. Taken together, we propose that CD24 expression on thymic antigen-presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.     

Incomplete TCR-β allelic exclusion accelerates spontaneous autoimmune arthritis in K/BxN TCR transgenic mice

Allelic exclusion of antigen receptor loci is a fundamental mechanism of immunological self-tolerance. Incomplete allelic exclusion leads to dual T-cell receptor (TCR) expression and can allow developing autoreactive αβ T lymphocytes to escape clonal deletion. Because allelic exclusion at the TCR-β locus is more stringent than at the TCR-α locus, dual TCR-β expression has not been considered a likely contributor to autoimmunity. We show here that incomplete TCR-β allelic exclusion permits developing thymocytes bearing the autoreactive, transgene-encoded KRN TCR to be positively selected more efficiently, thereby accelerating the onset of spontaneous autoimmune arthritis. Our findings highlight dual TCR-β expression as a mechanism that can enhance the maturation of autoreactive pathogenic T cells and lead to more rapid development of autoimmune disease.