Type 1 Diabetes: Cellular, Molecular & Clinical Immunology

Chapter 1 - Primer: Immunology and Autoimmunity
Stephanie C. Eisenbarth and Dirk Homann

Updated 8/08, slides updated 8/07 Click to download Powerpoint slide set

Introduction
Immunologists over the past two decades have solved a series of fundamental genetic and biochemical questions related to immune function. Discoveries include elucidation of the mechanism by which antibody and T cell receptor diversity is generated (1);(2) the characterization of a series of lymphokines essential for lymphocyte activation (3), the characterization of cell surface lymphocyte molecules (and in many cases their function), characterization of molecules of the innate immune system including a large a diverse family of patern recognition receptors (4;5), molecules mediating what are termed “second” signals of T lymphocyte activation (6), the crystal structure and peptide-binding properties of human leukocyte antigen (HLA) class II and class I molecules (known as major histocompatibility complex or MHC in animals) and characterization of CD4 T cell subsets with divergent functions (7-10). The field of immunology is now focused on defining mechanisms by which the immune system discriminates between self and non-self antigens and therefore by necessity, the determinants of autoimmunity. For some diseases and for some animal models, answers to a subset of the basic questions concerning autoimmunity posed in Table 1.1 are now available. It is clear that a single genetic or triggering mechanism to explain all autoimmune diseases affecting even a single tissue will be inadequate. For example myasthenia gravis whichresults from autoantibodies to the acetylcholine receptor, can be triggered by the drug penicillamine or a thymoma. However, for most patients with myasthenia gravis no triggering factor has been defined.

Normal Immune Response
In many respects, the modern age of immunology began with the independent descriptions by Talmage and Burnet in 1957 of the clonal selection theory (11). “The basic concept was of the immunologically competent cell; that is a cell which is susceptible to specific stimulation by contact with the appropriate antigenic determinant. The main possibilities of reaction are: (a) destruction, especially if physiologically immature; (b) proliferation without essential change of character – to what we would now call memory cells; and (c) proliferation and antibody production as a clone of plasma cells.” In 1957 the mechanism underlying the huge diversity (which is fundamental to the clonal selection theory) of immunological reactivity of single lymphocytes was unknown. It is now understood that B andT cells bear on their surface unique antigen receptors created by genetic recombinatorial processes.
The receptor of B cells is the immunoglobulin molecule(antibody), which can recognize soluble protein antigens. For each B cell its antibody receptor has the same specificity as the antibodies that will eventually be secreted by the mature plasma cell, the final fate of the B cell. Antibody genes are created by a combinatorial process in which any of a family of multiple variable-region gene segments (V) combine with short joining (J) and diversity (D) gene segments and then with the constant-region gene segments of immunoglobulin. A similar process in T cells, results in the pairing of rearranged alpha and beta (or gamma and delta) chains and produces an estimated 107 -108 different TCRs (T cell receptors) in adult humans (12). A number of DNA cleavage and repair enzymes are responsible for VDJ recombination of both the immunoglobulin and T cell receptor genes, including recombination-activating genes (RAG-1 and RAG-2). These enzymes are essential in recombination and therefore lymphocyte development. Indeed in RAG deficient mice and patients with Omenn Syndrome, lymphocyte development does not occur resulting in severe immunodeficiency (13). Following gene rearrangement in B cells, heavy and light chains of immunoglobulin are joined resulting in billions of different antibodies/B cell receptors (differing by their amino acid sequence). Antibody genes (but not T cell receptor genes) also undergo somatic hypermutation once B cell development is complete- a process of V region gene mutation in the secondary lymphoid organs that increases the affinity with which antigens are recognized by clones of B lymphocytes (called “affinity maturation”).  Lastly, antibodies can also undergo class switching to modify and specify an immune response.  Class switching involves gene rearrangement in the constant Fc region of the antibody/B cell receptor, which switches the default mu locus (IgM) antibody isotype for either the alpha, gamma or epsilon loci (IgA, IgG or IgE, respectively).  The recently described enzyme, activation-induced deaminase (AID) is responsible for the last two important steps of antibody diversification (14), (15), however, how AID directs somatic hypermutation and class switching is an important area of ongoing research (16).
Antigen Presenting Cells and MHC
Exogenous antigens are taken into a cell by specific receptors (e.g., binding of antigen to surface immunoglobulin of B cells, immunoglobulin Fc receptor-mediated uptake of immune complexes) or non-antigen-specific mechanisms (e.g., endocytosis). Such antigens are then cleaved into peptides and coupled to class II MHC molecules for presentation on the cell surface of antigen presenting cells forrecognition by T cells (CD4-positive T cells). Endogenous antigens synthesized by the cell (e.g., self proteins or viral proteins), are cleaved by peptidases in a proteosome complex, transported into the endoplasmic reticulum by specific peptide transporters (TAP1& TAP2) (17), coupled to class I MHC molecules and then presented on the cell surface for recognition by T cells bearing CD8 accessory molecules.

An important difference exists in the way in which B and T cells recognize their cognate antigen. Whereas the B cell receptor can recognize whole proteins in solution, typical (alpha/beta) T cells respond only to peptide antigen bound to class I or class II molecules of the major histocompatibility complex (MHC) on antigen presenting cells (APCs). Although APCs (e.g. macrophages, B cells, and dendritic cells) play a role in clearance of pathogens and other particles, a crucial element of APCs is the ability to endocytose proteins, process these antigens into smaller peptides and then present peptides in the groove of MHC molecules.  MHC molecules were initially called “transplantation antigens” as they determine the ability of a donor to accept a transplanted graft. HLA/MHC molecules function by binding short peptides, usually 9-10 amino acids in length, and presenting these peptides to the T cell receptor. T cells expressing a T cell receptor complex that includes the co-receptor CD4 interact with class II molecules (DR, DP, and DQ of man, H2-A and H2-E of the mouse [see Table 1.2]). The CD8 co-receptor performs a similar function for T cells that interact with class I molecules (HLA A, B, and C of man; K and D molecules of the mouse). Figure 1.1 illustrates the uptake, cleavage, and presentation of an external antigen to a CD4-positive T cell.

Figure 1.1. Antigen uptake and presentation by antigen presenting cells (APC) with peptide in the groove of a major histocompatibility complex (MHC) class II molecule (e.g., HLA DR, DP or DQ in humans) to a naive CD4+ T cell.

HLAclass II genes are also calledimmune response genes; these molecules are highly polymorphic, differing between individuals in their amino acid sequence (18). Each different sequence is given a number for man and letters+/-numbers for mice.  For instance DQB1*0302 is a DR4 associated diabetogenic allele of man and I-A^g7 an analogous diabetogenic allele of the NOD mouse.  Allelic HLA differences are inherited in a Mendelian fashion. The sequence differences determine which peptides can be bound and presented. Thus, such inherited differences in HLA molecules between individuals can determine which antigen an individual can respond to, and importantly, which autoimmune disorder they are likely to develop. As modeled above, the developing immune system has the potential to respond to essentially every foreign antigen, but also to every self-antigen.

Figure 1.2. APC:TCell Interactions. A number of surface molecules on the APC and T cell can interact during activation. In addition to TCR (T cell receptor) ligation, co-stimulatory molecules such as B7.1 and B7.2 on the APC must ligate CD28 to achieve full T cell activation. CTLA-4 competitively binds B7 molecules and results in a dampening of the T cell response. Peripheral interactions provided by adhesion molecules such as LFA-3 and CD2 assist during MHC: TCR ligation. The T cell also provides activating signals to the APC such as CD40:CD40 Ligand interactions that result in immunoglobulin class switching in B cells and cytokine production in dendritic cells.

T Cell Activation and the Innate Immune System
In addition to the requirement of peptide presentation in the context of MHC molecules (“signal one”), a T cell requires an activation signal from the antigen-presenting cell (see Figure 1.2). This “second signal” can be provided by cell surface molecules CD80 or CD86 (B7.1 and B7.2, respectively). Additional B7 family members (such as B7.h) and other co-stimulatory molecules are also capable of providing this second signal (19). Thus, recognizing its cognate peptide in the groove of the MHC molecule is not enough to activate a naive T cell and therefore is also not enough to break tolerance to self-peptides. On the contrary, if a T cell recognizes its cognate peptide on the surface of an antigen-presenting cell (APC) without appropriate co-stimulation, tolerance or anergy is induced. The requirement for a second signal is a potent mechanism of preventing non-specific or self-specific T cell inflammatory reactions. However, this leads to the question of how the APC knows when to activate a T cell with a second signal in order to initiate an appropriateimmune response (20).  Work over the past ten years has started to answer this question.
One T cell can recognize up to a million different peptides with its receptor (see section on “Target Antigens”) but it would obviously be disadvantageous for the immune system to activate a T cell in the absence of a real threat (e.g. infection) (21). This puzzle was recently solved with the discovery of pattern recognition receptors (PRR) and in particular, Toll-like receptors (TLRs) first in Drosophila and then in mammals(19). PRRs are considered a part of the Innate Immune system (as opposed to the Adaptive Immune System); Innate Immunity reacts faster to infections and is comprised of phagocytic cells, the complement system and a number of soluble inflammatory mediators such as antibacterial peptides and cytokines (see Table 1.3). PRRs are located on APCs, T cells (22) and some epithelium (23) and recognize highly conserved sequences within pathogen structures or PAMPs (pathogen-associated molecular patterns). For example, lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, signals through TLR4. The eleven TLRs that have been identified provide for a broad spectrum of responsiveness to pathogens, including bacteria, viruses and fungi. Therefore, when an APC encounters an invading pathogen, it not only engulfs it for antigen presentation, but also is stimulated via PRRs to upregulate MHC and co-stimulatory molecule expression and to produce a host of pro-inflammatory cytokines (e.g., IL-1, IL-6 and IL-12) (24). The combination of these changes in the APC enables effective stimulation of naive T cells and is the link between the Innate and Adaptive Immune systems (see Figure 1.3)(25), (26).  Molecules such as LPS can be crucially important not only for induction classical Th1 mediated disease (e.g., anti-tuberculosis response), but depending upon the dose administered, Th2 disorders as well (e.g., asthma) (27).

Figure 1.3. T Cell Activation by an Activated APC. Innate immune system dependent adaptive immune activation: A series of pattern recognition receptors (e.g., Toll-like receptors or TLRs) are present on antigen presenting cells. They recognize common molecules of infectious agents (pathogen associated molecular patterns or PAMPs) and stimulate the antigen-presenting cell. Subsequently, MHC and B7 molecules are unregulated on the surface of the APC and interact with the T cell receptor and CD28, respectively. Furthermore, interleukins are produced, such as IL-12, which help determine T cell differentiation (“signal 3”). The T cell requires multiple signals for activation; in the absence of both signal 1 and 2, TCR ligation results in anergy rather than activation.

LPS (Lipopolysaccharide); NOD (Nucleotide-binding and Oligomerization domain-containing) PGN (Peptidoglycan), DC (Dendritic Cell), LTA (Lipoteichoic Acid); LBP (LPS Binding Protein); Phagocytes (Monocytes, Macrophages & Neutrophils); MBP (Mannan-binding Protein) = MBL (Mannose-binding Lectin).
The NALPs represent a large family of intracellular molecules related to NOD (nucleotide-binding oligomerisation domain molecules that are able to sense multiple Pathogen Associated Molecular Patterns that gain access to the intracellular compartment as well as signals of cell stress (e.g. extracellular ATP, uric acid) often resulting in the secretion of potent inflammatory molecules such as IL-1(28).  Mutations of these molecules result in a series of autoinflammatory diseases (28) but are implicated in multiple inflammatory responses (e.g. uric acid crystals and gout(29); silicosis (30), asbestosis (31) and even adaptive immune responses enhanced by the adjuvant Alum (32).
For further readings on the information in Table 1.3 see (33-36).
B Cell Activation
Once an antigen-specific T cell has been appropriately activated, it in turn helps activate the humoral arm - the B cell. Helper CD4+ T cells initiate the majority of humoral responses, although there are a few exceptions with certain classes of pathogens, but we will focus on the former here. T cell help is initiated within the lymph nodes or spleen when an antigen-specific armed T cell recognizes its antigen peptide in MHC II on the surface of the B cell (whose Ig receptor recognized the same pathogen, internalized it and presented fragments of the pathogen on class II). This is termed Linked Recognition because it ensures that the T and B cells recognize the same pathogen although not necessarily the same epitope. Engagement of the TCR induces upregulation of CD40 Ligand on the surface of the T cell and the secretion of Bcell activators, such as IL-4, IL-5 and IL-6. The combination of these cytokines stimulates the B cell to proliferate, secrete antibody and, depending on the cytokines produced by the effector T cell, isotype switch from producing IgM to IgA, IgG or IgE antibodies. Therefore, this is another stage at which tolerance to self-peptides is censured or can be broken. If an autoreactive B cell does not encounter an activated T cell specific for the same self-protein, then no autoantibodies can be produced (unless the antigen is a large repeating molecule and T cell independent). This control step could be circumvented in autoimmunity, however, through the chance meeting of an armed T cell specific for a foreign peptide which recognizes a B cell that has phagocytosed and presented that T cell’s antigen on MHC, but has antibodies specific for a self antigen. Activation of that B cell would lead to the production of autoantibodies(see Table 1.4).
Chemokines
The immune system is a distributed network of organs, tissues, cells and extracellular factors. Functional integration of these components faces a particular challenge as the principal sentinels, regulators and effectors of immune function are often highly mobile single cells. The regulated spatio-temporal positioning of these cells is achieved by adhesion molecules such as integrins and selectins as well as chemo-attractant cytokines (chemokines) and their receptors that function as a “molecular address system” to regulate trafficking of specific cells into, out of and within defined anatomic microenvironments (37-41). Accordingly, chemokines have been implicated in a wide variety of pathological states including infectious disease, cancer, allergy, autoimmunity, and transplant rejection (42-47).
The family of chemokines consists of a large number of structurally related and mainly secreted molecules that share a defining tetracysteine motif. According to a recent systematic classification (48), chemokines are divided into four distinct subfamilies based on the configuration of their aminoterminal cysteine residues (Table 1.4). CC chemokines (CCL1-28) harbor two adjacent cysteines whereas these residues are separated by a single, non-conserved amino acid among the CXC chemokines (CXCL1-16). The sole CX3C chemokine (CX3CL1) contains three amino acids between the corresponding cysteine residues and C chemokines (XCL1 and 2) lack one of the first two cysteines present in the other subfamilies. Overall, chemokines distinguished according to these structural criteria interact with specific members of corresponding chemokine receptor subgroups. Nevertheless, due to promiscuity among certain members of the CXC and CC subfamilies, some chemokines are capable of binding more then one chemokine receptor and vice versa. Furthermore, small alterations in the amino termini of many chemokines can lead to pronounced changes in bioactivity and are the basis for aspects of protease-mediated regulation of chemokine function (37;41). It should be noted that an older and complementary classification distinguishes inducible (inflammatory) and constitutive (homeostatic) chemokines. However, this distinction is not without problems since the constitutive expression patterns of many chemokines under conditions of immune homeostasis have not been precisely defined. Indeed, “constitutive chemokines” can be upregulated during inflammation while some “inducible chemokines” are apparently expressed in the absence of injury, infection or other inflammatory stimuli (37).
The defining function of chemokines, demonstrated in numerous in vitro experiments, is their capacity to induce the directed migration of locomotive cells by establishing a spatial gradient. While the existence of comparable chemokine gradients in vivo remains a matter of debate, chemokines exhibit a host of additional functions including control of lymphopoiesis and lymphoid organogenesis, alterations of leukocyte adhesive properties by modulation of integrins as well as regulation of lymphocyte differentiation, proliferation, apoptosis, cytokine release and degranulation (40-42;49;50).
Chemokines and Type 1 Diabetes
Work conducted over the past decade has implicated as many as half of all known chemokines in the pathogenesis of T1D (51). Published observations range from correlative data obtained by molecular profiling of islet cells, or infiltrating T cells to successful therapeutic intervention by means of experimental chemokine/chemokine receptor blockade (Table 1.4). For example, early work by Bradley et al. has shown that pancreas-infiltrating CD4+T cells produce a wide range of chemokines including CCL2, CCL3, CCL4, CCL5, CCL7, CCL12, CXCL10 and XCL1 (52). and specified that a high CCL3:CCL4 ratio in the pancreata of NOD mice is associated with destructive insulitis while a lower CCL3:CCL4 ratio was observed in diabetes-resistant NOR mice (53). A possible pathogenic role for CCL17 and CCL22 was deduced from the CCR4+ phenotype of islet-infiltrating CD4+T cells as well as corresponding chemokine neutralization studies (54). More recently, the importance of CXCR3-binding chemokines CXCL9 and CXCL10 has been documented by the absence of T1D induction in CXCR3-/- mice or mice treated with a neutralizing antibody specific for CXCL10 (55;56). In addition, transgenic expression of CXCL10 in beta-cells, although not associated with spontaneous diabetes development, leads to accelerated virus-induced T1D onset (57), a process that may be amplified through CXCL10 production by islet-specific T cells. Nevertheless, CXCL10-blockade alone may not be sufficient under experimental conditions associated with enhanced inflammatory alterations (U. Christen, personal communication) and may thus require the therapeutic targeting of additional chemokines. For example, CCL5 is upregulated in the pancreata and islets of prediabetic mice(55;56) and neutralization of the CCL5 receptor CCR5 can reduce beta-cell destruction and T1D incidence (58).
Taken together, the wide-ranging observations derived from experimental model systems and clinical studies emphasize the complex regulation of T1D pathogenesis by different chemokines and suggest a multiplicity of potential targets for prevention and amelioration of disease. Yet an integration of these observations into a coherent perspective on T1D pathogenesis has been hampered by the fact that the relevant cellular sources of these chemokines have for the most part not been identified. Furthermore, the majority of published chemokine expression analyses are limited to quantitation of chemokine transcripts and corresponding data on chemokine protein expression is often scarce. Consequently, proposed pathogenetic mechanisms are at times somewhat speculative and the precise contribution of individual chemokines as well as chemokines at large to T1D development remains at present incompletely defined.

Programmed Cell Death and Central Tolerance
Because the immune effector mechanisms that have evolved are capable of rapidly killing cells, the potential for response to self is a consequence of having a potent immune system (analogous to having firearms in a house to protect oneself from burglars - weapons that may nevertheless accidentally kill family members). As stated by Burnet, “Autoimmune disease can be defined as a condition in which structural or functional damage is produced by the reaction of immunocytes or antibodies with normal components of the body . . . the central theme is the emergence of ‘forbidden clones’ of pathogenic immunocytes and the various ways by which these can arise and find ways of escaping the normal controls”. Such a pathogenic mechanism underlies the disease of mice with mutations or knockouts of the AIRE (Autoimmune Regulator) gene (see below).
At a functional level, the breaking of “tolerance” is fundamental to concepts of autoimmunity. The first and perhaps most vital stage of tolerance induction to self-antigens occurs in the thymus during T cell development. It was previously believed, that certain peripheral tissue-restricted proteins or certain proteins that are exclusively expressed only after a particular developmental stage could not be expressed in the thymus. Self-reactive T cells are deleted in the thymus by reacting strongly to self-antigens presented by thymic APCs. Therefore, if certain antigens could not be expressed in the thymus, the process of selection would be “blind” to such self-reactive T cell clones, which could therefore escape negative selection. These clones would then have to be controlled or deactivated in the periphery (see next section). However, the thymus and other lymphoid organs actually contain “self-antigen-presenting cells” (59). For example, insulin message and protein are present in the thymus, lymph node, and spleen. Functional studies suggest that minute quantities of such self-antigens are important for the maintenance of central tolerance (60). More recent work has identified a gene responsible for this ectopic expression of self-antigens in the thymus - AIRE or Autoimmune Regulatory gene (59). Mutations of the AIRE gene on chromosome 21 in fact cause APS-I (Autoimmune Polyendocrine Syndrome), which presents with a spectrum of autoimmune diseases such as Addison’s disease, hypoparathyroidism, mucocutaneous candidiasis, hepatitis, and type 1 diabetes (61;62). Anderson and colleagues went on to show that many of the target self-antigens in APS-1 are not expressed in the thymus of mice lacking AIRE. It is likely that the AIRE gene has a number of different effects on central tolerance in addition to influence on expression of “peripheral” antigens in the thymus.  These important discoveries emphasize the critical role central tolerance plays in preventing autoimmune disease by removing potentially self-reactive lymphocytes from the T cell repertoire (63).
Peripheral Tolerance and Regulatory T Cells
In addition to the concept of “forbidden” clones arising because of mutations that bypass normal regulatory events, it has become clear that, given the appropriate context of antigen presentation, autoimmune responses can be generated to essentially all proteins. For example, subcutaneous injection of human insulin (the current therapy for type 1A diabetes) almost invariably leads to low levels of anti-insulin autoantibodies. For most destructive autoimmune disorders, a characteristic group of autoantibodies arise (see Table 1.5). These antibodies frequently react with a series of molecules derived from the same subcellular localization. For lupus erythematous, it is the ribonucleoprotein complex; for type 1 diabetes, insulin secretory granules and islet synaptic-like microvesicles. This suggests that once tissue destruction commences, in the context of an inflammatory lesion (e.g., insulitis), tolerance is broken to a series of autoantigens. This highlights the importance of another form of tolerance that controls the expansion of autoreactive lymphocytes after T cell development and selection are concluded - peripheral tolerance.

Whereas central tolerance occurs in the thymus during T cell development, peripheral tolerance occurs in secondary lymphoid organs (e.g., lymph nodes and spleen) after T cell development is concluded. This form of tolerance regulates the activation of naïve T cells via two mechanisms: anergy and regulatory T cells. Anergy involves the clonal inactivation of T cells with the potential to respond to self-antigens, resulting in cells that are resistant to activation upon antigen encounter. The second form of immune regulation can occur by T regulatory cells (Tregs). This is an emerging field within immunology and many different types of Tregs have been described with only a few over-arching principles (see Table 1.6). In general, “natural Tregs” can be identified by the surface expression of CD25 (the IL-2 receptor), CTLA-4, GITR, CD62L and OX40(64) and lack of the IL7(CD127 receptor)(36) and intracellular expression of FoxP3. Foxp3, unlike these other markers, is primarily (?exlusively for mouse) found in regulatory T cells and is required fort the development of CD25+ Tregs in the thymus (65), but for human T cells can also be acquired. Scurfy mice lack a functional Foxp3, which results in a deficiency of the protein scurfin. These mice succumb to a lethal autoimmune syndrome that mimics the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome in man (also termed XPID syndrome). In the human disease infants die of overwhelming autoimmunity and can have neonatal diabetes. Autoimmunity in both man and the murine model results from a deficiency in CD4+CD25+ regulatory T cells. Studies using the scurfy mice have identified IL-10 as one mechanism by which Foxp3+ Tregs may control wasting diseases such as IBD (Inflammatory Bowel Disease). How they limit aberrant T cell proliferation is not yet clear. Interestingly, during normal immune responses to pathogens, Tregs may be held in check by the triggering of pattern recognition receptors on APCs, resulting in the production of IL-6 and possibly other molecules capable of inhibiting suppressor T cell activity (66).
Much work is currently underway to elucidate the development and effector mechanisms utilized by Tregs and these studies should shed light on important pathways used by the immune system to regulate itself and prevent autoimmunity. Indoleamine 2,3-dioxygenase (IDO) has been a recent focus of numerous studies due to its role in the suppression of T cell responses. IDO is an enzyme that degrades tryptophan possibly resulting in either T cell depletion of an essential amino acid or the production of downstream metabolites that enhance APC suppressor function.  IDO has been implicated as a negative regulator of autoimmune disease in animal models such as systemic lupus erythematosus, multiple sclerosis and diabetes mellitus (67). In addition, this enzyme may be one mechanism used by Tregs to exert their suppressor effects.
The suppressive cytokine TGFbeta, is also used by Tregs to inhibit peripheral T cells. In fact creation of a transgenic mouse with a T cell receptor that recognizes an islet insulin peptide (insulin B:9-23) suppresses type 1 diabetes of the NOD mouse led to T cells of the transgenic that suppress diabetes through a TGFbeta depedent mechanism via T cell production of TGFbeta which acted in both an autocrine and paracrine manner (40).  T cell receptors from a clone recognizing the same peptide, but which were pathogenic, when used to produce transgenic mice resulted in diabetes (41). Thus for these T cell receptors it appears their phenotype in terms of pathogenicity is determined by the receptor sequence with fidelity when transgenic mice are created.

The Genetics of Autoimmunity
The general concept that autoimmunity develops in the setting of genetic susceptibility, and in particular in association with a series of specific HLA alleles, applies to most human autoimmune disorders. It is important to note, however, that the same HLA allele may protect from one and yet be associated with another disorder (e.g., the HLA DR2 associated DQ allele, DQA1*0102, DQB1*0602 is rare in patients with type 1 diabetes but is associated with multiple sclerosis (70;71)). It appears that susceptibility encoded by alleles within the HLA region do not globally influence the development of autoimmunity but rather influence the likelihood of specific disorders.
An alternative manner by which HLA alleles may determine susceptibility to autoimmune disease is by altering the developing T cell repertoire. This may be particularly important for diseases associated with what have been termed “superantigens” such as Kawasaki’s disease. Superantigens are molecules that bind outside of the peptide binding “groove” of class I and class II molecules and trigger whole families of T cells by binding to common sequences of T cell receptors (bearing specific Vbeta chains). Superantigens thereby activate large numbers of T cells (more than a usual antigen which interacts only with clones of T cells with a specific complementary binding site).  Thus, for T cell responses to superantigens, it is predicted that responding cells will bear T cell receptors of whole families (e.g. Vbeta8.2), but the T cell receptor antigen-binding region (“complementarity determining region”) will differ in amino acid sequence.
Recent experiments with transgenic mice have provided convincing evidence that the predilection of T cells for self-antigens is due to the way the T cell receptor repertoire is derived. Developing thymocytes express a multitude of T cell receptors, and only the infrequent thymocyte that expresses a receptor with low affinity for MHC antigens presented in the thymus matures further; the selection signal is supplied via engagement of the T cell receptor complex. This selection on self-MHC with self peptides (termed positive selection) is potentially dangerous in that T cells with an autoreactive nature can be selected. However, those developing autoreactive thymocytes with a very strong affinity for self MHC transduce a signal via their T cell receptor that leads to cell death rather than further differentiation, termed negative selection. The MHC repertoire seems to play an important role in this process of TCR selection. For example, Santamaria and coworkers have produced a transgenic mouse with a T cell receptor that targets islets and causes diabetes. However, multiple class II alleles, when crossed onto this transgenic mouse background, delete the autoreactive T cells and prevent disease (72;73).
In autoimmune diseases studied to date, concordance of identical twins is usually between 30% to 70%(74;75). Concordance of non-identical twins is often in the range of 5% and similar to the risk of siblings. Furthermore, data strongly suggests that alleles of genes outside the major histocompatibility complex contribute to disease susceptibility, in that monozygotic twins have a higher disease concordance for many autoimmune disorders (e.g., multiple sclerosis and type 1 diabetes) than HLA (MHC) identical siblings. For type 1 diabetes however, extreme risk can be identified for siblings who have the highest risk HLA DR/DQ genotype (DR3/4;DQ2/DQ8) and have inherited both HLA haplotypes identical by descent with their proband sibling. A risk as high as 80% for activating islet autoimmunity can be identified(76) with diabetes following several years after the appearance of islet autoantibodies.  Alleles of a number of genes influencing multiple autoimmune disorders are now well established including PTPN22, CTLA-4, IL2 receptor.  A single amino acid change to PTPN22 (R620W) that codes for a lymphocyte specific phosphatase, results in increased suppression of T cell receptor signaling, and is associated with diabetes, rheumatoid arthritis, Graves’ disease and other autoimmune disorders (77;78). 
In animal models, a number of alleles influencing disease susceptibility have been defined (79). In humans, for example, autoimmunity is associated with complement deficiency in the case of lupus erythematosus (80), with alleles of insulin in type 1 diabetes (81;82) and nucleoside phosphorylase deficiency in hemolytic anemia. It is likely that complement deficiencies contribute to autoimmunity by altering processing of antigen-antibody complexes and/or activation of Fc receptors. Insulin gene polymorphisms influence insulin synthesis in the thymus and therefore may influence “tolerance” to insulin (83). T cells are particularly sensitive to nucleotide metabolites and it is thought that nucleoside phosphorylase deficiency may be associated with autoimmunity secondary to T cell toxicity in hemolytic anemia. The genes underlying two remarkable syndromes of autoimmunity APS-I (Autoimmune Polyendocrine Syndrome Type 1) and XPID (X-linked Polyendocrinopathy, Immune dysregulation and Diarrhea) are now cloned. For both syndromes mutations of transcription factors lead to multiple autoimmune disorders including type 1 diabetes (see section on “Programmed Cell Death and Tolerance”). With refined techniques for genetic mapping and the knowledge provided by the genome project it is likely that in the next decade progress will be made in the definition of alleles underlying genetic susceptibility for common autoimmune disorders. However, even as genes causing autoimmunity are discovered their mechanism of action will not be immediately clear.
Triggering of Autoimmunity
A large group of experimental autoimmune disorders can be induced by immunization with self-proteins. To induce disease, and even to generate an immune response, such molecules are usually injected in a depot form with adjuvants that activate antigen-presenting cells.

For example, a model of multiple sclerosis follows immunization with myelin basic protein (84). There are conceptually similar models for experimental autoimmune thyroiditis (immunization with thyroglobulin), experimental myocarditis (immunization with myosin peptides), and experimental autoimmune oophoritis (immunization with the oocyte sperm cell receptor) (85). Once disease is induced in these animal models, T cell clones reacting with immunizing antigens are sufficient to transfer disease. In that disease can be induced by simple immunization with self-molecules, it is clear that T cells and B cells reactive to self exist in normal animals. Thus, the context of immunization determines whether pathogenic clones are expanded.  Recent crystal structure analysis of MHC with a bound autoantigen also suggests that the TCR may bind in an unusal configuration in autoimmune disease in which only part of the autoantignen is actually recognized (see Figure 1.4) (58). In part, the ease of inducing autoimmunity raises the question as to what mechanismspreventexpansion of autoimmune clones.

Figure 1.4.  T cell receptors from patients with multiple sclerosis binding shifted to N-terminus of cognate peptide (open ball P5 position of peptide in MHC). [A: DRB1*0101; B:DRB5*0101; C:DRB1*1501].

In animal models, both genetic (e.g., T cell immunodeficiency gene on chromosome 4 of the BB rat) and environmental manipulations of the immune system (neonatal thymectomy, injection of anti-RT6 antibodies in nonlymphopenic BB rats, and neonatal therapy with cyclosporine A or injection of poly-IC into RT1-U rats) lead to autoimmunity (86). It appears that neonatal thymectomy induces autoimmunity by decreasing a specific subset of “regulatory” T lymphocytes (see “Programmed Cell Death & Tolerance” section) (87).
Table 1.7 presents an “etiologic” classification of human autoimmunity based upon identified triggering factors. Infectious agents (88), neoplasms, and drugs have all been found to induce specific autoimmune diseases. Tumors that induce autoimmunity are characterized by the expression of specific autoantigens. One of the best-characterized oncogenic autoimmune syndromes results in cerebellar degeneration. Oncogenic cerebellar degeneration is associated with specific anti-Purkinje cell antibodies. It is induced by ovarian carcinomas expressing what have been termed CDR (cerebellar degeneration related) antigens (89). Additional oncogenic autoimmune disorders include pemphigus associated with lymphoma, myasthenia gravis and pure red cell aplasia associated with thymomas, and retinopathy associated with small-cell carcinoma. It is thought that, similar to the induced autoimmune disorders of animals described above, presentation to the immune system by tumor cells of self-peptides induces “remote” autoimmunity.
The drug penicillamine (dimethylcysteine) is associated with a large number of different autoimmune disorders, including myasthenia gravis, bullous pemphigoid, lupus erythematosus, polymyositis, and dermatomyositis. In addition to overt disease, penicillamine induces antibodies such as anti-insulin autoantibodies in the absence of overt pathology. It is hypothesized that penicillamine may induce autoimmunity by haptenation of multiple proteins.
One of the best examples of autoimmunity induced by food antigens is celiac disease, or gluten sensitive enteropathy (90). Celiac disease is characterized by marked lymphocytic infiltrates of the small bowel associated with villous atrophy. Ingestion of the wheat protein gliadin leads to the production of anti-transglutaminase autoantibodies and destruction of the villi. The disease is “cured” by elimination of wheat gluten from the diet. Like most autoimmune disorders, celiac disease occurs only in the presence of specific HLA alleles; in particular, with DQ alpha and beta sequences (DQA1*0501 and DQB1*0201) which occur in individuals with either a DR5 (DQA1*0501) and DR7 (DQB1*0201) HLA haplotypes in trans or in individuals with DR3 haplotypes (DQA1*0501/DQB*0201). There is now considerable evidence that the enzyme transglutaminase that is a very important target of autoimmunity in celiac disease acts on gliadin peptides to deamidate glutamine residues and this is “essential” for the generation of pathogenic peptides stimulating T cell responses (91).  It is also hypothesized that gliadin peptides may form covalent bonds with transglutaminase, thus acting as a hapten for immune activation.
Autoimmunity associated with mononucleosis is related to the unique ability of the virus to directly infect B cells (92). The Epstein-Barr virus (EBV) stimulates B cell proliferation and polyclonal antibody production. These polyclonal antibodies reacting with self -antigens can lead to disease. The proliferation of B cells is self-limited in the presence of an effective T cell response. Although pathogens may trigger autoimmune processes, infections can also suppress autoimmunity. For example, multiple viral infections in NOD mice prevent diabetes, as does a single injection of complete Freund’s adjuvant. Therefore the nature and timing of infection is likely to be critical in the triggering of autoimmune processes.
Target Antigens
The antibody and T cell responses characteristic of autoimmune disorders appear to be “antigen-driven.” The amino acid sequences of autoantibodies are mutated relative to immunoglobulin germ-line sequences and autoantibodies are usually of high affinity. In addition, the large number of different autoantibodies to different autoantigens for even a single autoimmune disease suggests that whatever the initial lesion leading to autoimmunity, the immune response “spreads” to a series of antigenic epitopes and molecules of the involved tissue. For example, large families of antibodies react with islet autoantigens during the beta cell destruction associated with type 1 diabetes. To date, these autoantibodies target molecules within two islet secretory organelles: insulin secretory granules (e.g., insulin, proinsulin, ICA512 (IA-2), ZnT8, carboxypeptidase H, GM2-1 ganglioside) and synaptic-like microvesicles where gamma amino butyric acid (GABA) is stored (e.g. glutamic acid decarboxylase). The detection of a family of autoantibodies for each autoimmune disease complicates the elucidation of which autoantigen (if any) is central to disease pathogenesis. In the past, the major criteria for defining such autoantigens rested primarily on the ability to immunize with the given antigen and create disease. This is probably an inadequate criterion to define pathogenic autoantigens of spontaneous autoimmune disorders of man and animal models.
It is known that for many tissues, immunization with several different autoantigens can induce disease (e.g., myelin basic protein and proteolipid protein for experimental autoimmune encephalitis). With the development of molecular genetic techniques, more definitive tests of an antigen’s disease relevance are now possible. For example, genes for specific autoantigens can be introduced into ectopic tissues by creating transgenic mice with the gene coding for the autoantigen coupled to a promoter directing ectopic expression. If, for example, destruction of the ectopic tissue is induced, the single autoantigen is sufficient to target autoimmunity. In a similar manner, induction of thymic expression of an autoantigen may be sufficient to induce tolerance to the antigen (see section on “Central Tolerance”). If all autoimmunity is blocked in such transgenic mice, then the given autoantigen is essential for disease pathogenesis. Introduction of autoantigen synthesis by transplanted tissues (e.g., adenovirus gene transfer systems) may allow a series of autoantigens to be rapidly tested to determine whether they are “sufficient” for tissue destruction. Just as important, gene knockouts can be used to directly test the relevance of specific antigens. Baekkeskov and coworkers have found that NOD mice lacking GAD65 develop diabetes. Thus, GAD65 is not essential, though GAD67 may play a role. Of note a knockout of the insulin 2 gene accelerates and a knockout of insulin 1 gene prevents most diabetes of the NOD mouse (93) while knocking out both insulin genes, and replacing insulin with a mutated insulin in the key insulin B chain 9-23 sequence, prevents almost all anti-islet autoimmunity (68).
Autoantibodies usually react with conformational epitopes of their target antigen, and for many autoimmune disorders autoantibodies to multiple different epitopes of even single antigens are targets. In contrast, T lymphocytes target relatively short peptides (8-12 amino acids in length). Peptides that have only one or two similar amino acids have been shown to activate the same T cell receptor. Thus there is enormous potential for a given T cell receptor to react with peptides from multiple different molecules and some of these molecules will have no “obvious” sequence homology (21).  This suggests that many molecules may be “molecular mimics” (peptide mimotopes) and may be one mechanism of initiation autoimmune responses. In that most individuals do not have autoimmune disease, the immune system usually deletes or appropriately regulates autoreactive T cells.
Effector Mechanisms
In most immune disorders, both humoral and cellular arms of the immune system contribute to disease pathogenesis. Autoantibody-mediated disorders include autoimmune hemolytic anemia, immune-mediated thrombocytopenia (ITP), myasthenia gravis, Lambert-Eaton myasthenic syndrome, pemphigus, and Graves’ disease. For each of these disorders, if the mother has autoantibodies, a transient neonatal disease may follow transplacental passage of antibodies. 
Autoantibodies can induce pathology by blocking the function of specific receptors (e.g., hypothyroidism (94) or “geriatric” hypoparathyroidism (95)), by stimulating receptors (Grave’s disease, hypoglycemia associated with anti-insulin receptor autoantibodies), by direct cytotoxicity (hemolytic anemia), by opsonizing cells (hemolytic anemia and ITP), or by deposition of immune complexes (lupus nephritis).  There is also evidence that autoantibodies and B lymphocytes can enhance T cell mediated disorders and thus anti-B lymphocyte therapties (e.g. anti-CD20) are being studied or used in a series of presumably T cell mediated disorders (72).
Cell-mediated immunity is usually associated with tissue destruction (e.g., type 1 diabetes or thyroiditis) either through direct cellular cytotoxicity or by indirect cytotoxic mechanisms (e.g., delayed-type hypersensitivity). The classic pathway of allogeneic tissue rejection is mediated by direct recognition of antigenic peptides complexed with MHC class I molecules by CD8-positive cytotoxic T cells; killing by cytotoxic T cells involves direct cellular contact between T cells and the target cell. The function and expansion of clones of such cytotoxic T cells are dependent upon “help” provided by CD4-positive T cells that respond to antigenic stimulation by releasing a series of lymphokines, including IL-2 .
CD4-positive T cells have been subdivided based on the cytokines they produce (96) into multiple groups of “helper cells”: Th0, Th1, and Th2 and Th17. Th0 cells constitute a small subset of cells in the thymus postulated to react to autoantigens resulting in the production of IL-2 but also low levels of interferon gamma (IFN-g). Th1 cells mediate delayed type hypersensitivity reactions and produce IL-2 and IFN-gamma. Th2 cells produce IL-4, IL-5 and IL-13 and are associated with allergic diseases. Th17 cells secrete IL17 and are strongly associated with multiple autoimmune and inflammatory diseases(10).
An additional mechanism of cell-mediated destruction of target cells involves indirect cytotoxicity mediated by CD4-positive (Th1) lymphocytes. Such CD4-positive lymphocytes, following recognition of their target antigen presented in the context of a class II MHC molecule, secrete a group of cytokines (e.g., interferon gamma), which in turn activates cells such as macrophages to release IL-1, nitric oxide, and free radicals. Each of these molecules can directly kill selected target tissues (97). This second mechanism may be primarily responsible for the autoimmune beta cell destruction of type 1 diabetes and for destruction of xenogeneic tissue transplants. For example, islet specific CD4-positive clones (as will be discussed in later chapters) are sufficient to transfer diabetes (98).
Therapy of Autoimmunity
A series of novel therapies for the prevention and treatment of autoimmunity are being studied. One can broadly divide these into therapies which are antigen-specific and those which are antigen-nonspecific. Examples of antigen-specific therapies include prevention of diabetes in NOD mice by parenteral or oral administration of insulin (99;100), MHC-binding peptide therapies for prevention of experimental autoimmune encephalitis (101) and T cell or peptide vaccination strategies (102). Another form of antigen-specific therapy includes the removal of a disease-inciting antigen (if it is identified). For example, celiac disease is successfully treated by the removal of the wheat protein gliadin from the diet. Studies of the effects of eliminating bovine milk products in neonates are underway for infants at high genetic risk of type 1 diabetes, though recent epidemiologic studies have implicated early introduction of cereals to infants may also increase the risk of developing autoimmune diabetes (103;104).
For endocrine glands it is possible to suppress cellular activity through feedback inhibitory circuits (e.g., administration of thyroxine or insulin inhibits thyroid or beta cells, respectively). Therapy with insulin not only prevents development of diabetes in BB rats and NOD mice (105), but also prevents lymphocytic infiltrates into the islets and beta cell destruction. Protection by insulin in the BB rat requires metabolically active insulin and induction of hypoglycemia with concomitant inhibition of insulin secretion by beta cells. In contrast, non-metabolically active peptides of insulin prevent diabetes in NOD mice. A single report suggests that glucocorticoid therapy in patients with anti-adrenal autoantibodies leads to loss of autoantibodies and prevention of Addison’s disease (106).
Antigen nonspecific therapies include drugs such as rapamycin, cyclosporine A, glucocorticoids, some anti-T cell monoclonal antibodies, IL-2-diptheria toxin conjugates (107;108), and FK506 (tacrolimus). A recent trial of a single course of anti-IL2 receptor antibody followed by chronic mycophenolate mofetil therapy failed to slow loss of C-peptide secretion in patients with new onset diabetes (ADA 2008 oral presentation Trialnet).  This suggests that potentially acceptable “moderate” broad immunosuppression will not be sufficient in type 1 diabetes.  The disadvantage of non-antigen specific therapies is the likely suppression of important immune functions with increased risk of infection and malignancy. In addition, non-selective drugs have unique toxicity unrelated to immunosuppression, such as osteoporosis and diabetes with glucocorticoids and renal toxicity, which is associated with cyclosporine A. Nevertheless, it is clear that potent immunosuppressive drugs such as cyclosporine A, which act by blocking cytokine production by T cells, can prevent certain forms of autoimmunity. When it is possible to use such drugs at low doses with marked therapeutic effect (e.g., cyclosporine A for psoriasis), they become important therapeutic agents. It is also hoped that certain strategies of short-term immunosuppression may lead to a state of long-term tolerance, thereby minimizing the deleterious effects of immunosuppression. For example, treatment with specific monoclonal antibodies directed against the T cell co-receptor CD4 can lead to long-term engraftment of both heart and islet transplants; non-activating anti-CD3 antibodies can cause long-term remission of diabetes in NOD mice and multiple studies now document preservation of islet beta cell function in man for approximately 2 years (109;110).
Another proposed antigen-specific therapy of autoimmunity involves immune deviation, which is thought to induce tolerance following oral administration of antigen. It is hypothesized that the feeding of autoantigens induces T cell production of suppressive cytokines such as TGF-beta upon subsequent encounters with antigen (111). Studies on the mechanism underlying oral tolerance are most advanced for experimental autoimmune encephalitis, where in vivo antibodies to TGF-beta block induction of ‘oral tolerance’ (112). For the most part however, studies of oral tolerance in man have failed to achieve clinical benefit.
The trimolecular complex (MHC molecule, peptide antigen, and T cell receptor) underlying the specificity of T cell responses is an obvious target for therapy of autoimmunity. In experimental disorders with a very limited T cell receptor response, such as experimental autoimmune encephalitis (almost exclusive use of T cells bearing Vbeta8.2 T cell receptor chain), targeting of a specific family of T cell receptors is efficacious in preventing disease. Another approach being pursued is the production of peptides that bind to MHC alleles associated with disease. For example, a peptide binding to a high-risk allele for type 1 diabetes, DQA1*0301/DQB1*0302, might prevent diabetes by preventing the binding of “diabetogenic” peptides.
One of the most exciting pathways for the prevention of autoimmunity in animal models goes under the rubric of “immunologic vaccination.” It has been found that a series of spontaneous and experimental autoimmune disorders can be prevented by administering target autoantigens or peptides of given autoantigens. A single injection of the insulin B chain peptide, amino acids B:9-23, can prevent type 1 diabetes for the life of an NOD mouse (113). Multiple routes of antigen administration have been used successfully to prevent disease in animal models including subcutaneous injection with or without adjuvant, oral or nasal mucosal administration and administration of DNA constructs coding for the autoantigen. Direct production and infusion of regulatory T cells targeting islet antigens has shown promise in animal models (114).
Unfortunately “ Immunologic vaccination” for type 1 diabetes to date has not been effective. For example an altered peptide ligand of insulin peptide B:9-23 in new onset patients failed to slow loss of C-peptide. In addition there is a risk that the peptide will actually exacerbate disease. Large trials of oral insulin and low dose parenteral insulin injections did not delay progression to diabetes overall (115),although the majority of patients in the oral insulin trial who expressed insulin autoantibodies above 80nU/ml (initial trial entry criteria) did have a significant delay in the development of diabetes.  This significant effect (delay of several years in progression) requires confirmation as it is a “subgroup” analysis (93) and a repeat Trialnet study of oral insulin is underway.  Development of improved assays to measure T cell function and autoreactive T cell frequencies will be important in improving the risk benefit ratio for such therapies. Two general assay formats, namely ELISPOT assays (116) and “tetramer” (also termed “multimer”) (117) assays have already greatly impacted studies of T cell responses to infections and are likely to be important for autoimmunity. The ELISPOT assays rely upon culturing lymphocytes and then detecting individual cells and the cytokines they produce. One form of tetramer assay utilizes avidin to couple together four biotin labeled MHC molecules containing a relevant peptide. By having four MHC molecules the affinity for reaction with the receptors of T cells is greatly increased. Such molecular complexes are labeled with fluorescent dyes and are able to bind to the T cell receptors specific for the restricting MHC element and the peptide. Using tetramers and a fluorescent cell sorter, autoreactive T lymphocytes can be identified. It would be a major advance if one could rapidly evaluate therapies for autoimmune disorders in terms of effects upon the pathogenic autoimmune T cells with ELISPOT and/or tetramer analysis (118).

Figure 1.5."Tetramer" for T cell analysis. Tetramers are so-called because avidin binds four biotinylated class II HLA molecules (DQ in this example). The DQ molecule with peptide, especially as a multimer as indicated, then binds to the T cell receptor of T cells specific for that DQ and specific peptide.

Conclusion
Autoimmunity is a result of the failure of a number of immunological processes, from genetically associated HLA risk factors to environmentally derived signals that may overcome tolerance and regulatory control mechanisms. Dissecting the numerous components leading to overt disease is necessary before effective and specific therapies can be developed. Many recent advances, such as the discovery of Toll-like receptors, and the “re-discovery” of regulatory T cells, will greatly aid in our understanding and therefore in our ability to treat the underlying causes of autoimmune disorders.

Reference List - links to PubMed available in Reference List.

Chapter 1 Powerpoint slide set - Updated 8/07

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