WO2004084822A2 - Methods for inducing immune tolerance - Google Patents

Abstract

The regulatory mechanisms that influence resistance versus susceptibility to progressive infection by microbial pathogens are of fundamental importance to understanding pathogenesis and to developing effective vaccines. Susceptibility to progressive infection in the lepromatous form correlated with the local expression of genes encoding members of the leukocyte immunoglobulin-like receptor (LIR) family, including LIR-3, -4, -7, and - 8. The gene expression profiles provided a clear and consistent distinction between the different clinical courses of the disease. The present invention shows that gene expression technology may be useful for predicting the clinical course of leprosy and perhaps other infectious diseases. Further, the invention provides methods of treatment of inflammatory or infectious diseases based on regulation of LIR-7 expression or activity.

Description

METHODS FOR INDUCING IMMUNE TOLERANCE

RELATED APPLICATION DATA

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e), to U.S. Provisional Application No. 60/456,789, filed March 21, 2003, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION [0002] The invention relates generally to immunology and more specifically to the leukocyte immunoglobulin-like receptor (LIR) family and induction of tolerance in a subject.

BACKGROUND INFORMATION [0003] For many pathogens, some individuals are resistant to infection and develop limited or no disease, while others are highly susceptible, developing progressive infection. The clinical manifestations of leprosy comprise a spectrum that correlates with the type of immune response to the pathogen Mycobacterium leprae.(l) At one end of the spectrum, the tuberculoid form (T-lep) is characterized by limited, self-curing disease with few bacteria and the local expression of type 1 cytokines characteristic of strong cell- mediated immunity. In contrast, lepromatous leprosy (L-lep) patients present clinically with disseminated lesions and high bacterial loads, with expression of type 2 cytokines in lesions typically associated with humoral immunity and suppression of cell-mediated immune responses. To gain insight into the mechanisms by which the host response to infection is regulated, we examined the gene expression profiles in leprosy lesions. Since it is important for staging the disease to biopsy the skin lesions of leprosy patients, and previous studies have indicated that differences in patients' immune responses could be discerned from study of lesions but not from peripheral blood, (4) we were able to compare patterns of expression of ~12,000 genes directly in lesions, where the clinical outcome is determined.

[0004] Immune system cellular activity is controlled by a complex network of cell surface interactions and associated signaling processes. When a cell surface receptor is activated by its ligand a signal is sent to the cell, and, depending upon the signal transduction pathway that is engaged, the signal can be inhibitory or activatory. For many receptor systems cellular activity is regulated by a balance between activatory signals and inhibitory signals. In some of these it is known that positive signals associated with the engagement of a cell surface receptor by its ligand are downmodulated or inhibited by negative signals sent by the engagement of a different cell surface receptor by its ligand.

[0005] The biochemical mechanisms of these positive and negative signaling pathways have been studied for a number of known immune system receptor and ligand interactions. Many receptors that mediate positive signaling have cytoplasmic tails containing sites of tyrosine phosphatase phosphorylation known as immunoreceptor tyrosine-based activation motifs (IT AM). A common mechanistic pathway for positive signaling involves the activation of tyrosine kinases which phosphorylate sites on the cytoplasmic domains of the receptors and on other signaling molecules. Once the receptors are phosphorylated, binding sites for signal transduction molecules are created which initiate the signaling pathways and activate the cell. The inhibitory pathways involve receptors having immunoreceptor tyrosine based inhibitory motifs (ITLM) which, like the ITAMs, are phosphorylated by tyrosine kinases. Receptors having these motifs are involved in inhibitory signaling because these motifs provide binding sites for tyrosine phosphatases which block signaling by removing tyrosine from activated receptors or signal transduction molecules. While many of the details of the activation and inhibitory mechanisms are unknown, it is clear that functional balance in the immune system depends upon opposing activatory and inhibitory signals.

[0006] One example of immune system activity that is regulated by a balance of positive and negative signaling is B cell proliferation. The B cell antigen receptor is a B cell surface immunoglobulin which, when bound to antigen, mediates a positive signal leading to B cell proliferation. However, B cells also express Fc.gamma.RJIbl, a low affinity IgG receptor. When an antigen is part of an immune complex with soluble immunoglobulin, the immune complex can bind B cells by engaging both the B cell antigen receptor via the antigen and Fc.gamma.RIIbl via the soluble immunoglobulin. Co-engagement of the Fc.gamma.RIIbl with the B cell receptor complex downmodulates the activation signal and prevents B cell proliferation. Fc.gamma.RIIbl receptors contain MTLM motifs which are thought to deliver inhibitory signals to B cells via interaction of the ITIMs with tyrosine phosphatases upon co-engagement with B cell receptors.

[0007] The cytolytic activity of Natural Killer (NK) cells is another example of immune system activity which is regulated by a balance between positive signals that initiate cell function and inhibitory signals which prevent the activity. The receptors that activate NK cytotoxic activity are not fully understood. However, if the target cells express cell-surface MHC class I antigens for which the NK cell has a specific receptor, the target cell is protected from NK killing. These specific receptors, known as Killer Inhibitory Receptors (KIRs) send a negative signal when engaged by their MHC ligand, downregulating NK cell cytotoxic activity.

[0008] Clearly, the immune system activatory and inhibitory signals mediated by opposing kinases and phosphatases are very important for maintaining balance in the immune system. Systems with a predominance of activatory signals will lead to autoimmunity and inflammation. Immune systems with a predominance of inhibitory signals are less able to challenge infected cells or cancer cells. Isolating new activatory or inhibitory receptors is highly desirable for studying the biological signal(s) transduced via the receptor. Additionally, identifying such molecules provides a means of regulating and treating diseased states associated with autoimmunity, inflammation and infection.

[0009] For example engaging a newly discovered cell surface receptor having ITIM motifs with an agonistic antibody or ligand can be used to downregulate a cell function in disease states in which the immune system is overactive and excessive inflammation or immnunopathology is present. On the other hand, using an antagonistic antibody specific to the receptor or a soluble form of the receptor can be used to block the interaction of the cell surface receptor with the receptor's ligand to activate the specific immune function in disease states associated with suppressed immune function. SUMMARY OF THE INVENTION [0010] The regulatory mechanisms that influence resistance versus susceptibility to progressive infection by microbial pathogens are of fundamental importance to understanding pathogenesis and to developing effective vaccines. To gain insights into such mechanisms, gene microarray technology was used to define expression profiles of ~12,000 genes in lesions of leprosy, a disease that presents clinically as a spectrum in which resistance correlates with the level of cell-mediated immunity to the pathogen. In tuberculoid leprosy, a self-limited form of the disease, expression was elevated for a set of pro-inflammatory genes encoding type 1 cytokines, chemokines and proteins involved in antigen presentation. Strikingly, susceptibility to progressive infection in the lepromatous form correlated with the local expression of genes encoding members of the leukocyte immunoglobulin-like receptor (LIR) family, including LLR-3, -4, -7, and -8. The gene expression profiles provided a clear and consistent distinction between the different clinical courses of the disease. The data presented herein suggest that gene expression technology may be useful for predicting the clinical course of leprosy and perhaps other infectious diseases.

[0011] Disclosed in the present invention are methods of predicting the clinical course of an autoimmune, inflammatory or infectious disease in a subject by detecting gene expression cytokine profiles indicative of Thl/type 1 or Th2/type 2 immune responses and correlating the profiles with disease progression. In a related aspect, such profiles are predictive of outcome in subjects with microbial infections and autoimmune diseases.

[0012] In a related aspect, such diseases include Addison's Disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), Behcet's disease, chronic fatigue syndrome, Crohn's disease and ulcerative colitis, diabetes, fibromyalgia, Goodpasture syndrome, graft versus host disease, Graves' disease, Guillain-Barre syndrome, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, myositis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, atopic dermatitis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, vitiligo, and Wegener's granulomatosis. [0013] In one aspect, the disclosed methods would include obtaining a biological sample of a test and control subject, treating the samples with a chaotropic agent to release nucleic acids, extracting extraneous biological materials to purify an appropriate nucleic acid fraction, reverse transcribing the nucleic acid fraction, synthesizing probes from the resulting transcripts and hybridizing these probes to prefabricated surface immobilized gene arrays. In this way, resulting signals from the arrays can be used to generate informative sets of differentially expressed genes between test and control samples.

[0014] The present invention also relates to a method for diagnosing a response or survival in a subject having an infection, e.g., a microbial infection, or inflammatory disease, e.g., autoimmune disease, by detecting a gene expression profile of particular cytokines (e.g., IL-10 or 12) or genes in the LIR family. In particular, the profile of expression of LIR-7 may be of value.

[0015] Methods are also disclosed for differentiating clinical manifestations of leprosy including correlating immune responses to Mycobacterium leprae infection using patient lesion samples, where the manifestations can be characterized by the determination of type 1 cytokine expression and cell mediated immunity or by the determination of type 2 cytokine expression and humoral immunity and suppression of a cell-mediated immune response.

[0016] In one aspect, determination of type 1 cytokine expression correlates with the tuberculoid form (T-lep) of leprosy.

[0017] In another aspect, determination of type 2 cytokine expression correlates with the lepromatous form (L-lep) of leprosy.

[0018] Disorders mediated by autoimmune disease associated with failure of a negative signaling LIR to down regulate cell function may be treated by administering a therapeutically effective amount of a modulating agent (e.g., agonistic antibody or ligand) of LIR family members to a patient afflicted with such a disorder. Disorders mediated by disease states associated with suppressed immune function can be treated by administering a soluble form of the negative signaling LIR. Conversely, disorders mediated by diseases associated with failure of a activatory signaling LIR can be treated by administering an agonistic antibody of the activatory receptor. Disorders mediated by states associated with autoimmune function can be treated by administering a soluble form of the receptor.

[0019] Such disorders may be characterized by a hyporesponsive or hyperresponsive immune reaction or a Thl/type 1 or Th2/type 2 dominant immune response. In a related aspect, the disease may be characterized by a hyporesponsive immune reaction where a modulating agent inhibits LIR-7 activity associated with a Th2/type 2 dominant immune response, thereby inhibiting LIR-7 increases in activatory signaling.

[0020] In another related aspect the disease may be characterized by a hyperresponsive immune reaction where modulating agent stimulates LIR-7 activity associated with a Thl/type 1 dominant immune response to down regulate cell function.

[0021] The present invention further relates to LIR-7 as a target for therapy by modulating agents, including small molecule and biological therapeutic agents. For example, small molecule agonists or antagonists of LIR-7, or antibodies, may be utilized in the present invention.

[0022] Further, methods are disclosed for identifying modulating agents (e.g., agonists and antagonists) which include contacting cells with test agents in the presence of toll-like receptor (TLR) agonists and allowing such agents to incubate with ceils for a sufficient time to determine the production of TLR agonist-induced type 1 or type 2 cytokines. Subsequently, the amount of the cytokine produced in the presence and absence of the test agents can be determined, and where there is a difference in the production of cytokines in the presence and absence of the test agents such a difference is indicative of the modulation of LIR-7 by such agents.

[0023] In a related aspect, the cells are incubated with test agents in the presence and absence of an anti-LIR-7 antibody or interferon gamma.

[0024] In another related aspect, the cells may be myeloid or lymphoid, and include monocytes and eosinophils. In another aspect, the methods are envisaged that use TLR agonists selective for TLR2/1 or TLR4, and may include assaying for modulation of FcR gamma or DAP 12 expression.

[0025] Exemplary methods and compositions according to this invention are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1 A-C show two unsupervised data analyses separates leprosy patients into clinically relevant subclasses based on their gene expression patterns

[0027] Figures 2A-B show two supervised analyses coupled to permutation analysis indicate that the gene expression differences between T-lep and L-lep patient groups are statistically significant.

[0028] Figures 3A-B show differentially expressed genes in T-lep and L-lep lesions.

[0029] Figures 4A-C show LIR-7 is highly expressed in L-lep lesions and downregulates type 1 cytokines produced during the innate immune response.

DETAILED DESCRIPTION OF THE INVENTION

[0030] For many pathogens, some individuals are resistant to infection and develop limited or no disease, while others are highly susceptible, developing progressive infection. The clinical manifestations of leprosy comprise a spectrum that correlates with the type of immune response to the pathogen Mycobacterium leprae. (1) At one end of the spectrum, the tuberculoid form (T-lep) is characterized by limited, self-curing disease with few bacteria and the local expression of type 1 cytokines characteristic of strong cell- mediated immunity. (2, 3, 3) In contrast, lepromatous leprosy (L-lep) patients present clinically with disseminated lesions and high bacterial loads, with expression of type 2 cytokines in lesions typically associated with humoral immunity and suppression of cell- mediated immune responses. To gain insight into the mechanisms by which the host response to infection is regulated, the gene expression profiles in leprosy lesions were examined. Since it is important for staging the disease to biopsy the skin lesions of leprosy patients, and previous studies have indicated that differences in patients' immune responses could be discerned from study of lesions but not from peripheral blood, (4) patterns of expression of ~ 12,000 genes directly in lesions were compared, where the clinical outcome is determined.

[0031] DNA microarray data of the skin biopsies were obtained from six T-lep and five L- lep patients, who were clinically diagnosed and classified according to the criteria of Ridley (1) (supplemental figures). The data was analyzed using two unsupervised learning algorithms, principal component analysis and hierarchical clustering analysis, individually on the eleven samples. Principal component analysis was performed to capture the main trends in gene expression arrayed on the chip. (5)

[0032] Figures 1 A, B, and C show two unsupervised data analyses separates leprosy patients into clinically relevant subclasses based on their gene expression patterns. (A) Principal component analysis shows that the gene expression pattern of T-lep patients is distinct from the L-lep patients. All T-lep patients (T1-T6), except patient T6 (marked with an arrow), formed a group with positive values whereas L-lep patients (L1-L5) had negative values and formed a second group. These patient designations are shown in italics. (B) After verification that patient T6 had been misclassified it was regrouped within the correct L-lep subclass, and is now labeled as L6 and indicated by an arrow. The corrected patient groupings are not italicized. (C) Hierarchical clustering analysis is consistent with the principal component analysis. The different gene expression patterns of T-lep and L-lep patients divide them into two distinct groups that cluster on separate branches of a dendrogram. The original (italics) and corrected clinical subclass of each patient is shown.

[0033] As shown in Figure 1 A, the first principal component, or strongest gene expression trend, reveals that the L-lep patients have distinct gene expression from the T-lep patients. All five L-lep patients formed a group with negative Eigen values. Of the six T-lep patients, five had positive Eigen values, but patient T6 had a negative value. [0034] The second unsupervised algorithm was performed, hierarchical clustering, to explicitly group together patient samples with similar gene expression patterns. (6) The clustering algorithm revealed two main groups, one containing the L-lep patients and the other the T-lep patients as depicted by the dendogram in Figure lC. Once again, and consistent with the principal component analysis, patient T6 was clustered with the L-lep group. The patient record was reexamined and the histology of a biopsy taken on the same day was observed to be consistent with L-lep. Upon correction of the classification of patient T6 to L-lep (L6), the principal component analysis (Figure IB) and the hierarchical clustering (Figure 1C) showed a clear and totally consistent distinction between T-lep and L-lep patients. The ability of gene expression analysis to identify the clinical T-lep and L- lep classifications, and to expose the misclassified sample, demonstrated the power of the technology, and confirmed that lesions of T-lep and L-lep patients have distinct gene expression profiles.

[0035] To determine if the observed differences in gene expression between the T-lep and L-lep patients were statistically significant, permutation analysis was performed. (7) The T-lep/L-lep grouping (T1-T5 as T-lep group and L1-L6 as L-lep group, based on clinical diagnosis) was systematically compared to all other 461 possible patient groupings and it was ascertained that fewer than 1% (p<0.01) of the permuted groupings have more differentially expressed genes than the defined T-lep/L-lep patient grouping using the Student's t-test (Figure 2A). This established that despite the relatively small number of samples examined in this study, the differences between T-lep and L-lep patients are not likely to have been due to chance. Figure 2 shows two supervised analyses coupled to permutation analysis indicate that the gene expression differences between T-lep and L-lep patient groups are statistically significant. (A) Determination of the number of differentially expressed genes between subgroups. The cumulative number of genes (y- axis) with Student's t-test p-values less than various threshold levels (x-axis) were calculated for the T-lep/L-lep grouping and plotted (black). Every possible permutated grouping was also generated and tested (see Methods). The mean (green), median (red), 10% (blue) and 1% (yellow) confidence levels for these permutated groupings are shown. Less than 1% of the permutated groupings manifest more distinction in gene expression than the defined T-lep / L-lep patient grouping, indicating that the grouping is statistically significant. (B) Prediction accuracy using weighted gene voting and leave one out cross validation. Using the T-lep/L-lep grouping, the weighted gene voting algorithm correctly assigned the subclasses of 8 out of 11 samples with high confidence (prediction strength >0.4, see Methods). Permutation analysis shows this to be statistically significant since less than 1% of all other possible groupings (4 of 462, indicated by the arrow) were able to correctly assign 8 samples with a prediction strength >0.4, demonstrating that the differences in gene expression between T-lep and L-lep patients are statistically significant and can be used to predict the subclasses of the disease.

[0036] The detection of the diagnostic mislabeling indicates the power of gene expression profiles of leprosy patients to predict the clinical subclasses (T-lep, L-lep) of the disease. To evaluate whether gene expression profiles are robust enough correctly to assign the subclasses of unknown samples, a class prediction analysis was performed. Using 'leave one out cross validation' (LOOCV), (7) the most differentially expressed 100 genes between all but one of the samples to predict the class of the withheld sample were used. Using the correct T-lep/L-lep grouping, the classes of all eleven samples correctly (100% accuracy) were able to be predicted. Eight out of the eleven assignments had high confidence (prediction strength > 0.4). The probability of permutated groupings to assign eight samples correctly with high confidence (prediction strength >0.4) was less than 1% (4 out of 462 groupings) (Figure 2B). Taken together, these supervised prediction results and the unsupervised principal component analysis and hierarchical clustering results above demonstrate that T-lep and L-lep lesions are quite distinct at the gene expression level. It will be of interest in future to learn whether distinctions in expression profiles of T-lep and L-lep classification subgroups can be used to predict disease complications that cause patient morbidity, such as nerve damage.

[0037] In order to identify the most informative differentially expressed genes between the two leprosy subclasses, each gene was ranked by the probability that the means of its expression values are statistically distinct between the T-lep and L-lep patients using the Student's t-test. The fold change in mean expression of each gene between the two groups was also calculated and this value was used as a secondary ranking. Attention was focused on genes that exhibited a fold change of >1.5 and p <0.05. A total of 371 genes were found to be comparatively upregulated in T-lep lesions, and 622 were upregulated in L-lep lesions. When these genes were organized into categories based on function or cellular distribution using the OMLM and Locus Link databases at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov), the gene expression patterns that separated patients into clinically relevant subclasses spanned a wide range of functional categories, including immune, signal transduction, apoptosis, cell cycle, adhesion, structural, and housekeeping genes (Figure 3 A), likely reflecting the mixture of cell types in lesions and the type of clinical presentation. For example, the upregulation of keratins and desmoglein-3 in T-lep lesions is consistent with the histologic thickening of the epidermis. In L-lep lesions, the elevation of genes involved in neuronal function raises the possibility that such changes may reflect the damage to peripheral nerves that is typical of such lesions.

[0038] Figure 3 shows differentially expressed genes in T-lep and L-lep lesions. (A) Breakdown of differentially expressed genes in leprosy patients. A list of differentially expressed genes (p < 0.05 and a fold change > 1.5) in T-lep and L-lep patients was compiled and organized into categories based on proposed function or cellular location according to information available in the Locus Link and OMIM databases. The percentage of genes in each category is shown for both T-lep and L-lep patients. (B) Relative expression of immune genes in T-lep and L-lep patients. Hierarchical clustering software was used to visualize the differences in T-lep and L-lep expression of genes with relevance to various sub-categories of the immune response. The expression data from each patient for the genes indicated is represented by a color gradient from red (relatively high expression) to green (relatively low expression).

[0039] The tissue granuloma is the site where the battle between the host response and the pathogen is joined. Previous work characterizing, both types of leprosy has shown that these granulomas contain roughly equivalent numbers of T cells and monocytes, (8) and suggested that the differences in disease manifestation may reflect two opposing gene expression programs that influence the type of host response. By RT-PCR, T-lep lesions were found to express elevated levels of type 1 cytokines associated with cell-mediated immunity, whereas L-lep lesions predominantly expressed type 2 cytokines . (2, 9) The present microarray analysis of genes in the immune category confirms the type 1/type 2 paradigm (Figure 3B), with T-lep lesions differentially expressing genes encoding type 1 cytokines including lymphotoxin-a, (2) IL-7, (2,10) and IL-15, (11) whereas L-lep lesions differentially expressed the type 2 cytokine IL-5. (2, 9) IL-4 and IL-10 were not found to be differentially expressed in L-lep lesions by the gene chip analysis but were confirmed by quantitative PCR (data not shown), indicating that the microarrays tend to underestimate differences. Microarray analysis also confirmed the previously reported upregulation of CDlb (12) and signaling lymphocytic activation molecule (SLAM) (13) in T-lep lesions. Interestingly, mRNAs for two anti-microbial molecules were also upregulated in T-lep lesions, secretory leukocyte protease inhibitor (SLPI) and cathepsin G, (14, 15) which may contribute to the control of infection.

[0040] Understanding the mechanism of immunologic unresponsiveness and tolerance remains a fundamental problem in immunology. In leprosy, the presence of CD4+ and CD8+ T-regulatory cells that secrete type 2 cytokines has been shown to correlate with immunologic unresponsiveness in L-lep patients. (3) The present data revealed a striking and unexpected finding: the elevated expression of inhibitory receptors in L-lep lesions, including several members of the leukocyte immunoglobulin-like receptor family (LIR). LIRs are a family of receptors expressed on both myeloid and lymphoid cells. The LIR family can be subdivided into two main groups: transmembrane molecules with two to four internal ITLM signaling motifs (LIR-1, LIR-2, LLR-3, LIR-5, LLR-8) and transmembrane molecules with short cytoplasmic domains and no ITIMs (LIR-6, LIR-7, LIR-9). (16) Instead, the latter interact with ITAM-containing adaptor proteins through a charged arginine residue within their transmembrane domain, such as FcRg and DAP 12, both of which were also elevated in L-lep lesions. The ligands for activating LLRs are completely unknown and the function of LIRs is unclear; however these receptors have been shown to increase calcium flux, serotonin release, and production of cytokines from monocytes. (17-19)

[0041] LIR-7 was of greatest interest because it was the most differentially expressed of the LIR genes, expressed 5.4-fold more in L-lep than in T-lep lesions. To confirm the results obtained by microarray analysis measured the expression of LIR-7 mRNA in lesions obtained from 12 additional T-lep and L-lep patients by qPCR was measured. Figure 4A shows upregulation of LIR-7 in 4/6 L-lep lesions, whereas no expression was detected in T-lep samples (0/6). This pattern was remarkably similar to data from our initial microarray analysis, sharing almost identical p-values.

[0042] Figure 4 shows LIR-7 is highly expressed in L-lep lesions and downregulates type 1 cytokines produced during the innate immune response. (A) Measurement of LIR-7 mRNA in leprosy lesions by microarray and qPCR. For qPCR, cDNA was isolated from 12 new leprosy patients (6 T-lep, 6 L-lep) and analyzed for expression of LIR-7 mRNA. Reactions were done using the TaqMan Universal PCR Master Mix and a gene-specific primer and probe set at a final concentration of 500nM and lOOnM, respectively. Q-PCR data represents relative expression of LIR-7 in arbitrary units (AU), determined using the DDCT method as previously described. (30) The differences in LIR-7 expression observed by microarray and qPCR were both statistically significant (p=0.027 and p=0.028). (B) Regulation of LIR-7 expression on monocytes by TLRs. Primary monocytes isolated from healthy donors were cultured in the presence or absence of ligands for TLR4 (LPS, 10 ng/ml) or TLR2/1 (19kD lipopeptide, 5 mg/ml). Resulting cells were stained with control rat IgG2a (thin line), or rat-anti human LIR-7 mAbs (thick line) as previously described. (17) Data is shown as percentage of LIR-7+ cells relative to the isotype control and is representative of at least 3 experiments. (C) Engagement of LIR-7 modulates monocyte production of IL-12 and IL-10. Primary monocytes were isolated as described above and incubated in the presence of isotype control or rat anti- human LIR-7 mAbs followed by a goat anti-rat IgG crosslinking antibody (Jackson Laborotories, Westgrove, PA) as previously described. (17) Cells were then cultured in media containing 100 U/ml IFN-g and serially diluted amounts of ligands for TLR2/1 (19kD lipoprotein (32)) or TLR4 (LPS). Supernatants were harvested and analyzed for production of IL-12 and IL-10 by ELISA.

[0043] The hypotheses that LIR-7 is regulated in lesions by activation of Toll-like receptors (TLRs) and that cytokines affect its expression was also tested. As shown in Figure 4B, the expression of LIR-7 protein on primary monocytes was significantly repressed by ligands for TLR2/1 and TLR4. This effect was not dependent on TLR- activated cytokines, as a panel of type 1 and type 2 cytokines had no effect on LIR-7 expression (data not shown). This suggests that type 1 cytokines in T-lep lesions, by enhancing TLR expression and activation, ultimately inhibit LIR-7 expression whereas in L-lep lesions, type 2 cytokines that can inhibit TLRs may allow for increased LIR-7 expression and subsequent activation of this inhibitory receptor.

[0044] Because the balance of type 1 and type 2 cytokines produced during the innate immune response plays an important role in instructing the development of the ensuing adaptive response, (20) the effect of LIR-7 on TLR-dependent production of IL-12 and IL-10, two cytokines well-characterized for their roles in potentiating type 1 and type 2 immune responses, respectively was examined. (21) As shown in Figure 4C, the production of IL-12 in response to two different TLR ligands was almost completely abrogated in the presence of activating anti-LIR-7 antibodies. At the same time, the level of IL-10 release was either unaffected or increased, thus increasing the ratio of IL-10 to IL-12. These data suggest that LIR-7 on antigen-presenting cells and macrophages may influence the balance of cytokines produced by T cells during the innate response, diverting it from the pro-inflammatory program required for the development of cell- mediated immunity and the control of infection.

[0045] Although LIR-7 has previously been thought to be an activating receptor because of its association with the ITAM-containing adaptor protein FcRg, the data suggested that, in terms of monocyte function, it has inhibitory function on type 1 cytokine responses. Other ITAM-containing proteins have also been shown to mediate inhibitory functions. Ligation of the FcgRl has been shown to repress IL-12 production and enhance IL-10 release. (22, 23) Our data suggest that LIR-7 on antigen presenting cells may belong to a group of receptors that utilize ITAM-dependent pathways to negatively regulate type 1 cytokine production, diverting T cells to the type 2 cytokine pattern that in the context of an in racellular pathogen presents as immune unresponsiveness or tolerance.

[0046] Although T cells in L-lep patients are unresponsive in terms of type 1 cytokine production, previous data indicate that T cells from L-lep patients produce type 2 cytokines and can provide help to B cells. (3) One third of the immune response genes (24/60) that were highly expressed in L-lep lesions encoded proteins relevant to the humoral immune response, represented by mRNAs for immunoglobulin heavy and light chains and the CD 19 and CD22 lineage markers was observed. Immunohistology demonstrated clusters of CD 19+ and CD22+ cells in L-lep lesions, suggesting the presence of early lymphoid follicles in these lesions. This finding was unanticipated because although the serum of L-lep patients has elevated anti-M. leprae immunoglobulins, previous studies had not detected such B cell responses in the lesions themselves.

[0047] In previous studies of microbial infection, microarray analyses have been used to characterize responses of normal peripheral blood monoculear cells to distinct classes of pathogens in vitro (24, 25) or mice challenged with different antigens. (26) In the case of cancer, genome-scale gene expression analysis of different tumors and stages have been used to classify leukemia, (7) breast cancer, (27) and melanoma, (28) and in some cases to predict survival. Our data suggest that it should be feasible to use such methodology to diagnose and predict outcome in patients with microbial infection, whether from ordinary community-acquired infection or from a bio-terrorist attack.

[0048] Analysis of gene expression profiles in leprosy lesions was highly correlated with the clinical course of the disease, and indeed may be useful in understanding the dichotomous disease states in leprosy and shed light on mechanisms of host resistance and susceptibility to infection. Unknown/undetected characteristics of M. leprae infection, such as the expression of immune response genes associated with humoral immunity within L-lep lesions were identified. Additionally, the identification of elevated inhibitory receptor expression in L-lep lesions suggests drug targets for therapies designed to unmask immunologic unresponsiveness. Thus, modern genomics can reveal the sets of genes that correlate with protective responses, or inappropriate responses leading to tolerance, providing unanticipated insights into pathogenesis and targets for therapy.

[0049] In one embodiment, a method of identifying an agent that modulates LIR-7 expression or activity in a cell can be performed, for example, by contacting a cell expressing LIR-7 with a test agent; and detecting altered expression or activity due to contact with the test agent as compared to expression or activity in the absence of the test agent. A test agent useful in a method of the invention can be any agent suspected of having the ability to alter, and preferably inhibit LIR-7 activity or expression, including a peptide, a polynucleotide, a small organic molecule, a peptidomimetic, an antibody or the like.

[0050] The invention includes antibodies immunoreactive with LIR-7 polypeptide or functional fragments thereof. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256:495, 1975). The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab'), (2) Fv and SCA fragments which are capable of binding an epitopic determinant on LIR-7.

[0051] An Fab fragment consists of a monovalent antigen binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

[0052] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

[0053] An (Fab') (2) fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab') (2) fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.

[0054] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. [0055] A single chain antibody ("SCA") is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.

[0056] As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen, such as a LIR-7 polypeptide, to which the paratope of an antibody, such as an LIR-7 specific antibody, binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0057] As is mentioned above, antigens that can be used in producing LIR-7 specific antibodies include LIR-7 polypeptides or LIR-7 polypeptide fragments. The polypeptide or peptide used to immunize an animal can be obtained by standard recombinant, chemical synthetic, or purification methods. As is well known in the art, in order to increase immunogenicity, an antigen can be conjugated to a carrier protein. Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). In addition to such carriers, well known adjuvants can be administered with the antigen to facilitate induction of a strong immune response.

[0058] Monoclonal antibodies used in the method of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0059] The term "immunometric assay" or "sandwich immunoassay", includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which maybe developed in the future. These are intended to be included within the scope of the present invention.

[0060] Monoclonal antibodies can be bound to many different carriers and used to detect the presence of LIR-7. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.

[0061] In performing the assays it may be desirable to include certain "blockers" in the incubation medium (usually added with the labeled soluble antibody). The "blockers" are added to assure that non-specific proteins, proteases, or anti-heterophilic immunoglobulins to anti-LIR-7 immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results. The selection of "blockers" therefore may add substantially to the specificity of the assays described in the present invention.

[0062] It has been found that a number of nonrelevant (i.e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGl, IgG2a, IgM, etc.) can be used as "blockers". The concentration of the "blockers" (normally 1-100 μg/μl) may be important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen. [0063] In using a monoclonal antibody for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the LIR-7 antigen for which the monoclonal antibodies are specific. The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having LLR-7 is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

[0064] As a rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of monoclonal antibody can vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m . Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors known to those of skill in the art.

[0065] For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.

[0066] For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are ^ιπIn, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹T1.

[0067] A monoclonal antibody useful in the method of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe.

[0068] Other useful fragments of LIR nucleic acids are antisense or sense oligonucleotides which include a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to a target LIR mRNA (sense) or LIR DNA (antisense) sequences. Such fragments are generally at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to create an antisense or a sense oligonucleotide based upon a cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

[0069] Binding antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block translation (RNA) or transcription (DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block LLR expression.

[0070] In one embodiment antisense or sense LLR oligonucleotides used in binding procedures may encompass oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Oligonucleotides having sugar linkages resistant to endogenous nucleases are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.

[0071] Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO₄ - mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by inserting he antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retroviral vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

[0072] Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugating the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor, or block entry of the sense of antisense oligonucleotide or its conjugated version into the cell.

[0073] Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. [0074] In still a further aspect, the present invention provides antibodies that specifically bind LIR polypeptides, i.e., antibodies bind to LIR polypeptides via an antigen-binding site of the antibody (as opposed to non-specific binding). Antibodies of the present invention may be generated using LIR polypeptides or immunogenic fragments thereof. Polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York 1980; and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.

[0075] Included within the scope of the present invention are antigen binding fragments of antibodies which specifically bind to an LIR polypeptide. Such fragments include, but are not limited to, Fab, F(ab'), and F(ab') (2). Antibody variants and derivatives produced by genetic engineering techniques are contemplated as within the presented invention.

[0076] The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al., Nature 332:232, 1988; Lie et al. PNAS 84:3439, 1987; Larrick et al. Bio/Technology 7:934, 1989; and Winter and Harris TIPS 14:139, 1993.

[0077] As mentioned above, antibodies of the present invention are useful in in vitro or in vivo assays to detect the presence of LIRpolypeptides and in purifying an LIR polypeptide by affinity chromatography. [0078] Additionally, antibodies capable of blocking an LIR from binding to target cells may be used to inhibit a biological activity of an LLR polypeptide. More specifically, therapeutic compositions of an antibody antagonistic to one or more LLR family members having the ITLM motif may be administered to an individual in order to block the interaction of a cell surface LIR with its ligand. The result is an activation of immune function and is particularly beneficial in disease states in which the immune system is hyporesponsive or suppressed. Conversely, therapeutic compositions of an antibody antagonistic to one or more LIR family members lacking the ITIM motif may be used to obtain the opposite effect and be beneficial in disease states in which the immune system is overactive and excessive inflammation or immunopathology is present.

[0079] The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1

MATERIALS AND METHODS

Preparation of RNA from leprosy patient samples

[0080] Leprosy patients were evaluated at the Los Angeles County Hansen's Disease Clinic and classified as T-lep or L-lep according to the criteria of Ridley and Jopling. (1) Frozen skin biopsies taken from 11 patients were treated with guanidinium-isothiocyanate (GTC) buffer to extract total RNA, as previously described. (29) RNA was digested with DNase I (Promega, Madison, WI) for 2 hours to remove contaminating genomic DNA and extracted with phenol chloroform. Quality of the RNA was confirmed using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).

Synthesis of cRNA probes and hybridization

[0081] To generate the cRNA probes, 2-5 _μg of total RNA was reverse transcribed into double-stranded cDNA using HPLC-purified T7-(dT)₂₄ primers (Integrated DNA Technologies, Coralville, LA) and the Superscript cDNA synthesis system (Life Technologies, Grand Island, NY) according to the manufacturers' instructions. The resulting products were used as templates to synthesize biotinylated cRNA probes using the Bioarray RNA Transcript Labeling Kit (Enzo, Farmingdale, NY). The probes were purified and fragmented, and hybridized to the Affymetrix Hu95Av2 Genechip (Affymetrix, Santa Clara, CA) at the UCLA Microarray Core Facility. Hybridization images were read using a Hewlett-Packard Gene-array Scanner (Affymetrix, Santa Clara, CA), and inspected for uniformity. Expression values for each probe set and background binding levels were determined using Affymetrix Microarray Suite software. The intensities across multiple chips were normalized to a target intensity of 2500 using global normalization scaling.

Unsupervised gene expression analyses

[0082] Principal component analysis was performed using all -12,000 genes arrayed on the DNA chips as previously described ⁵. The data was first centered by subtracting the mean expression level of each gene across all the samples. For hierarchical clustering analysis, (6) -2,200 genes were used with significant variation across the samples (standard was also deviation (σ) > 1000 and a coefficient of variation (σ/mean) > 0.3). Genes were also required to have Affymetrix present calls in at least half of the experiments.

Definition of differentially expressed genes in lesions

[0083] To identify the most informative set of differentially expressed genes between the two leprosy subclasses, each gene was ranked by the probability that the means of its expression values are statistically distinct between the T-lep and L-lep patients using the Student's t-test. The fold change was also calculated in mean expression of each gene between the two groups and used this value as a secondary ranking. Attention was focused on genes that met a designated criteria: p <0.05 and a fold change of >1.5. Genes from this list were used to create training sets for the supervised permutation analysis and class prediction tests described below. Permutation analysis

[0084] To determine the statistical significance of our results, we systematically considered all possible groupings were systematically considered and subsequently the frequency at which a grouping yielded a result equal to or better than that of the defined T- lep/L-lep grouping was ascertained. For all 462 possible patient groupings (with 5 patients in one group, 6 in the other; (11 !/(6! x 5!))) the mean and various significance levels for the number of genes with Student's t-test p- values greater than a sliding threshold level and compared these to the respective numbers for T-lep/L-lep grouping were calculated. (7)

Prediction algorithm

[0085] For gene expression based prediction the weighted gene voting algorithm of Golub et al.(7) was used with minor modifications and leave-one-out cross-validation. For the weighting factor we used the same measure of differential expression as used in the

Student's t-test, t = where μ;, σ;, and m represent the mean, standard

deviation, and sample number of the i'th sample group, respectively was used. For

prediction strength (PS),

score for the unknown sample based on its expression values x_g for the top 50 genes (g) and the t-test gene weights (t_g) for these genes, and the denominator is the absolute value of the expected voting score for an ideal member of either class

for all g) was used. When support vector machine (SVM) analysis in place of weighted voting was used comparable results (data not shown) were observed. Assignment of genes to functional categories

[0086] Genes that met the established criteria for differential expression (described above) were organized into categories based on function or cellular location as described in the OMIM and Locus Link databases at the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov). In some cases, expressed sequence tags (ESTs) that did not match entries in either database were identified using the Celera Discovery System (www.celera.com).

(1) qPCR cDNA obtained from leprosy lesions was subjected to qPCR analysis to measure levels of LIR-7 mRNA. Primers and probe sets for LIR-7 (IDT, Coralville, IA) were designed using Primer Express (Applied Biosystems, Branchburg, New Jersey). PCR reactions were done in a total volume of 50 μl containing the TaqMan Universal PCR Master Mix (Applied Biosystems) and the gene-specific primer and probe set used at a final concentration of 500nM and lOOnM, respectively. Each reaction was run in duplicate in an MJR Opticon machine and analyzed using Opticon 1.05 software (MJ Research, Waltham, MA). Relative expression levels were determined using the ΔΔCT method, as previously described. (30)

Flow cytometry

[0087] Primary monocytes were isolated from peripheral blood of healthy donors as previously described, (31) and cultured for 24 hrs in the presence or absence of ligands for TLR4 (LPS, 10 ng/ml) or TLR2/1 (19kD lipopeptide, 5 μg/ml). Resulting cells were blocked in 20% human serum on ice and stained with control rat IgG2a (Pharmingen, San Diego, CA), or rat-anti human LIR-7 mAbs as previously described. (17) Data was analyzed using WinMDI 2.8 (Joseph Trotter, Scripps Research Institute, San Diego, CA).

In vitro stimulation of monocytes

[0088] Primary monocytes were isolated as described above and incubated in 96-well microtiter plates (5xl0⁴/well) in the presence of isotype control or rat anti-human LIR-7 mAbs for 30 min on ice prior to addition of a crosslinking goat anti-rat IgG mAb (Jackson Laborotories, Westgrove, PA), as previously described. (17) Cells were then cultured for 24 hrs in media containing 100 U/ml IFN-γ (Endogen, Woburn, MA) and serially diluted amounts of TLR agonists LPS (TLR4) or 19kD lipoprotein (TLR1/2). (32) Supernatants were harvested and analyzed for production of IL-12 and IL-10 by ELISA (Pharmingen). Antibodies used for tissue culture were azide-free and contained <1 pg/ml of LPS contamination, as determined by Limulus-Amoebocyte Assay (BioWhittaker, Walkersville, MD).

Reference List

1. D. S. Ridley and W. H. Jopling, hitJ.Lepr. 34, 255-273 (1966).

2. M. Yamamura et al., Science 254, 277-279 (1991).

3. P. Salgame et al., Science 254, 279-282 (1991).

4. R. L. Modlin et al, Proc.Natl.Acad.Sci.U.S.A. 85, 1213-1217 (1988).

5. O. Alter, P. O. Brown, D. Botstein, Proc.Natl.Acad.Sci.U.S.A 97, 10101-10106 (2000).

6. M. B. Eisen, P. T. Spellman, P. O. Brown, D. Botstein, Proc.Natl.Acad.Sci.U.S.A 95, 14863-14868 (1998).

7. T. R. Golub et al., Science 286, 531-537 (1999).

8. R. L. Modlin et al., J.Immunol. 132, 3085-3090 (1984).

9. M. Yamamura et al., J.Immunol. 149, 1470-1475 (1992).

10. P. A. Sieling et al., J.Immunol. 154, 2775-2783 (1995).

11. D. Jullien et al., J.Immunol. 158, 800-806 (1997).

12. P. A. Sieling et al., J Immunol 162, 1851-1858 (1999).

13. V. E. Garcia et al., J Immunol 167, 5719-5724 (2001).

14. P. S. Hiemstra et al., Infect.Immun. 64, 4520-4524 (1996).

15. E. P. Reeves et al., Nature 416, 291-297 (2002).

16. L. Borges and D. Cosman, Cytokine Growth Factor Rev. 11, 209-217 (2000). 17. H. Nakajima, J. Samaridis, L. Angman, M. Colonna, J.Immunol. 162, 5-8 (1999).

18. N. Tedla et al., Proc.Natl.Acad.Sci.U.S.A 100, 1174-1179 (2003).

19. L. Borges, M. Kubin, T. Kuhlman, Blood 101, 1484-1486 (2003).

20. A. O'Garra, Immunity. 8, 275-283 (1998).

21. G. Trinchieri, Nat.Rev.Immunol. 3, 133-146 (2003).

22. F. S. Sutterwala, G. J. Noel, R. Clynes, D. M. Mosser, J.Exp.Med. 185, 1977- 1985 (1997).

23. F. S. Sutterwala, G. J. Noel, P. Salgame, D. M. Mosser, J.Exp.Med. 188, 217- 222 (1998).

24. Q. Huang et al., Science 294, 870-875 (2001).

25. C. E. Belcher et al., Proc.Natl.Acad.Sci.U.S.A 97, 13847-13852 (2000).

26. S. Ehrt et al., J.Exp.Med. 194, 1123-1140 (2001).

27. C. M. Perou et al., Nature 406, 747-752 (2000).

28. M. Biftner et al., Nature 406, 536-540 (2000).

29. P. Chomczynski and N. Sacchi, Anal.Biochem. 162, 156-159 (1987).

30. L. Monney et al., Nature 415, 536-541 (2002).

31. J. R. Bleharski et al., J Immunol in press, (2003).

32. H. D. Brightbill et al, Science 285, 732-736 (1999). [0089] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed:

1. A method of treating an autoimmune, inflammatory or infectious disease in a subject comprising administering to the subject, a therapeutically effective amount of an agent that modulates the expression or activity of leukocyte immunoglobulin-like receptor 7 (LIR-7).

2. The method of claim 1, wherein the agent is selected from the group consisting of an antibody, a peptide, a peptidomimetic, a protein, a nucleic acid and a small molecule.

3. The method of claim 2, wherein the agent is an antibody.

4. The method of claim 3, wherein the antibody is a monoclonal antibody.

5. The method of claim 4, wherein the monoclonal antibody is therapeutically labeled.

6. The method of claim 5, wherein the therapeutic label is selected from a radioisotope, a drug, a lectin, or a toxin.

7. The method of claim 1 , wherein the disease is characterized by a hyporesponsive or hyperresponsive immune reaction.

8. The method of claim 7, wherein the immune response is characterized as a Thl/type 1 or Th2/type 2 dominant immune response.

9. The method of claim 7, wherein the disease is characterized by a hyporesponsive immune reaction and the modulating agent inhibits LLR-7 activity associated with a Th2/type 2 dominant immune response, whereby inhibiting LIR-7 increases activatory signaling.

10. The method of claim 9, wherein the disease is a skin disease.

11. The method of claim 10, wherein the skin disease is atopic dermatitis.

12. The method of claim 7, wherein the disease is characterized by a hyperresponsive immune reaction and the modulating agent stimulates LIR-7 activity associated with a Thl/type 1 dominant immune response to down regulate cell function.

13. The method of claim 12, wherein the disease exhibits excess inflammation or immunopathology.

14. The method of claim 13, wherein the disease is selected from AIDS, Addison's Disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), Behcet's disease, chronic fatigue syndrome, Crohn's disease and ulcerative colitis, diabetes, fibromyalgia, Goodpasture syndrome, graft versus host disease/graft rejection, Graves' disease, Guillain-Barre syndrome, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, myositis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, vitiligo, or Wegener's granulomatosis.

15. The method of claim 12, wherein the disease is from a skin disease.

16. The method of claim 15, wherein the skin disease is psoriasis.

17. The method of claim 1, wherein the administering is parenteral, oral or topical.

18. The method of claim 16, wherein parenteral administration is by subcutaneous, intramuscular, intraperitoneal, intracavity, transdermal, or intravenous injection.

19. A method of identifying a modulating agent for leukocyte immunoglobulin- like receptor-7 (LIR-7) comprising:

a) contacting a cell with a test agent in the presence of a toll-like receptor (TLR) agonist;

b) allowing the agent to incubate with the cell for a sufficient time to determine the production of a TLR agonist-induced type 1 or type 2 cytokine; and c) determining the amount of the cytokine produced in the presence and absence of the test agent,

wherein a difference in the production of a cytokine in the presence and absence of the test agent is indicative of the modulation of LIR-7 with the test agent.

20. The method of claim 19, further comprising contacting the cell with a test agent in the presence and absence of an anti-LIR-7 antibody.

21. The method of claim 19, wherein the cell is myeloid or lymphoid.

22. The method of claim 21, wherein the cell is a monocyte.

23. The method of claim 21, wherein the cell is an eosinophil.

24. The method of claim 19, wherein the TLR agonist is selective for TLR2/1 or TLR4.

25. The method of claim 19, further comprising assaying for modulation of FcR gamma or DAP 12 expression.

26. The method of claim 19, wherein the cytokine is IL-12 or IL-10.

27. The method of claim 20, further comprising contacting the cell with interferon gamma.

28. A method of identifying a leukocyte immunoglobulin-like receptor-7 (LIR- 7) agonist comprising:

a) contacting a cell with a test agent in the presence of a toll-like receptor (TLR) agonist;

b) allowing the agent to incubate with the cell for a sufficient time to determine the production of a TLR agonist-induced cytokine; and c) determining the amount of a type 1 cytokine produced in the presence and absence of the test agent,

wherein a decrease in the production of the type 1 cytokine in the presence of the test agent is indicative of an LIR-7 agonist.

29. The method of claim 28, further comprising contacting the cell with a test agent in the presence and absence of an anti-LIR-7 antibody.

30. The method of claim 28, wherein the cell is myeloid or lymphoid.

31. The method of claim 30, wherein the cell is a monocyte.

32. The method of claim 30, wherein the cell is a eosinophil.

33. The method of claim 28, wherein the TLR agonist is selective for TLR2/1 or TLR4.

34. The method of claim 28, further comprising assaying for modulation of FcR gamma or DAP 12 expression.

35. The method of claim 28, wherein the cytokine is IL-12 or IL-10.

36. The method of claim 35, wherein a decrease in IL-12 production in the presence of the test agent is indicative of an LIR-7 agonist.

37. The method of claim 35, wherein there is an increase in IL-10 production in the presence of the test agent.

38. The method of claim 29, further comprising contacting the cell with interferon gamma.

39. A method of identifying a leukocyte immunoglobulin-like receptor-7 (LLR- 7) antagonist comprising: a) contacting a cell with a test agent in the presence of a toll-like receptor (TLR) agonist;

b) allowing the agent to incubate with the cell for a sufficient time to determine the production of a TLR agonist-induced cytokine; and

c) determining the amount of a type 1 cytokine produced in the presence and absence of the test agent,

wherein an increase in the production of the type 1 cytokine in the presence of the test agent is indicative of an LIR-7 antagonist.

40. The method of claim 39, further comprising contacting the cell with a test agent in the presence and absence of an anti-LIR-7 antibody.

41. The method of claim 39, wherein the cell is myeloid or lymphoid.

42. The method of claim 41 , wherein the cell is a monocyte.

43. The method of claim 41, wherein the cell is a eosinophil.

44. The method of claim 39, wherein the TLR agonist is selective for TLR2/1 or TLR4.

45. The method of claim 39, further comprising assaying for modulation of FcR gamma or DAP 12 expression.

46. The method of claim 39, wherein the cytokine is IL-12 or IL-10.

47. The method of claim 39, wherein an increase in IL- 12 production in the presence of the test agent is indicative of an LIR-7 antagonist.

48. The method of claim 40, further comprising contacting the cell with interferon gamma.

49. A method of predicting the clinical course of an autoimmune, inflammatory or infectious disease in a subject comprising detecting a gene expression cytokine profile indicative of Thl/type 1 or Th2/type 2 immune responses and correlating the profile with disease progression.

50. The method of claim 49, wherein the profile is predictive of outcome in subjects with microbial infections.

51. The method of claim 49, wherein the profile is predictive of outcome in subjects with an autoimmune disease.

52. The method of claim 51 , wherein the disease is selected from Addison's Disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), Behcet's disease, chronic fatigue syndrome, Crohn's disease and ulcerative colitis, diabetes, fibromyalgia, Goodpasture syndrome, graft versus host disease, Graves' disease, Guillain-Barre syndrome, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, myositis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, vitiligo, or Wegener's granulomatosis.

53. The method of claim 49, further comprising:

i) obtaining a biological sample of a test and control subject;

ii) treating the samples with a chaotropic agent to release nucleic acids;

iii) extracting extraneous biological materials to purify an appropriate nucleic acid fraction;

iv) reverse transcribing the nucleic acid fraction obtained in step (iii);

v) synthesizing probes from the transcripts of step (iv); and

vi) hybridizing the probes of step (v) to prefabricated surface immobilized gene arrays, wherein the resulting signals from the arrays are used to generate informative sets of differentially expressed genes between test and control samples.

54. The method of claim 53, further comprising application of a weighted gene voting algorithm.

55. The method of claim 54, wherein candidate genes are organized based on function or cellular location.

56. A method of differentiating clinical manifestations of leprosy comprising correlating immune response to Mycobacterium leprae infection using patient lesion samples, wherein the manifestations can be characterized by the determination of type 1 cytokine expression and cell mediated immunity or by the determination of type 2 cytokine expression and humoral immunity and suppression of a cell-mediated immune response.

57. The method of claim 56, wherein determination of type 1 cytokine expression correlates with the tuberculoid form (T-lep) of leprosy.

58. The method of claim 57, wherein the T-lep form is further characterized by limited self-curing disease accompanied by low bacterial load.

59. The method of claim 57, wherein there is an increase in type 1 cytokine expression.

60. The method of claim 59, wherein there is an increase in the expression of lymphotoxin-alpha, IL-7, IL-12 or LL- 15.

61. The method of claim 56, wherein determination of type 2 cytokine expression correlates with the lepromatous form (L-lep) of leprosy.

62. The method of claim 61, wherein the type 2 cytokine is JL-5, IL-4 or IL-10.

63. The method of claim 61 , wherein the L-lep form is further characterized by patients presenting clinically disseminated lesions accompanied by high bacterial load.

64. The method of claim 61 , further comprising determination of the expression of leukocyte immunoglobulin-like receptors (LIRs), FcR gamma, or DAP 12.

65. The method of claim 64, wherein the gene is LIR-7.

66. The method of claim 65, wherein increased expression of LIR-7 in patient lesion samples is predictive of L-lep.

67. The method of claim 58, further comprising determination of the expression of genes selected from the group consisting of CDlb, signaling lymphocytic activation molecule (SLAM), secretory leukocyte protease inhibitor (SLPI), and cathepsin G.

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FIG. 1A FIG. 1B

Original Correctc d

low ' βi high

Relative expression FIG. 1C 2/5

Probability (p-value)

FIG. 2A

Correctly assigned samples (prediction strength > 0.4)

FIG. 2B 3/5

T-lep patients L-lep patients

Functional category #genes %total #genes %total

Immune response 29 7.82 60 9.65

Pro-inflammatory 12 3.23 1 0.16

T cells / antigen presentation 7 1.89 2 0.32

Anti-inflammatory 0 0.00 12 1.93

B cell response 1 0.27 24 3.86

Chemotaxis 4 1.08 6 0.96

Innate 5 1.35 15 2.41

Adhesion/structural 33 8.89 45 7.23

Apoptosis 9 2.43 2 0.32

Growth factor/hormone 12 3.23 23 3.70

Heavy metal binding 6 1.62 0 0.00

Neuronal 12 3.23 25 4.02

Signaling/gene regulation 52 14.02 115 18.49

Housekeeping 145 39.08 163 26.21

Unknown 73 19.68 189 30.39

Total 371 100.00 622 100.00

FIG. 3A

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FIG. 3B 5/5

FIG.4B

□ Control Ig ■ -LIR-7

TLR2/1 ligand (ng/ml) TLR4 ligand (ng/ml)

FIG. 4C