Review Cytokines & Chemokines

Th1/Th2 cells in inflammatory disease states: therapeutic implications

1. T cells and inflammation

Ronald B Moss†, Thomas Moll, Mohammad El-Kalay, Connie Kohne, William Soo Hoo, Jeffrey Encinas & Dennis J Carlo

2. T helper cell differentiation 3. T cells and allergic disease

Telos Pharmaceuticals LLC, San Diego, CA, USA

4. T cells and HIV infection 5. T cells and autoimmune disease 6. T cells and cardiac disease 7. T cells and cancer 8. T cells and vaccines 9. Theories on natural modulation of T helper cell immunity 10. Restoring the balance 11. Expert opinion and conclusion

Inflammation is initiated as a protective response by the host, but can often result in systemic pathology. Among cells of the immune system, T lymphocytes play a major role in the inflammatory response. T cell inflammation is characterised histologically by an infiltration of mononuclear cells. Key regulators of this response are a subset of T lymphocytes called T helper (Th) cells. These cells secrete soluble mediators called cytokines, which orchestrate the immune response. The appropriate regulation of Th cell immunity is critical in the control and prevention of diverse disease states. This review will focus on the role of Th cells in the inflammatory process involved in allergic disease, diabetes, infectious disease, rheumatoid arthritis, heart disease, multiple sclerosis and cancer. In the area of autoimmunity, in particular, a basic understanding of Th cells and cytokines has contributed to the development of clinically efficacious biological agents. This review also examines current and novel treatment strategies under investigation at present that regulate Th cell immunity, which may result in better treatments for immune-mediated diseases. Keywords: allergy, autoimmunity, cancer, CpG, heat-shock proteins, infectious disease, inflammation, T cell immunoglobulin mucin domain, T helper, TIM-1, TIM-3 Expert Opin. Biol. Ther. (2004) 4(12):1887-1896

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T cells and inflammation

Inflammation is an attempt by the host to protect itself from foreign pathogens. The typical inflammatory reaction is initiated by an ‘insult’ with a non-self antigen, such as a local bacterial infection, which then prompts immune cells to the area of infection to produce inflammatory messenger proteins called cytokines. Cytokines amplify the inflammatory reaction through the recruitment of different cell types of the immune system (Table 1). Initially, neutrophils, natural killer cells and macrophages, cells of the innate immune system, are involved in the inflammatory response. Subsequently, adaptive immunity plays an essential role in the propagation of the inflammatory response. B cells produce antibodies, while T cells are involved primarily in the cell-mediated immune response. The inflammatory reaction is a highly regulated process involving a multitude of different cells of the immune system, which, under normal circumstances, is designed to minimise tissue damage by attempting to clear the inflammatory ‘insult’. However, dysregulation of an inflammatory reaction can result in diverse disease states [1]. The acute form of inflammation has been classically described by the physical signs of pain (dolor), redness (rubor), heat (calor) and swelling (tumour). Although the inflammatory processes can be characterised into distinct types, once injury has occurred, multiple forms of inflammation may be involved in the same pathological process [1]. Inflammation can be divided into different response types; allergic, cytotoxic, immune complex, and T cell-mediated. Allergic inflammation involves mast

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Th1/Th2 cells in inflammatory disease states: therapeutic implications

Table 1. The innate and adaptive immune response. Immune response

Characteristics

Components

Innate

Immediate, nonspecific

Neutrophils, macrophages, natural killer cells

Adaptive

Gradual build-up, highly-specific, long-lasting

T cells, B cells, antibodies

pe

B cells Th2

Th1

B cell CTLs

Infected cell

Th1 cytokines promote CTL responses to intracellular pathogens (viruses, bacteria)

Th2 cytokines promote B cell secretion of antibodies to neutralise circulating pathogens

CTL contact with infected cell causes the death of the cell and prevention of disease replication

Neutralising antibodies produced by B cells bind to pathogens and prevent infection

Figure 1. The adaptive immune response. The adaptive immune response involves the generation of Th1 and Th2 cytokines. CTL: Cytotoxic T lymphocyte; Th: T helper.

cells, basophils and IgE. Cytotoxic antibodies, as well as complement activation, are involved in another form of inflammation, which can result in the destruction of platelets (thrombocytopenia) and can be associated with systemic lupus erythematosus (SLE). Immune complexes are critical to yet another type of inflammation, rheumatoid arthritis (RA), which is also accompanied by complement activation. Finally, T cells and their mediators are involved in inflammation. Histologically, T cell inflammation is characterised by an infiltration of mononuclear cells. Key regulators of this response are a subset of T lymphocytes called T helper (Th) cells. These cells secrete cytokines – soluble mediators that orchestrate the immune response. The appropriate balance of Th cell immunity is therefore paramount to our understanding of health and disease. Activated T cells are known to be major effector cells in the maintenance of health and in immune-mediated diseases. Th cells can be further differentiated into two subtypes; Th1 and Th2 cells, which are distinguished by the different cytokines they produce (Figure 1) [2,3]. Th1 cells produce IL-2, TNF-α, and IFN-γ, and are associated with cell-mediated immunity against intracellular pathogens, as well as being involved in delayed-type hypersensitivity skin reactions. Th1 cells also stimulate chemokines, which provide an important link between the recruitment of inflammatory cells and adaptive immunity. Chemokines are pivotal in stimulating leukocyte migration from the blood to the tissues and can be secreted in response to Th1 cytokines, including IL-1 and TNF. 1888

Interestingly, Th1 cells have been associated with the induction of autoimmune diseases. Specifically, autoimmune diseases such as RA and multiple sclerosis (MS) are thought to result partially from an augmentation of Th1 cells, and are associated with increases in inflammatory cytokines, such as TNF-α, IFN-γ and IL-2 (Figure 2). In contrast, Th2 cytokines, such as IL-4, -5 and -10, are involved in the control of extracellular helminth infections and circulating pathogens by enhancing antibody-mediated immunity (Figure 1). However, they are also associated with allergic diseases including asthma, allergic rhinitis and eczema (atopic dermatitis) (Figure 2). This dichotomy between Th1 and Th2 immunity has been demonstrated in animal models, but remains somewhat controversial in human disease. However, in humans, the ability to modulate cytokine production has already become a useful therapeutic strategy to treat autoimmune diseases. Strategies under investigation at present that can selectively shift the balance between Th1 and Th2 cells may be more effective in treating immune-mediated diseases. 2.

T helper cell differentiation

Th cell differentiation involves a complex chain of events [4]. In this process, three signals are involved in the skewing of Th cells to Th1 or Th2 phenotypes. One signal involves the ligation of the T cell receptor by pathogen-derived peptides, which are presented by major histocompatibility complex

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Moss, Moll, El-Kalay, Kohne, Soo Hoo, Encinas & Carlo

Th1 cell

Th2 cell

Inappropriate Th1 responses result in:

Inappropriate Th2 responses result in:

Autoimmune disorders • Multiple sclerosis • Psoriasis • Rheumatoid arthritis • Type I diabetes

Asthma

Inability to clear extracellular infections

Transplant rejection

Susceptibility to HIV

Lack of Th1 cytokines results in the inability to fight intracellular pathogens (viruses, bacteria)

Lack of Th2 cytokines results in the inability to neutralise invading viruses and bacteria

Allergic disorders

Figure 2. Th1 and Th2 cytokine imbalance in various disease states. Th: T helper.

(MHC) class II molecules on the surface of specialised antigen-presenting cells (APCs), such as dendritic cells. For optimal activation, the primed APC also requires cross talk with T cells, which involves the ligation of CD40 by CD40L on T cells. A second signal involves the triggering of costimulatory molecules, such as CD28, on T cells by CD80/CD86 after ligation by pattern recognition receptors such as the Tolllike receptors (TLRs). Lastly, T cell polarising signals, such as cytokines, are involved in skewing the Th response into Th1 or Th2. Members of the TLR family recognise conserved microbial patterns (pathogen-associated molecular patterns), such as bacterial lipopolysaccarides, microbial DNA and RNA, and are involved in activating the signalling pathways that result in immune responses [5]. TLRs have homology to the Drosophila Toll protein and the human IL-1 receptor family. TLRs consist of 10 human members where each TLR is involved in recognising a distinct molecular structure. For example, TLR 9 is the receptor for stimulating sequences of DNA containing unmethylated CpGs (discussed later in this review). The activation of TLRs drives Th cells primarily into Th1 patterns, although some recent data suggests that some TLRs (TLR 2 and 4) may also have the ability to polarise the immune response towards a Th2 phenotype [6,7]. However, in general, Th2 priming is thought to occur as a default pathway when TLR signals are absent or when Th2-type receptors are activated.

At the molecular level, Th1 signalling involves the activation of the (signal transducer and activator of transcription) STAT4, which is followed by the activation of another transcription factor, T-Bet (T box expressed in T cells), which induces the production of Th1 cytokines such as IFN-γ. Antigen stimulation resulting in activation of the Th2 pathway involves a cascade of transcription factors including STAT6, C-maf and GATA-3 [8]. 3.

T cells and allergic disease

Despite a greater understanding of the pathogenesis of asthma, it continues to be a great public health problem worldwide. It is estimated that 20 million Americans have asthma, and its prevalence has increased by > 80% in the past two decades [9]. In the US, asthma results in ∼ 500,000 hospitalisations and 5000 deaths per year. The pathological features of allergic asthma include denudation of airway epithelium, collagen deposition, oedema, mast cell activation and inflammatory cell infiltration. Although a variety of cell types are involved in allergic inflammation, there is substantial evidence that infiltration of Th2-like bronchoalveolar T lymphocytes is common in the lungs of allergic asthmatics, as well as increased levels of Th2 cytokines, including IL-4, -5, and -10 [10-12]. Most notably, the level of Th2 cytokines appears to correlate with the severity of disease [13]. Increased cytokine production then

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leads to the activation of other cell types, such as eosinophils, which are associated with lung injury in asthma. The pivotal role of cytokines in lung injury involved in asthma is also supported by animal models of asthma. Indeed, knockout mice that lack the Th1 transcription factor T-bet develop spontaneous lower airway disease changes consistent with human asthma [14]. Inhaled and systemic steroids are the treatment of choice for asthma at present. These agents work therapeutically by nonspecifically suppressing airway inflammation. Indeed, steroids may decrease CD4+ T cell infiltration and Th2 cytokine production in asthmatics, and are considered the most effective treatment for asthma [15]. However, because they lack specificity, steroids have limitations that include significant shortand long-term side effects (growth retardation, osteo-necrosis, etc.), resulting in additional morbidity. As T cells appear to be central in the pathogenesis of asthma, cytokine modulation is a viable treatment strategy for this common clinical disease. As many cytokines are involved in asthma, strategies that inhibit multiple Th2 cytokines may be more efficacious than approaches that target a single cytokine. 4. T

cells and HIV infection

The immunopathogenesis of AIDS can be characterised by a virally induced immune suppression, resulting from infection with human immunodeficiency virus (HIV), with a characteristic deficiency of CD4 + T cells and HIV-specific cell-mediated immunity. T cell inflammation is a component of this disease, and some studies have suggested that progression of disease is accompanied by a polarisation to Th2 immune responses. Indeed, HIV-infected individuals commonly have allergic and upper airway sinus disease [16]. The concept of a Th1 to Th2 switch during HIV infection is based on studies which demonstrated that Th1 immunity correlated with slower progression and longer survival of HIV-infected individuals [17,18]. Furthermore, Th2 clones were shown to be more susceptible to HIV-1 infection [19]. Most importantly, individuals who are exposed to HIV, but who remain infection-free, have strong Th1 cell-mediated immunity [20,21]. Protection may be afforded by Th1 cells, as they are critical to the development of effective killer cells (cytotoxic T lymphocytes [CTLs]) that lyse virus-infected cells [22]. Therefore, the enhancement of cell-mediated and Th1 immunity has been proposed as an appropriate strategy for preventative and therapeutic vaccination in order to counter the Th2 skewing observed in HIV disease [23]. 5.

T cells and autoimmune disease

T cells are critical in the pathogenesis of autoimmune diseases such as RA, MS and type 1 diabetes. Autoimmune diseases are thought to occur when there is a loss of tolerance to self-antigens and when Th1 cells are abnormally activated. 1890

RA is a common chronic inflammatory disease affecting ∼ 1% of adults, predominantly women. The long-term prognosis in RA is generally poor, and 80% of RA patients are disabled after 20 years with the disease [24]. Due to a progressive inflammatory synovitis, RA results in debilitating joint deformities. The synovial infiltrate in RA consists primarily of activated CD4+ T cells. The activated T cells, as well as other cells, produce predominantly Th1 cytokines. Existing treatments for RA nonspecifically target the inflammatory process via non-steroidal, anti-inflammatory drugs, such as cyclooxygenase-2 inhibitors, and diseasemodifying antirheumatic drugs (DMARDs), such as methotrexate. Steroids are also used to treat autoimmune diseases such as RA. In addition, biological DMARDs, which focus on inhibiting inflammatory Th1 cytokines (anti-TNF-α, TNF-α receptor), as well as other inflammatory cytokine inhibitors (IL-1), have also been developed. To their credit, these biological approaches to treat autoimmune diseases have been the most successful biological therapies so far that successfully modulate the cytokine milieu and have shown significant clinical benefit. However, it is difficult to ascribe all of the clinical benefits observed with TNF blockade to the downmodulation of a single cytokine such as TNF. This approach however, has significant clinical efficacy in certain patient populations, but may also be associated with an increased incidence of infectious complications, lymphoma, autoantibodies and other toxicities [25]. Other approaches of cytokine modulation to treat RA are being studied at present, including IL-12, -6 and -18 antagonists and IL-4 agonists [24]. Inflammatory T cells are also thought to be involved in the pathogenesis of MS. MS is a common disease of the CNS, affecting ∼ 1 million adults worldwide [26]. Animal models of MS support the important role of T cells in its pathogenesis. Vaccination of susceptible animals with myelin basic protein results in a relapsing-remitting, inflammatory, demyelinating CNS disease called experimental autoimmune encephalomyelitis (EAE). Experimentally, EAE can be adoptively transferred to naive animals through T cells that recognise myelin proteins. Thus, activated Th1 cells are thought to be important in the pathogenesis of MS. Indeed, the CNS lesions observed in MS are similar to those induced experimentally by Th1 cytokines in vivo [27]. These lesions typically contain Th1 inflammatory cytokines, activated T cells and mononuclear phagocytes. Existing treatments for MS include nonspecific therapies, such as steroids and the cytokine IFN-β. However, these therapies appear to be only marginally effective in treating the disease and, hence, additional treatment modalities are warranted [28]. Type 1 diabetes is another example of an autoimmunemediated disease and results from the destruction of insulin-producing β cells in the pancreatic islets. Patients with type 1 diabetes are typically treated with exogenous

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Moss, Moll, El-Kalay, Kohne, Soo Hoo, Encinas & Carlo

insulin. In spite of treatment, there is a high incidence of complications, including retinopathy, nephropathy, neuropathy and cardiovascular disease [29]. Th cells also appear to play a role in the pathogenesis of diabetes. For example, the predominant immune cells infiltrating the pancreas in diabetes have been shown to be T lymphocytes [29]. In addition, type 1 diabetes is associated with Th1 cells [30]. Indeed, recent attempts to suppress Th1 cytokines with nonspecific inhibitors of T cell activation have shown some clinical activity in type 1 diabetes [31]. 6.

T cells and cardiac disease

Cardiovascular disease remains the main cause of death in the US, Europe and parts of Asia. Although atherosclerosis results primarily from an accumulation of lipids and other materials in the arterial wall, attention has recently focused on the associated inflammatory processes. Inflammation of the artery walls leads to ischaemia of the heart, brain, extremities, and ultimately infarction. In atherosclerosis, lesions are infiltrated by activated T cells, as well as other inflammatory cells. CD4+ and CD8+ T cells, as well as macrophages, are found in the atherosclerotic lesions. These cells have been shown to produce a number of Th1-type cytokines, including TNF-α [32]. Inflammation is secondary to the release of cytokines by T cells in the disease process. Furthermore, TLRs, which modulate some forms of inflammation, are expressed in myocardial and arterial cells and, thus, may play a role in cardiovascular disease [33,34]. Indeed, new clinical markers, which broadly measure inflammation, such as C-reactive protein have recently been studied and offer prognostic utility, along with quantitation of lipid levels [35]. Therefore, limiting Th1 inflammation may be an additional treatment approach for the control of the inflammatory process in heart disease. 7.

mechanisms by which infections, and thus resulting cancers, are prevented are thought to be primarily Th2-mediated. In contrast, the regression of already established infections or cancers is thought to depend on the involvement of Th1 cells [39]. The ability to stimulate immunity against tumour cells or infectious agents associated with carcinogenesis is a laudable goal to treat and prevent cancer. 8. T

cells and vaccines

Vaccines have made a major public health impact against various infectious diseases by stimulating specific immunity against known pathogens [40,41]. During vaccination, Th cells and the cytokines they produce are critical components of protection against disease. Th1 cytokines promote ‘killer cells’, which are crucial in the fight against diseases caused by viruses (e.g., Vaccinia, HIV, influenza) and bacteria (e.g., Francisella tularensis, Mycobacterium tuberculosis, Salmonella typhi) that have entered host cells. Similarly, Th1 immune responses are pivotal against a variety of cancers and are critical for an effective cancer vaccine. In contrast, Th2-dependent antibody responses are the critical correlates of protection against certain types of bacterial or viral infections, bacterial toxins, and for the elimination of helminthic parasites. For example, Th2 cytokines are thought to be involved in the protection of a number of vaccines, including hepatitis B, diphtheria–tetanus–pertussis, measles–mumps–rubella and polio. In spite of the importance of Th1/Th2 immunity, the only approved adjuvant (alum) at present does not allow the preferential regulation of Th1/Th2 responses. In contrast, new approaches may preferentially modulate the Th1 or Th2 pathways and provide for more effective vaccines.

Theories on natural modulation of T helper cell immunity 9.

T cells and cancer

T cells, as well as other cells of the immune system, are thought to be involved in immune surveillance against cancer. Therefore, deficiencies in immune surveillance play a role in the development of cancer. Consequently, many cancer therapies have focused on stimulating Th1 immune responses with cytokines, which in turn function in part to activate so-called ‘killer cells’ of the immune system. Although some of these attempts to stimulate Th1 immunity have resulted in modest clinical regressions [36], Th2 cells have also been found to play an important and distinct role in tumour eradication [37]. Furthermore, T cells are thought to be critical in the design of effective vaccines, which combat infectious agents that cause cancer. Human papillomavirus (HPV), for example, is thought to be the major cause of cervical cancer worldwide. Papillomavirus vaccines result in significant effects on decreasing HPV infection and cervical neoplasia [38]. Although not completely understood, the

New theories have been proposed that synthesise the changing epidemiology and pathogenesis of immune-mediated diseases and Th cell polarisation. Two examples of HIV co-infection, as well as the so-called ‘hygiene hypothesis’, exemplify how differential Th responses may potentially impact on the incidence and pathogenesis of disease. As a result of the migration of African immigrants to Israel, an interesting hypothesis has emerged that may suggest a unique pathogenesis for HIV in developing countries. Parasitic infections are common in African countries and this theory suggests that an exacerbation of Th2-mediated HIV disease may be caused by co-infection with Th2-associated helminths. This hypothesis is based on the demonstration that many African immigrants displayed severe immune activation with a dominant Th2 profile and high levels of IgE and eosinophilia [42]. The observed Th2-dominant profile in these individuals was observed to be more extreme than in HIV-infected individuals in Western countries, and had been

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attributed to co-infection with intestinal worms or helminths [43]. Therefore, this severe skewing of the immune profile of individuals co-infected with helminths and HIV may result in a greater difficulty in treating or preventing HIV infection. Thus, eradication of parasitic infections or more potent stimulators of Th1 immunity may be required to treat or prevent HIV infection in parts of the world where parasites are endemic. Indeed, treatment of intestinal worms in one study has been shown to be associated with decreased virus levels in HIV-infected individuals [44]. A beneficial co-infection theory has emerged with respect to HIV-1-positive individuals co-infected with GB virus, a relative of hepatitis C. GB virus, also known as GBV-C, is a flavivirus and was first detected in patients with non-A, non-B hepatitis, but has not been associated with disease pathology. In a recent study, co-infection with GB virus was significantly associated with prolonged survival, while loss of detection of GB virus was associated with poor prognosis [45]. Interestingly, this increased survival with GB–HIV co-infection was associated with a Th1 profile, whereas progression was associated with Th2 cytokines [46]. Furthermore, co-infection with GB virus has been shown to enhance β-chemokines, which have previously been associated with Th1 cytokines [47]. The polarisation of Th1 and Th2 responses in various animal models, as well as human disease, coupled with a dramatic increase in allergic diseases, has resulted in the so-called ‘hygiene hypothesis’ [48]. Developmentally, it is believed that humans are born with a Th2 dominance, as this favours maternal–fetal compatibility. The natural exposure to viral and bacterial diseases during early childhood, however, may result in a skewing of the Th immune response predominantly towards Th1 responses. However, particularly in industrialised countries, where there is better hygiene, including vaccination and a reduction in microbial burden, weaker Th1 responses and a shift towards Th2 responses have resulted. The increase in Th2 responses, possibly due to better hygiene, appear to correlate with the observed increase in incidence of allergic diseases. This theory is supported in part by epidemiological data. For example, a recent study revealed a reduced incidence of allergic disease in children who had developed fever secondary to upper respiratory infections in early childhood [49]. As there are many observed exceptions to this theory, many have considered the ‘hygiene hypothesis’ to be an over simplification of a more complex phenomena. For example, this theory does not account for the fact that the prevalence of Th1 autoimmune diseases is also increasing. Furthermore, Th2-associated parasitic diseases are not typically associated with allergic diseases. However, as discussed later, recently discovered relationships between T cell immunoglobulin mucin domain (TIM)-1 molecules and hepatitis A suggest that the skewing of certain Th cell-mediated diseases, such as asthma, may be more related to changes in the epidemiology of specific infections and interactions with Th immunity than overall hygiene. 1892

10.

Restoring the balance

Based on our understanding of the central role of Th1 and Th2 cells in disease, it may become possible to restore the balance of the immune system with new agents that selectively modulate Th1/Th2 immunity. For Th1-mediated diseases, therapies which modulate the immune system towards more Th2-dominant responses (e.g., autoimmunity, transplantation and heart disease) may be useful. For Th2-mediated disease (e.g., allergic diseases) or infectious diseases, which require strong cell-mediated immunity, modulation towards a Th1 response would be warranted. Some new regulators of Th immunity that are under investigation are discussed below. 10.1 Heat-shock proteins: unidirectional modulation of

Th immunity Heat-shock proteins (HSPs, in particular HSP-60, -70, -90 and gp 96) are immunodominant antigens of bacteria that are upregulated by several forms of stress and are thought to be involved in the regulation of inflammation. Such proteins interact with APCs and are capable of stimulating strong Th1, as well as CTL, responses when utilised as adjuvants [50]. Specifically, immunisation of animals with HSP-70, a subfamily of HSPs, results in the stimulation of significant amounts of IL-1β, TNF-α and IL-12, thereby suggesting a Th1 bias. Furthermore, vaccination with peptide in combination with HSP-70 can result in the stimulation of antigen specific IFN-γ-secreting cells and CTLs that are associated with suppression of tumours in mice. HSP-70 is thought to bind to CD91, CD14, and TLRs 2 and 4 on the surface of APCs, thereby resulting in activation and maturation of these cells. The ability of HSPs to activate APCs makes them a potentially useful adjuvant for peptide vaccines against cancer or infectious diseases in order to stimulate Th1 responses [51]. Indeed, > 150 medical centres worldwide are studying autologous cancer-derived HSP–peptide complexes for the treatment of renal cell carcinoma and melanoma [52]. CpG: unidirectional modulation of Th immunity Although it has been noted for some time that bacterial, but not vertebrate, DNA stimulated the immune system [53], only recently has this observation been exploited for immune modulation. Oligodeoxynucleotides that contain immunostimulatory motifs of unmethylated CpG sequences influence the immune system by acting via TLR 9 on dendritic [54] and B cells, leading to the release of immunoglobulins and cytokines. CpGs have been shown to stimulate dendritic cells, and increase the expression of MHC class II and costimulatory molecules [55]. Specific sequences of oligonucleotides appear to stimulate both innate as well as antigen-specific immune responses, primarily in a Th1 direction. Immune responses to pathogens containing such sequences, therefore, may play a role in exacerbating autoimmune diseases that are Th1-mediated. For example, a study has linked heart disease in mice to Chlamydia infection containing pathogen-specific 10.2

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Moss, Moll, El-Kalay, Kohne, Soo Hoo, Encinas & Carlo

TIM-1

TIM-3

TIM-1 ligandexpressing cell

Macrophage Increased cell division Increased cell division

IFN-γ Amplification of Th1 cytokines

IL-2

IL-4 IL-5 IL-10 IL-13

Amplification of Th2 cytokines

Figure 3. Modulating the immune response. It may be possible to bidirectionally modulate Th immunity by the therapeutic use of the TIMs. Th: T helper; TIM: T cell immunoglobulin mucin domain.

CpG motifs [56]. Similarly, CpG oligonucleotides have been observed to be potent adjuvants for the activation of MS-type symptoms in animal models of EAE [57]. For diseases in which Th1 responses are beneficial, CpGs may have some therapeutic effects. Activation of the immune system with CpG resulted in the protection of mice against multiple pathogens, including Listeria monocytogenes, Francisella tularensis, Anthrax, Ebola, malaria, Leishmania and Schistosoma [58]. For allergic diseases, which are Th2-mediated, animal models have suggested some therapeutic activity of CpGs in ameliorating or preventing airway inflammation [59-61]. For vaccines, CpG complexes, when combined with protein antigens, have been shown to stimulate strong Th1 responses [62-64]. In summary, CpG sequences have been utilised in animal models and have been shown to stimulate strong vaccine and antiallergic responses. The testing of these molecules should provide proof of concept of the essential role of Th responses in ameliorating allergic disease, cancer, and also in enhancing vaccine immune responses. However, the clinical safety of CpGs is now under scrutiny, as a recent study in animals has suggested that repeated immunisations with this approach may result in immunotoxic and hepatotoxic effects [65]. The mechanism of these unwanted effects is unknown. Human studies using CpGs in the areas of cancer, vaccines and allergy are ongoing at present. TIMs: bidirectional modulation of immunity Recent studies examining the relationship of asthma susceptibility genes and new T cell-associated proteins have led to the identification of a new family of Th cell-expressed proteins, the TIMs [66,67]. The first TIM gene to be identified, TIM-1, is expressed by the kidney and liver, and was initially cloned as kidney injury molecule-1 (KIM-1) and hepatitis A virus (HAV) cellular receptor [68,69]. So far, the TIM family consists of eight genes on mouse chromosome 11B1.1, and three genes on the syntenic human chromosome 5q33.2, with no 10.3

other intervening genes. The three identified human TIM genes (TIM-1, TIM-3, TIM-4) are homologous to mouse TIM-1/TIM-2, TIM-3 and TIM-4 [70]. A recent study [66] defined a single chromosomal region in mice that conferred reduced Th2 responsiveness and protected against airway hyper-reactivity, characteristic of asthma. TIM-1 and TIM-3 were found to be strongly associated with this chromosomal region and with the differentiation to a Th2 or Th1 cell phenotype, respectively. TIM-1 mRNA was found to be expressed preferentially by Th2 cells, whereas TIM-3 mRNA was preferentially expressed by Th1 cells. Similar mRNA expression patterns for TIM-1 and TIM-3 were also observed in human cells [67,70]. These studies suggest that Th1 and Th2 cells might be regulated directly or indirectly by TIM-1 and/or TIM-3 (Figure 3). Human TIM-1 was initially identified as the cellular receptor for HAV. Several studies in both Europe and the US have noted that infection with HAV is associated with protection against the development of asthma in humans [71,72]. Recent studies characterised several TIM-1 polymorphisms in humans, and also demonstrated that HAV seropositivity protects against atopy, but only in individuals with a specific insertional mutation at amino acid position 157 of TIM-1 [73]. This particular TIM-1 allele was found to be expressed in 63% of Caucasians, 46% of Asians, and 64% of African-Americans in the population tested. In addition, another study suggested a direct correlation between certain TIM-1 allelic variants in exon 4 of the gene and susceptibility to atopic diseases [74]. Thus, in this study, the TIM-1 protein was found be associated with the control of atopy, but only in individuals who have had serological evidence of hepatitis A infection. The relationship between TIM-1 expression and atopy in hepatitis A seropositive individuals provokes the question of whether it would be possible to regulate TIM-1 expression in Th2-mediated diseases, such as asthma.

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The TIM proteins have also been shown to modulate Th1 immunity in animal models of autoimmunity, such as MS (EAE), as well as in transplant rejection, which are thought to be Th1-mediated. To study the role of TIM-3 in the development of a Th1-mediated autoimmune disease, Kuchroo et al., administered TIM-3-specific antibody to SJL mice that had been immunised to induce the development of EAE [67]. Administration of TIM-3-specific antibody increased severity and mortality in the treated animals. The therapeutic use of the TIMs has also been examined in islet cell allograft transplantation models, where rejection is thought to be Th1-mediated. In studies by Strom et al., treatment with a combination of donor-specific transfusion and anti-CD154 (anti-CD40L) was shown to result in long-term survival and tolerance of MHC-mismatched islet cell allografts [75]. In this model, immunisation with TIM-3/Fc prevented tolerance induction. Thus, the administration of TIM-3 in these models has been shown to exacerbate Th1-mediated diseases. Conversely, it may be possible to downregulate TIM-3 or upregulate TIM-1 to alleviate such Th1-mediated diseases. Finally, as the TIMs have been shown to preferentially modulate Th cell immunity, the potential exists to utilise

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them as adjuvants in combination with specific antigens in vaccines. In vivo administration of TIM-3/Fc during an ongoing immune response results in the hyperproliferation of antigen-activated T cells with the spontaneous production of Th1 cytokines [76]. This approach, in combination with pathogenspecific antigens, suggests the potential to modulate Th1 or Th2 immunity in the development of vaccines. In summary, the TIM family of molecules exemplifies the potential to bidirectionally modulate Th immunity to treat either Th1- or Th2-mediated diseases. 11.

Expert opinion and conclusion

T cells are critical in diverse disease states, and their polarisation to Th1 or Th2 cells has now been recognised as an integral part of the inflammatory response. Th1 proinflammatory cytokine antagonists have already become part of the standard care for the treatment of autoimmune disease. Newly described strategies that modulate Th1/Th2 immunity are being tested and offer the potential to treat diverse immune-mediated diseases, including allergy, autoimunity, cancer, heart disease, transplantation and infectious diseases.

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Ronald B Moss MD†, Thomas Moll PhD, Mohammad El-Kalay PhD, Connie Kohne, William Soo Hoo PhD, Jeffrey Encinas PhD & Dennis J Carlo PhD †Author for correspondence Telos Pharmaceuticals LLC, 10150 Meanley Drive, San Diego, CA 92131, USA Tel: +1 858 693 5117; E-mail: [email protected]