- Top of Page
OF THE INVENTION
The astonishing complexity of the immune system is its greatest strength. The 1012-1014 possible antibody specificities, the delicate interplay between the various regulatory and effector cells, the restriction of T cell responses according to MHC antigens; all these contribute to the ability of the host to effectively react against infectious agents and other antigens perceived as foreign. But this diversity has its drawbacks. Mistakes happen: the target of a response may turn out to be a normal self protein; inflammatory responses are misregulated; and normal responses are undesirably directed against grafts and transplanted cells. Under these circumstances, the complexity of the system makes diagnosis and therapy extremely difficult.
The profile of cytokines produced by CD4+ T cells during an immune response determines the nature of effector functions which develop and regulates the outcome of an immune response. Production of IL-2 and IFN-γ during Th1-dominated responses is associated with vigorous cell-mediated immunity, the induction of IgG2a and inhibition of IgE synthesis, and with resistance to intracellular pathogens. In contrast, the production of IL-4, IL-5 and IL-10 during Th2-dominated responses is associated with humoral immunity and protection from autoimmune pathology. Overproduction of Th2-cytokines by allergen-specific CD4+ T cells can result in the development of allergic disease and asthma, while Th1 cells have been associated with a variety of pro-inflammatory diseases.
One approach to immune associated diseases is immunotherapy. Immunotherapy has proven to be effective when used properly, and it is hoped that advances in immunologic intervention will further improve the efficacy. Alternative approaches have attempted to use cytokines to shift the immune response. IL-12, a heterodimeric cytokine produced by macrophages and dendritic cells, is potent in driving the development of Th1 cytokine synthesis in naive and memory CD4+ T cells. Other cytokines, such as IL-13 and IL-4, have been associated with the differentiation of T cells to a Th2 type.
Atopy, which includes asthma, allergic rhinitis, and atopic dermatitis, is a complex trait that arises as a result of environmentally induced immune responses in genetically susceptible individuals. The prevalence of all atopic diseases has dramatically increased in industrialized countries over the past two decades. Asthma is the most common chronic disease of childhood and affects more than 15 million individuals in the United States, leading to direct treatment costs exceeding $11 billion per annum. Epidemiological studies have suggested that the increase in asthma prevalence results from changes in hygiene and from reduced frequency of infections (e.g., tuberculosis or hepatitis A) within industrialized society. However, the specific molecular pathways that result in the increased asthma prevalence, and the genetic polymorphisms that confer asthma susceptibility are poorly understood.
Expression of asthma is influenced by multiple environmental and genetic factors that interact with each other in non-additive ways, complicating the identification of asthma susceptibility genes. Asthma susceptibility has been linked to several chromosomal regions, but with resolution no better than 5-10 cM, in which there are usually hundreds of candidate genes. Moreover, because the effects of genetic variation in any single gene are likely to have only modest effects in the overall pathogenesis of asthma, and because gene-gene and gene-environment interactions confound the analysis, the location of putative susceptibility genes to regions amenable to positional cloning has proven difficult to refine. Nevertheless, asthma susceptibility has been linked to chromosomes 5, 6, 11, 14, and 12. Of these, chromosome 5q23-35 has received the greatest attention because it contains a large number of candidate genes (11, 12, 13, 14, 15, 16, 17, 18), including IL-9, IL-12p40, the β-adrenergic receptor, and the IL4 cytokine cluster, which contains the genes for IL-4, IL-5, and IL-13. However, the large size of the linked region of 5q complicates its analysis, and a gene for asthma from this site has not yet been conclusively identified.
The genetic sequence of the human hepatitis virus A cellular receptor may be found in Genbank, accession number XM—011327. A related sequence is provided in Genbank, accession number BAB55044. Monney et al. (2002) Nature 415:436 describe cell surface molecules expressed on Th1 cells. U.S. Pat. No. 5,721,351, U.S. Pat. No. 6,204,371, U.S. Pat. No. 6,288,218 relate to sequences corresponding to a mouse TIM-3 allele.
- Top of Page
OF THE INVENTION
Genetic sequences of a gene family encoding polypeptides associated with immune function and cell survival are provided. These genes encode cell surface molecules with conserved IgV and mucin domains, herein referred to as T cell Immunoglobulin domain and Mucin domain (TIM) proteins. The locus comprising the TIM family is genetically associated with immune dysfunction, including asthma. Furthermore, the TIM gene family is located within a region of human chromosome 5 that is commonly deleted in malignancies and myelodysplastic syndrome. Polymorphisms are identified in TIM-1, TIM-3 and TIM-4, which can be associated with Th1/Th2 differentiation and airway hyperresponsiveness (AHR).
The nucleic acid compositions are used to produce the encoded proteins, which may be employed for functional studies, as a therapeutic, and in studying associated physiological pathways. TIM specific binding agents, including nucleic acids, antibodies, and the like, are useful as diagnostics for determining genetic susceptibility to atopy and asthma and as diagnostics for assessing tumor resistance to cancer therapy. TIM blocking agents find use as therapeutics in the treatment of immune dysfunction and disorders of cell survival, including malignancies.
BRIEF DESCRIPTION OF THE DRAWINGS
- Top of Page
FIG. 1a, HBA mice produce significantly less IL-4 than do BALB/c mice. Lymph node cells of mice immunized with 150 μg of KLH were harvested, B-cell depleted, and cultured in cDME with 10 μg/ml KLH. Supernatants were harvested after 96 hours and assessed for IL-4 levels by ELISA. Shown is a box plot of IL-4 levels (n=10 in each group), representing the full range of data, with the boxes encompassing the upper and lower quartiles, with the median of the data set shown inside each box. IL-4 levels produced by BALB/c cells and by (BALB/c×HBA) F1 cells are significantly higher than HBA IL-4 levels P<0.0001 (student's t-test). b, HBA mice produce significantly less IL-13 and IL-10 than do BALB/c mice. Data shown are the average values of cytokines produced by lymph node cell cultures from ten individual mice in each experimental group, ±S.D. HBA IL-13 and IL-10 levels were lower than either the BALB/c or (BALB/c×HBA) F1 values, P<0.0001. HBA IL-5 versus BALB/c, P<0.05. HBA IFN-γ versus BALB/c, P<0.001. c, Allergen-induced airway hyperreactivity is significantly greater in BALB/c than do HBA mice. Pulmonary airflow obstruction was measured, and data shown represent peak enhanced pause (Penh) values averaged among sensitized mice in each group at various methacholine concentrations, +S.E.M. (BALB×HBA) F1 demonstrate BALB/c phenotypes, while (BALB×DBA) F1 mice demonstrate DBA/2-like phenotypes.
FIG. 2. Regions of HBA chromosome 11 were inherited from DBA/2. HBA chromosome 11 contains two regions derived from DBA between hba-α2 and es-3, as delineated by SSLP markers. The regions of HBA chromosome 11 with DBA/2 genotypes are highlighted (in blue) in the diagram to the left. The markers (left) provide 2-5 cM resolution distal to D11Mit230. Where the mouse chromosome 11 has regions of highly conserved synteny with 5q23-35, additional markers were identified with informative polymorphisms between BALB/c and DBA/2, to provide 0.01-2 cM resolution in this region (right column markers). Single-stranded confirmation polymorphisms (SSCP) markers are shown in green to distinguish them from the SSLP markers, and the positions of particular genes of interest, denoted in red, are also shown. Where the arrangement of our marker map differs from the Chromosome Committee Reports and the MIT linkage map, our map concurs with previous linkage and physical maps.
FIG. 3a. IL-4 production by N2 mice is bimodal, with peaks corresponding to F1 and HBA phenotypes. As a means of evaluating the relative phenotypes of recombinant N2 mice in multiple experiments, we utilized an indexing function that allowed us to consolidate data from multiple experiments. A histogram shows a bimodal distribution of IL-4 index values for N2 mice with (BALB×HBA) F1, HBA homozygous, and recombinant haplotypes indicated. Distributions associated with the F1 and HBA haplotypes are distinct (P<0.0001, paired Student's t-test)-b. IL-4 regulation segregates with Kim1sscp. Recombinant N2 haplotypes were sorted by IL-4 index values into groups associated with high IL-4 phenotypes (index<0.35) and low IL-4 phenotypes (index>0.65). Each column of boxes represents a recombinant haplotype. Alleles for these haplotypes at loci between D11Mit269 to D11Mit154 are shaded according to genotype (dark=F1; pale ═HBA). High IL-4 production segregates with four haplotypes (left), and low IL-4 production segregates with four haplotypes (right). The IL4 phenotype is linked to Kim1sscp. c. AHR of N2 mice is bimodal, with peaks corresponding to F1 and HBA phenotypes. Index values calculated from Penh values are shown. The histogram shows a bimodal distribution of AHR index values for N2 mice. (BALB×HBA) F1, HBA homozygous, and recombinant haplotypes are indicated. Distributions associated with the F1 and HBA haplotypes are distinct (P<0.0001, paired student's t-test). d. AHR Regulatory Locus cosegregates with the IL-4 Regulatory Locus between D11Mit22 and D11Mit271. Recombinant N2 haplotypes were sorted by AHR index values into groups associated with high AHR phenotypes (index<0.35) and low AHR phenotypes (index>0.65). Each column of boxes represents a recombinant haplotype. Alleles for these haplotypes at loci between D11Mit269 to D11Mit154 are shaded according to genotype (dark=F1; pale=HBA). High AHR segregates with four haplotypes (left), and low IL-4 production segregates with three haplotypes (right). The AHR regulatory locus is linked to Kim1sscp, between D11Mit271 and D11Mit22.
FIG. 4 Mouse chromosome 11 interval containing Tapr is highly homologous to 5q33. In order to construct a composite map around the Tapr locus, we integrated available information from the mouse linkage, backcross, and radiation hybrid maps and identified a region of highly conserved synteny in current maps of the human genome (Human Genome Browser v3, UCSC, March 2001). ESTs located on a physical map of mouse chromosome 11 (left) are denoted by their accession numbers and aligned by homology to genes (center), which correspond to ESTs on human chromosome 5 (right). All known genes between KIAA0171 and Sgd are shown.
FIG. 5a,b,c. Identification novel TIM gene family and major polymorphisms in TIM-1 and TIM-3. Cloning of mouse TIM-1 and mouse TIM-2. Members of a gene family. Sequences of the mouse TIM gene family members are shown. Shaded boxes illustrate identity between two of the mouse TIM genes. Total RNA from conA-stimulated splenocytes was reverse transcribed using Gibco Superscript II. PCR products of full length Tim-3 cDNA were amplified, purified with Qiagen QIAquick PCR Purification reagents, and sequenced directly by Biotech Core (Mountain View, Calif.). PCR products for Tim-1 and Tim-2 cDNA were cloned into electrocompetent TOP10 cells with TOPO-XL cloning reagents (Invitrogen). Plasmids were purified with a standard alkaline lysis protocol. BALB/c and HBA plasmids were sequenced, as described. Homology of mouse TIM-1, rat KIM-1, and HAVcr-1. Identity with mouse TIM-1 is denoted by the shaded boxes. The approximate signal site is denoted by an open, inverted triangle and the Ig domain/mucin domain boundary is shown with a filled diamond. The predicted transmembrane domains are underlined. TIM-1 and TIM-3 sequences with major polymorphisms between BALB/c and HBA TIM-1 and TIM-3 shown.
FIG. 6. Tapr Regulates CD4 T cell IL-4 and IL-13 Responses. T cells from BALB/c DO11.10 mice produce higher levels of IL-4 and IL-13 in response to antigen than do T cells from HBA DO11.10 mice. Splenic CD4+ cells were isolated by positive selection with anti-CD4 magnetic beads and then cocultured with bone marrow-derived DCs and OVA. After seven days, the cells were restimulated. Supernatants were harvested after 18-24 hours of the secondary culture. Data represent mean cytokine levels detected at increasing concentrations of OVA, ±S.D. Detection of expression of TIM-1 mRNA in purified CD 4 T cells during priming and differentiation.
FIG. 7. Sequence alignment of Human and Mouse TIM protein sequences.
FIG. 8. Polymorphisms in human TIM-1
FIG. 9. SSCP polymorphism analysis of human TIM-1.
- Top of Page
OF THE EMBODIMENTS
Genetic sequences associated with immune function, including susceptibility to asthma, are provided. The sequences of murine Tim-1, Tim-2, Tim-3, and Tim-4, and the human counterpart sequences, are provided herein. The sequence of major polymorphisms are also provided. Genomic sequences demonstrate that these polymorphisms, including a deletion, are true polymorphisms, not splicing variants. Variants of the TIM-1 and TIM-3 sequences are associated with susceptibility to airway hyperreactivity and allergic T cell responses, and other variants associated with protection against these responses.
The extracellular domain of TIM gene family members contains two domains, an IgV domain and a mucin domain. This Ig/mucin structure is also found in MAdCAM (mucosal addressin cell adhesion molecule), which contains two Ig domains and a mucin domain; however, there is only a low level of homology between TIMs and MAdCAM. A similar degree of homology is also present between TIM-1 and TOSO (NP—005440.1 GI:4885641), a protein which, like TIM-1 is expressed on activated T cells, and which protects T cells from Fas-mediated apoptosis.
T cells express the TIM family of genes, which critically regulates CD4 T cell differentiation. Th1 cells preferentially express the TIM-3 protein, while Th2 cells preferentially express the TIM-1 protein. TIM-1 has been linked to airway hyperreactivity and TIM-3 to autoimmune disease, therefore the expression pattern on differentiating lymphoid cells and the kinetics of expression of TIM-1 on lymphoid cells reflect the function of these molecules.
In another aspect of the invention, methods are provided for determining susceptibility of an individual to developing asthma and atopic diseases, by determining the genotype at the Tim locus. Screening may analyze, for example, polymorphisms in any one of the TIM-1, TIM-3 or TIM-4 alleles provided herein, or otherwise determined. Methods are provided for screening such polymorphisms, e.g. SSCP analysis, size polymorphisms, and the like.
In another aspect of the invention, a method of screening for biologically active agents that modulate Tim gene or polypeptide function is provided, where the method comprises combining a candidate biologically active agent with any one of: (a) a Tim polypeptide; (b) a cell comprising a nucleic acid encoding a Tim polypeptide; or (c) a non-human transgenic animal model for Tim gene function comprising one of: (i) a knockout of an Tim gene; (ii) an exogenous and stably transmitted Tim gene sequence; or (iii) a Tim promoter sequence operably linked to a reporter gene; and determining the effect of said agent on Tim function.
The activity of TIM polypeptides may be modulated in order to direct immune function. TIM-1 is preferentially expressed in Th2 cells, and agents that modulate TIM-1 activity find use in the treatment of Th2 related disorders, including allergies, asthma, and the like. TIM-3 is preferentially expressed in Th1 cells, and agents that modulate TIM-3 activity find use in the treatment of pro-inflammatory immune diseases, including autoimmune diseases, graft rejection and the like.
Conditions of Interest
Atopic diseases are complex genetic traits that develop as a result of environmentally induced immune responses in genetically predisposed individuals. Both atopic and non-atopic individuals are exposed to the same environmental factors, but genetic differences that distinguish atopic from non-atopic individuals result in atopic disease in some individuals, manifested by allergic inflammation in the respiratory tract, skin or gastrointestinal tract, as well as by elevated serum IgE, eosinophilia and the symptoms of wheezing, sneezing or hives. In addition, allergic inflammatory responses are characterized by the presence of Th2 lymphocytes producing high levels of IL-4, IL-5, IL-9 and IL-13, which enhance the growth, differentiation and/or recruitment of eosinophils, mast cells, basophils and B cells producing IgE.
Allergens of interest include antigens found in food, such as strawberries, peanuts, milk proteins, egg whites, etc. Other allergens of interest include various airborne antigens, such as grass pollens, animal danders, house mite feces, etc. Molecularly cloned allergens include Dermatophagoides pteryonyssinus (Der P1); Lol pl-V from rye grass pollen; a number of insect venoms, including venom from jumper ant Myrmecia pilosula; Apis mellifera bee venom phospholipase A2 (PLA2 and antigen 5S; phospholipases from the yellow jacket Vespula maculifrons and white faced hornet Dolichovespula maculata; a large number of pollen proteins, including birch pollen, ragweed pollen, Parol (the major allergen of Parietaria officinalis) and the cross-reactive allergen Parjl (from Parietaria judaica), and other atmospheric pollens including Olea europaea, Artemisia sp., gramineae, etc. Other allergens of interest are those responsible for allergic dermatitis caused by blood sucking arthropods, e.g. Diptera, including mosquitos (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.) particularly black flies, deer flies and biting midges; ticks (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, e.g. the order Siphonaptera, including the genera Xenopsylla, Pulex and Ctenocephalides felis felis. The specific allergen may be a polysaccharide, fatty acid moiety, protein, etc.
Polymorphisms in a number of interacting/epistatic atopy genes are thought to result in enhanced susceptibility to allergic disorders. Genome wide scans have sought to identify the responsible genes by linking various parameters of allergy and asthma with polymorphic DNA markers of specific genes, usually repeat sequences of DNA (microsatellites that contain di- tri- and tetra-nucleotide repeats). These studies have identified several chromosomal regions as likely to be involved in the pathogenesis of atopy, but with resolution no better than 5-10 cM in which there are typically hundreds of candidate genes. Nevertheless, asthma susceptibility has been linked to chromosomes 5q23-31, chromosome 6p21, chromosome 11q13, and chromosome 12q (9-13) by two or more of these genome wide scanning studies.