SKIN-ASSOCIATED LYMPHOCYTES AND MARKERS

Abstract: Regulation of hematopoietic cell association with skin during chronic inflammation may be modulated by the use of agents which selectively identify and target skin associated leukocytes or inhibit the binding of a skin associated antigen (CLAM-1) with receptors present in cutaneous associated cells. The agents may be used for diagnosis of inflammations and malignancies associated with the presence of the CLAM-1 carrying hematopoietic cells at cutaneous sites. By employing lymphocytes or other antibodies or cytotoxic agents, inflammatory disorders mediated by cutaneous lymphocytes or other T-cells or skin associated T-cell malignancies may be treated. Cutaneous inflammation and lymphoid neoplasia can be characterized and modified by the agents which selectively identify and target skin-associated T-cells or which inhibit the binding of CLAM-1 with receptor(s) present in cutaneous associated cells.

Description: SKIN-ASSOCIATED LYMPHOCYTES AND MARKERS CROSS-REFERENCE TO RELATED APPLICATIONS This Application is a continuation-in-part of Application Serial No. 539,844 filed June 18, 1990. INTRODUCTION Technical Field The field of this invention concerns lymphocytic cells and markers associated with such cells. Background For animals, the hematopoietic system is a major line of defense against various traumas, such as injury, pathogens, neoplasia, and the like. The hematopoietic system is being intensively investigated today in order to understand how various cells respond to the numerous challenges which the body meets. As the hematopoietic system is studied, it is found that the various lineages of the hematopoietic system, lymphoid, myeloid and erythroid, comprise subsets which may be further divided into smaller subsets associated with unique functions. As an example, the lymphoid lineage is divided into B- and T-cells, where the T-cells are further divided into CD4 and CD8 cells, frequently referred to as helper cells and cytotoxic/suppressor cells. The location of the lymphoid cells in the body is found to be subject to substantial regulation. Referred to as "homing," it is found that lymphoid cells will have different surface markers depending upon the location of the cells and the tissue to which they bind or have an affinity. Thus, it is found that some lymphocytes will be directed to peripheral lymph nodes, while others will be directed to Peyer's patches associated with mucosal tissue, particularly in the gut. In many situations, one may wish to modulate the homing of hematopoietic cells, and hence the local representation of subsets of such cells, either by enhancing the number of cells directed to a particular site, reducing the number or preventing cells from being directed to that site, or in activating cells at the site. There is, therefore, substantial interest in understanding the manner in which cells are regulated to direct them to a particular site, the particular mechanism by which products are produced associated with such directing of cells, identification of the products associated with such direction, or induced or otherwise associated with the localization of cells. By understanding the regulation of trafficking of the cells, one may then be in a position to allow for the control and redirection of such trafficking. By identifying lymphocyte markers unique to lymphocytes that home to or are found in certain tissues or microenvironments of the body, one may be in a position to identify, target or modulate the function of lymphocyte subsets responsible for tissue-specific immune responses, for example, in the gut, skin or joints. Relevant Literature The HECA-452 monoclonal antibody was originally described as a marker of high endothelial differentiation that also reacted with a poorly characterized monocytoid cell population in tissue sections. Duijvestijn et al. , Am. J. Pathol. (1988), 130:147-155. Normal skin has been shown to have characteristic lymphoid components (Skin- Associated Lymphoid Tissue or SALT) by Smeilein, J. Invest. Dermatol. (1983), 8):125-165; rueger and Stingel, J. Invest. Dermatol. (1989), £2:325-575; and Bos. et al. , J. Invest. Dermatol. (1987), 88.J569-573. The enhanced ability of some lymphoid populations to migrate to cutaneous inflammatory lesions is described by Rose et al. , Cell. Immunol. (1976), 22:36-46; Issekutz et al.. ibid (1980), 5_4:79-86 and Issekutz et al. , Cell Immunol. (1986), 5jB:87-94. The propensity for certain T-cell malignancies to remain localized to skin has been reported by Abel, Dermatol. Clinics (1985) 3.:647; Edelson, Am. Acad. Dermatol. (1980), £ :89-106 and Miller et al. , New Encfl. J. Med. (1980) 303:89-92. van Der Valk et al. , Am. J. Surσ. Pathol. (1989), 12:97-106 report that HECA-452 monoclonal antibody reacted with PMNs and myeloid precursors in sections of bone marrow. SUMMARY OF THE INVENTION Methods and compositions are provided for diagnosis and therapy associated with skin-associated lymphocytic cells, as a result of lymphoid infiltration of inflam¬ matory lesions, malignancy or other conditions associated with lymphoid trafficking to skin. Specifically, identification and/or isolation of skin-associated or skin-homing lymphocyte subsets defined by CLAM-1, modifying the regulation of CLAM-1 or trafficking of cells associated with the CLAM-1 carbohydrate and/or protein determinants, or targeting of immunomodulatory or toxic agents to CLAM-1 + cells, can be used in the diagnosis and treatment of a variety of conditions associated with skin. Antibodies or receptors to CLAM-1 can be used to identify, isolate, and/or characterize CLAM-1 + cells for diagnostic purposes, or to deplete or modulate the function or localization of CLAM-1 expressing cells for therapeutic purposes. DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, skin homing or skin-associated leukocytes, particularly lymphocytes are identified, by detection of CLAM-1 expression, in blood, skin, or other sites, and characterized for diagnostic purposes. In addition, leukocytic involvement with the skin is modulated for prophylaxis and therapy. Particularly, by modulating the cutaneous interaction of leukocytes with cutaneous tissue, e.g. distribution, number, or functional properties of skin-associated leukocytic cells, particularly as to the binding of cutaneous receptors, one can diagnose or treat various conditions associated with leukocytic-skin involvement. Cell surface CLAM-1 or cross-reactive determinants thereof are characterized by binding to the monoclonal antibody HECA-452, by being present on -10-30% of circulating peripheral blood T-cells, 80-90% of T-cells infiltrating diverse chronic inflammatory lesions of the skin, but on <15% of T cells in most other sites of chronic inflammation, by being associated with an adhesion molecule 1 CD45R ° putative memory lymphocyte subset. CLAM-1 is also on myeloid cells, e.g., monocytes and neutrophils. CLAM-1 includes protein species characterized in addition by being highly glycosylated and by having a prominent component of molecular weight -200 kD on lymphocytes. The HECA-452 epitope of CLAM-1, as well as the ELAM-1 binding determinant, are sensitive to mild periodate treatment and to neuraminidase digestion, indicating that a carbohydrate component containing sialic acid or other neuraminidase sensitive sugars is a central defining feature of CLAM-1. CLAM-1 may also include non- protein glycosylated species, such as glycolipids, defined by HECA-452 and/or cutaneous associated receptors, e.g. ELAM-1 binding on lymphocytes, and by the cellular distribution on lymphocytes described above. Lymphocytes carrying CLAM-1 antigen are associated with cutaneous locations. Some or all of CLAM-1 is found to be a lymphocyte ligand for endothelial cell leukocyte adhesion molecule-1 (ELAM-1) , an activity that is sensitive to prior treatment of CLAM-1 with neuraminidase or periodate and that requires the presence of calcium ion. As used in this application, CLAM-1 on lymphocytes includes lymphocyte ELAM-1 ligands, cross-reactive with CLAM-1, whether recognized by HECA-452 or not, in that ELAM-1 is a vascular addressin for skin homing T cells and can be used as a soluble agent to identify or target CLAM- 1+ skin-associated or skin-homing lymphocytes. A number of proteins of different molecular weight are cross-reactive with CLAM-1 from tonsil lymphocytes, and, in particular, display the same or closely related neuraminidase-sensitive, HECA-452 and ELAM-1 binding carbohydrate determinants. These protein analogues found on other cells are analogous ligands and find analogous applications, as do glycolipids, defined carbohydrates or other analogues that exist or may be prepared. CLAM-1 protein may be obtained in its own unglycosylated form or partially glycosylated. It may be derived from any mammalian source, including murine, equine, ovine, feline, canine, bovine, primate, particularly human, etc. The CLAM-1 proteins (or ligand comprising the HECA-452 epitope) may be obtained in a variety of ways. The ligand may be obtained by lysing leukocytes, particularly lymphocytes, more particularly those associated with cutaneous sites, including neoplastic cells, such as cells associated with mycosis fungoides or other lymphoma associated with the skin and carrying the subject antigen. The lysate may be passed through an affinity column comprising the HECA-452 mAb and the captured ligand eluted in accordance with conventional techniques. If desired, by employing appropriate saccharidases, the core protein may be separated from the carbohydrate group, or by employing appropriate proteases, the carbohydrate may be separated from the core protein. If the CLAM-1 protein is to be prepared by recom- binant techniques, various means are available for isolating the gene for insertion into an appropriate expression vector. A cDNA library may be prepared from cells expressing the CLAM-1 antigen and the library subtracted with cDNA from a lymphocyte which does not express CLAM-1. The remaining genes after subtracting complementary cDNAs from the two types of cells, may then be screened for expression in a mammalian cell host and the resulting transformants screened with antibody specific for CLAM-1. Those cells positive for the CLAM-1 antibody may then be used to isolate the plasmid-encoded cDNA that encodes CLAM-1. The cDNA can be used for screening a genomic library for the gene expressing CLAM-1. To establish the identity of the recombinant protein and the natural protein, one may partially sequence the gene or the expression product of the gene and compare the recombinant sequence to the sequence determined from the protein that was isolated with the CLAM-1 antibody. The protein may be produced in accordance with conventional techniques growing CLAM-1 lymphocytes, lysing the cells, freeing a supernatant, and then purified to substantial purity (>95%) , e.g., using an affinity column with HECA-452. Alternatively, one may insert cDNA downstream from a gene, such as beta-galactosidase under the transcriptional control of a promotor functional in E. coli or other prokaryote, whereby fused proteins will be obtained. One may then screen the fused proteins for binding to CLAM-1 mAb. Those cells which produce protein which bind to the CLAM-1 mAb are used as a source for a DNA probe for identifying cDNA or genomic DNA in appropriate libraries of CLAM-l antigen positive cells or genomic human cell libraries. The carbohydrate group(s) associated with CLAM-l may be readily identified by cleaving the group using an appropriate endoglycosidase or beta-elimination. The resulting carbohydrate group is then analyzed by MS-FTIR and NMR for structure determination in accordance with known tree structures. Methods for enzymatically or chemically cleaving specific linkages are also available to identify the monomers of the carbohydrate. Alternatively, carbohydrate component(s) can be identified by comparing the reactivity of HECA-452 and/or ELAM-1 with known, previously characterized carbohydrate structures. It should be noted that certain cells which express high levels of the HECA-452-defined CLAM-l epitope, particularly neutrqphils, are characterized by abundant expression of αl,3-fucosylated polylactoseamine structures, including CD15 (Lewis Blood Group x) . Since the carbohydrate group of CLAM-l defined by monoclonal antibody HECA-452 and ELAM-1 recognition comprises sialic acid or other neuraminidase sensitive sugar components, one may screen sialylated structures related to CD15, i.e., sialyl Lewis x and related carbohydrates. The pattern of glycosyl markers varies with CLAM-l leukocytes. One may therefore separate or assay for the types of CLAM-l leukocytes by determining the glycosylation pattern using an anti-CLAM-1 antibody and an antibody to another glycosylation marker, e.g., Le . The carbohydrate may be obtained by cloning and expression of the appropriate glycosyl transferases which synthesize the glycosyl side chain. The glycosyl group can then be bound to or expressed on a glycolipid, e.g., diacylglycerol, which can be isolated to obtain the CLAM-l carbohydrate(s) . The glycoprotein may be further manipulated to provide for a soluble form, such as deleting the trans¬ membrane integrator sequence in accordance with conven¬ tional ways. These techniques include using the poly- merase chain reaction, employing appropriate primers, which delete all or a major portion of the transmembrane integrator sequence, in vitro mutagenesis, primer repair, or the like. Thus, one may prepare a truncated gene which expresses only the extracellular portion of the molecule. If desired, one may replace the transmembrane integrator sequence with the signal for linkage to a lipid for binding to the membrane, so as to allow for proper glycosylation and processing of the extracellular domain of the skin-associated antigen. After production of the surface membrane protein bound to the membrane of the cells, the soluble portion may be readily obtained by hydrolysis of the linkage to the lipid. Alternatively, fragments may be obtained which are able to compete with CLAM-l or the determinant thereof for binding to receptors for the cutaneous lymphocytes. Thus, CLAM-l may be prepared as fragments, with or without the presence of sugar side chains, by preparing the whole protein or portion thereof and by further degradation with protease, cyanogen bromide, or the like. With appropriate saccharidases or mild acidic hydrolysis all or fragments of the sugar side chain may be obtained. Alternatively, carbohydrate components of CLAM-l can be synthesized by conventional techniques or isolated from other tissue or fluid sources containing such component. These carbohydrate components may be screened by their ability to bind to ELAM-1 or comparable receptor. Soluble forms of the skin-associated antigen, fragments thereof, or synthetic analog of such fragments may be used to prevent binding of the lymphocytes to cutaneous vascular and endothelial, or other cutaneous cells, where it is desirable to prevent the infiltration of the lymphocytes into a cutaneous region. The soluble protein, fragments or analogs thereof, or carbohydrates may also be used for directing various agents to a cutaneous site to which CLAM-l antigen binds. Thus, one may direct a wide variety of agents to the site where ELAM-1 or other receptors for CLAM-l are present. Agents may include labels which allow for detection of such sites, such as radioactive labels, ligands associated with the up- or down-regulation of the receptor(s) or other functional components of the target tissue, cytotoxic agents, e.g., antibiotics, liposomes, which may be carriers of a wide variety of agents, including ligands, cytotoxic agents, or the like. Monoclonal antibodies may be used for many of the functions described for the soluble CLAM-l antigen or its fragments. Thus, monoclonal antibodies, such as HECA-452 or other antibodies which bind to CLAM-l antigen may be used to block the lymphocytes from trafficking to skin sites comprising the receptor. The monoclonal antibodies may be IgG, IgM, IgD or IgA. The CLAM-l antigen may be used for producing other monoclonal antibodies, having binding sites other than the HECA-452 binding site in accordance with conventional ways. The antigen may be used as an immunogen in an appropriate host, e.g. a mouse, and after one or more injections at appropriate sites, in combination with adjuvants, the spleen may be isolated and the splenocytes immortalized, conveniently by fusion with an immortalized myeloid cell line. Thus, a wide variety of monoclonal antibodies may be obtained, where the monoclonal antibodies may be screened for their ability to block binding to cutaneous cells., In addition, the monoclonal antibody may be modified by changing the constant region as to species and/or isotope or class. Thus, the antibodies may have murine or human constant regions. Alternatively, the monoclonal antibodies, ELAM-1, or the lectin domain thereof, may be used in turn as immunogens for the production of anti-idiotope antibodies which may serve to mimic the CLAM-l antigen and either block the CLAM-l antigen from binding to cutaneous, vascular or other cells or direct various agents, as pre¬ viously described, to such cells. The anti-idiotopic antibody producing cells may be readily screened for their ability to secrete antibodies which block binding of the HECA-452+ lymphocytes. By preparing tissue sections of inflamed skin which comprise the receptor and adding the anti-idiotopic antibodies, which are labeled with a reagent which provides for a detectable signal, to the tissue section, washing away non-specifically bound antibodies and detecting the binding by detecting the presence of the label, the desired antibodies may be identified. Such antibodies may be used for recognition of the CLAM-l receptor on inflamed endothelial cells (ELAM-1) , as well as other cutaneous receptors for CLAM-l. In addition, the anti-CLAM-1 antigen antibodies may be used for the diagnosis and/or treatment of malignancies associated with lymphocytic association with skin, exemplified by mycosis fungoides. In the case of mycosis fungoides, it is found that the skin associated antigen appears to be present during the patch/plaque or epidermotropic-stage mycosis fungoides, but not in the advanced stage of the tumor. Thus, the skin associated antigen antibodies may be used for staging mycosis fungoides. For treatment, agents specific for CLAM-l ELAM-1 may be bound to a therapeutic agent for mycosis fungoides, e.g., antibodies may be linked to various cytotoxic agents to be directed with the lymphocytes to the site of the mycosis fungoides lesion. The specific antibodies may be used for identifying the site of the skin associated antigen population in the case of neoplasms, by labeling the antibodies with an appropriate label, e.g. radiolabel or other label, for tomography, or other means of detection. Quantitation of serum levels of CLAM-l may be used as a serologic assay to follow the course of disease and the effectiveness of therapies. Also, by employing various labels, such as radioactive labels, cytotoxic agents, e.g. toxins, or the like, immunotoxins may be prepared for treatment of the neoplasm. Also, the antibodies may be used to deplete T-cell populations, particularly memory cells associated with the skin to inhibit detrimental activities of the T-cells. The T-cells may be killed with immunotoxins, antibodies to provide a complement or ADCC pathway for removing T-cells or the like. The presence of CLAM-l T-cells may be used as a diagnostic of cutaneous inflammatory disorders by establishing a particular phenotype associated with such disorder. Also, the CLAM-l T-cells may be isolated, expanded and returned to the host source in the treatment of the inflammatory disorder. Since the target of toxic CLAM-1-targeted therapies will often be skin-localized lymphocytes or malignant lymphoid cells, anti-CLAM-1 antibodies or other reagents that target CLAM-l may also be conjugated to photoactivatable toxins. After localization of the toxins to skin-associated lymphoid cells following systemic or local administration, the toxin is activated by exposure to light or uv irradiation of the appropriate wavelength, permitting localized precise regulation of the lymphocytotoxic effect. The light may be transmitted through the skin by means of optical fibers, through an incision, or the like. Also, the antibodies or receptor(s) for CLAM-l or fragments thereof can be used to target immunomodulatory agents to skin-associated lymphocyte subsets. For example, anti-CLAM-1 antibodies can be conjugated chemically to a cytokine such as interferon-7, and used to target such a cytokine to skin-associated lymphocyte populations involved in skin inflammatory or neoplastic reactions. Alternatively, chimeric proteins containing the antigen recognition site of anti-CLAM-1 antibodies (or the lectin domain of ELAM-1) and active domains of a given cytokine could be produced by expression of chimeric cDNAs in conventional ways. To enhance the number of CLAM-l antigen containing lymphocytes, one can isolate such cells using panning, affinity selection, or the like, to greatly concentrate the CLAM-1+ lymphocytes. Such cells or distinct subsets thereof, using one or more surface markers for selection of such subsets e.g., CD4 or CD8, may then be expanded in culture and returned to the host to enhance the CLAM-1+ lymphocyte population. Syngeneic cells need not be employed, where other lymphocytes, which may be accepted by the host, may be employed. Thus, one treatment would be to introduce into a host, CLAM-l antigen presenting cells for treatment of a condition responsive to such cells. The trafficking of the CLAM-1+ T-cells offers the opportunity to deliver various proteinaceous or non- proteinaceous agents to cutaneous sites. T-cells may be transformed by conventional ways with structural genes capable of integration into the genome or with episomally maintained constructs for constitutive or inducible expression of various agents or factors, such as immunomodulatory agents, cytokines or antiinflammatory agents. Alternatively, various agents may be introduced into the cell which would leak slowly from the cell, such as agents allowing for detection, e.g., radioisotopic agents, dyes, contrast agents or the like, or drugs for the treatment of diseases, at cutaneous sites where the T-cells will home. Alternatively, agents may be bound to the T-cell surface through antibodies or ligands having high affinity for surface membrane receptors for delivery to cutaneous sites. The various products may be formulated in con¬ ventional ways, using lyophilized products as appropriate, or suspensions, solutions or dispersions. Media may include physiologically acceptable media, such as saline, PBS, aqueous ethanol, and the like, generally, buffered at about physiologic pH. The concentration and dosage will vary widely depending upon the particular product, its efficacy, mode of administration, purpose of administration or treatment, etc. The products may be administered parenterally, e.g., intravascularly, etc. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Materials and Methods Tissues and Cell Preparation: Samples of snap-frozen tissues for immunostaining were obtained from the Department of Pathology's frozen tissue bank at Stanford University, except for synovial tissue and salivary gland specimens which were provided by Drs. S. Jalkanen (University of Turku, Finland) , and N. Wu (Stanford University) , respectively. Diagnostic classification of all specimens was done on the basis of standard pathologic, and as appropriate, clinical cri¬ teria. Tonsil lymphocytes and thymocytes were obtained by gentle mincing and washing of fresh, pathologically benign, pediatric tonsils or whole thymic lobes over type 304 steel scree (Tylenter, Mentor, Ohio) in RPMI-1640 media (GIBCO, Grand Island, N.Y.) with 2% FCS. The collected thymocytes were washed twice in the same media prior to use, whereas tonsil lymphocytes were further purified by centrifugation over Ficoll-Hypaque (Histopaque 1077; Sigma Chemical Co., St. Louis, MO), and then were washed twice prior to use. Peripheral blood buffy coats from normal adult donors were separated into high density [>98% polymorphonuclear leukocytes (PMNs) by morphology] and low density [peripheral blood mononuclear cells (PBMC) ; 80-85% lymphocytes and 15-20% monocytes] populations by Ficoll- Hypaque two-step gradient density sedimentation (Histopaque 1077 and 1119:Sigma) . The PBMC population was further separated into purified monocyte, lymphocyte (PBL) , and T-cell populations, as previously described (Gonwa et al. J. Immunol. (1983), 130:706-711) . Briefly, PBMC were adhered to plastic petri dishes (Falcon, Oxnard, CA) and separated into adherent (90-95%) monocytes) , and non-adherent (>95% lymphocytes) populations. T-cell enriched populations (95% CD3+) were obtained by rosetting the non-adherent cells with 2-aminoethyl isothiouronium bromide hydrobromide (Sigma)-treated sheep erythrocytes. This latter population was 99% CD3+ when analyzed by flow cytometry (FACS analysis, see below) using lymphocyte gates. The U937 human monocytoid cell line was originally obtained from the American Type Culture Collection (ATCC CRL 1598, Rockville, MD) . Activation of PBMC: PBMC were stimulated with phytohemagglutinin (PHA) at 1/100 final dilution of stock (GIBCO) or with 5-10 μg/ml Concanavalin A (Con A; Sigma) in RPMI-1640 media supplemented with 10% autologous serum. The cells were cultured at 1 x 10 /ml for 3-14 days in a humidified incubator with 10% CO_ at 37°C. In some experiments, cells from the same donor were incubated without mitogen under identical conditions. The culture media were periodically changed to maintain optimal conditions for cell growth. MAbs: The production of the HECA-452 mAb (rat IgM) has been described previously (Duijvestijn et al. , Am. J. Pathol. (1988), 130:147-155) . The OZ-42 (against a mouse cerebellar antigen; Pickford et al. , J. Neurocytology (1989), 18.:466-478 and MECA-79 (against the mouse peripheral lymph node addressin, Streeter et al. , J. Cell Biol.. (1988), 107:1853-1862) mAbs were used as species- and isotope-matched controls. Purified preparations of these mAbs were conjugated to fluorescein isothiocyanate (FITC) according to the method of Coding (J. Immunol. Methods (1976), 13:215-226) . The Leu 2 (CD8 MHC class I restricted T-subset) . Leu 3 (CD4, MHC class II restricted T-subset) , Leu 4 (CD3, T-lineage) , Leu 5 (CD2, T-lineage) , Leu 9 (CD7, T-lineage), Leu Ml (CD15, myeloid) , Leu M3 (CD14, monocyte/macrophage) Leu 18 (CD45R) , and Leu 12 (CD19, B-lineage) mAbs (both FITC-labeled and unconjugated) were obtained from Becton-Dickinson (Mountain View, CA) . The anti-transferrin receptor mAb 0KT9 (CD71) was obtained from Ortho Diagnostics (Raritan, NJ) , and FITC-conjugated 4B4 mAb (CD29, VLA β-chain) from Coulter Immunology (Hialeah, FL) . The anti-TCR-S-1 mAb was obtained from Dr. M. Brenner (Boston, MA; Groh et al. , J. Exo. Med. (1989), 169:1277-1294). The anti-LFA-1 β-chain mAb (TS1/18) , and the anti-LFA-3 mAb TS2/9 were obtained from Drs. T. Krensky and C. Clayberger (Stanford, CA) (Sanchez-Madrid et al.. PNAS USA (1989), 56:1063- 1072) . The anti-human H-CAM (CD44/Pgp-1) mAbs Hermes-3 and HZ-77 were produced as previously described (Goldstein et al. , Cell (1989) 56.:1063-1072) . All antibodies were used at saturating concentrations as determined by both FACS analysis and immunoperoxidase techniques. SUBSTITUTESHEET Flow Cvtometry: Cell populations (1 x 10 6 cells/test) were incubated with a primary unconjugated mouse mAb, washed twice in PBS, incubated with a phycoerythrin (PE)-conjugated anti- mouse IgG (Tago, Burlingame, CA) , washed with PBS, blocked (10 minutes) and washed with 5% normal mouse serum (NMS) 5% normal rat serum (NRS) in PBS, and then incubated with FITC-HECA-452 or control mAb. All antibody incubation periods were for 30 minutes at 4°C in the presence of 0.2% sodium azide. After washing twice, the stained cells were either analyzed immediately or fixed in 1% paraformaldehyde in PBS and saved at 4°C for later analysis. In some experiments, unconjugated HECA-452 or control mAb were used as initial antibodies followed by staining with PE-conjugated anti-rat IgG (Tago, heavy and light chain reactive), and then FITC-labelled mouse mAbs. These two staining techniques gave identical results. Flow cytometry analysis was performed on a FACStar (Becton Dickinson Immunocytometry Systems, Mountain View, CA) equipped with an argon laser which was operated at 488nm. The FITC and PE emissions were collected with 530/30 and 585/42 band pass filters, respectively. All data was collected in list mode, ungated. For analysis, gates were drawn to include the whole population excluding only doublets and larger cell aggregates, or were drawn around the lymphocyte, monocyte, and granulocyte regions as appropriate. Data analysis was done with Consort 30 software. The data are represented on contour plots with the lowest level at 2 or 3 cells and the contours drawn at 2, 4, 8,. 16, 32, 64 and 128. The delineation and quantitation of positively stained populations (i.e., placement of marker quadrants) was based on samples stained with isotope-matched antibody controls (positive quadrants with <0.7% cells in control plots). Electrophoresis/Western Blotting 7 LLyyssaatteess wweerree pprreeppaarreedd bbyy iinnccubating 2-4 X 10 cells in 1 ml lysis buffer (2% Nonidet P-40, 150mM NaCl, ImM MgCl , 10 μg/ml Aprotinin, and ImM phenylmethyl sulphonyl fluoride in 20mM Tris-HCl, pH 7.5) for 45 minutes at 4°C, followed by centrifugation at 10,000 x g for 30 minutes. Aliquots of these lysates were applied to 8% SDS-PAGE gels under reducing conditions. Proteins were transferred to nitrocellulose with a Biorad (Richmond, CA) transblot apparatus. After blocking non-specific protein binding with 100% horse serum for 30 minutes, primary and secondary antibody incubations [alkaline phosphatase conjugated anti-rat IgM (Zymed, So. San Francisco, CA) for HECA-452 and controls or anti-mouse IgG (Promega, Madison, WI) for H-CAM/CD44 mAbs] were done in a Miniblotter 25 staining apparatus (Immunetics, Cambridge, MA; 1 hour incubation at room temperature for each) . Washes between incubations were with TBST (lOmM Tris-HCl, 150mM NaCl, 0.05% Tween-20, pH 8.0). The alkaline phosphatase reaction was developed as described by Promega. For periodate oxidation experiments, strips of nitrocellulose with blotted proteins were treated with 20 mM sodium periodate in 50mM acetate buffer (pH 4.5)±250mM ethylene glycol for 40 minutes at room temperature in the dark. After washing with TBST, the treated nitrocellulose strips were stained as described above. Tissue Section Immunostaining: Serial, acetone-fixed, air-dried cryostat sections (5-6 μm) were prepared from snap-frozen tissue specimens and stained using a 3-stage immunoperoxidase technique. Sections were incubated serially (45 minutes in a humidified chamber with PBS washes in between) with primary mAbs, biotinylated secondary antibodies - goat anti-rat IgM (Kirkegaard & Perry Labs, Gaithersburg, MD) , or horse anti-mouse IgG (Vector Labs, Burlingame, CA) - as appropriate for the primary mAb, horseradish peroxidase- conjugated Streptavidin (Zmed) , and then developed with .05% 3,3-diaminobenzidine (Sigma) and 0.009% hydrogen peroxide in 50mM Tris/HCl (pH 7.5) for 10 minutes. Second and third stages included 5% normal human serum (NHS) to decrease background. After darkening the reaction with 0.5% copper sulfate in 0.9% NaCl for 5 minutes, sections were counterstained with 2% methylene blue, dehydrated, and coverslipped. Two-color tissue section or cytospin immunofluor- escence (HECA-452 vs. Leu 4 or other mouse mAbs) was accomplished using sequentially a 2-stage detection system for mouse IgG followed by an appropriate blocking step and then a 3-stage system for rat IgM. Briefly, sections were incubated (30 minutes in a humidified chamber for each incubation step) with a mouse IgG mAb followed by rhodamine-conjugated anti-mouse IgG (Sigma) . After blocking 5 minutes with 5% NMS/5% NRS in PBS, the sections were incubated with the primary rat IgM mAb (i.e., HECA- 452 or control) , followed by biotinylated anti-rat IgM (Kirkegaard and Perry Labs) in PBS with 2% NHS/5% NMS, and finally by FITC-conjugated avidin (Becton-Dickinson) . Immunohistologic Interpretation/Ouantitation: The immunoarchitecture of all cases was defined with CD3 (T-cell) , CD19 (B-cell) , and CD14 (macrophage/ mono- cyte) mAbs. In some cases, CD15 mAb was used to define tissue PMNs. Serial sections were then evaluated for HECA-452 vs. control mAbs. The number of HECA-452+ cells with lymphoid morphology were evaluated in T-cell zones, defined as areas containing at least 80% CD3+ cells: morphologically discernible PMNs, macrophages, endothelial cells, dendritic cells, fibroblasts and epithelial cells were not considered. An average of 590 cells (range: 434- 829) were counted in at least 5 separate fields. In small specimens or those with focal infiltrates, multiple sections at different levels of the tissue block were evaluated. Two color immunofluorescence analysis (CD3 vs. HECA-452 as described above) was used in 12 cases, including 5 cutaneous and 7 extra-cutaneous infiltrates, to check the accuracy of this evaluation, and in all instances similar results were obtained. The T- lineage lymphoma cases were evaluated differently. In these cases, the malignant cell population (which was usually intermixed with variable numbers of reactive cells) was determined by morphologic and immunophenotypic criteria (Dicker et al. , Am. J. Pathol. (1987), 128:181- 201) , and the malignant cells were evaluated for specific HECA-452 reactivity. A case was considered positive if 20% or more of the malignant population was clearly HECA 452+. Results Two-color flow cytometry was used to more precisely define patterns of HECA-452 among populations of peri¬ pheral blood leukocytes. The HECA-452 epitope is expressed on a sub-population of both CD3+ T-cells and CD19+ B-cells and on essentially all CD14+ monocytes. Similar analysis of isolated PMN populations also revealed essentially 100% surface reactivity. In six different donors the mean percentage (and range) of HECA-452+ T-cells was 16% (8-23%) . About 11% (6-14%) of peripheral B-cells were HECA-452+. However, HECA-452 staining intensity of these cells was quite low (barely above background) . Roughly similar subsets of both CD4+ (Class II MHC-restricted) and CD8+ (Class I MHC-restricted) T-cells were HECA-452+, 17% (10-23%) and 11% (9-15%) , respectively. A larger subset of T-cell receptor-5 bearing T-cells-32% (28-36%, 3 experiments) express the HECA-452 determinant. HECA-452 also stained about 10% of T-cells in suspensions of tonsil lymphocytes including both CD4+ and CD8+ T-cells. In thymocyte suspensions, only about 1% of CD2+ or CD7+ cells display the HECA-452 epitope. Western analysis of SDS-PAGE separated proteins indicated that the apparent M-. of HECA-452 antigen varied among the different cell types. Both tonsil lymphocytes and PBL showed predominant bands at about 200kD. These were barely detectable in lysates from the myeloid cell types. PBL also displayed a 125kD species similar to the predominant species displayed by monocytes and the monocytoid cell line U937. The 125kD band and PBL lysates may represent contamination by monocytes. In contrast to both monocytes and lymphocytes, PMNs showed multiple intense bands ranging from 75kD to l60kD, as well as a faint broad band at 55-60kD. The number of different specific bands identified with the HECA-452 mAb, as well as the broadness of these bands, are consistent with the protein(s) identified by this mAb being heavily glycosylated. Based on sodium periodate experiments (Spiro RG: Characterization of carbohydrate units of glycoproteins. In Methods in Enzvmology. Vol. VIII: Complex Carbohydrates, EF Neufeld V. Ginsberg, eds. Academic Press, New York, p. 26-49, 1966) indicated the involvement of carbohydrate in the HECA-452 epitope. Periodate treatment of HECA-452 antigen containing nitrocellulose strips from U937 lysate completely abrogated the ability of HECA-452 mAb to recognize all of the HECA-452 specific bands. Identical results were obtained using HECA-452 antigens from PMNs and tonsil lymphocytes (CLAM-l) . Periodate had no effect on control anti-HCAM/CD44 mAbs (Hermes-3 and H2-7) which are known to recognize carbohydrate independent determinants. As already indicated, there is no correlation of CLAM-l expression with MHC Class I or II restriction and both TCR-αj3 and TCR- 6 bearing T-cells contained a CLAM-1+ subset. Since memory T-cells (or previously activated T-cells) have been reported to be distinguishable from virgin T-cells by their level of adhesion molecules (LFA- 3/CD58, LFA-l/CDlla/18, H-CAM/Pgp-1/CD44, VLA (S-chain/CD29, CD2 molecules) and CD45R expression, the possibility that CLAM-l expression might be related to previous activation was investigated using two-color flow cytometry of purified T-cells and comparing expression of a CLAM-l antigen and the above described markers. Memory T-cells are reported to express higher levels of the adhesion cells and lower levels of the CD45R epitope than virgin T-cells. (Sanders et al. , J. Immunol. (1988), 140:1401-1407) . Essentially all the CLAM-1+ T-cells are found in the adhesion molecule , CD45R T-cell subset, indicating that most CLAM-l expression develops post- thymically as a consequence of activation. However, stimulation of PBMC with both PHA and Con A for 3-14 days did not result in an increase in the fraction of CLAM-1+ T-cells. After mitogen stimulation, CLAM-1+ T-cells manifest activation antigens (i.e., transferrin receptors/CD71) , indicating that the CLAM-1+ subset can respond to mitogens. Tissue section immunohistology was used to compare the distribution of CLAM-1+ T-cells in 54 specimens of normal/reactive lymphoid tissues and sites of chronic inflammation. Optimal visualization of CLAM-1+ lymphoid cells require the use of frozen-sections in a three stage biotin-avidin immunoperoxidase procedure and was critically dependent on the use of an appropriate rat IgM- specific second stage. HECA-452 staining patterns were similar in reactive tonsils and lymph nodes, showing staining of HEV dendritic cells (including follicular, paracortical and sinusoidal types) and a minor subset (10 ± 0.8% for six specimens, 3 lymph nodes, 3 tonsils) of small lymphocytes located in the paracortical region. B-cells in germinal centers and mantle zones appear to lack HECA-452 reactivity and two-color immunofluorescence histology (HECA-452 vs. CD3) confirm the T-cell nature of the paracortical HECA-452+ cells. In normal spleen, Peyer's patches and appendix, only rare HECA-452+ lymphoid cells were present, but when observed, the cells were in T-dependent areas. Similarly, only a small minority of lamina propria or gut intraepithelial lymphocytes appeared to express the HECA-452 antigen. PMNs present in splenic red pulp or gastrointestinal tract mucosa were HECA-452+ as were PMNs in other tissue sites. In agreement with the FACStar analysis mentioned above, HECA-452+ cells were also scarce in normal thymus with only about 1-2% positive cells scattered in both the cortex and medulla. Some of these positive cells had dendritic or macrophage morphology and two-color immunofluorescence analysis of both thymic frozen sections and cytospin preparations of thymocytes indicated that fewer than half the total number of HECA-452+ cells were of T-cell lineage. Among 22 non-cutaneous chronic inflammatory lesions, all of which had prominent, if not markedly predominant, T-cell infiltrates (including lesions of the gut, lung, synoviu , liver, kidney, salivary glands, heart, thyroid and periorbital soft tissue, see Table 1) , twenty-one had fewer than 10% HECA-452+ cells within the T-cell infiltrates. Table 1 Cases Studied: Diagnoses # Cases examined Normal/Reactive Lymphoid Tissues Lymph Node 3 Spleen 3 Gut-Associated Lymphoid Tissues 3 Tonsil Appendix 2 Peyers Patch 2 Thymus 3. 16 Inflammatory Lesions Gastrointestinal Tract Chronic gastritis/gastric 1 lymphoid hyperplasia Chronic duodenitis (non-specific 2 and gluten sensitivity) Chronic colitis (non-specific 3 and Crohn's disease) Lung Lymphoid hyperplasia (Inflammatory 1 Pseudo tumor) Interstitial pneumonitis 2 Interstitial pneumonitis and 1 vasculitis (Rheumatoid Lung) Synovium Rheumatoid arthritis 3 Heart Myocarditis 1 Chronic rejection 1 Liver Hepatitis 2 Table 1 Cases Studied: Diagnoses (continued) # Cases examined Skin Allergic contact dermatitis 5 Psoriasis 3 Drug eruption 2 Lichen planus 1 Pityriasis lichenoides et variolifor is acuta 1 Pityriasis rubra pilaris 1 Granuloma annulare 1 Chronic dermatitis, non-specific 2 Other Lymphocytic thyroiditis 1 Chronic renal rejection 1 Nasopharyngeal lymphoid hyperplasia 1 Periorbital lymphoid hyperplasia 1 Sjogren's Syndrome (minor salivary glands) 1 38 Overall, the non-cutaneous lymphoid tissues and inflammatory sites showed a mean (± S.E.) of 5.0 ± 1.0% HECA-452+ lymphocytes within T-cell areas. In striking contrast, the mean (± S.E.) fraction of HECA-452+ cells within the T-cell infiltrates of 16 varied inflammatory skin lesions (Table 1) was 85 ± 2.1% (P<0.0005. Students t-test) . Two-color tissue section immunofluorescence analysis of five of these cases confirm that the great majority of HECA-452+ cells co-express the pan T-antigen CD3. Although intraepidermal T-cells comprise only a minor fraction of the T-cell infiltrates in the skin lesions, this population appeared to be virtually 100% HECA-452+. Expression of the CLAM-l antigen was investigated in 59 cases of T-lineage lymphoma including 22 cases of peripheral T-lineage lymphoma, 14 cases of thymic (T- lymphoblastic) lymphoma, and 23 cases of mycosis fungoides (18 of patch/plaque- or epidermotropic-stage mycosis fungoides, and 5 of tumor-stage disease) . The neoplastic cells of 16/18 cases of patch/plaque mycosis fungoides expressed the CLAM-l antigen. All 14 lymph node or thymic based lymphoblastic lymphomas were HECA-452 negative, and of 22 peripheral T-lineage lymphomas, only two were HECA- 452+, both of which were cutaneous lesions. The neoplastic cells of five cases of advanced tumor-stage mycosis fungoides, including two cases which were demonstrated to be HECA-452+ in the patch/plaque stage, were also HECA-452 negative. The above data support the conclusion that a portion of recent thymic emigrants and virgin T-cells —initially -452 negative and non-tissue-selective in homing behavior —are first stimulated in the context of an immune reac¬ tion at a cutaneous site. Specific induction of the CLAM-l epitope occurs. Along with CLAM-l antigen induction, activation results in up-regulation of multiple general cell adhesion molecules characterizing the "memory" phenotype of CLAM-1+ cells. The result of this process is the observed phenomenon that nearly all skin- associated T-lymphocytes express the CLAM-l epitope. CLAM-l is a leukocyte ligand for endothelial cells. Binding of CLAM-l TO ELAM-1 CLAM-l was isolated from HL-60 cells or tonsil tissue extracts using a two-step procedure. NP-40 tissue extracts were prepared by incubation of cells at 10 7- lθ 8 /ml in Lysis Buffer (2% NP-40/20 mM Tris HC1, pH 8.0/150 mM NaCl/0.02% NaN 3 /5 mM CaCl 2 /5 mM MgCl 2 /l mM phenylmethylsulfonyl fluoride containing 10 μg/ml of each of aprotinin, leupeptin and pepstatin) for 30 in on ice. After centrifugation at 10,000 g for 30 min at 4 °C, the clarified lysate was passed over a column containing wheat germ agglutinin-coupled agarose (vector, Burlingame, CA) . The column was washed with Wash Buffer (containing 50 mM i8-octylglucoside/20 mM Tris-HCl, pH 8.0/150 mM NaCl/0.027 NaN 3 /5 mM CaCl 2 /5 mM MgCl 2 ) and specifically bound material was eluted with Wash Buffer containing 0.05 M N- acetyl glucosamine. A WGA-binding material (which contained all of the HECA-452 active material) was then SUBSTITUTESHEET passed through 2 ml affinity columns of Hermes-1 (anti- human CD44), rat IgM mAb control or HECA-452 coupled to Sepharose 4B (prepared according to manufacturer's specifications, Pharmacia) . Columns were then washed as above and specifically bound material eluted with Elution Buffer (50 mM β-octylglucoside/0.2 M acetic acid/150 mM NaCl/0.02% NaN 3 /5 mM CaCl 2 /5 mM MgCl 2 ) . 600 μl fractions were collected and neutralized with 1 M Tris-HCl pH 8.0. 5 μl samples of CLAM-l isolated from tonsil extract were absorbed onto glass well of slides (LabTek) by 20-fold dilution DMEM followed by a 2 hour incubation at RT. After blocking with 5% NBS/10 mM HEPES/DMEM pH 7.0 (CM) T*T AM—1 «fi Ll-2 cells were applied to each well (1.5 x 10 /0.15 ml) . After 25 min incubation at RT on a rotating shaker at 50 rp , the tops of the wells were removed and the slides washed three x DMEM and then fixed by incubation in 1.5% glutaraldehyde (Kodak) in DMEM. Vector transfected control Ll-2 vector cells did not bind. CELLS/UNIT AREA (ARBITRARY UNITS) L1-2 ELAM - 1 L1-2 VECT0R (Control) CLAM-l 289 ± 46 The binding of the ELAM-1 expressing Ll-2 transfec- tants to CLAM-l was shown to be blocked by CL2, a monoclonal antibody binding to ELAM-1. Control anti¬ bodies, Dreg-56 against the peripheral lymph node homing receptor, LECAM-1, and 30G12, against mouse T200, had no effect. In carrying out the determination, the Ll-2 cells expressing ELAM-1 were preincubated with the designated monoclonal antibody or medium and the antibodies were included in the assay. The assay was performed as described above with H-CAM(CD44) used as a control protein. With antibody CL2, the number of cells bound was about 36, while with the other antibodies or medium, the nu ber of cells range from about 207 to 242. With the control, the results with the three antibodies and medium ranged from about 26 to 38 cells bound per unit area. Ll-2 cells bound/unit area ANTIBODY Purified Molecule φ(medium) CL-2 Dreg-56 30G12 ** CLAM-l 139±12 12±5 105±21 139111 HCAM (CD44) N.T 7±3 17+7 14+4 CLAM-l and HCAM isolated from the myeloid cell line HL-60 * _ Ll.-2_ELAM-1 c„e-ll-s .bound,/.uni.t. area The above assay was repeated, except that HECA-452 was employed as to its effectiveness in inhibiting binding. The antibodies which were used besides HECA-452 was MECA-79, against the peripheral lymph node vascular addressin, or 2C2, against B220. The antibodies were used to pretreat the protein on the slide. With HECA-452, the number of cells per unit area was about 20, while with the other antibodies, the number of cells ranged from about 186 to 217. With the control protein, the number of cells range from about 21 to 22 with variations in the case of the controls of up to about 35%. ANTIBODY TREATMENT Molecule <f>(medium) MECA-79 HECA-452 2C2 * CLAM-l 217114 201114 2013 186111 (tonsil) HCAM (CD44) 2115 2218 2216 2117 (tonsil) CLAM-l 142112 N.T. 3814 152116 (HL-60) To demonstrate the calcium dependency, three different media were prepared: HBSS + 2mM Ca + +22; HBSS + 0.5mM EGTA; HBSS + 0.5mM EGTA + 2mM Ca +2 The results for the three media with CLAM-l were 195 1 23, 6 + 2, and 165 1 30, respectively, while for the control protein H-CAM, the results were 3 in each case. When CLAM-l derived from HL-60 was employed, the results were 361 1 25, 42 1 27, and 343 ± 11, respectively. The HECA-452 determinant was shown to be sensitive to neuraminidase by the following procedure. NP-40 human tonsil extracts were coated onto ELISA plates by dilution of 50 mM sodium bicarbonate, pH 9.0 overnight. They were then treated with neuraminidase (Vibro cholera. CalBioChem, San Diego, CA) at 5 mU/ml in 50 mM sodium acetate/100 mM NaCl/10 mM CaCl_ or buffer alone for 30 min at RT. After blocking in horse serum for 2h, plates were incubated with HECA-452 for 45 min. After washing in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% Tween-20) , plates were then incubated in horseradish peroxidase- conjugated rabbit anti-rat Ig for lh. Plates were developed after washing in TBST by incubation in OPD according to standard methods. Neuraminidase treatment resulted in complete loss of signal with HECA-452, but the reactivity of Meca-79 and Hermes-1 with their antigens, also present in tonsil extracts, were unchanged. Using the above described procedure, with CLAM-l, (tonsil) the following different conditions were employed: medium (DMEM) ; neuraminidase treatment; neuraminidase treatment in the presence of sialyllactose, a neuraminidase inhibitor; and neuraminidase buffer. The results were 66 1 9, 2 1 2, 56 1 12, 54 1 3. For the control protein H-CAM (tonsil) , the results varied from about 1 to 6 cells per unit area. The results for CLAM-l (HL-60) were N.T. , 4 1 1, 125 1 11, and 117 1 10, respectively. Using the procedure described previously for the Ll-2 ELAM-1 transfected cells, the ability of HECA-452 to inhibit binding to wheat germ lectin binding components of the myeloid cell line HL-60 was investigated. When the wheat germ agglutinin isolated membrane proteins from HL- 60 were added to the slide and the amount of binding of the proteins determined, it was found that in the presence of HECA-452, the amount of binding of the HL-60 proteins and H-CAM was about the same, while in the presence of MECA-79 or 2C2 or medium there was a much greater degree of binding of the HL-60 protein as compared to H-CAM. Differences in Glycosylation Profiles of CLAM-l T-Cells vs. Mveloid Cells Whole peripheral blood leukocytes, isolated by dextran sulfate sedimentation of erythrocytes, were stained for 2-color fluorescence analysis with HECA-452 (2nd-stage FITC anti-rat IgG) vs. anti-sialyl Le x antibody TT19 (2nd-stage phycoerythrin conjugated anti-mouse IgG, absorbed with rat IgG to specificity) . Flow cytometric profiles revealed coordinant expression of sialyl Le x and CLAM-l epitopes on monocytes and neutrophils, but weak or absent staining of sialyl Lex on CLAM-l+ T-cells, as well as on the bulk of CLAM-1~ B-and T-lymphocytes. Thus, although CLAM-l T-cells and myeloid cells share CLAM-l expression and ELAM-1 binding ability, the glycosylation patterns of surface determinants on the ELAM-1 binding myeloid vs. lymphoid cells are distinct. CLAM-l T- lymphocytes also express little or no CD15 (Le ) , the non- sialyated precursor of sialyl Le x . The results support the conclusion that although sialyl Le x and related SUBSTITUTE SHEET carbohydrates may serve as ligands for ELAM-1 and overlap with CLAM-l determinants on myeloid cells, ELAM-1-binding structures including CLAM-l on memory T-lymphocytes either lack the core sialyl Le x structure or express a modified form thereof. Antibodies produced by immunization with CLAM-l T-lymphocytes may be selected for their ability to discriminate between the structurally different forms of lymphocyte and myeloid CLAM-l. Thus, one may prepare antibodies which will identify other carbohydrate or protein determinants unique to CLAM-l T-cells not shared with other ELAM-1 binding cell types. The above results demonstrate the specificity of homing of CLAM-l leukocytes to cutaneous sites, their ability to bind to ELAM-1 present on cutaneous endothelial cells, the ability of antibodies to block the binding of CLAM-l and ELAM-1 and the sensitivity of such binding to treatment of CLAM-l cells with saccharide hydrolases or sugar degradative agents. By modulating the binding capability of CLAM-l cells to ELAM-1 or using such binding capability by employing the CLAM-l carbohydrate or protein determinant or fragments or analogs thereof cross- reactive with the binding epitope, by themselves or in conjunction with other agents, a wide variety of diagnoses and therapies may be performed. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for modulating lymphocyte interaction with cutaneous vascular endothelium or other cutaneous tissue, said method comprising: combining with a system comprising lymphocytes and cutaneous tissue an agent which regulates the cellular production of a skin-associated-epitope containing molecule, wherein said molecule is characterized by being a surface membrane molecule which binds to the antibody HECA-452 and/or to the endothelial cell lectin ELAM-1; or a moiety which is cross-reactive with said epitope; or an agent which modulates the binding of said epitope with its cutaneous receptor. 2. A method according to Claim 1, wherein said agent is an antibody which inhibits binding of said molecule with its cutaneous receptor. 3. A method according to Claim 2, wherein said antibody is HECA-452. 4. A method according to Claim 1, wherein said agent is a peptide or a carbohydrate cross-reactive with said epitope in binding to skin-associated vascular receptors or capable of inhibiting the binding of said epitope to skin-associated vascular receptors or other cutaneous tissue. 5. A method according to Claim 1, wherein said lymphocytes are T lymphocytes. 6. A method of staging a mycoid fungoides lesion, said method comprising: detecting the presence of skin-associated epitope- bearing atypical lymphocytes present in said lesion, SUBSTITUTE SHEET whereby the presence of said lymphocytes is indicative of the patch\plaque- or epidermotropic-stage of said mycoid fungoides. 7. A method of following the course of mycosis fungoides to determine a change in status, said method comprising: determining the level of a lymphocytic skin- associated surface membrane molecule in serum, as an indication of tumor burden. 8. A method for treating a mycoid fungoides lesion, said method comprising: contacting said lesion with an agent specific for CLAM-l and bound to a therapeutic agent for mycosis fungoides, wherein said CLAM-l specific agent is an antibody or fragment thereof specific for CLAM-l, or a soluble form of lectin ELAM-1 or other naturally occuring receptors for CLAM-l. 9. A method for detecting the presence of lymphocytes associated with cutaneous sites, said method comprising: determining the presence of CLAM-l antigen on the surface of said lymphocytes. 10. A method according to Claim 9, wherein said determining comprises detecting the binding of an antibody binding to CLAM-l, fragment thereof, or naturally occurring receptor for CLAM-l binding to a surface membrane molecule of said lymphocyte. 11. A method of isolating lymphocytes associated with cutaneous sites, said method comprising: combining a mixture of hematopoietic cells with an antibody or receptor specific for CLAM-l antigen; and separating cells specifically bound to said antibody or receptor from cells which are unbound or non- specifically bound. 12. A method according to Claim 11, comprising the additional step of administering said cells from said separation to a host suspected of suffering from a cutaneous inflammatory disorder. 13. A method for diagnosing cutaneous inflammatory disorders, said method comprising: isolating CLAM-1 + T cells from a physiological source; and characterizing said CLAM-1 + T-cells as to their phenotypic characteristics associated with an inflammatory disorder. 14. A method for reducing the population of skin- associated lymphocytes bearing the CLAM-l antigen, said method comprising: adding to a lymphocyte population a cytotoxic agent specific for CLAM-l antigen, whereby said cytotoxic agent binds to said CLAM-l antigen and kills lymphocytes comprising said CLAM-l antigen. 15. Receptor conjugates characterized by binding specifically to CLAM-l antigen and comprising a cytotoxic agent or a label for binding to a complementary member of a specific binding pair or a label capable of providing a detectable signal. 16. A substantially pure surface membrane glycoprotein characterized by being present on CLAM-1 + T-cells, binding to the monoclonal antibody HECA-452, being highly glycosylated, having a molecular weight as determined by gel electrophoresis of about 200kD and being present on about 10 to 30% of peripheral blood T-cells or the unglycosylated or partially unglycosylated protein or fragments thereof of at least 12 amino acids or intact carbohydrate side chain thereof. 17. A substantially pure carbohydrate characterized by having a composition of a carbohydrate bound to a protein according to Claim 16. 18. A carbohydrate according to Claim 17, further characterized by binding to the antibody HECA-452. 19. A transformed lymphocyte characterized by com¬ prising a surface membrane structure cross-reactive with the CLAM-l antigen and a structural gene capable of expression in said lymphocyte as a result of transformation of said lymphocyte with said structural gene, said gene expressing an immunomodulator, cytokine or antiinflammatory agent. 20. A method of treating a cutaneous disease susceptible to treatment with an immunomodulator, cytokine or antiinflammatory agent, said method comprising: administering to a patient having said disease lymphocytes comprising CLAM-l antigen and a structural gene capable of expression in said lymphocyte as a result of transformation of said lymphocyte with said structural gene, said gene expressing an immunomodulator, cytokine or antiinflammatory agent. 21. A monoclonal antibody specific for lymphocytic CLAM-l but not more than weakly reactive to other lineages bearing CLAM-l. 22. A method for separating subsets of hematopoietic cells comprising: combining a mixture of hematopoietic cells with a monoclonal antibody according to Claim 21; and separating cells bound to said monoclonal antibody from unbound cells. 23. A monoclonal antibody specific for CLAM-l on myeloid and/or monocytic cells, and non-reactive with lymphocytes.

Inventors: BERG ELLEN LAKEY, BUTCHER EUGENE C, PICKER LOUIS, BERG, ELLEN, LAKEY, BUTCHER, EUGENE, C., PICKER, LOUIS
Applicants: UNIV LELAND STANFORD JUNIOR, THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIO
Application year: 1991
Main classification: A61K39
IPC codes: C07K14