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Originally published online as doi:10.1189/jlb.0803394 on December 4, 2003

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(Journal of Leukocyte Biology. 2004;75:586-599.)
© 2004 by Society for Leukocyte Biology

Altering immune tolerance therapeutically: the power of negative thinking

Gérald J. Prud’homme1

Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital and University of Toronto, Ontario, Canada

1Correspondence: St. Michael’s Hospital, 30 Bond St., Room 2013CC, Toronto, ON, Canada M5B1W8. E-mail: prudhommeg{at}smh.toronto.on.ca


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ABSTRACT
 
The etiology of most human autoimmune diseases remains largely unknown. However, investigators have identified several negative regulatory mechanisms acting at the level of innate and/or adaptive immunity. Mutations resulting in a deficiency of some key regulatory molecules are associated with systemic or organ-specific inflammatory disorders, which often have a prominent autoimmune component. Genetic studies have implicated the negative regulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) and other regulatory molecules in human autoimmune diseases. In addition to CTLA-4, key inhibitory molecules include programmed death 1 and B and T lymphocyte attenuator. Transforming growth factor ß1 and interleukin-10 also play major anti-inflammatory and regulatory roles. Tumor cells and infectious agents use negative regulatory pathways to escape immunity. The therapeutic blockage of negative signaling (particularly of CTLA-4) increases immunity against tumor antigens but also induces or aggravates autoimmune diseases. It appears that under normal conditions, the immune system is under strong "negative influences" that prevent autoimmunity and that release of this suppression results in disease. Regulation involves communication between the immune system and nonlymphoid tissues, and the latter can deliver inhibitory or stimulatory signals. Recent studies reveal that the generation of negative signals by selective engagement of inhibitory molecules is feasible and is likely to be of therapeutic benefit in autoimmune diseases and allograft rejection.

Key Words: autoimmunity • CTLA-4 • diabetes • DNA vaccination • inflammation • inhibitory molecules • lupus • PD-1 • regulatory T cells


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INTRODUCTION
 
Despite intensive investigations over a period of decades, the reasons for the breakdown of immune tolerance, which results in autoimmune diseases, have remained elusive. This can be attributed to several factors such as the diversity of organ-specific and systemic autoimmune diseases, the incomplete understanding of tolerance mechanisms, and the numerous disparate immune defects that have been described. The establishment of the danger model [1 ] and the discovery of Toll-like receptors (TLR) and other pattern recognition receptors [2 ] have highlighted the fact that specific signals, usually related to infection, are necessary to initiate an immune response. The requirement for adjuvants in immunization against a wide variety of antigens provides ample support for these concepts. This could lead us to think that the simple absence of danger signals is sufficient to prevent autoimmunity. However, there are compelling reasons for believing that this is only part of the equation and that the immune system is poised to respond, sometimes explosively, if it is not continuously kept in check. The finding that the removal of key negative regulatory elements such as cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) [3 , 4 ] or transforming growth factor-ß1 (TGF-ß1) [5 ] in mice results in autoimmunity and rapidly fatal multi-organ inflammatory syndromes supports this view. In humans, therapeutic CTLA-4 blockade to promote anticancer immunity is associated with autoimmunity in several organs (dermatitis, enterocolitis, hepatitis, and hypophysitis) [6 , 7 ]. Surprisingly, some "danger signals" appear to be endogenous and always present. This may result from an inherent reactivity of T cells against self-peptide/major histocompatibility complexes (MHC), but the nature of this autoreactivity is not well characterized.

It can be hypothesized that under normal circumstances (in the absence of pathogenic agents), the immune system is under predominant "negative influences"; i.e., it is kept quiescent through a series of negative regulatory pathways. These can inhibit innate/inflammatory and adaptive responses. Presumably, only strong adjuvants (usually of infectious agents) can overcome this state by generating appropriate danger signals. This insures that healthy tissues are protected from unnecessary inflammation and injury and that there are few allergic responses to nonpathogenic, foreign antigens. To a large extent, this is consistent with the infectious-nonself hypothesis of Janeway Jr. and Medzhitov [2 ], although it adds an important regulatory dimension. The peripheral tissues themselves might generate some of these regulatory influences, and indeed, B7-family ligands of immunoinhibitory molecules are expressed in nonlymphoid organs. Cytokines are also likely involved, as TGF-ß1, which has pleiotropic, immunosuppressive activity, is produced in latent form by a wide variety of cells, adheres to extracellular matrix proteins, and is normally present in the plasma (reviewed in ref. [8 ]). Following activation, it may thus protect against autoimmunity at the local tissue and systemic levels. In effect, as Piccirillo and I first proposed a few years ago [8 ], potential target cells are not necessarily passive, and they might signal that they are healthy, just as they can signal that they are not. The expression of unaltered self-MHC I molecules, as an example, protects against natural killer (NK) cell-mediated killing through inhibitory receptors [9 , 10 ].

The complexity of the immune mechanisms that control or limit inflammation and/or autoreactivity is immense. Furthermore, a failure at almost any level can have deleterious consequences. It is interesting that there is strong evidence that malignant tumors and some infectious agents use these negative pathways to escape immunity, and this is an unfortunate and often devastating demonstration of the power of negative regulation. Obviously, it is not possible in this short review to discuss the many immunoregulatory influences that are known, but some key pathways are mentioned below.


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ALTERED NEGATIVE REGULATION OF INNATE AND ADAPTIVE IMMUNITY AND RELATED DISEASES
 
Negative regulation affects innate and adaptive immunity with some overlap, and defects in these regulatory pathways have been associated with specific diseases (Table 1 ). There are several molecules controlling innate responses and limiting inflammation. Some block complement activity [15 ], others inhibit inflammatory cytokines [e.g., interleukin (IL)-1 receptor antagonist; ref. 16 ], and some exert a wide variety of anti-inflammatory effects (e.g., TGF-ß and IL-10) [8 ]. Many other regulatory molecules exist, some of which have only recently been identified. This includes NOD proteins, which regulate the inflammatory response (recently reviewed in ref. [12 ]). Mutations of some NOD proteins result in autoinflammatory conditions such as FMF (pyrin mutations), NOMID (cryopyrin mutations), Crohn’s disease (NOD2 mutations), as well as other inflammatory disorders (Table 1) . The Tyro 3 receptor protein tyrosine kinases appear to be another important family [11 ]. They prevent macrophage and dendritic cell (DC) hyperactivity, and their deletion in mice results in T cell/B cell lymphoproliferative disease, broad-spectrum autoimmune disease, and early death [11 ]. The defective macrophages also fail to phagocytose and remove apoptotic bodies. Remarkably, the autoimmune disease has features of rheumatoid arthritis, SLE, pemphigus vulgaris, and Sjogren’s syndrome. This illustrates how dysregulation of mechanisms, which appear to control inflammation primarily, can result in general immune hyperactivity, aberrant presentation of autoantigens, and severe autoimmune syndromes.


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Table 1. Examples of Autoinflammatory Diseases Associated with Failed Negative Regulation

As is well known, NK cells express several negative regulatory molecules, such as killer-cell immunoglobulin (Ig)-like receptors, which characteristically bind to MHC class I molecules and deliver their negative signals through cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) [9 , 10 , 17 ]. In contrast, activating receptors of NK, B, and T cells and other lymphoid cells have cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAM) [9 , 10 , 17 18 19 ]. In many cases, the activating receptor occurs in pair with a closely related ITIM-bearing inhibitory molecule, and both receptors may bind the same or similar ligands [17 18 19 ]. Coaggregation of ITAM- and ITIM-containing receptors by extracellular ligands triggers inhibitory signals. The ITIM undergoes tyrosine phosphorylation by a Src family kinase, providing a docking site for phosphatases with a Src homology 2 (SH2) domain [17 18 19 ]. This includes the SH2 containing tyrosine phosphatase (SHP)-1 and SHP-2 and the SH2 domain containing inositol phosphatase (SHIP), which inhibit signaling pathways. In addition to NK cells, macrophages, DCs, T cells, B cells, mast cells, and granulocytes express diverse families of ITIM-bearing, negative regulatory molecules [17 ].

The SOCS represent an important family of intracellular molecules that can regulate the release of inflammatory cytokines [14 ]. Overexpression of SOCS1 inhibits Janus tyrosine kinase (JAK)1 and JAK2 and blocks interferon-{gamma} (IFN-{gamma}) signaling. Mice deficient in SOCS1 are subject to uncontrolled IFN-{gamma} stimulation and die a few weeks after birth with myelomonocytic infiltration in several organs [14 ]. These are just examples, and the list of negative regulatory molecules is constantly growing.

At the level of adaptive immunity, there is vast literature showing that negative selection of T-lineage lymphocytes first occurs in the thymus. Nevertheless, this process is still not completely understood, and recent studies show that autoantigens thought to be organ-specific are expressed in the thymus, leading to appropriate negative selection. Indeed, mutations of a single transcriptional activator gene, denoted autoimmune regulator, reduce the expression of some self-antigens in the thymus and hamper deletion of the corresponding self-reactive thymocytes [20 21 22 ]. This results in the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome [21 ], a rare, autosomal, recessive disorder characterized by an immune-mediated destruction of endocrine tissues and several other abnormalities. This confirms that a failure of central tolerance results in autoimmunity, although it is presently unclear whether this is relevant to other autoimmune disorders. It is possible that central tolerance deletes only high-affinity T cells, and others leave the thymus and are tolerized in the periphery. Self-reactive B lymphocytes are deleted or anergized in the bone marrow, and it is likely that disruption of this process is relevant to some autoimmune diseases such as SLE [23 ].

In the periphery, lymphocytes, DCs, and macrophages fall under the influence of stimulatory and inhibitory signals (Fig. 1 ). When T cells interact with the APCs in the process of antigen recognition, several pairs of molecules interact to stimulate a response. Most notably, this includes CD28/B7, ICOS/B7RP-1, and CD40L/CD40 interactions [26 , 27 ]. Conversely, inhibitory signals are delivered through CTLA-4/B7 [28 29 30 ], PD-1/PD-L1/L2 [31 32 33 34 35 36 37 38 ], and BTLA/B7x interactions [24 ]. Although CTLA-4 is primarily a T cell protein, T and B cells express PD-1 and BTLA. In addition, the presence in the milieu of inflammatory [e.g., IL-1, IL-12, tumor necrosis factor {alpha} (TNF-{alpha}), and IFN-{gamma}] and regulatory (e.g., TGF-ß1 and IL-10) cytokines can markedly alter responses. It seems probable that the sum of these stimulatory and inhibitory influences determines whether a T cell will respond as well as the type of response generated.



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Figure 1. Stimulatory and inhibitory signals in T cell/antigen-presenting cell (APC) interactions. In the process of T cell/APC interaction and antigen recognition, T cells are subject to signals generated through multiple stimulatory (shown in green) and inhibitory (shown in red) molecules. The figure shows some key molecular interactions, but several more are known, and they are thought to occur primarily within the immunological synapse. The positive signals include the classical signal 1 [T cell receptor (TCR) binding to the MHC/peptide complex] and signal 2 (the summation of multiple positive, costimulatory signals). Key costimulatory interactions include CD28/B7 and inducible costimulator (ICOS)/B7RP-1, and CD40L/CD40 interactions activate the APC and enhance signal 2. In addition, stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-18, IFN-{gamma}) in the milieu, produced by T cells, APCs, or other cells, contribute to the response. These effects are counteracted by multiple inhibitory signals generated through CTLA-4/B7, programmed death-1 (PD-1)/PD-ligand (L)1 or -L2, B and T lymphocyte attenuator (BTLA)/B7x (B7-H4), CD85/MHC I epitopes, and other negative costimulatory interactions [17 , 24 , 25 ]. Suppressive cytokines (e.g., TGF-ß1, IL-10) in the vicinity (produced by lymphoid or nonlymphoid cells) can act on the APC and the T cell to inhibit the response. Whether the T cell is activated or fails to respond likely depends on the sum of these positive and negative influences. Moreover, some cytokines may direct the response to a T helper cell type 1 (Th1; IL-12, IFN-{gamma}), Th2 (IL-4), or regulatory T cell (Tr; TGF-ß or IL-10) phenotype (not shown). Therapeutically, blocking inhibitory signals can enhance T cell responses [e.g., administration of monoclonal antibodies (mAb) against CTLA-4, PD-L1, or B7x], or enhancing negative signals can suppress them (see Table 4 ).

B cells are influenced by negative signals generated by the ITIM-bearing Fc receptor for IgG (Fc{gamma}R)IIB and several other ITIM-containing receptors including CD22, CD5, CD72, CD66a [carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)], Ig-like transcript (ILT)/CD85, PD-1, and leukocyte-associated Ig-like receptor-1 (reviewed in refs. [17 , 39 ]). A number of these inhibitory receptors (e.g., PD-1, CD66a, ILT) are also expressed by T cells and NK cells and in some cases, myeloid cells. Some of the receptors can also inhibit through ITIM-independent mechanisms. Defective signaling through these receptors can result in B cell hyper-responsiveness to B cell receptor (BCR) stimulation. Cross-linking Fc{gamma}RIIB with the BCR suppresses B cell activation, and null mutation of Fc{gamma}RIIB in C57BL/6 background mice results in a SLE-like disease with antinuclear antibodies and fatal glomerulonephritis [40 ]. Moreover, mutations of other molecules involved in B cell inhibitory pathways culminate in autoimmunity (reviewed in ref. [41 ]; Table 2 ). Thus, Lyn-deficient mice manifest SLE-like disease, and their B cells are hyper-responsive to BCR cross-linking. This results from a role of Lyn in inhibitory loops that attenuate BCR signaling, involving CD22 and Fc{gamma}RIIB. This Lyn-related defect might be expressed primarily at the level of peripheral, and not central, tolerance [41 ]. It is interesting that Lyn deficiency has been observed in some patients with SLE [48 ]. CD22 gene KO, not surprisingly, also results in a SLE-like disease [43 ]. Of note, SHP-1 and SHIP are essential for the inhibitory functions of Fc{gamma}RIIB in mice, and absent or decreased SHP-1 results in the autoimmune, motheaten phenotypes in mice [44 ]. In humans, low-affinity variants of Fc{gamma}Rs are associated with lupus nephritis [49 ].


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Table 2. B Cell Hyperactivity and SLE-Like Disease in Inhibitory Molecule Gene KO Mice

It is interesting that gene KO of TACI results in B cell hyperactivity, suggesting that this molecule inhibits B cell activation [45 46 47 ]. Conversely, administration of TACI–Ig interrupts B cell-activating signals generated through B-lymphocyte stimulator (BLys) and a proliferation-inducing ligand and protects lupus-prone mice against disease [50 ]. This is clinically relevant, as overrexpression of BLys is common in SLE patients [51 ].

Cells of hematopoietic origin express numerous ITIM-containing receptors, and a notable example is signal regulatory phosphatase {alpha}, which binds to the CD47, expressed on erythrocytes and virtually all other cells. Macrophages rapidly engulf and destroy red blood cells deficient in CD47 [52 ], and this might play a role in some clinical forms of hemolytic anemia [52 ]. Moreover, CD47-deficient NOD mice spontaneously develop severe, lethal autoimmune hemolytic anemia [53 ].

As in the thymus, apoptosis is an important event in the periphery, and this has been amply demonstrated in mouse models of defective apoptosis, particularly those involving Fas or Fas ligand deficiency [54 , 55 ]. These mice develop systemic autoimmune disorders characterized by T cell and B cell hyperplasia, increased inflammatory cytokine production, polyclonal hypergammaglobulinemia, autoantibody production, and immune complex disease. Bcl-2 transgenic mice also have defective B cell apoptosis and autoantibody production [56 ]. Presumably, in these cases, activated T cells or B cells fail to be deleted when they are no longer required, resulting in uncontrolled inflammation and autoimmunity.

Immune reactivity is further controlled by various types of Tr, which are still in the process of being characterized [13 , 57 58 59 60 61 62 63 64 ]. They can be broadly divided into two subsets, i.e., the natural Tr cells of CD4+CD25+ phenotype, which constitute 5–10% of peripheral T cells, and the stimulation-induced (or adaptive) Tr cells identified in various models of inflammation, alloreactivity, or autoimmunity. Natural CD4+CD25+ Tr cells play an important role in limiting autoimmunity and appear to act by a cytokine-independent, but contact-dependent, mechanism (reviewed in ref. [57 ]). Depletion of CD25 (IL-2R{alpha} chain)-positive Tr cells results in autoimmune diseases, and their adoptive transfer protects against these conditions [57 ]. The gene Foxp3, encoding the scurfin transcriptional regulator, appears to be necessary for the differentiation of natural CD4+CD25+ Tr cells (reviewed in ref. [13 ]). A Foxp3 mutation in scurfy mice results in the absence of these Tr cells and early death from a multi-organ inflammatory disorder similar to the CTLA-4 or TGF-ß1 null phenotypes. Mutations of Foxp3 in humans also result in a severe autoimmune syndrome (IPEX) [13 ], usually characterized by enteropathy, polyendocrinopathy [thyroid disorders, type 1 (autoimmune) diabetes mellitus (T1D)], and eczema.

In contrast, induced (adaptive) Tr cells probably differentiate from CD4+CD25– T cells and act principally by secreting regulatory cytokines such as TGF-ß1 (Th3 cells) [58 ] or IL-10 and TGF-ß1 (Tr1 cells) [59 , 60 ]. The differentiation of Tr1 cells is likely dependent on some distinct DCs that promote IL-10 production and express tolerogenic costimulatory molecules [59 , 60 ]. This cytokine dependency appears to distinguish them from the natural Tr cells, but this question is not fully resolved. Indeed, there are some contradictory reports about the cytokine independency of natural CD4+CD25+ Tr cell activity, and this is an area where important future developments are likely to occur. In fact, IL-10 (±TGF-ß)-producing CD4+CD25+ Tr cells are protective in animal models of colitis [59 60 61 62 ].

A major limitation is that Tr cells cannot easily be defined on the basis of cell-surface markers, as activated T cells also express CD25, and other markers are not specific. However, the glucocorticoid-induced TNF receptor (TNFR) family-related gene (GITR) is predominantly expressed on CD25+CD4+ and CD25–CD4+ Tr cells [64 ], which regulate the mucosal-immune responses and intestinal inflammation. It is interesting that both types of GITR+ Tr cells express CTLA-4 intracellularly, proliferate poorly, and produce IL-10 and TGF-ß. The function of CTLA-4 in Tr cells remains controversial, and some studies support a role in suppression, and other studies do not [62 ]. At any rate, it appears that depletion of CD25+ Tr cells combined with CTLA-4 blockade synergize in promoting immunological tumor rejection [65 ], suggesting that the two pathways are at least partly independent.

A highly relevant, recent study [63 ] indicates that the injection of anti-CD3 antibodies in NOD mice induces Tr cells, which appear to mediate remission of T1D. CD3 antibody treatment resulted in the generation of CD4+CD25+ Tr cells, which acted by producing TGF-ß, and this effect was negated by CTLA-4 blockade. It is interesting to note that these Tr cells could be generated in mutant mice that lack natural CD4+CD25+ Tr, suggesting that they originate from a CD25– population. Furthermore, following treatment, there was long-lasting TGF-ß production by CD4+ cells. These findings might be relevant to clinical CD3 antibody immunotherapy of T1D, other autoimmune diseases, and allograft rejection. CD3-induced Tr cells may be similar to superantigen-induced Tr cells, as discussed later.


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CTLA-4 AND RELATED NEGATIVE REGULATORY MOLECULES
 
The negative regulator CTLA-4 is the best-studied and possibly the most potent T cell inhibitory molecule [28 29 30 ], and it is expressed by T cells after activation and like the positive costimulatory molecule CD28, binds to B7-1 (CD80) and B7-2 (CD86) on the membrane of APCs. It then down-regulates T cell reactivity by mechanisms that are not fully elucidated. CTLA-4 competes with CD28 for ligand binding but is also capable of delivering inhibitory signals (recently reviewed in refs. [25 , 66 ]). CTLA-4 is induced following TCR engagement but is mostly stored in lysosomes, where it is degraded in the absence of further stimuli [25 ]. However, upon TCR stimulation, CTLA-4 shuttles to the membrane and modulates the immunological synapse. The recruitment of CTLA-4 is proportional to the intensity of the synapse, suggesting that it regulates high-affinity T cell responses. Notably, cross-linking of CTLA-4 inhibits early signaling events and later events involving IL-2 secretion and proliferation. Furthermore, there is evidence that the engagement of CTLA-4 promotes secretion of TGF-ß1 by T cells [67 68 69 70 ] and that this contributes to reduced immunity against parasites [69 ]. Other negative receptors may have similar effects. Notably, Saverino et al. [70 ] studied the effects of the human CD85 and CTLA-4 inhibitory receptors on the modulation of cell-mediated, immune responses in vitro. They report that cross-linking of CD85 or CTLA-4 molecules on cultured T cells using specific mAb and goat anti-mouse antiserum inhibits antigen-specific T cell proliferation. It is interesting that they found that inhibition was always paralleled by increased production of IL-10 and TGF-ß, and IL-2, IFN-{gamma}, and IL-13 were decreased. Blocking antibodies to both inhibitory receptors enhanced T cell proliferations and production of IL-2, IFN-{gamma}, and IL-13. The mechanisms responsible for the increased production of TGF-ß or IL-10 after the cross-linking of inhibitory receptors have not been elucidated.

Studies of CTLA-4 null mice established the negative costimulatory nature of this molecule beyond doubt [3 , 4 ]. As mentioned previously, these mice develop a rapidly fatal, lymphoproliferative disorder, where polyclonal T cell activation is observed as early as 5–6 days after birth. The mice die at 3–4 weeks of age with blast cell infiltration of the heart, pancreas, liver, and lung. There is general activation and proliferation of T cells as wells as hypergammaglobulinemia. The CD4 T cell subset is more severely affected by this mutation, and the activated T cells produce several cytokines [IL-2, IL-4, IL-6, IFN-{gamma}, granulocyte macrophage-colony stimulating factor (GM-CSF)]. In vitro, the proliferation of the naïve T cell during primary stimulation is only moderately increased, but it is markedly increased upon restimulation. Thus, CTLA-4 is most important in controlling previously activated T cells.

CTLA-4 is underexpressed in autoimmune, diabetes-prone NOD mice [71 ]. In humans, some polymorphisms of the gene, which alter CTLA-4 levels or function, increase susceptibility to T1D and other autoimmune diseases [72 73 74 75 ]. CTLA-4 blockade with mAb provokes autoimmunity in mice [76 , 77 ] and exacerbates T1D and experimental autoimmune encephalomyelitis (EAE; Table 3 ) [29 , 78 ].


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Table 3. Autoimmune Diseases Associated with CTLA-4 Gene Polymorphisms or Aggravated by Inhibitory Receptor Blockade

Several genetic studies provide a strong indication that CTLA-4-related dysfunction is a major factor in some autoimmune diseases. For instance, Ueda et al. [74 ] recently identified polymorphisms of the CTLA-4 gene relevant to Graves’ disease, Hashimoto’s thyroiditis, and T1D. It is interesting that disease susceptibility mapped to a noncoding area, which is associated with lower mRNA levels of a soluble, alternative splice form of CTLA-4. This molecule is found in human serum and has inhibitory effects on T cell responses in vitro [86 ]. In NOD mice, there was also reduced expression of a splice form, but in this case, it encoded a molecule missing the B7-binding domain that might mediate ligand-independent suppression. These findings suggest that the regulation of CTLA-4 expression, including a soluble version, is finely tuned and that reduced expression of splice variants predisposes to autoimmune disease. Moreover, polymorphisms of the CTLA-4 coding sequence also appear to be relevant. Thus, Kouki et al. [73 ] reported that CTLA-4 gene polymorphism at position 49 in exon 1 (G is detrimental instead of A) reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves’ disease. However, the differences between alleles were only moderate. In any case, in addition to T1D and thyroiditis, polymorphisms of CTLA-4 in coding or adjacent-noncoding regions have been associated with an increased incidence of several autoimmune diseases including MS [80 ], MG [82 ], RA [84 ], AHA [79 ], SLE, and other autoimmune diseases (reviewed in ref. [80 ]; Table 3 ).

PD-1 is another negative regulatory molecule that has recently attracted much attention and has relevance to disease [25 , 31 32 33 34 35 36 37 38 ]. Its cytoplasmic segment contains an ITIM and an immunoreceptor tyrosine-based switch motif, and it probably acts by mechanisms quite different from CTLA-4. Nevertheless, there are important similarities, as PD-1, like CTLA-4, is expressed following T cell activation and binds members of the B7 family. Although CTLA-4 binds to B7-1 and B7-2, expressed mostly by professional APCs, PD-1 binds to PD-L1 (B7-H1) and PD-L2 (B7-DC), expressed by APCs and cells of some organs or tissues such as the heart, lung, liver, and placenta [35 , 87 , 88 ]. Endothelial cells [31 ], keratinocytes [31 ], islet cells [33 ], and central nervous system (CNS) cells [32 ] also express PD-L1, and in these sites, its expression is often enhanced by IFN-{gamma} stimulation. Notably, PD-L1+ human endothelial cells suppressed T cell cytokine production [31 ]. It is also noteworthy that PD-1 is expressed by B cells and may contribute significantly to the regulation of these cells.

The expression of PD-L1 by lymphoid and nonlymphoid cells may have important consequences toward tolerance. Indeed, gene KO of PD-1 results in a systemic, lupus-like disease in C57BL/6 mice, presumably involving B cell dysregulation [42 ], or an organ-specific autoimmune cardiomyopathy in BALB/c mice [89 ]. In humans, a polymorphism of the PD-1 gene is associated with SLE [83 ]. Moreover, in view of their distribution, these ligands might exert regulatory effects in conditions as diverse as vascular diseases, skin inflammatory diseases, organ-specific autoimmune diseases, allograft rejection, and graft-versus-host disease (GVHD).

In one study, PD-L1/Ig was found to have an inhibitory effect in vitro as well as in vivo in a cardiac allograft model [36 ]. PD-L1/Ig administration in CD28 null recipients or in conjunction with immunosuppression in fully MHC-disparate combinations markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment. Thus, PD-L1/L2, expressed by cardiomyocytes or other cells, may be protective at the level of the target peripheral tissues. In GVHD, blockade of PD-1 engagement by anti-PD-1 mAb or PD-L1/Ig aggravated disease by an IFN-{gamma}-dependent mechanism [34 ]. In this case, PD-L1/Ig enhanced responses, unlike another study [36 ], possibly because it was constructed differently (it had a mutated mouse IgG2a-Fc segment instead of a human IgG1-Fc segment) and masked PD-1 without signaling. In most studies, anti-PD-1 antibodies and PD-L1/Ig similarly enhanced responses, suggesting that PD-1 signaling was blocked. However, PD-L1 might also bind to a stimulatory receptor (not yet characterized). Indeed, there is evidence that PD-L1/L2 binds to stimulatory and inhibitor ligands, and mutations that abolished PD-1-binding capacity could still costimulate proliferation and cytokine production of T cells from normal and PD-1-deficient mice [38 ].

It is most interesting that PD-L1 expression in situ has been identified in a variety of human tumors [35 ]. It was found positive on most carcinomas of the colon, breast, stomach, lung, liver, bladder, ovary, cervix, larynx, thyroid, and salivary glands. Moreover, in mice vaccinated against tumor antigens, PD-L1 expression by tumors imparted resistance against immune rejection [37 , 90 ]. This effect could be mediated, at least in part, by a ligand other than PD-1. Remarkably, Dong et al. [90 ] found that tumor-associated PD-L1 increased apoptosis of antigen-specific human T cell clones in vitro, and this effect was mediated largely by one or more receptors other than PD-1. Although further studies are required, these findings suggest that many tumors can escape immunity by expressing PD-L1. There is also considerable evidence that tumors can escape by producing TGF-ß and/or other immunosuppressive mechanisms [91 , 92 ]. Indeed, reducing the effects of TGF-ß might be of therapeutic benefit [91 ]. It seems increasingly probable that tumors survive immunity against tumor antigens by a Darwinian-like selection process, involving acquired expression of immunosuppressive molecules. At any rate, there are few alternative hypotheses to explain why so many malignant tumors express these inhibitory molecules. It follows from this hypothesis that clinically detectable tumors will be highly resistant to immunity, and this has been amply demonstrated in clinical studies.

Further confirming the relevance to tolerance, PD-1/PD-L1 pathway blockade with mAb rapidly induces T1D in NOD mice [33 ] and exacerbates myelin oligodendrocycte glycoprotein-induced EAE in mice [32 ]. It is interesting that PD-L1 was expressed in inflamed islets in T1D and expressed increasingly in the CNS of mice in parallel with the severity of EAE. Treatment of NOD mice with PD-L1/Ig is detrimental [85 ], suggesting that it blocks PD-1 signaling or binds to a stimulatory ligand.

The most recent addition to the inhibitory receptor family is BTLA [24 ]. This molecule shares many similarities with CTLA-4 and PD-1. Its expression is induced upon activation of T cells and B cells. Early on, it is expressed by Th1 and Th2 cells, but in highly polarized cells, expression is retained by Th1 cells only [24 ]. BTLA has two cytoplasmic ITIMs and is subject to inducible tyrosine phosphorylation and association with SHP-1 and SHP-2. Its ligand is a new member of the B7 family, variously termed B7x [24 ], B7-H4 [93 ], or B7S1 [94 ], which uncharacteristically for this family, appears to be anchored to the membrane by a glycosylphosphatidylinositol link [94 ]. APCs and nonlymphoid cells express B7-H4, and mRNA expression can be detected in many tissues, although some of these are negative for B7-H4 protein by immunohistochemistry [93 ]. BTLA null mice have increased T cell responses and more severe EAE [24 ]. It is striking that injection of B7-H4/Ig fusion constructs attenuates T cell responses [93 ], and mAb that bind to B7-H4 enhance immunity [93 , 94 ] and aggravate EAE [94 ]. B7-H4 (B7x), like PD-L1, is expressed by tumor cells and may inhibit antitumor immunity [95 ] (Fig. 2 ).



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Figure 2. Tumors escape immunity by a variety of mechanisms. Tumor-associated antigens (TAAs) are usually poorly immunogenic, and the tumor cells lack positive, costimulatory molecules. Moreover, these cells can stop expressing the TAA or become defective in their ability to present MHC/peptide complexes (denoted != in the figure). Even in situations where T cells are strongly activated against a TAA, their activity can be blocked by inhibitory signals originating from the tumor. This includes production of inhibitory cytokines (particularly TGF-ß) and the expression of B7 family molecules [PD-L1, B7x (B7-H4)], which engage T cell immunoinhibitory molecules such as PD-1 and BTLA. In addition, the tumor-derived cytokines (e.g., TGF-ß and IL-10) and probably other signals can induce DCs to differentiate to a tolerogenic phenotype. These DCs promote the differentiation of Tr cells, rather than effector T cells and down-regulate the antitumor immune response. Tr cells can be of the natural phenotype (CD4+CD25+), probably suppressing effector cells by direct cell contact, or of the induced (adaptive) phenotype, probably inhibiting by secreting regulatory cytokines (TGF-ß and IL-10). Tumor resistance to immunity can be reduced, at least in part, by blocking PD-1/PD-L1 interactions, neutralizing regulatory cytokines, or depleting CD4+CD25+ Tr cells. Tumors rarely express B7, but CTLA-4 blockade with mAb presumably reduces the threshold for T cell activation at the time of TAA presentation by the APC. Tumor cells can also escape immunity by other mechanisms, as listed in the figure. IDO, Indoleamine 2,3-dioxygenase.

Recent studies suggest that deficiencies of PD-1 or BTLA produce much less-severe phenotypes than CTLA-4 deficiency and are characterized by limited autoimmunity or a propensity to autoimmunity. This suggests that PD-1 and BTLA are involved in the "fine tuning" of immune responses, diminishing their magnitude without necessarily having a dramatic effect. In autoimmune diseases, negative regulatory signals are unlikely to be totally absent, except in rare cases, but rather, are diminished. This appears to be the case relative to susceptibility to diabetes and thyroiditis [74 ], where CTLA-4 signaling is altered but not absent. CTLA-4 gene KO can be seen as an extreme exaggeration of these natural phenotypes. It seems likely that autoimmunity will result only when a diminution in any of these inhibitory signals is combined with other factors predisposing to autoimmunity, whether they are environmental or genetic. Moreover, the expression of PD-L1/2, and perhaps also B7-H4, by cells in several tissues or tumors suggests that they mediate lymphocyte-tissue interactions, which down-regulate immunity. This might be the last line of defense that cells have against autoimmune destruction.


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ENHANCING TUMOR IMMUNITY BY CTLA-4 BLOCKADE
 
There are many potential reasons why tumor cells escape immunity [37 , 65 , 90 91 92 , 95 96 97 ] (Fig. 2) . First, in terms of antigen expression, tumor cells are very similar to normal cells [97 ]. When they do overexpress antigens, these are often self-molecules (TAAs), for which there is at least some degree of tolerance. Moreover, they lack costimulatory molecules, which could promote the direct activation of T cells, and the introduction of B7 into tumor cells is sufficient in some cases to lead to tumor elimination. In any case, it is difficult to conceptualize why the immune system would attack tumor cells bearing only self-molecules, except in the context of an autoimmune disease. Nevertheless, tumor cells do express mutant molecules that can be targeted and give rise to rejection. If an antitumor response is initiated against mutated or wild-type proteins, the tumor cells might escape by down-regulating expression of the antigen, MHC molecules, or other components required for antigen presentation [96 , 97 ]. Conversely, as mentioned previously, they can up-regulate the expression of immunoinhibitory molecules. In addition, authors [92 ] have reported that tumors can block T cell immunity by depleting tryptophan. T cells require high levels of tryptophan, and tumor cells deplete it by producing the enzyme IDO. Tumor cells might also resist apoptosis or induce apoptosis of responding T cells [96 ]. All these mechanisms can be documented in human tumors and/or experimental models, although it is not easy to establish their clinical significance [97 ]. It is highly likely that future studies will demonstrate more escape mechanisms. It is interesting that there is increasing evidence that normal cells can use some of the mechanisms used by tumor cells to resist autoimmunity. The immune system may neglect tumor cells, but several studies of anti-CTLA-4 blockade demonstrate that the potential for reactivity exists. It has been proposed that CTLA-4 blockade, performed by injecting anti-CTLA-4 mAb, lowers the threshold for T cell activation so that autoreactive T cells are activated [28 , 30 ]. These antibodies might also reduce the activity of a population of CTLA-4-expressing Tr cells. Some of the effector T cells generated have specificity for tumor antigens, and others react to normal tissues in an autoimmune manner [6 , 7 , 28 , 30 ]. In mice, CTLA-4 blockade has led to the rejection of transplanted tumors of many types (carcinomas, lymphomas, and sarcomas), provided they have sufficient immunogenicity. Indeed, CTLA-4 blockade is of little use in poorly immunogenic tumors, but when applied with concurrent vaccination with, for example, irradiated GM-CSF-producing tumor cells, it is effective against B16 melanoma [98 ], carcinoma of the mouse prostate [99 ], and mammary carcinoma [100 ]. Effective responses are highly dependent on CTL generation and can lead to autoimmunity against normal cells bearing the antigen (melanocytes->autoimmune vitiligo; prostate antigen->prostatitis). Clinical trials of CTLA-4 blockade revealed antitumor responses, but autoimmunity was a very significant drawback [6 , 7 ]. Notably, anti-CTLA-4 mAb treatment induced extensive lymphocytic infiltration and tumor necrosis in melanoma patients previously immunized with GM-CSF-secreting tumor cells but was minimally effective in patients immunized against defined melanocytic antigens [6 ]. In a landmark study [7 ], investigators treated 14 patients with metastatic melanoma by intravenous administration of a fully human anti-CTLA-4 antibody combined with vaccination against two MHC class I-restricted peptides from the gp100 melanoma-associated antigen. This treatment induced tumor regression in three patients (21%) but was associated with serious autoimmunity in close to half of the patients. Lesions included dermatitis, vitiligo, enterocolitis, hepatitis, pneumonitis, and hypophysitis. Most patients, however, had only one organ or tissue involved by autoimmunity. Some patients in these studies developed antinuclear antibodies, antithyroglobin antibodies, and rheumatoid factors [6 , 7 ]. Although only some patients responded, these studies document that antitumor responses can be generated by CTLA-4 blockade. Furthermore, they confirm that CTLA-4 has regulatory properties in humans similar to those in mice and that blockade breaks immune tolerance to various self-antigens. These autoimmune responses are diverse and are not just against the antigens used for vaccination, which is reminiscent (albeit in much milder form) of the phenotype of CTLA-4 null mice. Treatment yielded an increased number of CD4 cells with an activated phenotype, and there was no depletion of CTLA-4+CD4+CD25+ cells (regulatory phenotype). There are very few other examples, if any, where tolerance has been broken in such a dramatic way in humans by blocking the activity of a single receptor.


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INFECTIOUS AGENTS ACTING NEGATIVELY
 
I first alluded to danger signals as agents that turn on innate immunity, but infectious agents can also act on inhibitory molecules in many ways that are beneficial to them. This capacity of infectious agents to subvert the immune system has been documented extensively. Decreased immunity sometimes results from Th1/Th2 shifts. However, bacteria, viruses, and parasites can induce regulatory cytokine production (particularly of TGF-ß1) and various Tr cells [101 ], which on the one hand promotes survival of the pathogen but on the other hand, probably limits associated immunopathology. This is likely a viable solution, acquired through evolution, when the immune system is incapable of completely eliminating an infectious agent.

Pathogenic viruses, for example, produce cytokines (e.g., vIL-10) or anticytokines (e.g., soluble receptors for TNF, IL-1ß, IFN-{gamma}, IFN-{alpha}/ß, chemokines, and IL-18) that have anti-inflammatory effects [102 ]. In addition, some viruses can inhibit IFN signaling, antigen processing or presentation, complement activation, and other immune mechanisms [102 , 103 ]. Some inhibitory, pathogen-related effects may be mediated through DCs and/or altered activity of Tr cells. Yersenia V-antigen induces IL-10-mediated suppression by a TLR2- and CD14-dependent mechanism [104 ]. In fact, IL-10 protects against inflammatory bowel disease in various models and is obviously a key anti-inflammatory cytokine [61 ]. This is relevant to Crohn’s disease, which as mentioned above, is an important inflammatory disease associated with NOD2 mutations. Similarly, Bordetella pertussis induces IL-10-producing Tr1 cells through filamentous hemagglutinin (FHA), which acts on DCs [105 ]. FHA-stimulated DCs produce increased amounts of IL-10 and reduced amounts of IL-12.

Some bacteria can act on specific receptors to turn off T cells. Notably, Boulton and Gray-Owen [106 ] reported that Neisseria gonorrhoeae have a suppressive effect on the host-immune response. They observed that N. gonorrhoeae Opa proteins bound to CEACAM1 expressed by CD4+ T lymphocytes. CEACAM1 is an ITIM-bearing receptor that associates with the tyrosine phosphatases SHP-1 and SHP-2 and down-regulates responses. They propose that this mechanism accounts for the poor immunity and immunological memory against this pathogen.

As noted previously, Tr cells have been implicated in the persistence of infection. Recently, investigators [107 ] found that CD4+CD25+ regulatory T cells control the persistence of Leishmania major in the skin after healing in resistant C57BL/6 mice. During the course of infection, the CD4+CD25+ T cells accumulate in the dermis, where they suppress effector T cells. This suppression is mediated by IL-10-dependent and -independent mechanisms. It is interesting that although they block elimination of the pathogen, some Tr cells appear to have a beneficial effect by limiting immunopathology, as exemplified in mice in Helicobacter pylori gastritis [108 ] and Helicobacter hepaticus-induced colitis [62 ].

Bacterial superantigens (SAg) also induce Tr cells [109 ]. It is well known that repeated SAg injections in mice induce partial deletion and anergy of responding T cells. However, it now appears that the anergic T cells also have strong, suppressive activity. Staphylococcal enteroxin A or enterotoxin B induced these cells in TCR-transgenic mice bearing appropriate SAg-binding TCR-Vß elements. It is interesting that SAg-induced Tr cells were much more potent than natural CD4+CD25+ Tr cells and were identified in the CD25+ and CD25– subsets of CD4+ cells. SAg-induced Tr cells suppressed by cell contact and cytokine-mediated mechanisms. Notably, a mixture of anti-CTLA-4, anti-TGF-ß, and anti-IL-10 receptor antibodies (but not each antibody alone) partially reversed suppression of SAg-induced Tr cells and totally reversed suppression of natural CD4+CD25+ Tr cells. These findings reveal similarities and differences between the two Tr cells types and also suggest that suppressive mechanisms dependent on CTLA-4 and cytokines are additive. It seems likely that SAg-producing bacteria are protected from immunity by this powerful regulatory mechanism. Moreover, SAg stimulation appears to yield Tr cells that are similar to those induced by CD3 antibody injection [63 ]. In both cases, there is massive activation of T cells and production of high levels of several cytokines, perhaps explaining this similarity.

Not surprisingly, CTLA-4 blockade enhances the immunity induced by vaccination against some agents. This was the case, for example, in vaccination against cryptococcal [110 ], mycobacterial [111 ], or leishmanial antigens [112 ]. However, CTLA-4 blockade is not always beneficial in infectious diseases, as it sometimes exacerbates inflammatory lesions associated with infection [112 ]. Thus, the timing of any intervention has to be carefully considered.


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ALTERING NEGATIVE SIGNALS IN AUTOIMMUNE DISEASES
 
To prevent allograft rejection or autoimmunity, immunologists have frequently resorted to the elimination of positive signals by costimulatory receptor blockade (e.g., with CTLA-4/Ig) [113 ] or the neutralization of inflammatory cytokines with mAb or soluble cytokine receptors (e.g., TNFR-{alpha}/Ig) [114 ]. In contrast, to stimulate antitumor immunity, they have delivered cytokines such as IL-2 [115 ]. These approaches have met with some clinical success, although there is a need for more specific and more powerful therapies. Evidently, the ultimate therapy would be free of adverse effects (unlike IL-2 therapy) and turn on or shut off only the relevant immune response, but this remains an elusive goal. In this respect, the manipulation of negative signals, by increasing or decreasing them as required, provides a promising avenue for the future. The first indication that this was feasible came from the field of tumor immunity, as described above. However, in the case of autoimmunity, it is necessary to increase, rather than decrease, negative signals with ligands designed for this purpose. The administration of B7-H4/Ig, binding to the negative regulatory molecule BTLA, has already been mentioned as a recently developed approach for down-regulating immune responses. However, CTLA-4, which is a more potent regulatory molecule, has been a favorite target.


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ENGAGING CTLA-4 TO ATTENUATE ALLOREACTIVE OR AUTOIMMUNE RESPONSES
 
An alternative approach to immunotherapy involves delivery of molecules that bind selectively to CTLA-4 (Table 4 ) or CD28. Unfortunately, B7-1 and B7-2 bind (with different affinities) to CTLA-4 and CD28. Thus, B7/Ig fusion proteins clearly engage CD28 and CTLA-4, although it is interesting that they generally have immunostimulatory effects. The reasons for this stimulatory dominance are unclear, but I hypothesize that this is related to CTLA-4 blockade by soluble B7/Ig, as a mutated B7/Ig molecule not recognizing CD28 (but still binding to CTLA-4) retained a stimulatory capacity (my unpublished observations). Engaging CTLA-4 has proved difficult, as antibodies mask CTLA-4 and prevent negative signals, as alluded to above. Nevertheless, selective engagement of CTLA-4 in a way that delivers negative signals has been achieved with cell-bound, single-chain antibodies or mutated B7 molecules.


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Table 4. Selective Engagement of CTLA-4 to Depress Immunity

Some authors [116 ] designed single-chain antibodies (scFv) recognizing CTLA-4 specifically and demonstrated that in membrane-bound forms, they inhibit T cell responses. Coexpression of anti-CTLA-4 scFv with anti-CD3 and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated CD4+ and CD8+ T cells [116 ]. Inhibition was only effective when the CTLA-4 ligand was coexpressed on the same cell membrane as the TCR ligand. T cells from DO11.10 and 2C TCR transgenic mice were also negatively regulated in response to MHC/peptide complexes. Notably, Hwang et al. [117 ] reported that targeted ligation of CTLA-4 in vivo by a membrane-bound, anti-CTLA-4 antibody (7M) prevented rejection of allogeneic tumor cells. Additionally, the CTLA-4-positive T cells that encountered 7M+ tumor cells in vivo were hyporesponsive, with reduced cytolytic activity and secretion of cytokines (IL-2 and IFN-{gamma}), when restimulated in vitro with tumor cells bearing their target antigen. Thus, selective engagement of CTLA-4 down-regulates T cell activity in vitro and in vivo. A caveat is that it may be difficult to deliver a selective CTLA-4 ligand in a clinically relevant setting, as soluble forms are not inhibitory. It is also unknown whether the host could generate a neutralizing, immune response against the scFv fragment, although this should be attenuated by CTLA-4 engagement. Nevertheless, cells that are transplanted for therapeutic purposes (including stem cells and islet cells) could be engineered to express an appropriate, membrane-bound CTLA-4 ligand. This could be an effective way of preventing rejection in cases where there are allogeneic or autoimmune responses against the transplanted cells.

The CTLA-4 ligand can also be carried to a cell or target tissue as part of a bispecific antibody [118 , 119 ]. Notably, Vasu et al. [119 ] administered an anti-CTLA-4 antibody that was coupled to an antibody specific for the thyrotropin receptor. This bispecific antibody (BiAb) accumulated in the thyroid and prevented development of EAT in mice immunized with mouse thyroglobulin. Lymphocytes from BiAb-treated mice showed a significant reduction in their ability to proliferate and to produce IL-2, IFN-{gamma}, and TNF-{alpha} in response to thyroglobulin stimulation. Furthermore, treated mice had lower antithyroglobulin antibodies and lymphocytic infiltration of the thyroid. CD4+CD25+ regulatory T cells were increased in numbers and appeared to exert suppression by secreting TGF-ß1. These findings suggest that the engagement of CTLA-4 expressed on activated, autoaggressive T cells in close proximity to the thyroid can increase the number of regulatory T cells and their ability to produce TGF-ß1 with a reduction in inflammatory cytokine release and amelioration of EAT.


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MUTANT B7 MOLECULES
 
CTLA-4 can also be engaged by mutated B7-1 molecules, which differ only slightly from the wild-type molecule and hence, are not likely to be strongly immunogenic. Indeed, murine [122 ] and human [123 ] B7-1 can be mutated to produce molecules that do not bind CD28 (or have very low affinity) but still bind to CTLA-4. Guo et al. [122 ] generated a panel of these B7-1 (murine) mutants, from which I selected a suitable candidate capable of binding CTLA-4 but not CD28 for these studies. It results from a single amino acid substitution of B7-1, i.e., W88 > A, and is designated B7-1wa.

For DNA vaccination studies, we constructed expression plasmids encoding native B7-1 or B7-1wa, which are expressed on the cell membrane, or secreted Ig-containing variants of these molecules [120 ]. In a matrix-bound state, B7-1wa–Ig inhibited CD3-mediated T cell activation, unlike unmutated B7-1–Ig, which was stimulatory. In vivo, we found that DNA covaccination with B7-1wa cDNA blocked induction of immunity against a xenoantigen and reduced ongoing autoimmune responses against insulin in NOD mice with T1D (insulin-dependent diabetes mellitus).

In these experiments, we inoculated NOD mice with plasmids encoding preproinsulin (PPIns; an important target antigen in T1D) alone, B7-1wa alone, or both molecules (bicistronic plasmid or two plasmids) [120 ]. The spleen cells of mice injected with blank, B7-1, or B7-1wa plasmids responded equally well to insulin. In contrast, the spleen cells of NOD mice inoculated with the B7-1wa/PPIns vector had essentially no response to insulin in vitro. IFN-{gamma} and IL-4 secretion was severely depressed. The response to glutamic acid decarboxylase 65 (GAD65; another key target antigen) was not significantly altered, suggesting antigen specificity of tolerance induction. Cell-mixing experiments revealed that the spleen cells of insulin-tolerant mice could not suppress the anti-insulin response of spleen cells of naive NOD mice. IL-10 and TGF-ß1 have frequently been implicated as suppressive cytokines in this type of assay [8 ], but in this case, blocking antibodies against either had no effect. However, in more recent DNA vaccination studies, where we used an insulin-GAD65 fusion protein as a target antigen, we noted an increase in IL-10 production upon antigenic stimulation (unpublished observations). Thus, at present, it appears that the mechanism of tolerance induction could involve T cell anergy or generation of IL-10-producing regulatory T cells, depending on the target antigen or other factors.

In any case, the most important outcome of this study was that the NOD mice inoculated with pB7-1wa/PPIns, but neither pB7-1wa nor pPPIns alone, had significantly ameliorated insulitis and a disease incidence ~60% lower than control mice up to 34 weeks of age when the experiments were terminated. Recently, Yigang Chang et al. (manuscript in preparation) obtained similar findings in a DNA vaccination study but with a modified approach. They administered genes encoding membrane-bound insulin and a chimeric-soluble B7-1wa/Fc/CD40L fusion protein into prediabetic, female NOD mice. This ameliorated autoimmune diabetes, and presumably, the CD40L segment attaches this molecule to APCs and might also activate these cells in a beneficial way. I hypothesize that in all such studies, B7-1wa must somehow be membrane-bound, as I have found soluble B7-1wa/IgG1 to be immunostimulatory (unpublished observations).

It is interesting that therapy with CTLA-4–Ig, a well-studied, immunoinhibitory molecule, is not always beneficial in T1D [29 ]. The reason is unclear, but CTLA-4–Ig binds to B7-1 and B7-2 on APCs, blocking in T cell-positive (CD28-mediated) and -negative (CTLA-4-mediated) signals. Thus, one effect contradicts the other, and indeed, CTLA-4–Ig administration sometimes paradoxically aggravates autoimmunity. This approach avoids this limitation, as B7-1wa only engages CTLA-4.

A potential caveat is that molecules equivalent to B7-1wa may not be found in humans. However, a mutant human B7-1 molecule (W84>A) has properties very similar to murine B7-1wa [123 ] and is a potential candidate. Moreover, recently described members of the B7 superfamily, such as PD-L1 and B7-H4, which bind to other negative regulatory molecules of T cells, might also be effective. As an alternate approach, some investigators have generated selective ligands by molecular shuffling. Lazetic et al. [121 ] shuffled segments of CD80 genes originating from several species and produced costimulatory molecules that bind specifically to CD28 [CD28-binding protein (CD28BP)] or CTLA-4 (CTLA-4BP). In accord with studies of other selective ligands, CD28BP was immunostimulatory, and CTLA-4BP was inhibitory. Thus, CTLA-4BP inhibited a human mixed leukocyte reaction and enhanced IL-10 production, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. The amino acid sequence of CTLA-4BP is 96% identical with that of human CD80 and provides insight into the residues that are critical in CTLA-4 binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.


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CONCLUDING REMARKS AND FUTURE PROSPECTS
 
It is rarely clear which stimuli initiate autoimmune diseases, but there is considerable evidence that defects in negative signaling, involving CTLA-4 or other receptors, predispose to these conditions. In mice, deletion of CTLA-4 is particularly devastating, and this is relevant to human immunology, inasmuch as patients treated with CTLA-4-blocking antibodies develop autoimmune lesions. Experimentally, the mutation or blockade of other negative costimulatory molecules, such as PD-1 and BTLA, also predisposes to autoimmunity or aggravates autoimmune diseases. Tumors acquire the ability to suppress immunity at least in part by using these pathways, particularly through PD-L1 expression and secretion of regulatory cytokines. Furthermore, it appears that some normal tissues protect themselves from autoimmunity through similar pathways, although this question has not been studied sufficiently. There are a large number of negative regulatory molecules expressed by almost all cells of the immune system and at least some nonlymphoid cells. Indeed, the immune system and nonlymphoid tissues interact in complex ways that are only beginning to be understood. The mutation of these inhibitory genes or their regulatory elements can have serious detrimental effects on tolerance. In some cases, mutations affect anti-inflammatory molecules, resulting in autoinflammatory disease. Inflammation, in turn, is a fertile ground for the aberrant presentation of antigen and activation of low-affinity T cells, thereby promoting the development of autoimmunity.

If negative regulation is the natural way to prevent autoimmunity, it follows that clinical interventions should be directed at increasing negative signals. Recent studies suggest that this possible, but this approach needs to be further developed. Conversely, tumor immunity is greatly augmented by blocking negative signals. Whether we wish to increase or decrease negative signals, there will be serious risks involved. This has been clearly shown in cancer therapy, and evidently, a limitation of blocking CTLA-4 with mAb is that the effect is not closely antigen-specific. Ideally, the negative signals should be blocked only in tumor-reactive lymphocytes, but to my knowledge, this has not been achieved. The enhancement of negative signals in autoimmune diseases is also potentially treacherous, as the delivery of negative signals is finely regulated, and it will be very difficult to correctly re-establish normal immune homeostasis. DNA vaccines offer an interesting possibility, as local antigen recognition can be coupled to the delivery of negative signals, and presumably, other responses are unaffected. This endows the therapy with an antigen specificity, which would otherwise be difficult to achieve. Conversely, systemic therapy with molecules such as B7-H4/Ig, suppressing responses to most antigens, may be feasible, as the effects are likely to be less dramatic than those obtained by CTLA-4 engagement. The future in this area looks bright, provided immunotherapists persist with their negative ideas.


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ACKNOWLEDGEMENTS
 
The Juvenile Diabetes Research Foundation International, the Canadian Diabetes Association, and the National Cancer Institute of Canada funded these studies.

Received August 21, 2003; revised October 29, 2003; accepted November 2, 2003.


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