METHODS AND COMPOSITIONS FOR USING MHC CLASS II INVARIANT CHAIN POLYPEPTIDES AS A RECEPTOR FOR MACROPHAGE MIGRATION INHIBITORY FACTOR

Abstract

Methods and compositions for using the MEW class II invariant chain polypeptide, Ii (also known as CD74), as a receptor for macrophage migration inhibitory factor (MIF), are disclosed. These include methods and compositions for using this receptor, as well as agonists and antagonists of MIF which bind to this receptor, or which otherwise modulate the interaction of MIF with CD74 or the consequences of such interaction, in treatment of conditions characterized by locally or systemically altered MIF levels, particularly inflammatory conditions and cancer.

Claims

1. A method for screening compounds for an agonist or antagonist of MIF comprising: contacting an WIC class II invariant chain (Ii) polypeptide with MIF in the presence and absence of a candidate compound, and comparing the interaction of the MIF and said Ii polypeptide in the presence of said candidate compound with their interaction in the absence of said candidate compound, whereby a candidate compound that enhances the interaction of said MIF with said Ii polypeptide is identified as an agonist of MIF, and a candidate compound that inhibits the interaction of said MIF with said Ii polypeptide is identified as an antagonist of MIF; wherein said Ii polypeptide comprises the complete Ii amino acid sequence of FIG. 5 (SEQ ID. NO:2) or an MIF-binding fragment thereof.

2. An agonist or antagonist of MIF identified by the method of claim 1.

3. An agonist or antagonist according to claim 2 which is an antibody or antigen-binding fragment thereof.

4. The agonist or antagonist according to claim 3 wherein said antibody is an anti-CD74 antibody.

5. An anti-CD74 antibody of claim 4 selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody and a chimeric antibody.

6. A method of inhibiting an effect of MIF on a cell comprising on its surface an MEW class II invariant chain (Ii) polypeptide which binds MIF and thereby mediates said effect of MIF, said method comprising: contacting said cell with an antagonist of MIF wherein said antagonist inhibits binding of MIF to said Ii polypeptide.

7. A method according to claim 6 wherein said antagonist is an antibody or fragment thereof which binds to said Ii polypeptide.

8. A method according to claim 7, wherein cell is present in a mammal and wherein said antagonist is administered to the mammal in a pharmaceutical composition.

9. A method according to claim 8, wherein said mammal suffers from a condition characterized by systemic or local MIF levels elevated above the normal range in mammals not suffering from such a condition.

10. A method according to claim 9, wherein said mammal suffers from an inflammatory disorder and said antagonist is administered in an amount effective to treat the disorder.

11.-12. (canceled)

13. A method of inhibiting an activity of MIF, said method comprising: contacting MIF with an antagonist of MIF, wherein said antagonist of MIF comprises a MEW class II invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF.

14. A method according to claim 13, wherein said MEW class II invariant chain (Ii) polypeptide or fragment thereof which binds to MIF is a soluble form of said polypeptide or fragment thereof.

15. A method according to claim 14, wherein said soluble form of said polypeptide or fragment thereof comprises the extracellular binding domain of said polypeptide or a portion thereof.

16. A method according to claim 13, wherein the MIF to be inhibited is in a mammal and wherein said antagonist of MIF is administered to the mammal in a pharmaceutical composition.

17. A method according to claim 16, wherein said mammal suffers from a condition characterized by systemic or local MIF levels elevated above the normal range in mammals not suffering from such a condition.

18. A method according to claim 17, wherein said mammal suffers from an inflammatory disorder and said antagonist is administered in an amount effective to treat the disorder.

19.-20. (canceled)

21. A method of purifying MIF comprising: contacting a sample comprising MIF with an MHC class II invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF under conditions that promote the specific binding of MIF to the Ii polypeptide or fragment thereof, and separating the MIF:Ii polypeptide complex or MIF:Ii polypeptide fragment complex thereby formed from materials which do not bind to the Ii polypeptide or fragment thereof.

22. (canceled)

23. A method of assaying for the presence of MIF comprising: contacting a sample with an MHC class H invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF under conditions that promote the specific binding of MIF to said Ii polypeptide or a fragment thereof, and detecting any MIF:Ii polypeptide complex or MIF:Ii polypeptide fragment complex thereby formed.

24. A method for reducing an effect of MIF on a cell comprising on its surface an MEW class II invariant chain (Ii) polypeptide which binds MIF and thereby mediates said effect of MIF, said method comprising: providing to said cell an antisense nucleic acid molecule in an amount effective to reduce the amount of said Ii polypeptide produced by said cell, wherein said antisense nucleic acid molecule specifically binds to a portion of mRNA expressed from a gene encoding said MEW class II invariant chain (Ii) polypeptide and thereby decreases translation of said mRNA in said cell.

25.-28. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1A-FIG. 1E illustrate high affinity binding of MIF to THP-1 monocytes. a, Alexa-MIF shows full retention of dose-dependent MIF biological activity as assessed by activation of the p44/p42 (ERK-1/2) MAP kinase cascade, visualized by western blotting of cell lysates using antibodies specific for phospho-p44/p42 or total p44/p42; and b, suppression of p53-dependent apoptosis induced by serum starvatuin (CM: complete medium, SFM: serum-free medium). MIF or Alexa-MIF were added at 50 ng/ml. Data shown are MeanSD of triplicate wells and are representative of 3 independent experiments. Further evidence for the retention of native structure by Alexa-conjugation was provided by the measurement of MIF tautomerase activity using L-dopachrome methyl ester as substrate.sup.25. No difference in the tautomerase activity of Alexa-MIF versus unconjugated MIF was observed (Alexa-MIF: OD.sub.475=0.275 sec.sup.1 g.sup.1 protein, versus rMIF: OD.sub.475=0.290 sec.sup.1 g.sup.1; P=NS) c, Flow cytometric analysis shows the binding of Alexa-MIF to THP-1 monocytes is markedly enhanced by IFN- [sic] treatment. Competition for Alexa-MIF binding was performed in the presence of 1 g/ml unlabeled, rMIF d, Direct visualization of Alexa-MIF binding to THP-1 monocytes by confocal microscopy THP-1 cells were grown on cover slips, incubated with INF (1 ng/ml) for 72 hrs and stained with Alexa-MIF (left panel) or Alexa-MIF plus excess, unlabeled rMIF (right panel). Cell bound Alexa-MIF was rapidly internalized upon shifting cells from 4 to 37 for 15 mins (right panel). Magnification: 630 e, Binding characteristics of Alexa-MIF to IFN-activated, THP-1 monocytes. The inset shows the binding data transformed by Scatchard analysis, indicating two distinct binding activities; one with K.sub.d=3.710.sup.8 m the other with K.sub.d=3.510.sup.5, data are representative of 3 independent experiments.

[0020] FIG. 2A-FIG. 2D show that Ii is a cell surface binding protein for MIF a, Sequential cycles of fluorescence-activated cell-sorting of COS-7 cell transfectants shows enrichment for MIF binding activity b, Diagrams indicating structure of Ii (35 kDa isoform), and three of ten representative cDNA clones with MIF binding activity. IC, TM, and EC are the intracellular, transmembrane, and extracellular domains. M1 and M17 refer to two sites of alternative translation initiation. c, Flow cytometry analysis of MIF binding to Ii-expressing cells. Enhanced binding of Alexa-MIF to li-transfected versus control vector-transfected COS-7 cells (left panel), inhibited binding of Alexa-MIF to Ii-transfected COS-7 cells incubated with anti-Ii mAb (clone LN2) versus an isotypic mAb control (con mAb) (middle panel), and enhanced binding of Alexa-MIF to IFN-stimulated, THP-1 monocytes incubated with anti-Ii mAb (clone LN2) versus an isotypic mAb control (right panel). The data shown are representative of at least three independent experiments. The anti-Ii mAb, LN2 (PharMingen), is reactive with an epitope residing within 60 amino acids of the extracytoplasmic, C-terminus of the protein d, MIF binds to the extracellular domain of Ii in vitro. rSJ-Ii protein was prepared in a coupled transcription and translation reaction utilizing plasmids encoding Ii fragments of different lengths. Protein-protein interaction was assessed by measuring bound radioactivity in 96-well plates that were pre-coated with MIF (n=6 wells per experiment). The data shown are representative of three experiments, showing that MIF binding is severely compromised with vectors expressing Ii fragments of amino acids 1-72 or 1-109 versus robust binding with vectors expressing Ii fragments of amino acids 1-149 or 1-232 (full-length).

[0021] FIG. 3A-FIG. 3B illustrate Ii mediation of MIF stimulation of ERK-1/2 (p44/p42) phosphorylation in COS-7 cells a, ERK-1/2 phosphorylation is induced by MIF in COS-7 cells transfected with Ii vector (COS-7/Ii) or control vector (COS-7N). Cells were treated without or with various doses of rMIF for 2.5 hrs and analyzed for phospho-p44/p42 and total p44/p42 by western blotting b, There is dose-dependent inhibition of MIF-induced ERK-1/2 phosphorylation by anti-Ii mAb. COS-7 cells were transfected with an Ii vector and stimulated with 50 ng/ml MIF for 2.5 hrs in the presence of an isotypic control mAb or an anti-Ii mAb (clone LN2) at different doses. In control experiments, anti-Ii showed no effect on ERK-1/2 phosphorylation in the absence of added MIF (data not shown).

[0022] FIG. 4A-FIG. 4D illustrate western blots of MIF-induced phosphorylation a, MIF dose dependently stimulates ERK-1/2 (p44/p42) phosphorylation in human Raji B cells, as visualized by western blotting for phospho-p44/p42 b, There is inhibition of MIF-induced ERK-1/2 phosphorylation in Raji cells by anti-Ii mAb also. Raji cells were stimulated with 50 ng/ml of MIF for 2.5 hrs in the presence of an isotype control antibody (Con Ab) or the two anti-Ii mAbs, B741 or LN2, each added at 50 g/ml. c, Anti-Ii inhibits MIF-induced Raji cell proliferation quantified by .sup.3H-thymidine incorporation d, Anti-Ii inhibits MIF-induced proliferation of human fibroblasts also. Antibodies were added to a final concentration of 50 g/ml. The results shown are the MeanSD of triplicate assays and are representative of at least three separate experiments. Anti-Ii antibodies showed no effect on cell proliferation in the absence of added MIF (data not shown).

[0023] FIG. 5 shows the complete nucleotide sequence (SEQ ID NO: 1) and longest translated amino acid sequence (beginning at nt 8; SEQ ID NO:2) of the human mRNA for the Ii polypeptide (HLA-DR antigens associated invariant chain p33 [GenBank Accession Nr. X00497 M14765]), as reported in Strubin, M. et al. The complete sequence of the mRNA for the HLA-DR-associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. EMBO J., 3, 869-872 (1984).

DETAILED DESCRIPTION

[0024] The following abbreviations are used herein: Alexa-MIF: Alexa 488-MIF conjugate, ERK: extracellular-signal-regulated kinase, MHC class II-associated invariant chain (CD74), INF: interferon-, mAb: monoclonal antibody, MIF: macrophage migration inhibitory factor.

[0025] Utilizing expression cloning and functional analyses, we have identified as a cellular receptor for MIF the Class II-associated invariant chain, Ii (CD74).sup.10. MIF binds to the extracellular domain of Ii, a Type II membrane protein, and Ii is required for MIF-induced cellular effects, including for instance, activation of the ERK-1/2 MAP kinase cascade and cell proliferation. These data provide a mechanism for MIF's activity as cytokine and identify it as a natural ligand for Ii, which has been previously implicated in signaling and accessory functions for immune cell activation.sup.11-13. We linked the fluorescent dye Alexa 488.sup.14 to recombinant MIF by standard techniques, verified the retention of biological activity of the conjugate (FIG. 1A, B), and conducted binding experiments with a panel of cell types known to respond to MIF. By way of illustration, using flow cytometry, we observed high-affinity binding of Alexa-MIF to the surface of the human monocytic cell line, THP-1. This binding activity was induced by activation of monocytes with interferon-y (IFN), and was competed [sic] by the addition of excess, unlabeled MIF (FIG. 1C). Confocal microscopy and direct visualization of IFN-treated monocytes at 4 C. showed surface binding of Alexa-MIF, and cell-bound Alexa-MIF was internalized upon shifting temperature to 37 C. (FIG. 1D). Quantitative binding studies performed with increasing concentrations of Alex a-MIF revealed two apparent classes of cell surface receptors (FIG. 1E). The higher affinity binding activity showed a K.sub.d of 3.710.sup.8 M and 3.110.sup.4 binding sites per cell, and the lower affinity binding showed a K.sub.d of 3.510.sup.4 M and 4.910.sup.4 sites per cell.

[0026] To identify the MIF receptor, we prepared cDNA from IFN-activated THP-1 monocytes and constructed a mammalian expression library in the lambdaZAP-CMV vector.sup.15. Library aliquots representing a total of 1.510.sup.7 recombinants were transfected into COS-7 cells, which we had established previously to exhibit little detectable binding activity for MIF, and the transfectants were analyzed by flow cytometry for Alexa-MIF binding. Positively-staining cells were isolated by cell sorting, and the cDNA clones collected, amplified, and re-transfected into COS-7 cells for additional rounds of cell sorting (FIG. 2A). After four rounds of selection, single colonies were prepared in E. coli and 250 colonies were randomly picked for analysis. We sequenced 50 clones bearing cDNA inserts of 1.61 Kb and observed that 10 encoded the Class II-associated invariant chain, Ii (CD74), a 31-41 kD Type II transmembrane protein.sup.16. While the isolated clones differed with respect to their total length, each was in the sense orientation and encoded a complete extracellular and transmembrane domain (FIG. 2B).

[0027] To confirm that Ii is a cell surface binding protein for MIF, we analyzed the binding of Alexa-MIF to COS-7 cells transfected with an Ii expression plasmid (FIG. 2C). Binding was inhibited by excess, unlabeled MIF (data not shown), and by an anti-li mAb directed against the extracellular portion of the protein. Anti-Ii mAb also inhibited the binding of Alexa-MIF to IFN- stimulated THP-1 monocytes. The inhibition by anti-Ii mAb of Alexa-MIF binding to THP-1 monocytes was significant, but partial, consistent with the interpretation that Ii represents one of the two classes of cell surface receptors for MIF revealed by Scatchard analysis (FIG. 1E). [.sup.35S]-Ii protein prepared by a coupled transcription and translation reticulocyte lysate system bound to MIF in vitro, and the principal binding epitope was localized to a 40 amino acid region contained within the Ii extracellular domain (FIG. 2D).

[0028] To verify the functional significance of MIF binding to Ii in an exemplary system, we examined the activity of MIF to stimulate ERK-1/2 activation and cellular proliferation in different Ii-expressing cells. We observed an MIF-mediated increase, and a dose-dependent, anti-li mAb-mediated decrease, in ERK-1/2 phosphorylation in Ii-transfected COS-7 cells (FIG. 3). Irrespective of Ii gene transfection however, we could not detect any proliferative effect of MIF on this monkey epithelial cell line (data not shown). We then examined the activity of MIF to induce ERK-1/2 activation and downstream proliferative responses in the human Raji B cell line, which expresses a high level of Ii.sup.19. MIF stimulated the phosphorylation of ERK-1/2 in quiescent Raji cells, and each of two anti-li mAbs blocked this stimulatory effect of MIF (FIG. 4A, B). Of note, the inhibitory effect of anti-Ii on ERK-1/2 phosphorylation was associated with a significant decrease in the MIF-stimulated proliferation of these cells (FIG. 4C). Additionally, we confirmed the role of the MIF-Ii stimulation pathway in cells outside the immune system. MIF extends the lifespan of primary murine fibroblasts.sup.8, and both MIF's mitogenic effects and its induction of the ERK-1/2 signal transduction cascade have been best characterized in this cell type.sup.7. Fibroblasts express low levels of IP.sup.20 and we observed that anti-Ii significantly inhibited both ERK-1/2 phosphorylation and the mitogenic effect of MIF on cultured fibroblasts (FIG. 4D and data not shown).

[0029] In prior experiments, we have experienced considerable difficulty in preparing a bioactive, .sup.125I-radiolabelled MIF, and have observed the protein to be unstable to the pH conditions employed for biotin conjugation. By contrast, modification of MIF by Alexa 488 at a low molar density produced a fully bioactive protein which enabled identification of MIF receptors on human monocytes, and the expression cloning of Ii as a cell surface MIF receptor. These data significantly expand our understanding of Ii outside of its role in the transport of class II proteins, and support recent studies which have described an accessory signaling function for Ii in B and T cell physiology.sup.10-13.

[0030] These findings provide a first insight into the long sought-after MIF receptor, although additional proteins are likely involved in some MIF-mediated activities. For instance, like MIF, Ii is a homotrimer.sup.23, and the Ii intracellular domain consists of 30-46 amino acids, depending on which of two in-phase initiation codons are utilized.sup.16. Monocyte-encoded Ii has been shown to enhance T cell proliferative responses, and this accessory function of Ii has been linked to a specific, chondroitin-sulphate-dependent interaction between Ii and CD44.sup.11 We have observed an inhibitory effect of anti-CD44 on ERK-1/2 phosphorylation, but not MIF binding, in Ii-expressing cells. This is consistent with the inference that MIF-bound Ii is a stimulating ligand for CD44-mediated MAP kinase activation. CD44 is a highly polymorphic Type I transmembrane glycoprotein.sup.24, and CD44 likely mediates some of the downstream consequences of MIF binding to Ii.

[0031] Interference in the signal transduction pathways induced by MIF-Ii interaction, for instance by providing antagonists or inhibitors of MIF-Ii interaction, offers new approaches to the modulation of cellular immune and activation responses to MIF. Agents active in this regard (agonists and antagonists and other inhibitors) have predicted therapeutic utility in diseases and conditions typified by local or systemic changes in MIF levels.

[0032] The specific binding interaction between MIF and the class II invariant chain polypeptide, also makes convenient the use of labeled MIF reagents as Trojan horse-type vehicles by which to concentrate a desired label or toxin in cells displaying cell surface Ii. Briefly, a desired label or toxic entity is associated with an MIF ligand (for instance, by covalent attachment), and the modified MIF ligand then is presented to cells displaying cell surface-localized Ii, which class II invariant chain polypeptide binds to and causes the internalization of the modified MIF ligand, thus causing the operative cell to become specifically labeled or toxicated. The Ii-displaying cells may be exposed to the modified MIF ligand in vitro or in vivo, in which latter case Ii-displaying cells may be specifically identified or toxicated in a patient. A wide variety of diagnostic and therapeutic reagents can be advantageously conjugated to an MIF ligand (which may be biologically active, full length MIF or an Ii-binding fragment thereof, or a mutein of either of the preceding and particularly such a mutein adapted to be biologically inactive and/or to be more conveniently coupled to a labeling or toxicating entity), providing a modified MIF ligand of the invention. Typically desirable reagents coupled to an MIF ligand include: chemotherapeutic drugs such as doxorubicin, methotrexate, taxol, and the like; chelators, such as DTPA, to which detectable labels such as fluorescent molecules or cytotoxic agents such as heavy metals or radionuclides can be complexed; and toxins such as Pseudomonas exotoxin, and the like.

[0033] Methods

[0034] MIF and Antibodies.

[0035] Human recombinant MIF was purified from an E. coli expression system as described previously.sup.22 and conjugated to Alexa 488.sup.14 by the manufacturer's protocol (Molecular Probes, Eugene Oreg.). The average ratio of dye ligand to MIF homotrimer was 1:3, as determined by matrix-assisted laser-desorption ionization mass spectrometry (Kompact probe/SEQ, ICratos Analytical Ltd, Manchester, UK). Anti-human Ii monoclonal antibodies (clones LN2 and M-B741) were obtained from PharMingen (San Jose Calif.).

[0036] Flow Cytometry, Scatchard Analysis, and Confocal Microscopy.

[0037] THP-1 cells (2.5105 cells/ml) were cultured in DMEM/10% FBS with or without IFNI, (1 ng/ml, R&D Systems, Minneapolis, Minn.) for 72 hrs. After washing, 5105 cells were resuspended in 0.1 ml of medium and incubated with 200 ng of Alexa-MIF at 4 C. for 45 mins. The cells then were washed with ice-cold PBS (pH 7.4) and subjected to flow cytometry analysis (FACSCalibur, Becton Dickinson, San Jose, Calif.). In selected experiments, THP-1 monocytes or COS-7 transfectants were incubated with Alexa-MIF together with 50/ml of an anti-Ii mAb or an isotypic control mAb. For Scatchard analysis, triplicate samples of IFN-treated, THP-1 cells (110.sup.6) were incubated for 45 mins at 4 C. in PBS/1% FBS together with Alexa-MIF (0-1.5 M, calculated as MIF trimer), washed 3 with cold PBS/1% FBS, and analyzed by flow cytometry using CellQuest Software (Becton Dickinson, San Jose, Calif.).sup.29. The specific binding curve was calculated by subtracting non-specific binding (measured in the presence of excess unlabeled MIF) from total binding. Confocal fluorescence microscopy of Alexa-MIF binding to cells was performed with an LSM 510 laser scanning instrument (Carl Zeiss, Jena Germany). THP-1 cells were incubated with INF for 72 hrs and washed 3 with PBS/1% FBS prior to staining for 30 mins (4 C.) with 2 ng/l of Alexa-MIF, or Alexa-MIF plus 50 ng/l unlabeled, rMIF.

[0038] cDNA Library Construction, Expression, and Cell Sorting.

[0039] cDNA was prepared from the poly(A).sup.+ RNA of IFN-activated, THP-1 monocytes, cloned into the lambdaZAP-CMV vector (Stratagene, La Jolla, Calif.), and DNA aliquots (2.5 g/ml) transfected into 1510.sup.6 COS-7 cells by the DEAE-dextran method.sup.30. The transfected cells were incubated with Alexa-MIF for 45 min at 4 C., washed, and the positively-staining cells isolated.sup.31 with a Moflo cell sorter (Cytomation, Fort Collins, Colo.). In a typical run, 1.510.sup.7 cells/ml were injected and analyzed at a flow rate of 110.sup.4 cells/sec. Recovery was generally 90%. Plasmid DNA was extracted from sorted cells using the Easy DNA kit (Invitrogen, Carlsbad, Calif.) and transformed into E. coli XL-10 gold (Stratagene, La Jolla, Calif.) for further amplification. Purified plasmid DNA then was re-transfected into COS-7 cells for further rounds of sorting. After 4 rounds of cell sorting, 250 single colonies were picked at random and the insert size analyzed by PCR. Clones with inserts >1.6 Kb were individually transfected into COS-7 cells and the MIF binding activity re-analyzed by flow cytometry.

[0040] In Vitro Transcription and Translation.

[0041] Using a full-length Ii cDNA clone as template, three truncated (1-72aa, 1-109aa, 1-149aa) and one full-length (1-232 aa) Ii product were generated by PCR and subcloned into the pcDNA 3.1N5-HisTOPO expression vector (Invitrogen). The complete nucleotide sequence of an exemplary Ii cDNA clone and the putative Ii polypeptide forms that it encodes are presented in FIG. 5. The fidelity of vector construction was confirmed by automated DNA sequencing and the constructs then used as template for coupled transcription and translation using the TNT Reticulocyte Lysate system (Promega, Madison Wis.). The binding of [.sup.35S]-labeled Ii to immobilized MIF was assessed by a 3 hr incubation at room temperature, as recommended by the TNT protocol.

[0042] Activity Assays.

[0043] The dose-dependent phosphorylation of ERK-1/2 was measured by western blotting of cell lysates using specific antibodies directed against phospho-p44/p42 or total p44/p42 following methods described previously.sup.7. MIF-mediated suppression of apoptosis was assessed in serum-deprived, murine embryonic fibroblasts by immunoassay of cytoplasmic histone-associated DNA fragments (Roche Biochemicals, Indianapolis, Ind.).sup.8. Proliferation studies were performed by a modification of previously published procedures.sup.7,8. Human Raji B cells (American Type Tissue Culture, Rockville, Md.) were cultured in RPMI/10% FBS, plated into 96 well plates (500-1000 cells/well), and rendered quiescent by overnight incubation in RPMI/0.5% serum. The cells were washed, the RPMI/0.5% serum replaced, and the MIF and antibodies added as indicated. After an additional overnight incubation, 1 Ci of [.sup.3H]-thymidine was added and the cells harvested 12 hrs later. Fibroblast mitogenesis was examined in normal human lung fibroblasts (CCL210, American Type Tissue Culture) cultured in DMEM/10% FBS, resuspended in DMEM/2% serum, and seeded into 96 well plates (150 cells/well) together with rMIF and antibodies as shown. Isotype control or anti-Ii mAbs were added at a final concentration of 50 g/ml. Proliferation was assessed on Day 5 after overnight incorporation of [.sup.3H]-thymidine into DNA.

[0044] As will be apparent to a skilled worker in the field of the invention, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the invention may be practiced otherwise than as specifically described herein.

REFERENCES

[0045] [Endnotes:] [0046] 1. Weiser, et al., Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor, Proc. Natl. Acad. Sci. USA, 86, 7522-7526 (1989). [0047] 2. Metz, et al., Cytokine Reference Vol. I. Ligands Review: MIF, eds. Oppenheim, et al., Academic Press, San Diego, 703-716 (2000). [0048] 3. Bozza, et al., Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis, J. Exp. Med., 189, 341-346 (1999). [0049] 4. Swope, et al., Macrophage migration inhibitory factor: cytokine, hormone, or enzyme?, Rev. Physiol. Biochem. Pharmacol., 139, 1-32 (1999). [0050] 5. Calandra, et al., MIF as a glucocorticoid-induced counter-regulator of cytokine production, Nature, 377, 68-71 (1995). [0051] 6. Bacher, et al., An essential regulatory role for macrophage migration inhibitory factor (MIF) in T cell activation, Proc. Natl. Acad. Sci. USA, 93, 7849-7854 (1996). [0052] 7. Mitchell, et al., Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF), J. Biol. Chem., 274, 18100-18106 (1999). [0053] 8. Hudson, et al., A proinflammatory cytokine inhibits p53 tumor suppressor activity, J. Exp. Med., 190, 1375-1382 (1999). [0054] 9. Kleemann, et al., Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jabl, Nature, 408, 211-216 (2000). [0055] 10. Cresswell, et al., Assembly, transport, and function of MHC class II molecule, Arum. Rev. Immunol., 12, 259-293 (1994). [0056] 11. Naujokas, et al., The chondroitin sulfate form of invariant chain can enhance stimulation of T cell responses through interaction with CD44, Cell, 74, 257-268 (1993). [0057] 12. Naujokas, et al., Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development, Immunity, 3, 359-372 (1995). [0058] 13. Shachar, et al., Requirement for invariant chain in B cell maturation and function, Science, 274, 106-108 (1996). [0059] 14. Panchuk-Voloshina, et al., Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates, J. Histochem. Cytochem., 9, 1179-1188 (1999). [0060] 15. Wang, et al., Expression cloning and characterization of the TGF-fl Type III receptor, Cell, 67, 797-805 (1991). [0061] 16. Strubin, et al., Two forms of the Ia antigen-associated invariant chain result from alternative initiations at two in-phase AUGs, Cell, 47, 619-625 (1986). [0062] 17. Sant, et al., Biosynthetic relationships of the chondroitin sulfate proteoglycan with Ia and invariant chain glycoproteins J. Immunol., 135, 416-422 (1985). [0063] 18. Calandra, et al., The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor, J. Exp. Med., 179, 1895-1902 (1994). [0064] 19. Hansen, et al., Internalization and catabolism of radiolabelled antibodies to MI-IC class-II invariant chain by B-cell lymphomas, Biochem. J., 320, 293-300 (1996). [0065] 20. Koch, et al., Differential expression of the invariant chain in mouse tumor cells: relationship to B lymphoid development, J. Immunol., 132, 12-15 (1984). [0066] 21. Bendrat, et al., Biochemical and mutational investigations of the enzymatic activity of macrophage migration inhibitory factor (MIF), Biochemistry, 36, 15356-15362 (1997). [0067] 22. Thurman, et al., MIF-like activity of natural and recombinant human interferon-gamma and their neutralization by monoclonal antibody, J. Immunol., 134, 305-309 (1985). [0068] 23. Ashman, et al., A role for the transmembrane domain in the trimerizationof the MEW Class II-associated invariant chain, J. Immunol. 163, 2704-2712 (1999). [0069] 24. Lesley, et al., CD44 and its interaction with extracellular matrix, Adv. Immunol., 54, 271-335 (1993). [0070] 25. Calandra, et al., Protection from septic-shock by neutralization of macrophage migration inhibitory factor, Nature Med., 6, 164-170, (2000). [0071] 26. Bernhagen, et al, An essential role for macrophage migration inhibitory factor (MIF) in the tuberculin delayed-type hypersensitivity reaction, J. Exp. Med., 183, 277-282 (1996). [0072] 27. Mikulowska, et al., Macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of collagen type II-induced arthritis in mice, J. Immunol., 158, 5514-5517 (1997). [0073] 28. Lan, et al., The pathogenic role of macrophage migration inhibitory factor (MIF) in immunologically induced kidney disease in the rat, J. Exp. Med., 185, 1455-1465 (1997). [0074] 29. Palupi, et al., Bovine J3-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry, J. Biotech., 78, 171-184 (2000). [0075] 30. D'Andrea, et al., Expression cloning of the murine erythropoietin receptor, Cell, 57, 277-285. [0076] 31. Yamasaki, et al., Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor, Science, 241, 825-828 (1988). [0077] 32. Chesney, et al., An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of murine lymphoma, Mol. Med., 5, 181-191 (1999).

[0078] All publications and patent applications mentioned in the specification are herein incorporated by reference to the same extent as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference. The discussion of the background to the invention herein is included to explain the context of the invention. Such explanation is not an admission that any of the material referred to was published, known, or part of the prior art or common general knowledge anywhere in the world as of the priority date of any of the aspects listed above.

[0079] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and that this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.