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Published online before print December 4, 2003
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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 |
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Key Words: autoimmunity • CTLA-4 • diabetes • DNA vaccination • inflammation • inhibitory molecules • lupus • PD-1 • regulatory T cells
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INTRODUCTION |
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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 |
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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-
(IFN-) signaling. Mice deficient in SOCS1 are subject to uncontrolled IFN- 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 
(TNF-), and IFN-] 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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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 FcRIIB with the BCR suppresses B cell activation, and null mutation of FcRIIB 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 FcRIIB. 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 FcRIIB in mice, and absent or decreased SHP-1 results in the autoimmune, motheaten phenotypes in mice [44
]. In humans, low-affinity variants of FcRs are associated with lupus nephritis [49
].
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Cells of hematopoietic origin express numerous ITIM-containing receptors, and a notable example is signal regulatory phosphatase , 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 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 |
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, and IL-13 were decreased. Blocking antibodies to both inhibitory receptors enhanced T cell proliferations and production of IL-2, IFN-, 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-, 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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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- 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--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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ENHANCING TUMOR IMMUNITY BY CTLA-4 BLOCKADE |
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autoimmune vitiligo; prostate antigenprostatitis). 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. |
INFECTIOUS AGENTS ACTING NEGATIVELY |
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Pathogenic viruses, for example, produce cytokines (e.g., vIL-10) or anticytokines (e.g., soluble receptors for TNF, IL-1ß, IFN-, IFN-/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 |
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/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. |
ENGAGING CTLA-4 TO ATTENUATE ALLOREACTIVE OR AUTOIMMUNE RESPONSES |
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), 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-, and TNF- 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 |
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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- 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 |
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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 |
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Received August 21, 2003; revised October 29, 2003; accepted November 2, 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
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.