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US20150168398A1 - Methods for identifying patterns of ifn induced expression and use in diagnosis, monitoring and therapy - Google Patents

Methods for identifying patterns of ifn induced expression and use in diagnosis, monitoring and therapy Download PDF

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US20150168398A1
US20150168398A1 US14/401,655 US201314401655A US2015168398A1 US 20150168398 A1 US20150168398 A1 US 20150168398A1 US 201314401655 A US201314401655 A US 201314401655A US 2015168398 A1 US2015168398 A1 US 2015168398A1
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ifn
sample
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protein
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John Clayton Hall
Livia Casciola-Rosen
Antony Rosen
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Johns Hopkins University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • Sjögren syndrome is a chronic autoimmune inflammatory disease that targets exocrine glands, particularly salivary and lacrimal glands.
  • Significant evidence implicates IFNs in the pathogenesis of SS including the following: (i) increased levels of circulating IFNs in the plasma of SS patients; (ii) expression of IFN-regulated genes in minor salivary gland biopsies from SS patients; (iii) the presence of a prominent IFN signature in circulating monocytes and peripheral blood mononuclear cells (PBMCs) from SS patients; and (iv) the enrichment of plasmacytoid dendritic cells in SS salivary glands.
  • PBMCs peripheral blood mononuclear cells
  • IFN signatures have been observed in other rheumatic diseases, including dermatomyositis (DM), polymyositis, scleroderma, and systemic lupus erythematosus (SLE).
  • DM dermatomyositis
  • SLE systemic lupus erythematosus
  • IFN- ⁇ type II IFN
  • Quantification of the specific markers of both type I and type II IFN activity is essential to define the origin of the IFN-induced protein expression pattern seen in cells and tissues, and there exists a need for improved methods for identifying specific markers of both type I and type II IFN activity which will lead to improved disease subsetting, monitoring, and identification of improved therapies.
  • the inventors initially defined the genes induced by type I and type II IFNs in a human submandibular gland (HSG) epithelial cell line. Unexpectedly, it was found that the majority of genes that are highly up-regulated by IFN- ⁇ are also highly induced by IFN- ⁇ . In contrast, there was a substantial group of genes that are highly induced by IFN- ⁇ only. To determine whether type I or type II IFN activity was present in minor salivary gland biopsies from patients with SS, precise probes were selected and validated that report on the distinct IFN pathways. Protein expression in minor salivary gland biopsies from SS patients and controls was evaluated by immunoblotting and immunohistochemistry.
  • IFN-regulated proteins were expressed at high levels in SS patients in a pattern consistent with the activity of both type I and type II IFN. However, there was heterogeneity between patients, with evidence of type I IFN-preferential or IFN- ⁇ -preferential patterns. The dominant pattern in SS was quite distinct from that seen in DM, where a more prominent type I IFN pattern was evident. In SS, the cellular distribution of probes of different IFN pathways was also noteworthy: IFN- ⁇ -specific probes were localized to salivary epithelial cells and inflammatory cells in adjacent regions, whereas IFN- ⁇ -preferential markers were expressed mostly in salivary epithelial cells in regions that also demonstrate IFN- ⁇ activity.
  • the present invention provides a method for establishing the IFN expression profile of a tissue comprising: a) preparing a protein lysate from the tissue sample of the subject; and b) analyzing the tissue lysate of a) for the presence of at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins.
  • at least one marker of type I IFN activity e.g. MDA5 or IFIT3
  • the present invention provides a method for establishing the IFN expression profile of a tissue from a subject suspected of having a rheumatic disease comprising: a) preparing a protein lysate from the tissue sample of interest from the subject; b) analyzing the tissue lysate of a) for the presence of at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5, plus a loading control protein (e.g.
  • ⁇ -actin or vinculin proteins ⁇ -actin or vinculin proteins
  • the present invention provides a method for establishing the IFN expression profile of a tissue comprising: a) obtaining mRNA from the tissue sample of interest from the subject; and b) analyzing the mRNA of a) for the presence of mRNA encoding at least one of marker of type I IFN activity (e.g. MDA5 or IFIT3), plus at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5.
  • marker of type I IFN activity e.g. MDA5 or IFIT3
  • the present invention provides a method for establishing the IFN expression profile of a tissue from a subject suspected of having a rheumatic disease comprising: a) obtaining mRNA from the tissue sample of interest from the subject; b) analyzing the mRNA of a) for the presence of at least one marker of type I IFN activity (e.g.
  • MDA5 or IFIT3 markers of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5; c) comparing the levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 mRNA in the subject sample to the levels of the same mRNA in a control sample; and d) determining that the subject has evidence of type I IFN or type II IFN activity when the expression levels of one or more of the mRNAs of interest are increased when compared to the levels in the control sample.
  • the present invention provides a kit for establishing the IFN expression profile of a tissue from a subject, comprising: one or more ligand(s) for determining the levels or concentration of at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 in a tissue sample from a subject.
  • ligand(s) for determining the levels or concentration of at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 in a tissue sample from a subject.
  • the present invention provides in vitro use of an antibody binding at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 for establishing the IFN expression profile of a tissue from a subject suspected of having evidence of IFN- ⁇ or IFN- ⁇ activity comprising: a) preparing a protein lysate from the tissue sample of the subject; b) analyzing the tissue lysate of a) for the presence of at least one marker of type I IFN activity (e.g.
  • MDA5 or IFIT3 MDA5 or IFIT3
  • at least one marker of type II IFN activity GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins, and a loading control protein (e.g.
  • ⁇ -actin and vinculin c) normalizing the expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins in the subject sample to the expression levels of ⁇ -actin or vinculin in the same sample; d) comparing the normalized expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 protein in a patient sample against the same proteins in a control sample; and e) determining that the subject has evidence of type I IFN or type II IFN activity when the expression levels of one or more of the proteins are increased at least 3 standard deviations over the levels in the control sample.
  • the present invention provides a device adapted for carrying out any of the methods described herein, comprising: a) an analyzing unit comprising one or more antibodies binding at least one of MDA5 or IFIT3, and at least one of GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins, the unit being adapted for determining the level of at least one of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in a first and second sample from an individual; and b) an evaluation unit for comparing the determined level in the first sample with the level in the second sample whereby a rheumatic disease can be diagnosed, the unit comprising a database with the levels of at least one of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3,
  • FIG. 1 depicts temporal analysis of IFN- ⁇ and IFN- ⁇ responses in HSGs. Expression of the 416 IFN-inducible transcripts at (A) 4, (B) 12, (C) 24, and (D) 48 hours. Data are presented as log 2 fold change in expression relative to untreated cells.
  • the transcripts in each group are represented as follows: IFN- ⁇ specific (open squares), IFN- ⁇ / ⁇ responsive (circles), IFN- ⁇ specific (open triangles), and uninduced (inverted open triangles). The percentage of transcripts in each group is listed. The dashed lines indicate a twofold increase in expression.
  • FIG. 2 depicts the analysis of IFN-inducible gene expression using self-organizing maps (SOMs).
  • SOMs self-organizing maps
  • FIG. 3 is a Western blot validating probes of IFN- ⁇ and IFN- ⁇ activity.
  • Equivalent amounts of protein lysates from HSGs cultured for 4, 12, 24, or 48 hours in the absence or presence of IFN- ⁇ (1,000 U/mL) or IFN- ⁇ (50 ng/mL) were analyzed by Western blotting.
  • Antibodies against IFN- ⁇ -specific, IFN- ⁇ -specific, and IFN- ⁇ / ⁇ -responsive molecules as defined by array were used. Vinculin is included as a loading control.
  • FIG. 4 shows distinct patterns of type I and type II IFN-induced proteins in SS and DM tissue biopsies.
  • IFN-induced protein expression from A and B was quantified by densitometry and normalized to the level of vinculin expression in the respective sample.
  • Vinculin-normalized expression values were median centered and subject to unsupervised hierarchical clustering to define patterns of IFN-induced protein expression in individual patients.
  • FIG. 5 depicts colocalization of markers of type I and type II IFN activity in SS salivary gland biopsies.
  • Representative images from one control (A and D) and two SS (B, C, E, and F) biopsies are shown. Staining with isotype control antibodies was negative in all samples (data not shown).
  • the asterisks denote areas of inflammation, and the arrows designate salivary gland epithelial cells that express both IFIT3 and GBP2. (Scale bars, 50 ⁇ m.)
  • FIG. 6 depicts distinct patterns of IFN activity in minor salivary gland biopsies from SS patients.
  • IFIT3 type I IFN
  • GFP1 type II IFN
  • FIG. 7 depicts vinculin-normalized IFIT3 and GBP1 expression values for SS patients and controls used for establishing a cutoff for a positive value.
  • Vinculin-normalized expression values are indicated on the Y-axis. The mean expression levels in each group are indicated (bars). Dotted lines indicate 3 S.D. from the mean expression levels of the IFN low group.
  • the autoimmune rheumatic diseases are a complex group of disorders that display considerable heterogeneity in phenotype, immune response, disease course, and response to therapy. Defining whether distinct molecular subgroups exist may facilitate novel disease classification and allow more precise selection of therapy.
  • the IFN pathways are particularly relevant in this regard.
  • a cassette of molecules whose expression is strongly induced by, and highly specific for, type II IFN activity (e.g., CXCL10, GBP1, GBP2, GBP5, IRF1, IL18BP, INDO, RARRES3, SERPING1, UBD and WARS) across all time points after IFN exposure is provided.
  • type II IFN activity e.g., CXCL10, GBP1, GBP2, GBP5, IRF1, IL18BP, INDO, RARRES3, SERPING1, UBD and WARS
  • these markers should be included in analyses of IFN responses in tissues, to either confirm, or rule out, IFN- ⁇ activity.
  • the present inventive methods also highlight the importance of recognizing the cellular and kinetic heterogeneity of tissues when interpreting gene expression patterns in health and disease. Definition of specific markers of pathways, which maintain their specificity across a range of times, is both feasible and useful.
  • the present inventive methods use these tools to quantify the origins of IFN signatures in normal and diseased human tissues. Because multiple pathways may be activated simultaneously in diseased tissue, the inventive methods utilize a combination of IFN- ⁇ -preferential probes with IFN- ⁇ -specific probes was used to quantify the activity of the different IFN pathways. Heterogeneity in IFN pathway activation was observed in different SS salivary glands. In most patients, evidence of both type I and II IFN activity was present. In some patients, there was evidence of either predominant type I (patient SS6) or type II IFN activity (patient SS7). Further analysis has demonstrated considerable heterogeneity of IFN activity in SS minor salivary gland biopsies. In a separate study, 25 of 53 patients (47%) have measurable IFN activity.
  • the methods of the present invention are simple and quantitative, require very small amounts of tissue, provide an integrated readout of pathways active in the target tissues, and have the potential for automation. These inventive methods facilitate a more precise classification of patients based on activity of specific pathways in the target tissue.
  • the inventive methods can be used as a molecular diagnostic to more precisely delineate disease subsets, and assist in selecting patients for therapy or for monitoring effectiveness.
  • sample encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic, prognostic or monitoring assay.
  • the patient sample may be obtained from a healthy subject, a diseased patient including, for example, a patient having associated symptoms of a rheumatic disease.
  • a sample obtained from a patient can be divided and only a portion may be used for diagnosis, prognosis or monitoring. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis.
  • the definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, urine, saliva, amniotic fluid, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • a sample comprises a salivary gland sample.
  • a sample of muscle tissue is used.
  • a sample comprises a blood or serum sample.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations.
  • Samples further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.
  • providing a sample and “providing a biological (or patient) sample” are used interchangeably and mean to provide or obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from a patient, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, can also be used.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ .-carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an .alpha.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of ordinary skill in the art recognizes that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • the labels may be incorporated into the KIT nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
  • antibody includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies).
  • the term “antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab′, F(ab′).sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).
  • antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
  • An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.
  • an immunoglobulin has a heavy and light chain
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
  • Light and heavy chain variable regions contain four framework” regions interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs).
  • V H refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab.
  • V L refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • a “chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a “humanized antibody” is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Fully human antibody refers to an immunoglobulin comprising human hypervariable regions in addition to human framework and constant regions.
  • Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., McCafferty et al., 1990, Nature 348:552-554; Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol.
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos.
  • Epitope or “antigenic determinant” refers to a site on an antigen to which an antibody binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
  • Antibodies can be used to detect proteins stimulated by type I and/or type II IFN in the methods of the invention.
  • the detection and/or quantification of proteins stimulated by type I and/or type II IFN can be accomplished using any of a number of well recognized immunological binding assays.
  • a general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988) and Harlow & Lane, Using Antibodies (1999). Other resources include see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Ten, eds., 7th ed. 1991, and Current Protocols in Immunology (Coligan, et al. Eds, John C. Wiley, 1999-present) Immunological binding assays can use either polyclonal or monoclonal antibodies.
  • Commonly used assays include noncompetitive assays (e.g., sandwich assays) and competitive assays.
  • competitive assays the amount of genes expression products stimulated by type I and/or type II IFN present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) expression product displaced (competed away) from an anti-expression product antibody by the unknown present in a sample.
  • Commonly used assay formats include immunoblots, which are used to detect and quantify the presence of protein in a sample.
  • LISA liposome immunoassays
  • Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
  • the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
  • the labeling agent may be a labeled for gene products stimulated by type I and/or type II IFN or a labeled anti-type I or type II IFN antibody.
  • the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody/antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the labeling agent.
  • the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent compounds (e.g., fluorescein isothiocyanate, Texas red, rhodamine, fluorescein, and the like), radiolabels, enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), streptavidin/biotin, and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Chemiluminescent compounds may also be used.
  • the present invention provides in vitro use of an antibody binding one at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 for establishing the IFN expression profile of a tissue from a subject suspected of having a rheumatic disease comprising: a) preparing a protein lysate from the tissue sample of the subject; b) analyzing the tissue lysate of a) for the presence of at least one marker of type I IFN activity (e.g.
  • MDA5 or IFIT3 markers of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5; c) comparing the expression levels of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the subject sample to the expression levels of the same proteins in a control sample; and d) determining that the subject has evidence of IFN- ⁇ or IFN- ⁇ activity when the expression levels of one or more of the proteins are increased at least 3 S.D. over the levels in the control sample
  • control sample or “reference sample” means a sample from a subject known not to have a rheumatic disease or immune disorder.
  • comparing encompasses comparing the level of the peptide or polypeptide comprised by the sample to be analyzed with a level of a suitable reference level specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample or a ratio of amounts is compared to a reference ratio of amounts.
  • the comparison referred to in the methods of the present invention may be carried out manually or computer assisted.
  • the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program.
  • the computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format.
  • the present invention relates to a device adapted for carrying out the method of the present invention for diagnosing or predicting an outcome for a rheumatic disease
  • a device adapted for carrying out the method of the present invention for diagnosing or predicting an outcome for a rheumatic disease
  • MDA5 or IFIT3 MDA5 or IFIT3
  • at least one marker of type II IFN activity GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins
  • the unit being adapted for determining the level of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in a first and a second sample from an individual; and, b.
  • an evaluation unit for comparing the determined level in the first sample with the level in the second sample whereby a rheumatic disease progression or outcome can be diagnosed, the unit comprising a database with the level of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins in the first sample as compared to the second sample, the levels being, preferably, derived from an individual or a group of individuals known to have developed rheumatic diseases or more preferably derived from an individual or a group of individuals known not to have developed rheumatic diseases and a computer-implemented algorithm for carrying out a comparison step.
  • the reference levels are preferably derived from a sample of (reference) individuals as defined above. It will be understood by those of skill in the art that other proteins which are specifically induced by type I or type II IFNs can be used with the inventive methods.
  • the term “device” as used herein relates to a system comprising the aforementioned units operatively linked to each other as to allow the diagnosis or monitoring according to the methods of the invention.
  • Preferred detection agents which can be used for the analyzing unit are disclosed elsewhere herein.
  • the analyzing unit preferably, comprises the detection agents in immobilized form on a solid support which is to be contacted to the sample comprising the biomarkers the level of which is to be determined.
  • the analyzing unit can also comprise a detector which determines the level of binding ligand which is specifically bound to the biomarker(s). The determined level can be transmitted to the evaluation unit.
  • the evaluation unit comprises a data processing element, such as a computer, with an implemented algorithm for carrying out a comparison between the determined level and a suitable reference (e.g. a reference level, or the level of the marker in a first or second sample from the individual).
  • a suitable reference e.g. a reference level, or the level of the marker in a first or second sample from the individual.
  • Suitable references can be derived from samples of individuals to be used for the generation of reference levels as described elsewhere herein above.
  • the results may be given as output of parametric diagnostic raw data, preferably, as absolute or relative levels. It is to be understood that these data will need interpretation by the clinician. However, also envisaged are expert system devices wherein the output comprises processed diagnostic raw data the interpretation of which does not require a specialized clinician.
  • the present invention provides device adapted for carrying out any of the methods described herein, comprising: a) an analyzing unit comprising one or more antibodies binding at least one marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5, the unit being adapted for determining the level of at least one marker of type I IFN activity (e.g.
  • MDA5 or IFIT3 MDA5 or IFIT3
  • at least one marker of type II IFN activity GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins in a first and second sample from an individual
  • an evaluation unit for comparing the determined level in the first sample with the level in the second sample whereby an increase in IFN- ⁇ and/or IFN- ⁇ levels can be diagnosed, the unit comprising a database with the levels of at least one marker of type I IFN activity (e.g.
  • MDA5 or IFIT3 MDA5 or IFIT3
  • at least one marker of type II IFN activity GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins in the first sample to the second sample, and a computer-implemented algorithm for carrying out a comparison step.
  • the methods of the present invention can be used for diagnosis, prognosis and/or treatment monitoring of rheumatic diseases in a subject.
  • the amount of expression of genes induced by type I and/or type II IFNs expressed in a specific tissue of a subject can be used to determine the severity and type of immunological condition of the subject as well as determine whether an ongoing treatment is having an effect by comparing expression levels of genes induced by type I and/or type II IFNs before, during, and after treatment.
  • the determination of an increase in IFN- ⁇ and/or IFN- ⁇ levels can be diagnosed using the expression levels of at least marker of type I IFN activity (e.g. MDA5 or IFIT3), and at least one marker of type II IFN activity: GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins.
  • at least marker of type I IFN activity e.g. MDA5 or IFIT3
  • marker of type II IFN activity e.g. MDA5 or IFIT3
  • GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, GBP5 proteins e.g. MDA5 or IFIT3
  • the determination of an increase in IFN- ⁇ and/or IFN- ⁇ levels can be diagnosed using the expression levels of at least three, four, or 5 or more of MDA5, IFIT3, GBP1, GBP2, INDO, UBD, IRF1, RARRES3, WARS, CXCL10, IL18BP, SERPING1, and GBP5 proteins.
  • kits for diagnostic, prognostic or therapeutic applications may include any or all of the following: assay reagents, buffers, mRNA, PCR, qPCR probes, primers, antibodies, or the like.
  • the kit may, preferably, comprise standards, reference samples and control samples.
  • the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • inventive methods can be used to aid in the treatment of rheumatic diseases by identifying a particular expression profile of type I or type II IFN induced genes and then administering to the subject suitable medicaments for treatment including for example, recombinant antibodies which bind and neutralize IFNs in circulation, recombinant type I or type II IFN receptors which bind and neutralize circulating type I or type II IFN, other compounds which bind and inhibit the function of IFNs.
  • HSGs were cultured for 4, 12, 24, or 48 hours either with IFN- ⁇ (1,000 U/mL) or IFN- ⁇ (50 ng/mL), or without IFN added, and samples were collected in triplicate at each time point; 36 total samples were assayed.
  • Total RNA was extracted using TRIzol (Invitrogen). Additional purification was performed on RNeasy columns (Qiagen), and total RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies).
  • Biotin-labeled cRNA was prepared from total RNA according to the chip manufacturer's protocol (Illumina) cRNA was hybridized to Illumina Human HT12 v3 Expression BeadChips, and signal was detected with streptavidin-Cy3. Signal intensity quantification was performed using an Illumina BeadStation 500GX Genetic Analysis Systems scanner. The microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (Nucleic Acids Res 30(1):207-210 (2002)).
  • Transcripts i.e., Illumina probes considered to be differentially expressed between two groups of samples were those satisfying the following criteria: (i) Welch t test values of P ⁇ 0.01 (Bioinformatics 18(4):546-554 (2002)); (ii) Benjamini-Hochberg FDR of ⁇ 0.25 (35); (iii) a fold change of >2.0 (calculated using geometric means); and (iv) the expression value of the transcript is above the Illumina BeadStudio calculated background (detection P value of ⁇ 0.01) in all three samples in the group with the higher average expression level for that probe, thus avoiding false positives based on background noise and also reducing the number of statistical tests for the subsequent FDR calculation.
  • MSG salivary gland
  • Frozen minor salivary gland biopsies (for lysate generation) were obtained from eight SS patients and six controls. Salivary gland paraffin sections were obtained from four SS patients and three controls. A second set of minor salivary gland biopsies was obtained with informed consent from individuals enrolled in the Sjögren's International Collaborative Clinical Alliance [SICCA], funded under contract N01 DE-32636 by the National Institute of Dental and Craniofacial Research, with funding support from the National Eye Institute and Office for Research in Women's Health. All studies were performed under the approval of the JHU IRB protocol NA — 00079238. All patients satisfied the American College of Rheumatology Classification Criteria for Sjögren's syndrome Shiboski S C et al.
  • SICCA Sjögren's International Collaborative Clinical Alliance
  • Muscle biopsies were obtained from patients seen at the Neuromuscular Clinic at Johns Hopkins Hospital. Informed consent was obtained from every study subject, and all samples were collected under the auspices of Johns Hopkins Medicine Institutional Review Board-approved protocols. All patient samples were deidentified, with clinical and laboratory features linked only to the patient code. Surgical procedures were performed for patient management, and the research tissue samples were excess tissue obtained for routine diagnostic purposes. Frozen muscle biopsies were obtained from four patients with DM and three individuals whose biopsies were histologically normal. Histologic criteria for biopsies identified as DM were consistent with Bohan and Peter criteria (N Engl J Med 292(7):344-347; N Engl J Med 292(8):403-407 (1975)).
  • IFN- ⁇ Genes Most Strongly Induced by IFN- ⁇ are also Induced by IFN- ⁇ .
  • microarray studies were initially performed in an HSG cell line to define IFN- ⁇ and IFN- ⁇ responses in a disease-relevant cell type. Concentrations of IFN- ⁇ and IFN- ⁇ were selected that induced equivalent levels of Ro52 expression (IFN- ⁇ , 1,000 U/mL; IFN- ⁇ , 50 ng/mL) (data not shown).
  • IFN- ⁇ 1,000 U/mL
  • IFN- ⁇ 50 ng/mL
  • IFN-induced gene expression was analyzed at 4, 12, 24, and 48 hours. 416 mRNA transcripts were identified that were significantly induced [fold change, ⁇ 2.0; value of P ⁇ 0.01; false discovery rate (FDR), ⁇ 0.25] by IFN- ⁇ or IFN- ⁇ at one or more time points.
  • IFN- ⁇ -specific transcripts were induced eightfold (i.e., a log 2 fold change of 3) or less. Notably, however, >50% of IFN- ⁇ -responsive transcripts were induced exclusively by IFN- ⁇ ( FIGS. 1 C and D, squares). Of these, 10 were induced eightfold or greater at 24 hours and 23 were induced eightfold or greater at 48 hours; (iii) The majority of transcripts induced highly by IFN- ⁇ were also induced by IFN- ⁇ ( FIG. 1 A-D, circles).
  • IFN-inducible proteins were not detectable in untreated cells; (ii) as defined in the optimization phase, Ro52 was induced to equivalent levels by IFN- ⁇ and IFN- ⁇ at 24 and 48 hours; (iii) the IFN- ⁇ response was rapid, with maximal expression of MDA5, IFIT3, and Ro52 detected at 12 hours; this remained elevated through 48 hours; (iv) the IFN- ⁇ response occurred more slowly, with protein expression peaking at 48 hours; (v) MDA5 and IFIT3 were IFN- ⁇ specific at 12 hours but were IFN- ⁇ responsive (albeit at markedly lower levels than the IFN- ⁇ response) at 24 and 48 hours; (vi) GBP1 and GBP2 were robustly and specifically induced by IFN- ⁇ only; (vii) a low dose of IFN- ⁇ enhanced the expression of MDA5 and IFIT3 in IFN- ⁇ -treated cells, whereas IFN- ⁇ did not enhance the induction of GBP1 and GBP2 (not shown). Quantifying the expression of
  • Type I and Type II IFN Activity Is Prominent in Minor Salivary Gland Biopsies from SS Patients.
  • FIG. 4A In SS, striking increases in the expression of MDA5, IFIT3, GBP1, and GBP2 were evident in most patients, indicating both type I and type II IFN effects in most biopsies ( FIG. 4A ). Although six of eight patients had elevated expression of both IFN- ⁇ - and IFN- ⁇ -induced proteins, there was some variation in patterns between individual patients, with one patient exhibiting a predominantly IFN- ⁇ pattern ( FIG. 4A , lane 6) and one demonstrating a predominantly IFN- ⁇ pattern ( FIG. 4A , lane 7).
  • IFIT3 staining was observed in infiltrating inflammatory cells.
  • GBP2 was prominently expressed in the nuclei of infiltrating inflammatory cells.
  • GBP2 staining was also evident in salivary ducts, where the nuclei of both infiltrating inflammatory cells (strong staining) as well as duct epithelium were stained (moderate staining).
  • GBP2 and IFIT3 stained the same regions of the ducts that were surrounded by GBP2-positive inflammatory cells ( FIGS. 5 E and F). Isotype control antibody staining was negative for all samples (data not shown).
  • IFN pathway analysis in control and SS minor salivary gland biopsies Equivalent amounts of protein lysates from 29 control (Cont) and 53 SS (pSS) patient minor salivary gland biopsies were analyzed for IFN-inducible protein expression by Western blotting. IFIT3 was utilized to report of the activity of IFN- ⁇ and GBP1 was utilized to report on the activity of IFN- ⁇ . Vinculin is included as a loading control.
  • IFN pathway activity Distinct patterns of IFN pathway activity are evident in minor salivary gland biopsies from SS patients.
  • Vinculin-normalized expression values are indicated on the Y-axis. The mean expression levels in each group are indicated (bars). Dotted lines indicate 3 S.D. from the mean expression levels of the IFN low group.
  • IFIT3 and GBP2 were both expressed in salivary epithelial cells located in areas of significant inflammatory infiltrate, although the patterns differed. IFIT3 staining was enriched in ductal epithelial cells and did not stain inflammatory cells with similar intensity.
  • TLRs Toll-like receptors
  • TLR3 Toll-like receptors
  • type I IFN secretion and downstream pathways may enhance the type I IFN effect in the presence of relevant TLR ligands.
  • TLRs Toll-like receptors
  • some of the prominent ribonucleoprotein autoantigens targeted in autoimmune rheumatic diseases like SS can ligate and activate TLRs.
  • the existence of positively reinforcing interactions between the different IFN pathways in SS may provide important therapeutic opportunities. Quantifying IFIT3 and GBP2 expression in the target tissue during therapy with inhibitors of specific IFN pathways provides important tools for investigating the nature and direction of these reinforcing interactions.
  • the present inventive methods have defined probes that more precisely quantify the activity of different IFN pathways in tissues from various inflammatory rheumatic diseases. Using these inventive probes on human tissue has demonstrated that different rheumatic phenotypes associated with an IFN signature have distinct patterns of IFN activity, which are not evident using other analyses. Furthermore, heterogeneity in signatures exists even within a disease phenotype, which means that the probes provided herein are be useful markers of patient subsets, where specific IFNs play distinct roles. As new therapeutic agents that inhibit type I or type II IFNs become available, it will be essential to identify with precision the activity of that pathway in vivo at baseline and after therapy. The inventive methods and tools defined here can be used to accomplish this.

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