WO2008064031A2 - Potent activation of antigen presenting cells by the hepatitis a virus cellular receptor 1 and its role in the regulation of immune responses - Google Patents

Abstract

The present invention relates to the identification of IgAλ as a ligand for HAVCR1/TIM1, the receptor for Hepatitis A Virus. The invention provides compositions for inhibiting or promoting this interaction, and methods for influencing immunologic responses to antigens by administration of compounds that affect binding of IgAλ to HAVCR1/TIM1.

Description

POTENT ACTIVATION OF ANTIGEN PRESENTING CELLS BY THE HEPATITIS A VIRUS CELLULAR RECEPTOR 1 AND ITS ROLE IN THE REGULATION OF IMMUNE RESPONSES

BACKGROUND OF THE INVENTION

The Hepatitis A Virus (HAV) receptor 1 (HAVCR1/TIM1) is a type 1 integral membrane glycoprotein consisting of a characteristic six-cysteine Ig-like domain extended above the cell surface by a mucin-like domain that contains a variable number of threonine, serine, and proline (TSP) hexameric repeats (14). The monkey (14) and human (6) HAVCR1/TIM1 were the first identified members of the TIM family, an immunologically important group of receptors (18, 22, 23, 26) that is conserved in vertebrates. It has been shown that Tim-1 , the murine ortholog of HAVCR1/TIM1 , is preferentially expressed in Th2 cells and delivers a signal that enhances T-cell activation and proliferation, increasing airway inflammation and allergy (26, 43) whereas Tim-3, another murine TIM family member, is mainly expressed in Th1 cells and provides a negative costimulatory signal that leads to immune tolerance (34, 35). Additionally, Tim-3 ligand binding has been related to macrophage activation and to the development of autoimmune diseases (27)Polymorphisms in Tim-1 and Tim-3 confer susceptibility to the development of asthma and allergy (23).

[0001] Although HAV is a hepatotropic virus that causes acute hepatitis in humans, infection with HAV has been shown to greatly reduce the risk of developing asthma and allergy in man (20, 21). Because the gene encoding HAVCR1/TIM1 has been shown to be an important asthma and allergy susceptibility gene in man (7, 9, 23, 24), it appears that HAVCR1/T1M1 plays a critical role in regulating T cell differentiation (23) and the development of atopy (24). The present invention relates to the role of HAVCR 1 /TIM 1 in the activation of antigen presenting cells (APCs) leading to the regulation of the immune response. This invention also relates to the role of the HAVCR1/TIM1-activated APCs in the regulation of T cell differentiation, atopy, autoimmune disease, and cancer, and in the control of pathogen infections. [0002] In mice, Tim-1 has been shown to be an important T cell co-stimulatory molecule, which is preferentially expressed on Th2 cells (43). Cross-linking of mouse Tim-1 enhances T cell proliferation and cytokine production, and prevents the induction of respiratory tolerance, resulting in airway hyperreactivity (AHR), a cardinal feature of asthma (43). Tim-1 co-stimulation requires its cytoplasmic tail and a conserved tyrosine that can be phosphorylated (4). In humans, HAVCR1/TIM1 is expressed in Th2 cell lines, is associated with remission in patients with MS (17), and is highly expressed in kidneys (14) primarily after injury (11) or in tumors (45). Recently, mouse Tim-4, a TIM family member expressed on antigen- presenting cells (APCs), has been shown to be a ligand for Tim-1 (25). Humans have an orthologous TIM4 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Fig. 1. Soluble receptor constructs. Schematic representation of Fc fusion proteins used in the screening and evaluation of HAVCR1/TIM1 ligands. Constructs containing the human HAVCR1/TIM1 (6) IgV domain or whole ectodomain (IgV plus mucin) fused to the Fc and hinge regions of human IgGI were termed HAVCR1/TIM1 (IgV)-Fc and HAVCR1/TIM1 (lgV+muc)-Fc, respectively. Similar Fc fusion proteins containing the monkey HAVCR1/TIM1 IgV domain, mkHAVCR1/TIM1 (IgV)-Fc, or the IgV domain plus the 27 hexameric repeats of the mucin domain, mkHAVCR1/TIM1(lgV+muc)-Fc, were reported previously (39, 40). The PVR-Fc construct containing the whole poliovirus receptor ectodomain (IgV, IgCI, and lgC2 domains) fused to the same Fc tail (39) was used as a negative control.

[0004] Fig. 2. Expression cloning of HAVC1/TIM1 ligand. Cloning of PerroδD cells expressing an HAVCR1/TIM1 ϋgand. Upper panels: Naϊve PerroδD cells (column 1), PerroδD-Clone 1 cells expressing an HAVCR1/TIM1 ligand (column 2), PerroβD-Clone D1 cells expressing an irrelevant human cDNA(s) (column 3), and Perro6D-CD32 FcγRIl cells expressing human CD32 ( FcγRII) (column 4) were panned over plates coated with HAVCR 1/TIMI(IgV)-Fc (1st row) or with PVR-Fc (2nd row). PerroβD-Clone 1 cells bound only to plates coated with HAVCR1/TIM1 (IgV)-Fc indicating that these cells express an HAVCR1/TIM1 ligand. Negative control PerroθD and Perro6D-Clone D1 cells did not bind to plates, and positive control Perro6D-CD32 FcγRII cells bound to both plates via their Fc receptors. Images were obtained with an inverted microscope at an original magnification of 5OX. Images are representative of 3 independent experiments. Lower panels: Beads coated with HAVCR1/TIM1 (IgV)-Fc (3rd row) or control PVR- Fc (4th row) were incubated with naive or transfected PerroδD cells attached to plates. Beads coated with HAVCR1/TIM1 (IgV)-Fc, but not with PVR-Fc, bound to Perro6D-Clone 1 cells expressing an HAVCR1/TIM1 ligand but not to naive PerroδD and Perro6D-Clone D1 cells. Both beads bound to Perro6D-CD32 FcγRII cells via Fc receptors. Binding was performed for 30-45 min followed by extensive washing. Images were obtained with an inverted microscope at an original magnification of 100X. Images are representative of 5 independent experiments.

[0005] Fig. 3. PerroθD-Clone 1 cells express IgAλ at the cell surface. Cell surface ELISA was performed with PerroθD-Clone 1 cells (left panel) and untransfected PerroβD cells (right panel) stained with mAbs to Igα1 heavy chain (IgA), Igλ light chain, lgκ light chain, Igμ (IgM) heavy chain, human integrin α3, or M2 mAb against the FLAG epitope (as control). Values represent mean OD at 450 nm, and are representative of 3 experiments.

[0006] Fig. 4. IgAλ is a specific ligand of HAVCR1/TIM1. Binding of HAVCR1/TIM1 to IgA was inhibited by excess IgA and anti-HAVCR1/TIM1 mAb. Fig. 4A) IgA blocks binding of HAVCR1/TIM1 (IgV)-Fc to Perro6D-Clone 1 cells. Binding assay was performed as in Fig. 2, lower panels, but in the presence of excess purified human immunoglobulins. HAVCR1/TIM1 (IgV)-Fc coated beads were incubated with PerroδD-Clone 1 cells bound to plates in the presence of excess IgAiλslgA, human myeloma Ig λlight chain, IgM λ, opmedia (None). Human myeloma IgAI λ or slgA but not human myeloma Ig flight chain or IgM λ blocked binding of HAVCR1/TIM1 (IgV)-Fc coated beads to PerroβD-Clone 1 cells compared to media untreated control. Data are representative of three experiments. Micrographs were taken with an inverted microscope at 100X. Fig. 4B) Anti- HAVCR1/TIM1 mAb blocks binding of HAVCR1/TIM1 Fc fusion proteins to Clone 1 cells. PerroθD-Clone 1 cells were stained with HAVCR1/TIM1 (IgV)-Fc RPE or HAVCR1/TIM1 (lgV+muc)-Fc RPE in the absence (filled histogram) or presence (continuous line) of anti-HAVCR1/TIM1 mAb. PerroθD-Clone 1 cells were stained with isotypic control PVR-Fc RPE (broken lines). HAVC R 1 /Tl M 1 fusion proteins with or without the mucin region bound to PerroβD-Clone 1 cells specifically via the IgV domain since the anti-HAVCR1/TIM1 mAb blocked binding of both constructs. Control PVR-Fc, which contains the same Fc tail, did not bind to PerroδD-Clone 1 cells. Data are representative of 3 experiments.

[0007] Fig. 5. Expression of IgA in PerroθD cells transfected with plasmids coding for Igα1 and Igλ. PerroθD cells were cotransfected with Igαiand Igλ plasmids isolated from PerroδD-Clone 1 cells, and transfectants were selected with puromycin and termed PerrroδD- IgAI λ cells. An IgA capture ELISA detected secretion of IgA into the cell culture supernatant of PerrroδD- IgAI λ and positive control PerroβD-Clone 1 cells (IgA capture ELISA). A cell surface IgA ELISA detected IgA on the cell surface of PerroδD-Clone 1 cells but not Perro6D-lgAλ cells. Negative control PerroβD cells did not secrete human IgA or express it at the cell surface. Data represent mean OD at 450 nm + SD, and are representative of 2 experiments.

[0008] Fig. 6. In vitro receptor binding assay. Binding of IgAI λ to HAVCR1/TIM1 (IgV)-Fc or PVR-Fc captured on ELISA plates. Supernatants of Perro6D-Clone 1 , PerrroθD- IgAI. λ and PerroδD cells were titrated on the ELISA plates containing the captured soluble receptors. Binding of IgAI λ to the captured receptors was assessed by staining with peroxidase-labeled anti-human Igα1 antibody. IgAI λ in supernatants of PerroθD-Clone 1 and PerrroθD- IgAI. λ cells bound to HAVC R 1 /Tl M 1 (IgV)-Fc but not PVR-Fc. Supernatants of PerroδD produced background binding levels. Data represent mean OD at 450 nm + SD, and are representative of 3 experiments.

[0009] Fig. 7. Infection of African green monkey kidney cells in the presence of IgA. HAV (10⁵ TCID₅₀) was mixed with 10 μg of slgA (HAV+lgA), 10 μg of IgM (HAV+lgM), or alone (HAV) and titrated on 96-well plates containing GL37 cells. Viral titers were determined by ELISA assay using the method of Reed and Muench (33). Data are representative of 2 experiments and show the mean TCID50/ml and S. D. in bars.

[0010] Fig. 8. Neutralization of HAV by soluble HAVCR1/TIM1 in the presence of slgA. IgA enhanced significantly the neutralization of HAV by mkHAVCR1/TIM1 Fc fusion proteins. Fig. 8A) HAV (10⁵ TCID₅₀) was incubated with 5 μg of purified human slgA (HAV+lgA), 5 μg of purified human IgG (HAV+lgG), or media (NONE) overnight at 4⁰C and then treated with 40 μg of mkHAVCR1/TIM1 (IgV)-Fc (striped histogram) or PVR-Fc (closed histogram) for 2 h at 37⁰C. Fig. 8B) HAV (10⁵ TCID₅₀) was incubated with 5 μg of purified human slgA (HAV+lgA) or media (HAV) overnight at 4⁰C and then treated 4 μg of mkHAVCR1/TIM1 (lgV+muc)-Fc (open histogram) or PVR-Fc (filled histogram) for 2 h at 37⁰C. Residual infectious HAV was titrated on GL37 cells. Viral titers were determined by an endpoint ELISA assay using the method of Reed and Muench (33). Data are representative of 3 experiments and show the mean TCID₅o/ml and S. D. in bars.

Fig. 9: Human monocytes express HAVCR1/TIM1 ligand. Fig. 9A) PBMC purified from buffy coats of normal human blood donors were stained with HAVCR1/TIM1(lgV)-Fc RPE or PVR-Fc RPE. Cells were analyzed by flow cytometry gated on monocytes according to their characteristic forward and side scattering. Monocytes stain with HAVCR1/TIM1 (IgV)-Fc (open histogram) but not control PVRFc (filled histogram).

Fig. 9B) IgAI and Fc Rl are expressed at the cell surface of monocytes. Elutriated human monocytes from normal blood donors were stained with anti-human IgAI or anti- human Fc Rl mAbs or their corresponding isotype controls stained with RPE. Fig. 9C) Goat anti-human IgA blocks binding of HAVCR1/TIM1 (IgV)-Fc to monocytes. Elutriated monocytes were treated with goat anti-human IgA, control goat anti-rabbitt IgG, or media, and then stained with HAVCR1/T1M1 (IgV)-Fc RPE. Goat anti-human IgA (right panel, empty histogram) but not control goat antirabbitt IgG (middle panel, empty histogram) blocked binding of HAVCR1/TIM1(lgV)-Fc to monocytes compared to the media treated control (filled histograms). Control anti- HAVCR1/TIM1 mAb 3D1 also blocked binding of HAVCR1/TIM1 (IgV)-Fc to monocytes (left panel, empty histogram) compared to the media treated control (filled histograms).

Fig. 9D) HAVCR1/TIM1 soluble receptor form containing the Ig-like and mucin domain behaves similarly than constructs without the mucin domain. Elutriated monocytes were incubated with HAVCR1/TΪM1(lgV+mucin)-Fc previously treated with HAVCR1 /TIMI(IgV)-Fc, PVR-Fc, or anti-HAVCR1/TIM IgV mAb (open histograms) or negative control M1 mAb (filled histograms). Binding of HAVCR1/TIM1 (lgV+muc)-Fc was blocked by HAVC R 1 /Tl M 1 (IgV)-Fc and anti- HAVCR 1 /TIM- 1 mAb but not PVR-Fc indicating that HAVCR1/TIM1 (lgV+mucin)-Fc binds similarly to monocytes than HAVCR1/TIM1 (IgV)-Fc. Data are representative of A), B)₁ and D) 3 experiments or C) 2 experiments.

Fig. 10: HAVCR1/TIM1 activates human monocytes. Fig. 10A) Elutriated monocytes were treated in polypropylene tubes with HAVCR1/TIM1 (IgV)-Fc, PVR-Fc, LPS, or media and incubated 1-4 days at 37°C under CO₂. Monocyte activation was determined at days 1-4 by treating aliquots of monocytes for 4 h with WST-1 reagent, which. is cleaved into a water-soluble farmazan salt by mitochondrial succinate-tetrazolium reductase system and visualized by absorbance at 450 nm. Values represent the mean of triplicate samples and the bars are S. D.

Fig. 10B) Elutriated monocytes were treated as in a). After 2 days, activation of monocytes was analyzed by flow cytometry for enlarged/granular cells (high forward scatter and greater side scatter).

Fig. 10C) Anti-HAVCR1/TIM-1 and anti-lgA1 mAbs block activation of monocytes by HAVCR1/TIM1 (IgV)-Fc. Elutriated monocytes were treated with anti- HAVCR1/TIM-1, anti-lgA1 , or control M1 mAbs, or media, and incubated with HAVCR1/TIM1 (IgV)-Fc or media. After 6 days of culture at 37°C under CO₂, monocyte activation was assessed with WST-1 reagent and examination of absorbance at 450 nm. Maximum absorbance was reached after 4 h with WST-1 reagent, where there are significant differences between untreated or M 1 -treated monocytes and anti-HAVCR1/TIM1 or anti-lgA mAbs (P< 0.0001). Values represent the mean of triplicate samples and the bars are S. D. Anti-HAVCR1/TIM-1 and anti- lgA mAbs block HAVCR1/TIM1 (IgV)-Fc induced monocyte activation. A) to C) Data is representative of 4 experiments.

Fig. 11 : Monocytes activated with HAVCR1/TIM1 (IgV)-Fc express increased cell surface markers and excrete cytokines.

Fig. 11A) Treatment with HAVCR1/TIM1(lgV)-Fc activates monocytes, and increases expression of cell surface markers. Elutriated monocytes were treated with HAVCR1/TIM1 (IgV)-Fc, PVR-Fc, LPS, or media for 3 days in polypropylene tubes at 37°C under 5% CO₂. Cells were then stained with mAbs to CD40, CD14, CD11c, Class II, CD86, CD80, or CD83 (open histograms) or their respective isotype controls (filled histograms) and analyzed by flow cytometry. Fig. 11B) Treatment with HAVCR1/TIM1 (IgV)-Fc activates monocytes and increases production of IL-6, IL-10, and TNF-α. Cytokine production of elutriated monocytes were treated as described in a) for 4 days. Supernatants were evaluated by quantitative capture ELISA using anti-human IL-6, IL-10, or TNF-a mAbs. Significant differences were found between HAVCR1/TIM1 (IgV)-Fc and PVR-Fc treated monocytes regarding the secretion of IL-6 (P=O.0009), IL-10 (P=0.0005), and TNF-a (P=O.0006). Data represent the mean of triplicate samples and the bars are S.D.

Fig. 12: Mixed lymphocyte reactions (MLR), using monocytes treated with HAVCR1 /TIMI(IgV)-Fc, PVR-Fc, or media and cultured with purified allogeneic T cells from normal human blood donors. Elutriated monocytes in polypropylene tubes were treated with media, HAVCR1 /TIMI (IgV)-Fc, PVR-Fc, or LPS for 2 days as described above in the activation procedure, and then plated at a density of 2 x 10⁴ cells/well in 96-well cell culture plates (Corning). Allogeneic human T cells (2 x 10⁵ cells/well) purified from total lymphocytes using a T cells negative isolation kit (Dynal Biotech, inc.),were added to the 96-well plates containing the treated monocytes. After 6 day incubation, culture supernatants were removed and plates were pulsed for 16-18 h with 1μCi [³H]thymidine per well. The incorporated radioactivity was measured in a Beta Plate Scintillation Counter (Perkin Elmer Wallac). Data are presented as the average counts per minute (c.p.m.) of triplicate wells.

Fig. 12A) T cells cultured with monocytes treated with HAVCR 1 /Tl M 1 (IgV)-Fc incorporated approximately 5- and 8-fold more [³H]thymidine than those treated with media or PVR-Fc, respectively (PO.001).

Figs. 12B, 12C, 12D) T cell proliferation was also visualized by microscopic examination of the MLR cell cultures previous to the addition of [³H]thymidine. Few small clusters of T cells were observed in wells containing monocytes treated with media (Fig. 12B) or PVR-Fc (Fig. 12D) whereas an increased number of T cell clusters of larger size were observed in wells containing monocytes treated with

HAVCR1/TIM1 (IgV)-Fc (Fig. 12C).

DETAILED DESCRIPTION OF THE INVENTION

[0011] HAVCR1/TIM1 is the cellular receptor for HAV and an important atopy susceptibility gene in man (7, 9, 23, 24). The present invention relates to the identification of a ligand of human H AVC R 1 /Tl Ml Using an expression cloning strategy with a soluble form of the HAVCR1/TIM1 containing the HAVCR1/TIM1 Ig- like region fused to a human IgGI Fc tail [HAVCR 1/TIMI(IgV)-Fc] human IgAλ is found to be a specific ligand of HAVCR1/TIM1. The binding of HAVCR1/TIM1 (lgV)- Fc to IgAλ is specifically blocked with mAb to IgAI or Igλ light chain, with anti- HAVCR1/TIM1 mAb, or by treatment with excess IgAI λ or slgA, but not by IgM. [0012] HAVCR1/TIM1 is normally expressed at low levels in kidney and liver cells, and on the surface of some subsets of T cells. (17) [0013] The presence of IgA does not prevent HAV infection of African green monkey kidney cells suggesting that HAV and igA bind to different sites on the HAVCR1/TIM1 Ig domain. Moreover, IgA significantly enhances neutralization of HAV by soluble HAVCR1/TIM1 receptor forms indicating that binding of IgA to HAVCR1/TIM1 has a synergistic effect in the virus-receptor interaction. HAVCR1/TIM1 is sufficient for binding and alteration of HAV particles (39, 40), steps that are required for viral cell entry. It appears that IgA plays a role in vivo enhancing the interaction of the virus with the receptor under unfavorable infection conditions such as low receptor levels. Consequently, inhibition of the interaction of IgA with HAVCR1/TIM1 is expected to lower susceptibility to HAV infection and should be of therapeutic benefit in treatment of HAV by lowering the rate of infection of naive cells.

[0014] HAVC R 1 /Tl M 1 plays some roles in normal immune system function. Different alleles of HAVCR1/TIM1 result in differences in susceptibility to asthma and other atopic diseases in humans (26). An insertion of six amino acids into the receptor polypeptide at position 157 (termed "157insMTTTVP") is associated with decreased susceptibility to atopy.

[0015] Blocking the activation of HAVCR1/TIM1 by IgAλ, for example using a peptide fragment of IgAλ that acts as an antagonist, would be of benefit in preventing or treating allergy or asthma in a person having the susceptibility allele of HAVCR 1 /Tl M 1.

[0016] On the other hand, in a person having the "non-susceptible" allele of H AVC R 1 /Tl M 1 , peptides derived from IgAλ that are bivalent or multivalent for binding to HAVCR1/TIM1 and cross-link the receptor on the surface of T cells will act to provide protection against allergy or asthma.

[0017] Also, as shown below, binding of HAVCR1/TIM1 to IgAλ on the surface of monocytes results in proliferation of monocytes and their activation. Activated monocytes proliferate and differentiate into antigen presenting cells (APCs), including dendritic cells (DCs) that express IgA Fcα receptor on their surface. Exposure of T cells to monocytes activated by HAVCR1/TIM1 contacted with IgAλ in a mixed lymphocyte reaction results in increased proliferation of T cells. Thus, peptides derived from IgAλ that activate the receptor would be beneficial in general boosting of antigen-specific immune responses.

[0018] The cell entry and pathogenic process of HAV are poorly understood. Although it is well established that HAV is transmitted through the fecal-oral route, it is unknown whether there is a primary site of HAV replication in the digestive system or the input virus is transported to the blood and then reaches the liver, its target organ (41). HAV initially infects Kupffer cells, which are liver resident macrophages, and then extends to the hepatocytes (2, 19, 38). We have previously shown that HAVCR1/TIM1 is a receptor for HAV (6, 14) but the exact role of this receptor in pathogenesis of HAV needs to be fully elucidated. Soluble receptor forms of HAVCR1/TIM1 bind, alter, and neutralize HAV particles (39, 40), which indicates that additional co-receptors are not required for the initial steps in HAV cell entry. However, since IgA increased the receptor-mediated neutralization of HAV (Fig. 4), it appears that additional factors may enhance the cell entry process of HAV. Several possible mechanisms by which IgA enhances the interaction of HAV with HAVCR1/TIM1 can be envisioned. For instance, binding of IgA to HAVC R 1 /Tl M 1 may expose epitopes or provide scaffolding to the HAVCR1/TIM1 IgV virus-binding domain enhancing the virus-receptor interaction. We have previously shown that mkHAVCR1/TIM1 (IgV)-Fc cannot neutralize more than 0.5 log of HAV (39). However, in the presence of IgA, neutralization of HAV by mkHAVCR1/TIM1 (IgV)-Fc increased significantly (Fig. 8A) to levels not reached with this soluble receptor form alone. mkHAVCR1/TIM1 (lgV+mucin)-Fc, which contains a longer mucin with 27 hexameric repeats, neutralizes HAV more efficiently than mkHAVCR1/TIM1 (IgV)-Fc and induces the alteration of the viral particles (40), suggesting that the mucin domain provides structural support for the IgV domain of HAVC R 1 /Tl M 1 and/or binds to the viral particles. Since IgA also enhanced the neutralization of HAVCR1/TIM1 (IgV)-Fc, it is possible that IgA and the mucin domain may play a similar role providing the necessary scaffolding to the IgV domain of HAVCR1/TIM1. Interestingly, IgA also enhanced neutralization of HAV by a suboptimal concentration of mkHAVCR1/TIM1(lgV+mucin)-Fc (Fig. 8B) but not under saturating conditions. Since HAVCR1/TIM1 is expressed at low levels in most tissues (6, 14), except in testis and in kidneys (14) mainly after injury (1 1) or in tumors (45), it is possible that binding of IgA to HAVCR1/TIM1 may enhance its HAV-receptor function under the physiological conditions encountered by the virus. Moreover, the interaction of IgA with HAVCR1/TIM1 may play an important role in the pathogenesis of HAV by enhancing infection of cells that contain IgA and IgA-receptors at the cell surface such as Kupffer cells and hepatocytes.

[0019] Human IgA exists in multiple forms of two subclasses: IgAI, the most abundant serum subclass produced in the bone marrow as a monomer, and lgA2, the predominant mucosal subclass found in a polymeric form synthesized by local plasma cells before being secreted (15). The secreted IgA (slgA) is a dimeric form that contains two additional polypeptides, the J-chain and the secretory component, and functions at the first line of defense against pathogens. The function of serum IgA is poorly understood, it is not usually involved in humoral immune responses, does not activate complement, and regulates IgG-mediated phagocytosis, chemotaxis, bactericidal activity, oxidative burst, and cytokine release (15, 44). Serum IgA has both anti- (46, 47) and pro-inflammatory (44) effects. IgA is thought to be involved in suppressing the development of atopy (32). IgA deficiency is associated with increased susceptibility to autoimmune and allergic disorders (36), and expression of the myeloid specific IgA Fc receptor (CD89, also called FcαRI) is upregulated in eosinophils from allergic patients (28). Without being bound by any theory of the invention, the present inventors consider cells expressing HAVC R 1 /T I M 1 or natural soluble forms of this receptor (3) may crosslink IgA on antigen presenting cells (APC) and send a signal via FcαRI, resulting in the inhibition of the development of atopy. Since HAVC R 1 /T I M 1 is a significant atopy susceptibility gene(24), the present invention relates in part of the inventors' expectation that different allelic forms of HAVCR1/TIM1 have different effects on APC activation.

[0020] On monocytes, IgA binds to FcαRI or CD89, the myeloid specific IgA Fc receptor, expression of which is restricted to neutrophils, eosinophils, most monocytes/macrophages, interstitial dendritic cells (DCs), and Kupffer cells. Human monocytes express FcαRI but not the IgA binding transferrin receptor (CD71) (31 ) and do not express any other IgA receptor (29, 30). Normally, FcαRI functions as an inhibitory regulator, particularly in the absence of sustained FcαRI aggregation, but the interaction of HAVCR1/TIM1 with IgA bound to FcαRI may trigger sustained FcαRI receptor coaggregation, which results in ITAM-mediated cell activation, and proinflammatory activity (29, 30). Our results with HAVCR1/TIM1 and IgA may be similar to other studies showing that crosslinking of IgA at the cell surface of immature DC triggered the overexpression of co-stimulatory molecule CD86 and MHC class II, the production of IL-10, and increased allostimulatory activity (8). [0021] Since monocytes are precursors of DCs, the inventors expect that HAVCR1/TIM1 has important effects on both monocytes and DCs expressing IgA bound to FcαRI. The interaction of HAVCR1/TIM1 with IgA bound to FcαRI on human APCs cannot occur in mice since the biology of IgA is significantly different in humans (16, 44) and mice do not express FcαRI homologs (29, 30). Consequently, the mechanisms by which TIM family members regulate immune responses in mice and humans seems to have diverged significantly during evolution. [0022] Using an expression cloning strategy, IgAI λ is identified as a specific ligand of H AVC R 1 /T I Ml Further, the association between IgAI λ and HAVCR1/TIM1 , which could be specifically blocked by mAb against IgAI , Igλ light chain or HAVCR1/TIM1, or by treatment with excess IgAI λ, enhanced the interaction of HAVCR1/TIM1 with HAV. This result establishes that the interaction of IgA with HAVCR1/TIM1 plays a role in pathogenesis of HAV enhancing viral cell entry in cells expressing low levels of HAVCR1/TIM1. Finally, these results indicate that the interaction between HAVCR1/TIM1 and IgA represents a new pathway for the regulation of the immune responses by TIM family members. [0023] The present invention relates to methods to induce and/or enhance an immune response in humans using membrane bound or soluble forms of HAVCR1/TIM1. The methods of the invention include methods to activate human immune cells, including activation of monocytes and subsequently differentiated antigen presenting cells, including dendritic cells, using membrane bound or soluble forms of HAVCR1/TIM1. The methods of the invention also include methods to induce proliferation of T cells using membrane bound or soluble forms of HAVCR1/TIM1 and to bias the balance of the Th1 or Th2 immune response using membrane bound or soluble forms of HAVCR1/TIM1. Among the target cell types of the methods of the invention are those expressing IgA receptors, and in particular Fcα receptors.

APCs and/or myeloid cells contacted by HAVCR1/TIM1 may (Fig. 10A) or may not exhibit increased mitochondrial enzymatic activity as measured by the WST- 1 method, depending upon the context of the APC or myeloid cell in the body (for example whether it is present in a lymph node or in the circulation) as known to one of ordinary skill in the art. APCs and/or myeloid cells contacted by may (Fig. 10B) or may not HAVCR1/TIM1 exhibit increased cellular size and complexity as measured by forward and side scattering (FSC and SSC) FACS analysis, again depending upon the context of the APC or myeloid cell as known to one of ordinary skill in the art. APCs and/or myeloid cells contacted by said HAVCR1/TIM1 exhibit increased secretion of at least one cytokine selected from the group consisting of IL-6, IL-10 and TNF-α, the particular cytokine profile depending upon the context of the APC or myeloid cell as known to one of ordinary skill in the art. (See Fig. 11 B) Activated APCs and/or myeloid cells exhibits increased expression of at least one cell surface marker selected from the group consisting of CD40, CD14, CD11c, CD86 or MHC class Il molecules, the particular cell surface profile depending upon the context of the APC or myeloid cell as known to one of ordinary skill in the art. (See, Fig. 11A) Activated APCs and/or myeloid cells may induce (See Fig. 12A) or may inhibit proliferation of lymphocytes again depending upon the context of the APC or myeloid cell as known to one of ordinary skill in the art.

The present invention provides a method in which a membrane bound, particulated, or soluble form of HAVC R 1 /T I M 1 or peptides derived from this receptor is administered in an effective amount to activate APCs and/or myeloid cells in an individual in need to prevent or treat diseases comprising pathogen infections and autoimmune diseases, including asthma and atopic diseases.

The present invention also provides a method to screen for compounds, peptides, antibodies, or fragments of antibodies that inhibit the interaction of HAVCR1/TIM1 with APCs and/or myeloid cells and prevent their activation, or to screen compounds for inducing anergy by affecting the way in which APCs and/or myeloid cells contacted with HAVCR1/TIM1 respond in a context-dependent fashion. Isolation of APCs, myeloid cells, or precursors of these cells for use in such screening methods can be done using methods that are known to the art as described in Current Methods of Immunology (Coligan, J. E., A.M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, eds. 2006. Current protocols in immunology. R. Coico, series ed. New York: Wiley & Sons). Culture and maturation of APCs, myeloid cells, or precursors of these cells for use in methods of the present invention can be done using methods that are known to the art as described in Current Methods of Immunology.

[0024] The IgA utilized in the present invention is IgA that comprises a λ light chain or a part thereof. The IgA may be either intact IgA, or may be fragments of IgA such as F(ab), F(ab') or F(ab')2 fragments thereof. In some instances the IgA will be polymeric, joined by its J chain, or monomeric. An IgA fragment may be a "half antibody" or may be a single chain antibody. An IgA fragment may include only an antigen-binding arm of the antibody, or only the "framework" portion thereof, with or without a portion of the hinge region. Antibody fragments according to the invention may be bi- or multi-valent and joined by linker peptides or by non-peptide linkers. [0025] The IgA is preferably of human origin or humanized. Methods for preparing humanized antibodies are considered known in the art. [0026] A peptide is "derived from" IgA or from IgAλ or from another antibody if it represents a portion of the structure of IgA or IgAλ or other antibody. A peptide derived from IgA or from IgAλ or from another antibody may simply be a portion of the amino acid sequence of one of the polypeptides constituting the immunoglobulin. Alternatively, the peptide may include portions of both the light and heavy chains of the immunoglobulin, joined by a peptide or non-peptide linker. A peptide derived from IgA or IgAλ (or another antibody that specifically binds HAVCR1/TIM1) may be at least bivalent with respect to presentation of portions of the molecule that specifically bind to H AVC R 1 /Tl ML

[0027] A peptide derived from IgA or IgAλ (or another antibody that specifically binds HAVCR1/TIM1) or a fragment of IgA or IgAλ (or another antibody that specifically binds HAVCR1/TIM1) according to the invention may be made by treatment of isolated immunoglobulins, expressed in a cultured cell using recombinant DNA technology, or may be chemically synthesized or may be prepared using a combination of these technologies.

[0028] Furthermore, the IgA may be soluble, or may be utilized in a membrane bound form. Membrane bound forms may include IgA presented on the surface of a cell, for example bound to Fcα bound to the surface of an undifferentiated monocyte or other myeloid cell, or in particulated form, e.g. bound to an artificial membrane such as a liposome, or otherwise aggregated. Formulation of antibodies into artificial membranes, in the manner of other membrane-bound proteins, including liposomes having a lipid bilayer and exposing the antibody variable region on the surface of the liposome, is considered known in the art.

[0029] Additional compositions that may be utilized in the invention are those that interfere with the interaction of IgAλ with HAVCR1/TIM1. Such compositions may comprise an antibody that binds to HAVC R 1 /T I M 1 at a conformational epitope that includes the amino acid glutamine-90 or amino acids between or adjacent cysteine residues 3 and 4 (amino acids CSLFTC) in the wild-type human H AVC R 1 /T I Ml [0030] Compositions useful in some embodiments of the present invention comprise HAVCR1/TIM1 protein, or parts thereof that interact with IgAλ. The HAVCR1/TIM1 protein may be provided in soluble form, or bound to a membrane of a cell or in "particulated" form bound to an artificial membrane such as a liposome or otherwise aggregated.

[0031] The genes encoding HAVCR1/TIM1 and cDNAs from all of mouse, human and monkey are known (see, e.g. ref. 17). Thus, it is contemplated that HAVCR1/TIM1 proteins and parts thereof are easily expressed and purified using known recombinant DNA methods in eukaryotic and prokaryotic cells. For example, the variable region domain of HAVCR1/TIM1 can be expressed in CHO cells alone or as Fc fusion proteins and purified through protein A columns, or expressed in bacteria and purified by refolding from inclusion bodies. The methods for making mutations in HAVC R 1 /T I M 1 and expressing the mutant proteins are also considered known in the art.

[0032] The surfaces of HAVCR1/TIM1 that are contacted by IgAλ are described in the co-pending U.S. provisional application ser. no. 60/865,642, attorney docket no. 1173-1070PUS1 filed November 13, 2006 and entitled "Structure of TIM Family Members", hereby incorporated by reference in its entirety for all purposes. [0033] IgAλ binds to HAVCR1/TIM1 at an epitope including the CC loop of the receptor. This loop is formed by the portion of the receptor between the cysteine residues 3 (amino acid 34) and 4 (amino acid 39) of the receptor, which are disulfide bonded. It is contemplated that a fragment of HAVCR1/TIM1 comprising this portion of the receptor, or antibody that specifically binds to this portion of the receptor, such as the 3D1 antibody, can act as an inhibitor of HAVCR1/ T1M1. [0034] Formulation of proteins or peptides into pharmaceutical compositions for administration to humans or animals is considered known in the art. Formulation of membrane bound proteins into liposomes, including liposomes having a lipid bilayer structure exposing the protein on the surface of the liposome, are considered known in the art. [0035] Similarly, determination of dosage of proteins or peptides constituting amounts effective for treatment of diseases is considered known in the art. Typically in the art, an amount of the protein or peptide that provides from 0.1 to 5 μg/ml in the blood is used. A preferred dosage range in the present invention is one that profides from 0.5 to 2 μg/ml in the blood.

[0036] In one embodiment of the invention, cells expressing FcαRI are activated. The cells are contacted with IgAλ in soluble form, or by a peptide derived from IgA or a fragment of IgA, so that the IgAλ binds to FcαR I on the cell surface, or the IgAλ may already be bound to the surface of the cell via FcαR bound to the surface of said cell. The cell is then contacted with HAVCR1/TIM1 , resulting in activation of the cell.

[0037] In this embodiment of the invention, the cells are preferably monocytes, which differentiate into antigen presenting cells, preferably monocyte-derived dendritic cells, after activation as described above. Other cell types that can be activated using the methods of the invention include myeloid cells such as neutrophils, basophils, eosinophils, macrophages, interstitial dendritic cells and Kupffer cells as well as mast cells that express Fcα receptors. Eosinophils, basophils, and mast cells degranulate upon crosslinking of IgA at their cell surfaces inducing an anaphylactic response. In an embodiment of the invention, said degranulation will be prevented by inhibiting crosslinking of IgA by HAVCR1/TIM1 using anti-HAVCR1/TIM1 antibodies or other antagonists of the interaction of IgA with HAVCR 1 /Tl Ml

[0038] In preferred embodiments of the invention, the activated monocytes will be induced to secrete a variety of cytokines, or the amount of one or more cytokines that is secreted will be increased. Among the cytokines secreted by monocytes activated in the manner of the invention are IL-6, IL-10 and TNF-α. Assay for these cytokines is considered a test for monocyte activation, with increase levels of these cytokines indicating activation of monocytes.

[0039] Monocytes activated by the method of the present invention also typically increase their expression of the cell surface markers CD86 and MHC class Il molecules, indicating a differentiation into an antigen-presenting cell type. [0040] The present invention provides for methods for treating immune disorders, especially those that relate to atopy and allergy.

[0041] One of ordinary skill in the art understands that not every embodiment of the present invention will present all of the aspects of the invention or enjoy every advantage of the invention. Different embodiments of the invention may present different aspects of the invention and provide different advantages of the invention.

Examples:

[0042] The present invention is illustrated by the following working examples. The examples provided herein are illustrative only, and are not to be taken as limiting the invention, which is defined by the claims following.

General Materials and Methods Cells and virus

[0043] Chinese hamster ovary (CHO) cells deficient in the enzyme dihydrofolate reductase (dhfr-) were obtained from the American Type Culture Collection (ATCC). Perro6D cells derived from Canine Osteogenic Sarcoma D-17 cells (ATCC) transfected with EBNA-1 cDNA, are resistant to the antibiotic G418, and have an increased transfection efficiency for episomal plasmids containing an Epstein-Barr virus P1 origin of replication (42). African green monkey kidney GL37 cells were grown in EMEM containing 10% fetal bovine serum (FBS). The cell culture adapted HM-175 strain of HAV, obtained from S. Feinstone, FDA, was grown in GL37 cells. Human embryonic kidney 293-H cells were obtained from Invitrogen, Inc. [0044] Elutriated monocytes and lymphocytes as well as buffy coats were obtained from normal human donors at the NIH Blood Bank.

Antibodies and purified human immunoglobulins

[0045] Mouse anti-human Igα1 (IgAI), Igλ, and lgκ mAbs, and FITC-iabeled mouse anti-human CD14 mAb, were obtained from Southern Biotechnology, Inc. The anti-human IgA MAb was labeled with anti-mouse Fc RPE-labeled Fab fragments using Zenon technology as suggested by the manufacturer (Molecular Probes, Invitrogen). Purified human secretory IgA (slgA) was purchased from Serotec, Inc. Purified human myeloma IgAI λ, IgMλ, and Igλ were purchased from The Binding Site, Inc.

[0046] The following antibodies were obtained from BD Pharmingen, Inc.: R- Phycoerythrin (R-PE)-conjugated mouse anti-human CD83, CD11c, CD86, CD80 mAbs and specific isotype controls; Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD40 and HLA-DR, DP, DQ mAbs and their specific isotype controls. Anti-FLAG peptide mAbs M1 and M2 were purchased from Sigma Co. Anti- human integrin α.3 mAb was purchased from Invitrogen, Inc. Purified human secretory IgA was purchase from Serotec, Inc. Unlabeled goat anti-human Fc, IgG or IgA antibodies, and peroxidase-labeled goat anti-mouse IgG, IgM and anti-human IgG, IgM and IgA antibodies were purchased from KPL, Inc.

Fusion proteins

[0047] Schematic representations of the Fc fusion proteins used in the Examples below are shown in Fig. 1. All fusion proteins contain the same Fc and hinge fragments of human IgGI (Fc tail) and were constructed as described previously (39) with minor modifications. For these Examples, we constructed HAVCR1/TIM1 (lgV)- Fc, which contains the Ig-like region and three TSP repeats of the mucin-like region of HAVCR1/TIM1 tagged at the N-terminus with peptide DTKDDDK (FLAG) fused to the Fc tail. We also constructed HAVCR1/TIM1 (lgV+muc)-Fc, which contains the whole ectodomain (Ig-like plus mucin-like regions) of human HAVCR1/TIM1 fused to the Fc tail. We also used the following 3 previously constructed Fc fusion proteins. mkHAVCR1/TIM1 (IgV)-Fc, which contains the Ig-like region and two TSP repeats of the mucin-like region of monkey HAVCR1/TIM1 tagged at the N-terminus with peptide DTKDDDK (FLAG) fused to the Fc tail (39). mkHAVCR1/TIM1 (lgV+muc)-Fc, which contains the Ig-like region and twenty-seven TSP repeats of the mucin-like region of the monkey HAVCR1/TIM1 tagged at the N-terminus with peptide DTKDDDK (FLAG) (40). PVR-Fc contains the ectodomain of the poliovirus receptor fused to the Fc tail (39). All these Fc fusion proteins were produced in stably transfected Chinese hamster ovary (CHO) cells and purified on protein A columns as described previously (39). Generation of monoclonal antibodies against human HAVCR1/TIM1 [0048] Anti-HAVCR1/TIM1 hybridomas were generated by immunization of BALB/cByJ mice subcutaneously with HAVCR1/TIM1 (IgV)-Fc (Fig. 10) in CFA and boosting multiple times with HAVCR1/TIM1 (IgV)-Fc in PBS (100 μg). One day following the last boost, lymph node cells were fused with NS1 myeloma cells, cloned, and the hybridomas screened by cell surface staining of HAVCR1/TIM1 transfected CHO cells and for lack of reactivity with untransfected cells. Ten anti- HAVCR1/TIM1 specific hybridomas were subcloned. Hybridoma 3D1 (secreting mouse IgG 1κ) was chosen for further analysis on the basis of blocking the interaction of HAVC R 1 /Tl M 1 with an unidentified ligand and for robust staining.

Expression cloning of human HAVCR1 ligands

[0049] A human lymph node cDNA library (Edge Biosystems, Inc) constructed in pEAKδ, an episomal shuttle vector containing prokaryotic CoIEI and eukaryotic Epstein-Barr virus P1 origins of replication and selectable markers for ampicillin and puromycin, was transfected into PerroβD cells and transfectants were selected with puromycin. Briefly, PerroδD monolayers grown in 150-cm2 flasks were transfected with 2 μg of purified DNA from the human lymph node cDNA library or control pEAKδ and 30 μl of Fugeneδ as recommended by the manufacturers (Roche)., Transfectants were selected with 2 μg/ml of puromycin, detached from the plates with 0.5 mM EDTA in PBS, and panned (1 , 14, 37) 3 times to polystyrene Petri dishes coated with purified soluble HAVCR1/TIM1 (IgV)-Fc or PVR-Fc. To enrich for cells that did not bind to the Fc tail, transfectants were panned in the presence of excess of PVR-Fc and depleted of FcγRII (CD32) -positive cells using anti-mouse igG paramagnetic beads bound to anti-human CD32 mAb and magnetic separation. To rescue episomal plasmids from PerroθD-Clone 1 cells, Hirt supernatant DNA (12) was prepared and used to transform E. coli XL10-GOLD ultracompetent cells as suggested by the manufacturer (Stratagene). Ampiciliin-resistant colonies were selected, plasmids were purified, and nucleotide sequence of the inserted cDNAs was obtained by automated sequencing using the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems) and the ABI Prism (model 3100) analyzer (Applied Biosystems). The obtained nucleotide sequences were compared to GenBank using the BLAST program.

Bead binding assays

[0050] Purified soluble receptors, HAVC R 1 /Tl M 1 (IgV)-Fc or PVR-Fc, were covalently coupled to 6 micron blue carboxylated microparticles using the carbodiimide kit as suggested by the manufacturer (Polyscience, Inc.). For binding assays, cell monolayers were grown in 12-well plates and incubated with coupled beads in PBS -2 %FBS for 30-45 min at room temperature. Unbound beads were washed extensively and monolayers were examined under an inverted microscope at 50-100 X. For binding inhibition assays, coated beads were treated with 5 μg of purified mAbs or purified human myeloma immunoglobulins ( IgAI λ, IgMλ, and Igλ) before being added to cell monolayers.

Immunochemical detections

[0051] Expression of human CD32 on PerroθD cell transfectants and IgAλ on PerroβD-Clone 1 cells was analyzed by a cell surface ELISA on live cells grown in 96-well plates (13).

[0052] For the capture ELISA assay, 96-well ELISA plates were coated with 1 μg/ml goat anti-human IgA in bicarbonate buffer, washed, and blocked with 5% BSA in PBS. Two-fold dilutions of supernatants of PerroβD cell transfectants grown in the presence of 20 μg/ml puromycin were titrated on the plates in duplicates. Binding of IgA to the plates was detected by staining with 0.33 μg/ml mouse anti-human IgA mAb and 0.5 μg/ml goat anti-mouse peroxidase-labeled antibody. Plates were developed with TMB One-Component (KPL Inc.) substrate and read at 450 nm in an ELlSA plate reader. Measurement of human cytokines in monocyte culture supernatants was done in the Cytokine Core Laboratory at the University of Maryland by a quantitative capture ELISA using biotin-strepavidin-peroxidase detection (10).

In vitro receptor binding assay

[0053] For the in vitro assay to study binding of human IgA to HAVCR1/TIM1 ,

HAVCR1/TIM1 (IgV)-Fc or PVR-Fc (1 μg/ml) were captured on 96-well plates (Nunc, Inc.) coated with 1 μg/ml goat anti human IgGI Fc . Supernatants of dog cell transfectants were titrated on the plates and stained with peroxidase-labeled anti- human IgA and One-Component TMB. Absorbance at 450 nm was determined in an ELISA plate reader.

Flow Cytometry

[0054] Flow cytometry was done in a FACSCalibur cytometer and data was processed with the CellQuest program (Becton Dickinson). PBMCs were stained with HAVCR1/TIM1 (IgV)-Fc or PVR-Fc labeled with RPE-Fab to human Fc using the Zenon Technology as recommended by the manufacturer (Molecular Probes, Invitrogen, Inc.).

[0055] Detection of IgA on the surface of monocytes was performed with anti- human IgA mAb labeled with RPE-Fab to mouse Fc using the Zenon Technology. [0056] For specificity studies, monocytes were incubated for 30 minutes at room temperature with 5 μg of antibody or soluble receptor and then stained with HAVCR1/TIM1 (IgV)-Fc labeled with RPE as indicated above using Zenon Technology.

[0057] To study expression of activation markers, monocytes treated with media or 20 μg/ml HAVCR1/TIM1 (IgV)-Fc , PVR-Fc, or 1 μg/ml LPS for 3 day in polypropylene tubes at 37⁰C under 5% CO2 were stained with directly conjugated anti-CD40 (FITC), anti-CD80 (FITC), anti- HLA-DR, DP, DQ (FITC), anti-CD14 (FITC), anti-CD83 (R-PE) or anti-CD86 (R-PE) or their correspondent isotype controls.

HAV neutralization assays

[0058] The effect of IgA on HAV infection was analyzed by incubating 10⁵ TCID₅₀ of HAV with 10 μg of normal human slgA or IgM (negative control) in 0.5 ml of EMEM-10% FBS for 2 h at 37⁰C. Samples were diluted 1/4 in EMEM-10% FBS and filtered through a 0.22 μm sterile filter. Residual HAV infectivity was titrated in 96- well plates containing GL37 cells using 8 repetitions per 10-fold dilution, and plates were incubated for 10 days at 35⁰C under 5% CO2. Cells were fixed with 90% methanol and stained with anti-HAV antibodies, a secondary peroxidase-labeled antibody, and TMB one component substrate (KPL Inc.) as previously described (39). Viral titers were assessed using the Reed and Muench method (33). [0059] Soluble receptor-mediated neutralization of HAV was performed as described previously (40) with minor modifications. Briefly, 10⁵ TCID₅₀ of HAV was incubated with normal human slgA (5 μg), control normal human IgG (5 μg), or media in 0.5 ml of EMEM-10% FBS for 2 h at 37⁰C. For neutralization with the short form of the HAV receptor, 40 μg of mkHAVCR1/TIM1 (IgV)-Fc or negative control PVR-Fc were added and incubated overnight at 4⁰C. For neutralization with the long form of the HAV receptor, 4 μg of mkHAVCR1/TIM1 (lgV+muc)-Fc or PVR-Fc were added and incubated overnight at 4⁰C. Residual infectious HAV was titrated in 96-well plates containing GL37 or FRhK4 cells using 8 repetitions per 10-fold dilution. After 3 h adsorption, wells were washed 3 times with EMEM-10% FBS, and plates were incubated for 10 days at 35⁰C under 5% CO₂. Cells were fixed, and HAV titers were determined by ELISA as described above using the Reed and Muench method (33).

Activation of human monocytes

[0060] Activation of elutriated monocytes was measured using the WST-1 colorimetric assay as recommended by the manufacturer (Roche). Briefly, 5x 10⁶ monocytes were incubated in polypropylene tubes and treated with media or with 20 μg/ml of HAVCR1/TIM1 (IgV)-Fc, 20 μg/ml PVR-Fc, or 1 mg/ml of LPS for 1-4 days. All the Fc fusion protein preparations tested negative for endotoxin as indicated above. Aliquots of 100 ul were withdrawn daily and incubated with the WST-1 reagent for 4hs before reading absorbance at 450 nm. Alternatively, monocytes were incubated with fusion proteins in 96-well eel! culture plates (Corning) in the presence or absence of 50μg/ml of mAbs to test inhibition of activation. After 6 days incubation, WST-1 reagent was added to the monocytes and absorbance at 450 nm was determined each 60 min for 4 hours.

Statistical analysis.

[0061] P values between 2 treatments were calculated using unpaired t-test. Example 1: Expression cloning of HAVCR1/TIM1 ligands [0062] The only known ligand of HAVCR1/TIM1 is HAV, which usurps this receptor to enter the cell (5, 6, 14). To identify natural ligands of H AVC R 1 /T I M 1 , we used an expression cloning strategy in which PerroδD cells, a dog osteosarcoma cell line that is highly transfectable and allows maintenance of episomal plasmids containing a P1 origin of replication (42), were transfected with a human lymph node cDNA library cloned into the episomal shuttle vector pEAKδ. We used conditions that allowed incorporation of 1-10 plasmids per cell, and selected cell transfectants with 2 μg/ml puromycin. The PerroβD transfected cells were panned on Petri dishes coated with soluble forms of HAVCR1/TIM1 shown in Fig. 1. These fusion proteins included a construct called HAVCR1/TIM1 (IgV)-Fc, in which the IgV region of HAVC R 1 /Tl M 1 was fused to human IgGI Fc, as well as HAVCR1/TIM1 (lgV+muc)- Fc, in which both the IgV and mucin regions of HAVCR1/TIM1 were fused to human IgGI Fc. As a negative control, we used PVR-Fc (39), a construct containing the complete ectodomain of the poliovirus receptor consisting of 3 Ig domains fused to human IgGI Fc.

[0063] To enrich for cells that bound specifically to HAVCR1/TIM1 IgV and not to the Fc tail, the transfectants were first treated with excess PVR-Fc, which contains the same Fc tail as does HAVCR1/TIM1 (IgV)-Fc, and then panned to Petri dishes coated with HAVCR1/TIM1 (IgV)-Fc. We further depleted transfected cells expressing the FcγRII receptor (CD32) by magnetically depleting cells that stained with anti-human CD32 mAb. After this enrichment process, 20 transfected PerroβD cell clones were isolated and analyzed for the expression of HAVCR1/T1M1 ligands (Fig. 2). The Perro6D cell clones were tested by panning on coated Petri dishes and binding to colored latex beads coated with soluble receptors. The panning and latex bead assays gave similar results. Ten of the transfected PerroβD cell clones expressed CD32 and bound HAVCR1/TIM1 (IgV)-Fc and PVR-Fc (clone PerroθD- CD32 FcγRII is an example). Nine of the clones expressed neither CD32 nor bound soluble receptors (PerroθD-Clone D1 is an example). However, one clone, termed PerroβD-Clone 1 , specifically bound to HAVC R 1 /Tl M 1 (IgV)-Fc but not to PVR-Fc, did not express CD32, and expressed a ligand of HAVCR 1 /Tl M 1. Example 2: Human IgAiλ is expressed at the cell surface of PerroθD-Clone 1 cells

[0064] To identify the HAVC R 1 /T I M 1 ligand in the Perro6D-Clone 1 cells, plasmids were rescued from PerroβD-Clone 1 cells and used to transform E. coli A (12). Nucleotide sequence analysis revealed that the rescued plasmids encoded full-length human immunoglobulin heavy α1 and lightλ chains. A cell surface ELISA (Fig. 3) showed that PerroδD-Clone 1 cells expressed human IgAI and Igλ at the cell surface..^" PerroβD cells did not react with the mAbs raised against human IgAI or Igλ. The dog cells, Perro6D-Clone 1 and PerroβD cells, did not react with mAbs raised against human lgκ or IgM. As expected, negative control anti-human integrin and anti-FLAG mAbs did not stain the dog cells. These results confirmed that PerroδD-Clone 1 cells expressed human IgAI λ at the cell surface.

Example 3: HAVCR1/TIM1 binds specifically to IgAiλexpressed at the cell surface of PerroθD-Clone 1 cells

[0065] To assess the specificity of the binding of HAVCR1/TIM1 to IgAI λ, we blocked the binding of HAVCR1/TIM1 (IgV)-Fc coated beads to PerroβD-Clone 1 cells was blocked with excess of different human immunoglobulins. This experiment showed that myeloma IgAI λ and secretory IgA (slgA), a purified commercially available form of IgA that contains Igλand lgκ light chains, completely blocked binding of HAVC R 1 /Tl M 1 (IgV)-Fc beads to PerroθD-Clone 1 cells whereas IgMλ did not affect binding of the beads (Fig. 4A). Human myeloma Igλ did not block binding of the HAVCR1/TIM1 (IgV)-Fc beads to PerroβD-Clone 1 cells suggesting that the interaction of HAVCR1/TIM1 with IgA requires both the heavy and light immunoglobulin chains. Anti-human IgAI and Igλ blocked approximately 50% and 75% of the binding of HAVCR1/TIM1 (IgV)-Fc coated beads to PerroβD-Clone 1 cells, respectively, whereas negative control mAbs had no effect. Finally, flow cytometry analysis showed that anti-HAVCR1/TIM1 mAb 3D1 raised against the Ig- like region of HAVCR1/TIM1 almost completely blocked binding of RPE-labeled HAVCR1/TIM1 (IgV)-Fc and HAVCR1/TIM1 (lgV+muc)-Fc to PerroβD-Clone 1 cells (Fig. 4B), which indicated that the 3D1 epitope is involved in the interaction of HAVCR1/TIM1 with^" IgAλ. Taken together, these experiments proved that IgAλ binds specifically to the Ig-like domain of H AVC R 1 /T I Ml

Example 4: Human IgAI λ alone is sufficient for binding to HAVCR1/TIM1 [0066] To test whether IgAI λ expressed in the Perro6D-Clone 1 cells was sufficient for binding to HAVCR1/TIM1 and that no additional molecules from human or dog origin were required for the interaction, PerroδD cells were co-transfected with the plasmids coding for Igα1 and Igλ rescued from Perro6D-Clone 1 cells. Cell transfectants were selected with2 μg/ml puromycin and termed PerroδD- IgAI λ. To increase the expression of IgA, cells were grown in 20 μg/ml puromycin. A capture ELISA (Fig.5A) showed that Perro6D-lgA1λ and Perro6D-Clone 1 cells but not PerroδDcells secreted IgAI λ into the cell culture media. However, a cell surface ELISA (Fig. 5) showed that Perro6D-Clone 1 but not Perro6D- IgAI λ expressed IgAI λ at the cell surface..^"These results indicated that Perro6D-Clone 1 cells contain an IgA binding molecule at the cell surface not present in the majority of the PerroδD cells. This unexpected characteristic of binding of IgAI λ to the cell surface allowed us to select the PerroβD-Clone 1 cells by panning to Petri dishes coated with HAVCR 1/TIMI (IgV)-Fc.

[0067] Since our results showed that IgAI λ bound to the cell surface of PerroδD- Clone 1 cells but not to the cell surface of PerroδD-lgAiλ.it was of interest to determine whether IgAiλ was sufficient for binding to HAVCR1/TIM1. In vitro binding ELISA was used to analyze binding of IgA to HAVCR1/TIM1 (IgV)-Fc captured on ELISA plates coated with goat anti-human IgG Fc (Fig. 6). IgAI λ sercreted by Perro6D-lgA1λ and PerroδD-Clone 1 cells bound to HAVCR1/TIM1 (IgV)-Fc but not to control PVR-Fc. Similarly, human slgA and myeloma IgAλ bound to HAVCR1/TIM1 (IgV)-Fc but not to PVR-Fc. These data indicated that IgAI λ is sufficient for binding to HAVCR1/TIM1 and further confirmed that IgAλ is indeed a natural ligand of this receptor.

Example 5: Binding of IgA to HAVCR1/TIM1 does not block HAV infection [0068] Since our data showed that HAVCR1/TIM1 binds IgA, it was of great interest to determine whether IgA could block the interaction of HAV with H AVCR 1 /Tl M 1. HAV infects GL37 cells via HAVCR1/TIM1 (14). Therefore, this cell substrate was used for the experiment. It should be pointed out that the monkey HAVCR1/TIM1 (mkHAVCR1/TIM1 ) expressed in GL37 cells shares a high degree of homology with human HAVCR1/TIM1 (6) and also binds IgAλ. HAV (10⁵ TCID₅₀) was mixed with 10 μg of human slgA or control IgM, incubated at 37⁰C for 2 h, and titrated on 96-well plates containing monolayers of GL37 (Fig. 7). After 10 days incubation at 35⁰C, cells were fixed with methanol and stained with anti-HAV antibodies (42). Similar viral titers were obtained in the presence or absence of human slgA or control IgM indicating that presence of IgA did not affect infection of GL37 cells. This experiment also showed that our preparation of slgA did not contain anti-HAV antibodies. A one-step growth curve of HAV infection also showed that slgA did not affect infection of GL37 cells.

Example 6: Human IgA enhances the virus-receptor interaction [0069] The finding that IgA did not block HAV infection in GL37 cells suggested that IgA and HAV may bind to different epitopes on HAVCR1/TIM1. Since HAV grows poorly in cell culture and tends to bind unspecifically to cells and surfaces, it is extremely difficult to perform binding assays and almost impossible to quantitate meaningful results. Therefore, a soluble receptor neutralization assay that mimics the initial steps in HAV infection (39, 40) was used to further characterize the interaction of HAV with HAVCR1/TIM1 in the presence of IgA. mkHAVCRI /TIMI(IgV)-Fc (Fig. 1) neutralizes approximately 0.5 log of HAV infectivity by blocking receptor-binding sites on the viral particles whereas mkHAVCR1/T!M1 (lgV+mucin)-Fc (Fig. 1), which contains the 27 repeats of the mucin domain, neutralizes up to 1.5-2 log of HAV by altering the viral particles (40). The effect of IgA in the neutralization of HAV by mkHAVCR1/TIM1 (IgV)-Fc was tested (Fig. 8A). As expected, 40 μg of mkHAVCR1/TIM1 (IgV)-Fc neutralized approximately 0.5 log HAV compared to control PVR-Fc (P<0.0001). Treatment with 5 μg of IgG did not affect the HAV titers, which were similar to the titers obtained in the absence of IgG (None). However, in the presence of 5 μg slgA, mkHAVCR1/TIM1 (IgV)-Fc neutralized approximately 1 log of HAV, which was significantly higher than in the presence of control IgG (P<0.0001). HAV neutralization by mkHAVCR1/TIM1(lgV+mucin)-Fc in the presence of slgA was then tested (Fig. 8B). A suboptimal non-saturating concentration of 4 μg mkHAVCR1/TIM1 (lgV+mucin)-Fc within the linear range of neutralization (40) was used, which resulted in an expected reduction in the HAV titer of 0.5 log compared to PVR-Fc (P< 0.0001). Addition of 5 μg slgA increased significantly the neutralization of HAV to approximately 1.5 log compared to neutralization in the absence of IgA (P< 0.0001). These significant increases in the receptor-mediated neutralization of HAV clearly show that IgA enhanced the interaction of HAV with H AVC R 1 /T I M 1.

Example 7: HAVCR1/TIM1 binds preferentially to monocytes [0070] To study the effects of HAVCR 1 /Tl M 1 binding to IgA on human cells, human PBMC were stained with HAVC R 1 /T I M 1 (IgV)-Fc or with IgGI isotype control. Fig. 9A shows that monocytes were the predominant cell type that bound HAVC R 1 /Tl M 1 (IgV)-Fc, based on their characteristic size and granularity. Elutriated monocytes expressed surface IgA and FcαRI at the cell , the only IgA receptor on monocytes (Fig. 9B). The specificity of binding of HAVCR1/TIM1-Fc to the elutriated monocytes was examined (Fig. 9C). Goat anti-lgA antibody but not a control goat anti-rabbit antibody reduced the binding of HAVCR1/TIM1 (IgV)-Fc to monocytes by approximately 10-fold. Anti-HAVCR1/TIM mAb almost completely inhibited binding of HAVCR1/TIM1 (IgV)-Fc. It should be pointed out that an anti-lgA1 mAb minimally blocked binding of HAVCR1 /TIMI(IgV)-Fc to elutriated monocytes, which is consistent with our previous observation that anti-lgA1 mAb is not a good blocker and only partially prevented binding of beads coated with HAVCR1 /TIMI(IgV)-Fc to PerroθD-Clone 1 cells (Fig. 4B). These results clearly indicated that IgA is a ligand for HAVCR1 /TIM 1 (IgV)-Fc at the cell surface of the monocytes. To verify that soluble receptor forms of HAVCR1/TIM1 containing the mucin region also bound to monocytes, similar studies were performed with HAVCR1/TIM1(lgV+muc)-Fc, a construct containing the whole ectodomain of HAVCR1/TIM1 (Fig. 1). Binding of HAVCR1/TIM1 (lgV+muc)-Fc to monocytes was reduced with excess HAVCR1/TIM1 (IgV)-Fc by approximately 10-fold, and was minimally blocked by treatment with control PVR-Fc (Fig. 9D). Treatment with anti-HAVCR1/TIM1 mAb blocked binding of HAVCR1/TIM1(lgV+muc)-Fc to monocytes. These results indicated that HAVCR1/TIM1(lgV+muc)-Fc interacted with the monocytes primarily via its Ig-like domain, since HAVC R 1 /Tl M 1 (IgV)-Fc and anti-HAVCR1/TIM1 mAb 3D1 significantly blocked its binding to the monocytes.

Example 8: HAVCR1/TIM1 activates monocytes via binding to IgA [0071] To determine the immunological effects of the interaction of HAVCR1/TIM1 with IgA on monocyes, we examined the activation of monocytes with HAVCR1/TIM1 (IgV)-Fc was examined using a metabolic colorimetric assay based on the cleavage of the tetrazolium salt WST-1 to formazan by the succinate- tetrazolium reductase system in the mitochondria of metabolically active cells (Mosmann, 1983). Since our previous results showed that HAVCR1/TIM1 interacts with IgA primarily via its Ig-like region, the HAVCR1/TIM1 (IgV)-Fc construct was used to avoid confounding factors introduced by mucin-mucin interactions. Elutriated monocytes were treated with HAVCR1/TIM1 (IgV)-Fc, control PVR-Fc, or LPS in polypropylene tubes for 1-4 days. At different times after treatment, aliquots of the monocytes were stained with the WST-1 salt for 4 h and levels of the resulting formazan were quantitated by absorbance at 450nm in an ELISA plate reader (Fig. 10A). HAVCR1/TIM1 (IgV)-Fc induced peak activation 2 days post-treatment that was significantly higher (approximately 3-fold) than in monocytes treated with media or PVR-Fc- (P<0.0001) and 0.5-fold higher than in monocytes treated with LPS. An analysis of cell size and granularity of the cells two days after treatment with soluble receptors (Fig. 10B) showed that treatment with HAVC R 1 /T I M 1 (IgV)-Fc clearly activated the monocytes, whereas treatment with media or PVR-Fc did not, consistent with the WST-1 staining data. The activation of the monocytes with HAVCR1/TIM1 (IgV)-Fc was dependent on the interaction of HAVCR1/TIM1 with IgA, since anti-HAVCR1/TIM1 mAb or anti-lgA1 mAb completely and specifically blocked the monocyte activation (Fig. 10C). These results clearly showed that the interaction of HAVCR1/TIM1 (IgV)-Fc with IgA was responsible for the activation of the monocytes. Example 9: Monocytes treated with HAVCR1/TIM1 (IgV)-Fc increased the expression of cell surface activation markers and secreted cytokines [0072] The activation of the monocytes was accompanied by a distinctive pattern of expression of cell surface markers as shown by flow cytometry analysis using mAbs (Fig. 11A). Expression of CD40, CD14, and CD1 1c increased on monocytes activated with HAVCR1/TIM1 (IgV)-Fc or LPS compared to the expression of the same markers on monocytes treated with media or PVR-Fc. Expression of Class Il and CD86 increased approximately 10-fold in a small subpopulation of monocytes activated with HAVCR1/TIM1 (IgV)-Fc. Expression of CD80 and CD83 on monocytes did not increase with any of the treatments.

[0073] Analysis of cytokine expression by a quantitative ELISA (Fig. 11B) showed that the monocytes activated with HAVCR1/TIM1 (IgV)-Fc secreted higher levels of IL-6, IL-10, and TNF-α than did monocytes treated with media or PVR-Fc. This cytokine expression profile is consistent with the increased expression of activation markers at the cell surface of the monocytes and supports our finding that HAVCR1/TIM1 (IgV)-Fc activated monocytes.

Example 10: Mutation of the CC loop of HAVCR1/TIM1 eliminates IgAλ binding

The structure of the TIM family members predicted that the interaction of the CC with FG loops modulates the accessibility of the FG-loop and conformation of the BC-loop. To test this prediction, the CC'-loop of TIM1 was mutated by swapping amino acids SLFT found between the 3^rd and 4^th Cys residues of TIM1 for amino acids residues PYSG found in the same positions of human TIM4. Binding of IgA to the mutated TIM1 was then assessed.

The TIM1 mutant was prepared by overlapping PCR using the mutagenic oligonucleotide 5'-TGTCCCTACTCCGGTTGCCAAAATGGCATTGTCTGGACC-3¹. The resulting PCR fragment was cloned into the cDNA of TIMI-Fc, and the resulting mutant was termed TIM1 (Cys3-4)-Fc. The sequence of the mutant was verified by automatic nucleotide sequence analysis. CHO dhfr- cells were cotransfected with the TIM1 (Cys3-4)-Fc plasmid and a plasmid coding for the DHFR gene. CHO cell transfectants were selected in Iscove's media, and the expression of TIM1 (Cys3-4)- Fc was optimized with increasing concentrations of methotrexate. The TIM1 (Cys3- 4)-Fc protein was purified from the supernatants of the CHO transfectants using chromatography in protein A columns. A capture ELISA was used to test binding of monoclonal antibodies to HAVCR1/TIM1 (lgV+muc)-Fc or the cys 3-4 mutant that were expressed in transiently transfected cells. Peroxidase-labeled anti-mouse antibody was used as the secondary antibody for the ELISA. The attached table shows the reactivity of 3 anti-HAVCR1/TIM1 monoclonal antibodies (++++ is the maximum binding and - is nonreactive). M2 is an control anti-FLAG Mab which epitope was included in the mutant construct (the epitope is not present in the wt). MAbs 3D1 and 5E8 react with the region between the Cys 3-4 whereas mAb 1 D12 reacts against another epitope because the mutant and the wt are both positive. The 3 mAbs (3D1 , 1 D12, and 5E8) inhibit binding of IgA to HAVCR1/TIM1 as determined by FACS analysis.

TIMI-Fc or TIM1(Cys3-4)-Fc (1 μg/ml) were captured on 96-well plates (Nunc, Inc.) coated with 1 μg/ml goat anti-human Fc. Human secretory IgA (starting at 1 μg/ml) was titrated on the plates and stained with peroxidase-labeled anti-human IgA and One-Component TMB. Absorbance at 450 nm was determined in an ELISA plate reader. This in vitro binding assay clearly showed that TIM1(Cys3-4)-Fc did not bind IgA.

Table 1

Example 11 : Monocytes activated with HAVCR1 /TIMI(IgV)-Fc induce T cell proliferation

Mixed lymphocyte reactions (MLRs) were used to evaluate the effector function of monocytes activated with HAVCR1/TIM1 (IgV)-Fc. MLRs were performed using monocytes treated with HAVCR1/TIM1 (IgV)-Fc, PVR-Fc, or media and cultured with purified allogeneic T cells from normal human blood donors (Fig. 12). Elutriated monocytes in polypropylene tubes were treated with media, HAVCR1/TIM1 (IgV)-Fc, PVR-Fc, or LPS for 2 days as described above in the activation procedure, and then plated at a density of 2 x 10⁴ cells/well in 96-well cell culture plates (Corning). Allogeneic human T cells (2 x 10⁵ cells/well) purified from total lymphocytes using a T cells negative isolation kit (Dynal Biotech, inc.), were added to the 96-well plates containing the treated monocytes. After 6 day incubation, culture supernatants were removed and plates were pulsed for 16-18 h with 1 μCi [³H]thymidine per well. The incorporated radioactivity was measured in a Beta Plate Scintillation Counter (Perkin Elmer Wallac). Data are presented as the average counts per minute (c.p.m.) of triplicate wells.

T cells cultured with monocytes treated with HAVCR1/TIM1 (IgV)-Fc incorporated approximately 5- and 8-fold more [³H]thymidine than those treated with media or PVR-Fc, respectively (Fig. 12a) (P<0.001). As expected, T cells cultured without monocytes incorporated minimal levels of radioactivity. T cells treated with PHA as a positive control incorporated almost twice the amount of [³H]thymidine than T cells cultured with monocytes activated with HAVCR1 /Tl M 1 (IgV)-Fc. T cell proliferation was also visualized by microscopic examination of the MLR cell cultures previous to the addition of [³H]thymidine. Few small clusters of T cells were observed in wells containing monocytes treated with media (Fig. 12b) or PVR-Fc (Fig. 12d) whereas an increased number of T cell clusters of larger size were observed in wells containing monocytes treated with HAVCR1/TIM1 (IgV)-Fc (Fig. 12c). Similar MLR results were obtained regardless of whether HAVCR1/TIM1 (lgV)- Fc was present or washed out before the monocytes were cultured with the T cells. T cells alone cultured with HAVCR1/TIM1 (IgV)-Fc did not proliferate. These data clearly indicate that the monocytes activated with HAVCR1/TIM1 (IgV)-Fc are capable of inducing T cell proliferation. References

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Claims

Claims:What is claimed is:

1. A method for activating or increasing the activation of antigen presenting cells (APCs) and/or myeloid cells using membrane bound, particulated, or soluble forms HAVCR1/TΪM1 or peptides derived from this receptor to activate said cells.

2. A method for activating or increasing the activation of cells expressing FcαRI or CD89 comprising a) providing cells having IgA bound to the FcαRI at their surface; and b) contacting said IgA bound to said cells with membrane bound, particulated, or soluble forms of HAVCR1/TIM1 to activate said cells.

3. The method of claim 1 , wherein said cells are antigen presenting cells (APCs) and/or myeloid cells selected from the group consisting of neutrophils, basophils, eosinophils, monocytes, macrophages, dendritic cells, and Kupffer cells, and/or mast cells.

4. A method of increasing activation of APCs and/or myeloid cells expressing FcαRI (CD89) or other IgA receptors comprising a) providing APCs and/or myeloid cells having IgA bound to said FcαRI or other IgA receptors; and b) contacting said IgA bound to said APCs and/or myeloid cells with HAVCR1/TIM1 to activate said cells.

5. The method of claim 4, wherein said IgA is IgAI λ.

6. The method of claim 4, wherein said activated APCs and/or myeloid cells exhibit increased secretion of at least one cytokine selected from the group consisting of IL-6, IL-10 and TNF-α.

7. The method of claim 4, wherein said activated APCs and/or myeloid cells exhibits increased expression of CD86 or of MHC class Il molecules.

8. A method for treating an atopic disease comprising administering an effective amount of membrane bound, particulated, or soluble forms of IgAλ, or a fragment thereof, or a peptide derived from IgAλ, to an individual in need thereof.

9. The method of claim 8, wherein said atopic disease is asthma.

10. The method of claim 8, wherein said individual has an allele of an HAVCR1/TIM1 gene that confers susceptibility to allergy and atopy and a fragment of IgAλ, or a peptide derived from IgAλ that inhibits the binding of IgAλ to HAVCR1/TIM1 is administered.

11. A method for screening for compounds that inhibit the interaction of HAVCR1/TIM1 with IgAλ comprising contacting a cell expressing HAVCR1/TIM1, or a portion thereof that binds IgAλ, on its surface, or a substrate having HAVCR1/TIM1 , or a portion thereof that binds IgAλ attached thereto, with said compound, and measuring the binding of IgAλ, or a fragment thereof, to said cell or substrate.

12. A method for screening for compounds that inhibit the interaction of HAVC R 1 /Tl M 1 with IgAλ comprising contacting a cell expressing IgAλ, or a fragment thereof that binds HAV CR1/TIM1 on its surface, or an insoluble substrate having IgAλ, or a fragment thereof that binds HAVCR1/TIM1 attached thereto, with said compound, and measuring the binding of HAVCR 1 /Tl M1, or a fragment thereof that binds IgAλ, to said cell or substrate.

13. An antibody that specifically binds to HAVCR 1 /Tl M 1.

14. The antibody of claim 13 that is the 3D1 antibody.

15. The antibody of claim 13 that inhibits binding of IgAλ to an epitope comprising the portion of HAVCR 1 /Tl M 1 between cysteines 3 and 4.

16. The antibody of claim 13 that specifically binds the IgV domain of HAVCR1/TIM1.

17. A fragment and/or mutant of HAVCR1/TIM1 that binds to IgAλ at the cell surface of APCs and prevents activation of said APCs.

18. A fragment and/or mutant of HAVCR1/TIM1 that binds to IgAλat the cell surface of eosinophils, basophils, neutrophils, and mast cells and prevents crosslinking of CD89, degranulation, and anaphylactic response.

19. A method to enhance infectivity of HAV in cell culture for vaccine production comprising adding IgAλ or fragments of IgAλ that bind to HAVCR1/TIM1 to a culture of cells and HAV.

20. A polypeptide comprising a fusion of IgAλi or a fragment thereof that binds to HAVCR1/TIM1 and prevents HAV cell entry and infection.

21. A method for activating APCs and/or myeloid cells to prevent or treat an autoimmune disease comprising administering an effective amount of membrane bound, particulated, or soluble forms of H AVC R 1 /T I M 1 , or a fragment thereof, or a peptide derived from HAVCR1/TIM1 , to an individual in need thereof.

22. A method for activating APCs and/or myeloid cells to prevent or treat a pathogen infection administering an effective amount of membrane bound, particulated, or soluble forms of HAVCR1/TIM1, or a fragment thereof, or a peptide derived from H AVC R 1 /Tl M 1 , to an individual in need thereof.

23. A method for screening compounds for activity of inducing anergy in response to an antigen comprising: a) contacting an antigen presenting cell (APC) or myeloid cell cultured in a context such that contact with HAVCR1/TIM1 produces an anergic response with said compound, b) contacting said cultured APC or myeloid cell with HAVCR1/TIM1 or with a fragment of HAVCR1/TIM1 or a peptide derived therefrom; c) measuring at least one activity of said APC or myeloid cell selected from the group consisting of increased cellular size and complexity as measured by forward and side scattering (FSC and SSC) FACS analysis; increased secretion of at least one cytokine selected from the group consisting of IL-6, IL-10 and TNF-α; expression of at least one cell surface marker selected from the group consisting of CD40, CD14, CD11c, CD86 or MHC class Il molecules; and proliferation of lymphocytes contacted with said APC or myeloid cell; d) wherein finding an inhibition of said at least one activity by said compound identifies said compound as having activity of inducing anergy.

24. A method for screening for compounds that promote the interaction of HAVC R 1 /Tl M 1 with IgAλ comprising contacting a cell expressing

H AVC R 1 /Tl M 1, or a portion thereof that binds IgAλ, on its surface, or a substrate having HAVCR1/TIM1 , or a portion thereof that binds IgAλ attached thereto, with said compound, and measuring the binding of IgAλ, or a fragment thereof, to said cell or substrate.

25. A method for screening for compounds that promote the interaction of HAVC R 1 /Tl M 1 with IgAλ comprising contacting a cell expressing IgAλ, or a fragment thereof that binds HAV CR1/TIM1 on its surface, or an insoluble substrate having IgAλ, or a fragment thereof that binds

HAVC R 1 /Tl M 1 attached thereto, with said compound, and measuring the binding of H AVC R 1 /Tl M 1, or a fragment thereof that binds IgAλ, to said cell or substrate.