CD7

Abstract: The present invention relates, in general, to CD7 and, in particular, to a method of identifying individuals predisposed to autoimmune diseases based on CD7 analyses. The invention further relates to a method of treating patients suffering from autoimmune diseases by modulating CD7 expression and/or function. The present invention also relates to a method of identifying individuals predisposed to lipopolysaccharide (LPS)-induced shock, as well as to asthma or to other events and sequelae of infections mediated by TNF alpha and gamma-interferon (IFN- gamma ). The invention further relates to a method of treating patients suffering from septic shock, or preventing septic shock in patients predisposed to develop septic shock, by modulating CD7 expression and/or function. In addition, the invention relates to a method of treating patients suffering from asthma or from events and sequelae of infections mediated by TNF alpha or IFN- gamma , or preventing asthma or such events and sequelae of infections mediated by TNF alpha and IFN- gamma in patients predisposed to develop asthma or these events and sequelae, by modulating CD7 expression and/or function. The invention additionally relates to compositions suitable for use in such methods.

Description: CD7 The entire content of Provisional Application No. 60/061,343, filed October 8, 1997, is hereby incorporated by reference. TECHNICAL FIELD The present invention relates, in general, to CD7 and, in particular, to a method of identifying individuals predisposed to autoimmune diseases based on CD7 analyses. The invention further relates to a method of treating patients suffering from autoimmune diseases by modulating CD7 expression and/ or function. The present invention also relates to a method of identifying individuals predisposed to lipopolysaccharide (LPS)-induced shock, as well as to asthma or to other events and sequelae of infections mediated by TNFα and gamma-interferon (IFN-γ). The invention further relates to a method of treating patients suffering from septic shock, or preventing septic shock in patients predisposed to develop septic shock, by modulating CD7 expression and/ or function. In addition, the invention relates to a method of treating patients suffering from asthma or from events and sequelae of infections mediated by TNFα or IFN-γ, or preventing asthma or such events and sequelae of infections mediated by TNFα and IFN-γ in patients predisposed to develop asthma or these events and sequelae, by modulating CD7 expression and/ or function. The invention additionally relates to compositions suitable for use in such methods. BACKGROUND Considerable evidence is emerging that abnormal skewing of the T cell repertoire plays a central pathogenic role in rheumatoid arthritis (Weyand and Goronzy, Medical Clinics of N. America 81:55 (1997); Albani and Carson, Immunol. Today. 17:466-470 (1996)). The developing concept is that the over-representation of certain HLA-DR alleles in rheumatoid arthritis does not reflect the result of challenge to an arthritogenic antigen, but rather reflects the fact that certain DR4 alleles select in the thymus an abnormal T cell receptor (TCR) repertoire of autoreactive oligoclonal CD7-, CD4+ T cells that are present in certain DR4+ subjects regardless of whether or not they have rheumatoid arthritis (Schmidt et al, J. Clin. Invest. 97:2027- 2037, (1996)). A recent paper (Kouskoff et al, Cell, 87:811-822, (1996)) described the crossing of a TCR transgenic mouse (expressing a TCR transgene specific for Rnase peptide 41-61) with the NOD mouse. It was reported that the TCR coincidentally had reactivity with the unusual NOD Class II Aβ G7 , and that, up to 3 weeks of life, all single positive CD4+ and CD8+ thymocytes were clonally deleted in the thymus. After three weeks, however, th mic negative selection broke down, and TCR transgenic CD4+ T cells appeared in the periphery coincident with the development of synovitis indistinguishable from rheumatoid arthritis. Treatment of the mice with anti-T cell monoclonal antibodies abrogated the development of synovitis. Breeding studies showed that the predisposition gene was solely the Class II Aβ G7 (Kouskoff et al, Cell, 87:811-822 (1996)). These findings of arthritis in the TCR transgenic mouse are consistent with studies by Schmidt et al (J. Clin. Invest. 97:2027-2037 (1996)) and Weyand and Goronzy (Medical Clinics of N. America, 81:55 (1997)) in human rheumatoid arthritis, wherein a large population of PB and synovial T cells is decribed that is CD7-, that is autoreactive against self MHC antigens, and that has an oligoclonal TCR repertoire (Schmidt et al, J. Clin. Invest. 97:2027-2037 (1996)). Several lines of evidence suggest that the absence of CD7 on oligoclonal autoreactive T cells in rheumatoid arthritis may be significant. Ligation of CD7 on normal T cells and natural killer cells leads to increased T cell adhesion and IFN-γ production (Haynes et al, Proc. Natl. Acad. Sci. USA 76:5829-5833 (1979); Carrera et al, J. Immunol. 141:1919 (1988); Shimizu et al, J. Exp. Med. 175:577 (1992); Carrel et al, Eur. J. Immunol. 21:1195 (1991)). As with CD28, which plays a central role in regulation of T cell mediated immune responses, CD7 ligation of T cells is both able to induce a rapid calcium flux and able to function as a co-mitogen with CD3 for T cell activation (Carrera et al, J. Immunol. 141:1919 (1988); Shimizu et al, J. Exp. Med. 175:577 (1992); Carrel et al, Eur. J. Immunol. 21:1195 (1991); Ledbetter et al, Proc. Natl. Acad. Sci. USA 84:1384 (1987)). In addition, both human CD7 and CD28 have been shown to interact with the intracellular signal transduction molecule PI-3 kinase (Lee et al, Internatl. Immunol. 8:1195- 1203 (1996)). Ligation of CD7 also induces production of TNFα, in addition to IFN-γ (Lee et al, Internatl. Immunol. 8:1195-1203 (1996)). These two cytokines are important for LPS-induced shock syndrome, as well as mediation of Staphyloccus aureaus enterotoxin-mediated shock syndrome (Gutierrez-Ramos et al, Immunol. Today 18:329-334 (1997)). TNFα and IFN-γ are critical for mediation of a wide variety of immune responses, as well as for protection against a variety of infections. Moreover, serious sequelae to various infections, such as cerebral malaria, and various shock syndromes, are mediated by excessive production of TNFα and/ or IFN-γ (Lucas et al, J. Leuk. Biol. 61:551-558 (1997)). The data presented herein demonstrate that CD7 deficient mice have an antigen specific defect in IFN-γ production and have overproduction of double-positive thymocytes. Further, CD7 deficient mice are resistant to LPS-induced shock syndromes, and have LPS-induced defects of production of IFN-γ and TNFα in vivo. SUMMARY OF THE INVENTION The present invention relates to a method of identifying individuals predisposed to autoirrtmune diseases based on CD7 function and/ or expression analyses. The invention further relates to a method of treating patients suffering from autoimmune diseases by modulating CD7 expression and/ or function. The present invention also relates to a method of identifying individuals predisposed to lipopolysaccharide (LPS)-induced shock, as well as to other events and sequelae of infections mediated by TNFα and IFN-γ. The invention further relates to a method of treating patients suffering from septic shock, or preventing septic shock in patients predisposed to develop septic shock, by modulating CD7 expression and/ or function. In addition, the invention relates to a method of treating patients suffering from events and sequelae of infections mediated by TNFα or IFN-γ, or preventing such events and sequelae of infections mediated by TNFα and IFN-γ in patients predisposed to develop these events and sequelae, by modulating CD7 expression and/ or function. The invention additionally relates to compositions suitable for use in such methods. The invention also relates to CD7-knockout animals and to methods of screening drugs for therapeutic potential. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A-IC. Construction and identification of CD7 deficient mice. Fig. 1 A shows a schematic representation of two regions of mouse CD7 (mCDT) subcloned into the plasmid pPNT. pPNT encoded the neomycin and thymidine kinase genes used for positive and negative selection of transfected 129 strain embryonic stem cells. A null allele of mCD7 was accomplished by interrupting the mCD7 gene sequence in the 5' region of exon 3 (prior to the sequence encoding the transmembrane region) through insertion a stop codon and by insertion of the neomycin resistance gene. The 3' region of exon 3 was not included in this plasmid. The solid lines illustrate the region of mCD7 interrupted by the neo gene. The lower panel illustrates the structural organization of the mCD7 gene. Hatched lines represent regions where homologous recombination is required. Selected restriction sites are labeled: H3=HinDIII, EcoRI, BamHI, Xbal and Nde-1. Germline transmission and subsequent allele carrying offspring were identified by genomic southern blot of tail DNA. Fig. IB shows a genomic southern blot of tail DNA from three offspring digested with the restriction endonuclease EcoRI. Mouse number and genotype are labeled. (+)=wild type CD7, (-)=disrupted CD7. Lack of CD7 RNA was verified in Northern blot analysis of total RNA isolated from splenocytes and thymocytes of control (+/+) and CD7 deficient mice and probed with mouse CD7 cDNA (Fig. IC). Figures 2A and 2B. Functional responses of CD7 deficient splenocytes and thymocytes to mitogenic and antigenic stimulation. Fig. 2 A shows γ-IFN in pg/ml in culture supernatants from splenocytes stimulated in vitro as labeled. Fig. 2D shows TNF-α in pg/ml in culture supernatants from splenocytes stimulated in vitro as labeled. n= 8-/-, 8+/+ (mitogen-stimulated supernatants) and 6-/-, 7+/+ (tetanus toxoid- stimulated supernatants). *=p<0.02. Figures 3 A and 3B. CTL activity in CD7 deficient mice, antigen primed splenocytes. Fig. 3A shows the % specific lysis of 51 Cr labeled EG.7ova target cells by ovalbumin peptide-primed splenocytes from CD7 deficient and C57BL/6 control mice. Background killing of control EL-4 targets not expressing ovalburrύn is also shown. Fig. 3B shows the data in Fig. 3A expressed in lytic units (n=6 -/-, 6+/+) (*=p<0.03, **=p<.01). Figures 4A and 4B. Leukocyte subsets in CD7 deficient mice. Fig. 4A. Absolute numbers of thymocytes, splenocytes and bone marrow cells from 3 month old CD7 normal (+/+) and CD7 deficient (-/-) mice. Cells from 6 mice per group were prepared and counted as described herein. Data represent the absolute numbers of cells ± SEM. Fig. 4B. Thymocyte subset analysis from 3 month old CD7+/+ and CD7-/- mice. Thymocytes were stained with PE-conjugated anti-CD4 (Caltag, Burlingame, CA) and Tri Color-conjugated anti-CD8 (Caltag), and assayed by two-color flow cytometry on a Becton-Dickinson FACS-Star Plus Flow Cytometer. Data represent the absolute number of cells ± SEM per thymocyte subset (n=6 in each group). Figure 5. Survival of CD7 deficient mice treated with high dose LPS. C57BL/6 (CD+/+, solid circles, n=16) and CD7-/- mice (open circles, n=30) were injected i.p. with LPS (lOOmg/Kg). C57BL/6 (CD7+/+, open squares, n=3) and CD7-/- mice (solid triangles, n=3) were injected i.p. with saline. Mortality was assessed everyday for 7 days. *p<0.001 determined by Chi Square test. Figure 6. Survival of CD7 deficient mice treated with low dose LPS and D-galactosamine. C57BL/6 (CD7+/+, solid circles, n=10) and CD7-/- mice (open circles, n=10) were coinjected i.p. with LPS (lμg) and D-galactosamine (8mg) in saline. Mortality was assessed for 3 days. C57BL/6 (CD7+/+, n=3) and CD7-/- mice (n=3) were injected i.p. with LPS (lμg) or D-galactosarrύne (8mg) and no mortality was observed at 73 hours. *p<0.001 determined by Chi Square test. Figure 7. CD7 deficient animals have a significant reduction in TNFα response in vivo when treated with low dose LPS and D-galactosamine. Serum concentrations of TNFα at different time points after injected of LPS and D-galactosamine in C57BL/6 (CD7+/+) and CD7-/- mice, as described in Figure 6. One mouse at each time point was euthanized and the sera were collected for TNFα cytokine ELISA (Genzyme, Cambridge, MA). Data shown are a representative experiment of three experiments. Mean and standard deviation were determined be combining the results from the three experiments. Mean and standard deviation were determined by Student's T-test. Figure 8. Absence of an IFN-γ response in vivo by CD7 deficient animals treated with low dose LPS and D-galactosamine. Serum concentrations of IFN-γ at different time points after injection of LPS and D-galactosamine in C57BL/6 (CD7+/+) and CD7-/- mice, as described in Figure 6. One mouse at each time point was euthanized and the sera were collected for IFN-γ cytokine ELISA (Genzyme, Cambridge, MA). Data shown are a representative experiment of three experiments. Mean and standard deviation were determined be combining the results from the three experiments. * p<0.001 was determined by Student's T-test. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to CD7, particularly human CD7 which is a 40 kilodalton member of the Ig gene superf amily that is expressed on mature T and NK cells and on T, B, NK and myeloid lineage precursors in bone marrow (Haynes et al, Proc. Natl. Acad. Sci. USA 76:5829-33 (1979); Chabannon et al, J. Immunol. 149:2110-2113 (1992); Grumayer et al, Blood 77:64-8 (1991); Aruffo et al, EMBO J. 6:3313-6 (1987); Barcena et al, Blood 82:3401-14 (1993); Rabinowich et al, J. Immunol. 152:517-26 (1994); Cicuttini et al, J. Immunol. 151:29-37 (1993); Haynes et al, Immunol. Today 10:87-91 (1989)). CD7 is expressed early in T lymphocyte development before CD2, CD3, TCR, CD4 and CD8 (Haynes et al, Immunol. Today 10:87-91 (1989); Haynes et al, J. Exp. Med. 181:1445-1448 (1995)). Various studies suggest CD7 may be involved in T and NK cell activation and/ or adhesion. In TCRαβ T cells, crosslinking of CD7 leads to rapid increases in intracellular calcium concentrations (Ledbetter et al, Proc. Natl. Acad. Sci. USA 84:1384-8 (1987); Vandenberghe et al, J. Immunol. Methods 127:197-205 (1990)), induces CD7 cytoplasmic domain association with phosphotidylinositol-3- kinase (Lee et al, Internatl. Immunol. 8:1195-203 (1996); Ward et al, Eur. J. Immunol. 25:502-7 (1995)), synergizes with submitogenic amounts of CD3 monoclonal antibodies to induce T cell activation (Carrera et al, J. Immunol. 141:1919-24 (1988); Jung et al, Cell. Immunol. 141:189-9914 (1992)), modulates T cell adhesion (Shimizu et al, J. Exp. Med. 175:577 (1992)), and increases T cell IL-2 production and IL-2 receptor expression (Jung et al, Cell. Immunol. 141:189-9914 (1992)). In TCRγδ T cells , crosslinking of CD7 leads to cell activation and induction of TNF-α, TNF-β and GM-CSF mRNA (Carrel et al, Eur. J. Immunol. 21:1195 (1991)). In NK cells, crosslinking of CD7 leads to cell proliferation, IFN-γ production, increased ability to kill NK targets and increased cell adhesion to fibronectin via bl integrins (Rabinowich et al, J. Immunol. 152:517-26 (1994); Rabinowich et al, J. Immunol. 153:3504-13 (1994)). In bone marrow T and myeloid progenitor cells, crosslinking of CD7 leads to GM-CSF production (Hou et al, Blood 88:124-9 (1996)). The human CD7 gene has been cloned and distribution of human CD7 is known (Schanberg et al, Proc. Natl. Acad. Sci. USA 88:603 (1991); Haynes et al, Proc. Natl. Acad. Sci. USA 76:5829 (1979); Aruffo et al, EMBO J. 11:3313 (1987)). The present invention results, in part, from the realization that abnormalities in CD7 expression or function are involved in predisposition to forms of autoimmune disease, specifically human autoimmune disease. In addition, the invention results from the realization that CD7 regulates LPS-shock and other TNFα and IFN-γ mediated inflammatory and infectious disease syndromes. These realizations make possible methods of identifying individuals at increased risk of developing an autoimmune disease, for example, rheumatoid arthritis, and TNFα and IFN-γ mediated inflammatory and infectious disease syndromes, such as LPS-shock.. Appreciation for the involvement of CD7 in these diseases also makes possible methods of treating such diseases and methods of identifying agents for their potential use in pharmacological intervention strategies. Screening for Individuals at Increased Risk: The present invention relates to methods of identifying individuals at increased risk for developing an autoimmune disease or a TNFα and IFN-γ mediated inflammatory or infectious disease, based on polymorphisms in the CD7 gene, either alone or in addition to polymorphisms in IL-1 converting enzyme, ICAM-1, IFN-γ receptor, TNFRI, TNFRII, or CD28. IL-1 converting enzyme (ICE) deficient mice, ICAM-1, IFN-γ receptor deficient mice and TNFRI or II deficient mice all have been reported to be resistant to LPS induced shock (Gutierrez-Ramos et al, Immunol. Today 18:329-334 (1997), Pfeffer et al, Cell 73:457 (1993), Kamijo et al, J. Exp. Med. 178:1435-1440 (1993)) (Table 3). A first such method involves the use of genetic diagnostic techniques. For example, general mutation screening of the CD7 gene of a subject can be performed by such methods as direct sequencing or mismatch detection (see generally Nay lor et al, Human Molec. Genet. 2:11 (1993); Giannelli, Bailliere's Clinical Haematology 2/4:821 (1989); Green et al, Amer. J. Human Genet. 45(4 Suppl):A191 (1989); Dundar et al, J. Medical Genet. 32:154 (1995)) or single strand conformation polymorphism (SSCP)(Orita et al, Proc. Natl. Acad. Sci. USA 86:2766 (1989), Orita et al, Genomics 5:874 (1989), Ainsworth et al, Methods Molec. Biol. 31:205 (1994), Beier, Mammalian Genome 4:627 (1993), Hayashi et al, Human Mutation 2:338 (1993), Fan et al, Mutation Research 288:85 (1993), Hayashi, Genet. Anal. Tech. Appl. 9:73 (1992), Hayashi, PCR Methods and Applications 1:34 (1991)) or restriction fragment length polymorphism (RFLP) analysis (Boyle, ed. Current Protocols Human Genet. 1:2 (1994), Pourzand et al, Mutation Res. 288:113 (1993), Narayan, Ann. Clin. Lab. Sci. 21:291 (1991), Vineis et al, Int. J. Can. 47:26 (1991), Zijlstra et al, Prog. Clin. Biol. Res. 347:187 (1990)) after polymerase chain reaction (PCR) (Nollan et al, Scand. J. Clin. Lab. Invest. 221:116 (1995); Reischl et al, Molec. Biotech. 3:55 (1975); Gibbs, Curr. Opin. Biotech. 2:69 (1991); Arnheim, Genet. Engin. 12:115 (1990); Taylor et al, Curr. Opin. Biotech. 6:24 (1995)) or reverse transcription (RT)-PCR (Cane et al, Electroph. 16:329 (1995); Salmon, Diag. Molec. Path. 4:2 (1995); Holodnig, Clin. Lab. Med. 14:335 (1994); Briggs et ak, Sem. Nephr. 13:2 (1993)). Suitable methods for PCR amplification have been described (see, for example, PCR Technology: Principles and Applications for DNA Amplification, ed HA Erlich, Freeman Press, New York, NY (1992); PCR Protocols: A Guide to Methods and Applications, eds. Innis, Gelfland; Snisky, and White, Academic Press, San Diego, CA (1990); Mattila et al, Nucleic Acids Res. 19:4967 (1991); Eckert et al, PCR Methods and Applciations 1:17 (1991); PCR eds. McPherson, Quirkes, and Taylor, IRL Press, Oxford; and U.S. Patent 4,683,202). A further method for detection of genetic mutations is the amplification refractory mutation system (ARMS) described in EPO 0 332435. Sources of test samples include blood and buccal mucosa swabs. Alternatively, tissue samples from a subject can be screened for a mutant form of CD7 by in situ hybridization (Zhao et al, Cancer Res. 53:2901 (1993), Uner et al, Hemat./ Oncol. Clin. North America 8:771 (1994), Hopman et al, Molec. Biol. Rep. 19:31 (1994), Bentz et al, Leukemia 8:1447 (1994), Brown et al, J. Urology 152:1157 (1994), DeLellis, Human Path. 25:580 (1994)) and/ or immunohistochemistry (Jirtle et al, Carcinogenesis 15:1473 (1994), Calonje et al, Hemat./Oncol. Clin. North America 9:657 (1995), Szymas, Folia Neuropathol. 32:209 (1994), Yu et al, Histochem. J. 24:121 (1992), Miettinen, Ann. of Med. 25:221 (1993), Callea et al, Liver 12(4 Pt 2):290 (1992), Rahman et al, Malaysian J. Path. 13:17 (1991)). Suitable nucleic acid probes include CD7 cDNA (Aruffo et al, EMBO J. 11:3313 (1987)). Monoclonal antibody probes include, but are not limited to 3A1 (Haynes et al, Proc. Natl. Acad. Sci. USA 76:5829 (1979)). The invention includes antibodies specific for wild type and mutant forms of CD7 produced, for example, using art recognized techniques. Oligonucleotides specific for mutant CD7 gene forms can be identified and are also within the scope of the invention. Selecting Agents for Therapeutic Activity: Compounds can be selected for their ability to modulate (e.g., inhibit) CD7 expression and/ or function and thereby to serve as agents to prevent or treat autoimmune diseases and TNFα and IFN-γ mediated inflammatory and infectious diseases and disorders, including LPS-induced shock and associated tissue damage. Suitable compounds can be selected by assessing the ability of a test compound to inhibit antigen-specific IFN-γ production in vitro or assessing the ability of a test compound to inhibit LPS-induced death in vivo. Test compounds can be screened using, for example, in vitro cultures of human thymocytes, splenocytes, peripheral blood mononuclear cells (PBMC), purified T cells, lymph node cells or cell lines that express CD7, for their ability to modulate (e.g., decrease) CD7 expression by flow cytometry or cell-based ELISA using monoclonal antibodies to CD7 such as 3Ale (Coulter, Miami, FL). Test compounds can be screened for their ability to inhibit CD7-mediated functions by assessing for their ability to inhibit IFN-γ or TNF-α production by human PBMC stimulated with tetanus toxoid or their ability to inhibit death, liver injury, IFN-γ production or TNF-α production in both the high-dose LPS-induced and low-dose LPS+D-galactosamine models of shock in Jackson Labs C57BL/6 mice. CD7 deficient mice also have defects in CD3 monoclonal antibody triggered IFN-γ production in thymocytes. Accordingly, test compounds can be selected for ability to inhibit IFN-γ production triggered by CD3 monoclonal antibodies in C57BL/6 wild-type thymocytes. Examples of suitable inhibitors include a soluble recombinant CD7 protein (that is, any protein with the extracellular domain of CD7 with or without other attached amino acids to aid in the detection or purification of soluble CD7, such as 6-His tag, irnmunoglobulin heavy chain, secreted alkaline phosphatase, etc.) or mimetope thereof (eg., a proteinaceous imetope, or a DNA or RNA rriimetope, or other organic molecule) (see Szostak, TIBS 17:89 (1992); Tsai et al, J. Immunol. 150:1137 (1993)). Antibodies to CD7 (or antigen binding fragments thereof) can also be used, such as the monoclonal antibody 3Ale Compounds selected in accordance with the above-described approaches can be formulated with a pharmaceutically acceptable carrier or diluent to form a composition. Such compositions can be in dosage unit form. Compositions suitable for administration by, for example, injection, are, advantageously, sterile. The appropriate administration regimen will vary with the patient and the compound used and can readily be established by one skilled in the art. The data presented in the Examples that follow indicate that CD7 is important in a myriad of inflamrnatory, autoimmune and infections disease-mediated tissue damage settings in which TNFα and INF-γ are key mediators. Such settings include but are not limited to, malaria, Listeria monocytogenese infections, tuberculosis, leprosy infections, Herpes simplex and other Herpes virus infections, as well as inflamrnatory bowel disease, sarcoidosis, and rheumatoid arthritis. In addition, sensitivity to inhaled LPS has been implicated in causing environmental lung disease and asthma in certain humans, leading to lung LPS-induced inflammatory mediator release (10, 11). Compounds that modulate (e.g. inhibit) CD7 function and/ or expression can be used as useful therapeutics under these conditions as well. Optimum amounts to be administered depend on the agent, the patient and the effect sought. Such amounts can be readily determiend by one skilled in the art. It will be appreciated that alteration in the CD7 gene can protect in the above-described settings as well as other alterations in the CD7 gene can predispose to enhance CD7 activation and lung TNF-α and IFN-γ release leading, for example, to more severe LPS-induced asthma. Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow. (It should be noted that Bonilla et al (Internatl. Immunol. 9:1875 (1997)) have made CD7 deficient mice but did not observe the abnormalities described herein.) EXAMPLE I Characterization of CD7 Deficient Mice Experimental details Construction ofCD7 deficient mice. Disruption of the mouse CD7 gene was accomplished in 129 strain embryonic stem (ES) cells using described methods (Mohn, A., and Koller, B. H. 1995. Genetic manipulation of embryonic stem cells, In, DNA Cloning 4: Mammalian systems (D.M. Glover and B.D. Hames, Eds.) pp. 143-184. Oxford Univ. Press, New York). A homologous recombination cassette was constructed using the pPNT vector (Tybulewicz et al, Cell 65:1153-63) by inserting 1.6kb of the mouse CD7 gene ending midway through exon 3 (corresponding to the predicted membrane proximal extracellular domain), with the addition of an in-frame stop codon 5' of the neomycin resistance (neo ) gene (Figure 1A). This fragment was generated using PCR primers containing Notl restriction sites on both ends (5' primer S'-ATAAGAATGCGGCCGCTGTTGTAGCCAGAGTGGCTG-S' and 3' primer 5'- ATAAGAATGCGGCCGCTTACTGGGATCTGTATGCTTCTTGG-3') with the addition of an in-frame stop codon within exon 3. A 3' fragment of the mouse CD7 gene, beginning midway through intron 3 at the EcoRI restriction endonuclease site and extending to 2.5 kb 3' of exon 4, was inserted into the EcoRI site of the pPNT vector 3' of the neo gene and 5' to the herpes thymidine kinase gene. This fragment was derived from subcloned genomic DNA. ES cell clones with homologous recombination of the disrupted allele of CD7 were identified via Southern Blot analysis (Sambrook, J.E.F., Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual (1987). Cold Spring Harbor Press, Cold Spring Harbor, New York ) of Eco Rl digested genomic DNA due to a 2 kb restriction fragment length polymorphism resulting from the insertion of the neo gene. The probe used for Southern blotting was a 400 bp PCR generated fragment of genomic DNA immediately adjacent 5' to exon 1 (used in the homologous recombinant construct). The sequence of the primers used to generate this probe were: 5' primer 5'- TCAACTCCCTCCCGGCCTCT-3' and 3' primer 5'- AGC ACTGCCTGCTG AGTC A-3' . Chimeric mice were generated by insertion of 129 strain ES cells into C57BL/6 recipient embryos (Mohn, A., and Koller, B. H. 1995. Genetic manipulation of embryonic stem cells, In, DNA Cloning 4: Mammalian systems (D.M. Glover and B.D. Hames, Eds.) pp. 143-184. Oxford Univ. Press, New York). These chimeric offspring were backcrossed to C57BL/6 mice, and germline transmission of the disrupted CD7 allele was identified by Southern blot analysis of DNA purified from tail tissue and digested with EcoRI endonuclease. Tail DNA was extracted by incubating tail tissue in buffer containing 50 mM Tris pH 8.0, 100 mM EDTA, 0.5% SDS, and 1 mg/ml proteinase K at 55°C overnight. The samples were phenol/ chloroform extracted, ethanol precipitated with 3M sodium acetate pH 6.0 and resuspended in TE (lOmM Tris pH 8.0, 1 mM EDTA). All subsequent offspring were also screened for the disrupted CD7 allele by Southern blot analysis of DNA purified from tail tissue and digested with EcoRI endonuclease. Heterozygous offspring were intercrossed to generate CD7 deficient (-/-) and control (+/+) mice. Antibodies and reagents. Monoclonal antibodies Thyl.2-FITC (Anti- Thy-1, Becton Dickinson, Mountain View, CA), Ly5-FITC (anti-B220, Pharmingen, San Diego, CA), Lyt2-FITC (anti-CD8, Becton Dickinson, Mountain View, CA), and L3T4-PE (anti-CD4, Becton Dickinson, Mountain View, C A) CD8a-TRI (Caltag, South San Francisco, C A) were used at saturating titers for flow cytometry. Monoclonal antibodies PK136-FITC (anti-NKl.l, ATCC #HB191) and OX-12 (anti-rat kappa chain) ( Sera-Labs, Crawley Down, Sussex, England), were produced from hybridomas cultured in serum free media (SFM, Gibco, Grand Island, NY) and purified using affinity chromatography over a Staph protein A/G column (Pierce, Rockford, NY). Purified PK136 and OX-12 were subsequently fluorescein conjugated and saturating titers determined. Monoclonal antibodies E13 (Spangrude et al,./ Immunol 142:425-30 (1989)) (anti-granulocyte lineage), Ter 119 (Ikuta et al, Cell 62:863-74 (1990)) (anti-erythroid lineage) and F4/80 (ATCC #HB 198, Rockville, MD) (anti-macrophage subset) were cultured in DMEM supplemented with 10% FCS and tissue culture supernatants were used in saturating concentrations in flow cytometry. Indirect immunofluorescence assays andfloiv cytometr Analysis of cell surface phenotype was performed in both direct- and indirect-IF assays (Haynes et al, N. Engl. J. Med. 304:1319-23 (1981)). Cell suspensions were analyzed using a FACStar plus flow cytometer (Becton-Dickinson, Moutain View, CA). Preparation of cell suspensions. For all flow cytometry experiments, freshly obtained suspensions of peripheral blood and peritoneal wash cells were used. Femurs were dissected and flushed with 2ml ice cold RPMI 1640 to obtain bone marrow. Splenocytes and thymocytes were teased into RPMI media using tuberculin syringe plungers. Splenocytes were subsequently isolated by Hypaque-Ficoll (Mishell, B.B., and S.M. Shiigi. Selected Methods in Cellular Immunology (1980) W. H. Freeman and Co. New York) or Fico-Lite (Atlanta Biologicals, Atlanta, GA) density centrifugation. Peripheral blood was collected from the retroorbital plexus using a heparinized pasteur pipette while mice were under isoflurane anesthesia and PB erythrocytes were removed with Irnmunolyse™ (Coulter, Miami, FL) per manufacturer protocol. Cell suspensions were counted on a Coulter™ counter (Coulter, Miami, FL). Northern blot analysis ofCD7 mRNA expression Total cellular RNA was collected from control and CD7 deficient mice by lysis of freshly teased splenocytes and thymocytes using the TRIzol (Gibco-BRL Grand Island, NY) reagent per manufacturer protocol. 30 mg of total RNA was run on a 1.2% formaldehyde containing agarose gel in MOPS buffer as previously described (Sambrook, J.E.F., Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual (1987). Cold Spring Harbor Press, Cold Spring Harbor, New York). After transfer to Nylon membrane and UV crosslinking, this RNA was probed with mouse CD7 cDNA using QuickHyb (Stratagene, La Jolla, CA) per manufacturer protocol. Splenocyte mitogenic responses. For these mitogenic proliferation assays, single cell suspensions of freshly teased and Ficoll-Hypaque isolated splenocytes (Fico-Lite, Atlanta Biologicals, Atlanta, GA) were adjusted to 1X10 6 cells/ ml in RPMI 1640 supplemented with 10% fetal calf serum (FCS)(Hyclone Labs, Logan, UT) 5 X 10 "5 M 2-mercaptoethanol and lOμg/ml gentamycin. 1x10 cells (lOOμl) were incubated in the presence of either media, anti-CD3(2CH[32]), anti-CD3 plus anti-CD28 (37.5 [33]), phytohemagglutinin (PHA) (Murex Diagnostics Ltd., Dartford, England), conconavalin A (Con- A) (Sigma, St. Louis, MO) or lipopolysaccharide (LPS) (Sigma, St. Louis, MO) in round-bottom 96-well microtiter plates in triplicate and incubated at 37° C in a 10% C02 in air humidified environment for three (PHA) or four days. Four hours prior to harvesting, 0.4mCi H-thymidine in 20ml media was added to each well. Cells were harvested onto glass filters using a PHD sample harvester. Incorporation was determined by placing the filters in CytoScint scintillation fluid (ICN, Costa Mesa, CA) and counting with a scintillation counter (Tri-Carb 4000, United Technologies Packard, Downers Grove, IL). Thymocyte mitogenic responses. For these mitogenic proliferation assays, single cell suspensions of freshly teased thymocytes were adjusted to 1X10 6 cells/ml in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (Hy clone Labs, Logan, UT), 5 X 10 M 2-mercaptoethanol, 5ng/ml rIL-2 (R&D Systems, Minneapolis, MN) and lOmg/ml gentamicin. 1x10 cells (100 ml) were incubated in the presence of either media, anti- CD3(2C11[32]), phytohemagglutinin (PHA) (Murex Diagnostics Ltd., Dartford, England) or Con-A (Sigma, St. Louis, MO) in round-bottom 96- well microtiter plates in triplicate and incubated at 37° C in a 10% C02 in air humidified environment for three (PHA) or four days. Four hours prior to harvesting, 0.4mCi H-thymidine in 20ml media was added to each well. Cells were harvested onto glass filters using a PHD sample harvester. Incorporation was determined by placing the filters in CytoScint scintillation fluid (ICN, Costa Mesa, CA) and counting with a scintillation counter (Tri-Carb 4000, United Technologies Packard, Downers Grove, IL). Tetanus toxoid immunizations. Mice were immunized with 50mg tetanus toxoid (Wyeth-A erst Lab, Marietta, PA) in complete Freund's adjuvant (CFA) (Sigma, St. Louis, MO) on day 0 and boosted with 50mg tetanus toxoid in incomplete Freund's adjuvant (IF A) (Sigma, St. Louis, MO) on day 28 or 35 after the primary immunization. Mice were anesthetized with ketamine/xylazine for all immunizations. Sample collection. Blood was collected from the retroorbital plexus using a heparinized Natelson capillary tube (Baxter Healthcare Corporation, McGaw Park, IL) while mice were under isoflurane anesthesia. All samples were stored at -20 °C until assayed for anti-tetanus toxoid antibodies. ELISA. Enzyme-linked immunosorbent assay (ELISA) was used to determine the presence of anti-tetanus toxoid antibodies in serum samples. TT was suspended in CBC buffer (15 mM Na2Cθ3, 35 mM NaHCθ3, pH 9.6) at a concentration of 3mg/ml and plated to 96 well microtiter plates (Costar #3590, Cambridge, MA) at 50ml/ well. After overnight incubation at 4 °C, the contents of the wells was discarded and blocking buffer (CBC with 3% non-fat dry milk) was added at 200ml/ well. After incubating at room temperature for two hours, plates were stored at " 20 °C until used. ELISA plates were washed four times with ELISA wash buffer (PBS, 0.05% Tween-20, 0.1% sodium azide) before the addition of samples. All samples were diluted in serum diluent (PBS, 2.5% bovine serum albumin, 2.5% nonfat dry milk, 5% normal goat serum, 0.1% sodium azide, 0.05% Tween-20) and added to ELISA plates at 100ml/ well. After overnight incubation at 4 °C, plates were washed four times with ELISA wash buffer before the detection antibody was added. Alkaline phosphatase conjugated, goat anti- mouse IgG, -IgA, or -IgM (Southern Biotechnology Associates, Birmingham, AL) was diluted 1:1,000 (PBS, 0.05% Tween-20, 0.5% bovine serum albumin) and used as the detection antibody (100ml/ well). After incubation at room temperature for three hours, plates were washed 4 times with ELISA wash buffer and reacted with the alkaline phosphatase substrate p-nitrophenyl phosphate. After a 10 minute incubation, plates were read at 405 ran on a Titertek Multiscan Plus plate reader. Statistical analysis. The Student's t-test was used to determine the significance of differences between groups. NK cell activity. NK cell cytotoxicity was determined using a 4 hour 51 Cr release assay using YAC-1 mouse lymphoma cells as targets. Mice were injected i.p. with lOOmg poly I:C (Sigma, St. Louis MO) 24 hours prior to splenocyte harvest. Splenic mononuclear cells were obtained by Fico-Lite (Atlanta Biologicals, Atlanta, GA) density centrifugation. YAC-1 cells ( (33>X10 cells in 3ml medium) were labeled by addition of 300mCi Na2 51, Crθ4 (NEN/DuPont, Boston, MA) at 37°C for 4 hours. After extensive washing 5 X 10 3 labeled YAC-1 cells in 100ml were added to U-bottom 96- well plates. Splenic mononuclear effector cells were then added at 100:1, 33:1 and 10:1 effectoπtarget ratios in 100ml in triplicate. The plates were then spun at 200X G. for 3 min and incubated at 37° C for 4 hours in a humidified 5% Cθ2 atmosphere. After incubation 25ml of supernatant was harvested, plated onto a Luma-plate (Packard, Downers Grove, IL) and counted on a gamrna-counter. Control for spontaneous release using labeled YAC-1 cells in media only and for maximal release release using labeled YAC-1 cells in 1% Triton X-100 were also plated in triplicate. The percentage of 51 Cr release from target cells was calculated using the following formula: % specific lysis= 100((cpm experimental release - cpm spontaneous release)/ (cpm detergent lysis-cpm spontaneous release). CTL assays. Antigen specific CTL effector activity was assessed using splenocytes from ovalbumin peptide primed mice as previously described (Porgador et al, J. Immunol. 158:834-41 (1997)). CD7 -/- and C57BL/6 control mice were immunized intranasally with 60 μg ovalbumin peptide plus 0.5mg cholera toxin subunit (List Biological, Campbell, CA) in 15ml of PBS. This immunization was administered four times at 7 day intervals. 14 days after the final imrnunization, splenic mononuclear cells were Ficoll density separated (Fico-Lite, Atlanta Biologicals, Atlanta, Ga.) and placed in culture at 2 X 10 6 cells/ ml with 2 X 10 5 cells/ ml g-irradiated EG.7ova (Moore et al, Cell 54:777-85 (1988)) stimulator cells for 6 days. After culture, CTL activity was assessed with a 4 hour 51 Cr release assay using EG.7ova (Moore et al, Cell 54:777-85 (1988)), EL-4 (ATCC TIB 39) or P815 (ATCC TIB 64) mouse lymphoma cells as targets. These cells (1.5X10 cells in 1.5ml medium) were labeled by addition of 150mCi Na2 51 Crθ4 (NEN/DuPont, Boston, MA) at 37°C for 4 hours. After extensive washing 5 X 10 labeled cells in 100ml were added to U-bottom 96-well plates. Splenic mononuclear effector cells were then added at 100:1, 33:1 and 10:1 effector:target ratios in 100ml in triplicate. The plates were then spun at 200Xg for 3 min. and incubated at 37° C for 4 hours in a humidified 5% Cθ2 atmosphere. After incubation 25ml of supernatant was harvested, plated onto a Luma-plate (Packard, Downers Grove, IL).and counted on a gamma-counter. Control for spontaneous release using labeled EG.7ova cells in media only and for maximal release release using labeled EG.7ova cells in 1% Triton X-100 were also plated in triplicate. The percentage of 51 Cr release from target cells was calculated using the following formula: % specific lysis= 100 (cpm experimental release - cpm spontaneous release)/ (cpm detergent lysis-cpm spontaneous release). Lytic units were defined as the number of splenocytes required to achieve 30% specific lysis as previously reported (Roberts et al, Cancer Res. 47:4366-71 (1987)). Cytokine quantification. Quantities of cytokines present in culture supernatants were assessed using commercial sandwich cytokine ELISA assays per manufacturer protocol. IL-4, IL-10 and g-IFN ELISA assays were obtained from Pharmingen(San Diego, CA) while TNF-a, was obtained from Genzyme Corp. (Cambridge, MA). 3 H-Thymidine incorporation assays. Antigen specific proliferative responses were assessed using splenocytes from tetanus toxoid primed 3 mice in H-thymidine incorporation assays. For these assays, single cell suspensions of freshly teased spleen cells were incubated with ammonium chloride to lyse erythrocytes and were subsequently adjusted to 1X10 cells/ ml in RPMI 1640 supplemented with 10% FCS. lxlO 5 cells (100ml) were incubated in the presence of either media, or tetanus toxoid at lOOmg/ml, lOmg/ml or lmg/ml in round-bottom 96-well microtiter plates in triplicate and incubated at 37° C in a 10% C02 in humidified air 3 environment for six days. Twelve hours prior to harvesting, 0.4 mCi H- thymidine was added to each well. Cells were harvested onto glass filters using a PHD sample harvester. Incorporation was determined by placing the filters in CytoScint scintillation fluid (ICN, Costa Mesa, CA) and counting with a scintillation counter (Tri-Carb 4000, United Technologies Packard, Downers Grove, IL). Culture supernatants from identical conditions were harvested for cytokine analysis. Results Construction and initial characterization ofCD7 deficient mice. DNA from mice homozygous for the homologous recombinant CD7 gene was probed and characterized by Southern blot analysis (Figure IB). Absence of CD7 expression was documented by Northern blot analysis of thymus and spleen RNA, showing CD7 mRNA present in normal, +/+ mice and absent in CD7 deficient, -/- mice (Figure IC). These results were confirmed by RT- PCR analysis, where no full length CD7 cDNA was detectable. CD7 deficient mice bred normally, and histologic analysis of autopsy tissues (thymus, spleen, lymph node, brain, stomach, small intestine, large intestine, heart, skeletal muscle, salivary gland, kidney and pancreas) were normal. In particular, thymic cortical and medullary areas and B cell follicles and germinal centers in lymph node and spleen were present and normal. Thus, CD7 deficient mice did not have a SCID syndrome. Next, the absolute numbers of lymphoid subsets in CD7 deficient, -/- and normal, +/+ littermate controls in bone marrow, spleen, peripheral blood and thymus were determined. Whereas spleen, peripheral blood and bone marrow cell numbers were normal in CD7 deficient mice, thymocyte numbers in 3 month old were increased 60% compared to +/+ normal littermates (77 X 10 6 ± 9 X 10 6 CD7 deficient thymocytes vs 48 X 10 6 ± 6 X 10 normal +/+ thymocytes, p<0.02, n=6). However, the increase in CD7 deficient thymocyte numbers was transient, since 4 week, 6 month and 12 month old CD7 deficient mice did not have increased thymocyte numbers compared to control mice. Analysis of lymphoid subsets in spleen and peripheral blood showed no differences in percentage or absolute number of T-cells, B-cells, monocytes/macrophages, or NK cells (Tables 1 and 2), nor were differences noted in bone marrow granulocyte-, erythroid-, or B- lineage subpopulations. Table 1 Comparison of Lymphocyte Subsets in CD7 Deficient and Control Mouse Peripheral Blood CΛ c Average Total LY-5+ PBMC % Thy l.2 BPMC CD4+ PBMC CD8+ PBMC F4/80 PBMC 09 Mouse PBMC Mononuclear Cell no. no. no. no. no. in Source Number (xlO'/mm) (xlO'/mm 1 ) % (xlO'/mm 1 ) % (xlO'/mm 1 ) % (xlO'/mm') % (xlO'/mm 1 ) c H CD7 deficient (N=6) 5.06 + .90 37 + 9 1.97 + .62 31 + 6 1.46 + .36 21 + .5 .99 + .29 11 + 3 .50 + .07 5 + 2 .26 + .08 m Control (N=6) 5.39 + 1.03 33 + 10 1.94 + .60 27 + 6 1.52 + .56 16 + 4 .97 + .41 8 + 2 .43 + .18 4 + 2 .22 + .1 en x m m Shown is a quantitative analysis of peripheral blood mononuclear cell (PBMC) subsets as defined by mouse Thy 1 (T cell), mouse Ly-5 (B-cell). F4/80 H t (monocyte/macrophage) and mouse CD4 or CD8 (T cell subsets) expression. Data are expressed as average + standard error of the mean. The average total 31 σ- number of cells In each subset are also shown. Control mice were +/+ littermates of CD7 deficient -/- mice. C i m- ■ to O) Table 2 Comparison of Lymphocyte Subsets in CD7 Deficient and Control Mouse Spleen ω c DJ Mouse Spleen Average LY-5+ % Thy 1.2 CD4+ CD8+ F4/80+ CΛ PBMC Weight Total Cell Splenocytes Splenocytes Splenocytes Splenocytes Splenocytes H Source (grams) Number (xlO 7 ) % no. (xlO 7 ) % no. (xlO 7 ) % no. (xlO 7 ) % no. (xlO 7 ) % no. (xlO 7 ) C H m CD7 deficient (N=6) .063 + .018 4.01 ± 1.29 42 + 5 1.89 ± .63 50 ± 5 1.88 + .63 23 ± 2 .86 ± .30 21 + 4 .72 + .20 8 + 1 .28 ± .08 Λ Control (N=6) .068 + .016 3.40 + .73 43 + 3 1.5 + .39 43 + 2 1.47 + .33 20 + 2 .70 + .16 15 + 2 .47 + .08 9 + 1 .27 + .05 X m H Shown is a quantitative analysis of splenic lymphocyte subsets as defined by mouse Thy 1 (T cell), mouse Ly-5 (B-cell). F4/80 (monocyte/macrophage) and t mouse CD4 or CD8 (T cell subsets) expression. Data are expressed as average + standard error of the mean. The average total number of cells in each subset are c ι also shown. Control mice were +/+ littermates of CD7 deficient -/- mice. m r- In vivo antibody responses to antigen. CD28 is an Ig superfamily co- stimulatory molecule that, like CD7, associates with PI-3 kinase (Pages et al, Nature 369:327-9 (1994); Truitt et al, / Exp. Med 179:1071-6 (1994); Prasad et al, Proc Natl Acad Sci USA 91:2834-8 (1994); Aruffo et al Proc Natl Acad Sci U S A 84:8573-737-40). CD28 deficient mice, like CD7 deficient mice, developed normally and had normal lymphoid organs (Shahinian et al, Science 261:609-12 (1993)). However, CD28 deficient mice had defective B cell responses to antigen with a deficient of Ig isotype switching (Shahinian et al, Science 261:609-12 (1993)). In CD7 deficient mice, total serum Ig levels and serum Ig subclass levels (IgG, IgG2a, IgG2b, IgG3, IgA, IgM) were normal. In addition, when anti-tetanus toxoid specific antibody responses were tested, no differences were noted in serum anti-tetanus toxoid Ig isotypes between CD7 deficient and control mice. Thus, in CD7 deficient mice, no defects in T-dependent B cell Ig responses were apparent. H-thymidine incorporation and cytokine production assays ofCD7 deficient splenocytes and thymocytes. Since previous reports demonstrated a role for human CD7 as a co-mitogen for T lymphocyte mitogenic responses (Carrera et al, / Immunol 141:1919-24 (1988); Jung et al, Cell Immunol 141:189-9914 3 (1992)), H-thymidine incorporation responses by CD7 deficient splenocytes (to ConA, PHA, CD3, and CD3 + CD28 mabs) and thymocytes (to ConA, PHA and CD3 + rIL-2) were tested. In addition, proliferative responses to tetanus toxoid by splenocytes from CD7 deficient and control mice previously immunized with tetanus toxoid were assessed. Unlike the T cell triggering abnormalities seen in CD28 deficient mice (Shahinian et al, 3 Science 261:609-12 (1993)), no differences were found in H-thymidine incorporation between CD7 deficient and control mouse splenocytes or thymocytes in mitogen-stimulated or TCR-mediated T cell activation assays. Since work has demonstrated a role for CD7 in stimulation of cytokine production in human T cells (Carrel et al, Eur ] Immunol 21:1195-200 (1991)), cyokine levels in culture supernatants from mitogen and antigen stimulated splenocyte and thymocyte cultures described above were determined. Analysis of IL-4, IL-10, TNF-a and g-IFN supernatant levels demonstrated a significant difference between CD7 deficient and control mice only with g-IFN produced in the presence of tetanus toxoid-primed splenocytes stimulated in vitro by tetanus toxoid (0.7 ± 0.3 ng/ml vs 3.24 ± 0.7 ng/ml p<0.02, n=6-/-, 7+/+) (Figure 2A and 2B). g-IFN induced by CD3 stimulation of CD7 deficient splenocytes was also less than control; however, this difference did not reach statistical significance (g-IFN in CD7 deficient splenocytes, p=0.06). NK cell activity. Since CD7 has been implicated in regulating NK cell function (Rabinowich et al, / Immunol 152:517-26 (1994); Rabinowich et al, / Immunol 153:3504-13 (1994)), the ability of CD7 deficient splenocytes to lyse 51 Cr labeled YAC targets was tested. Splenocytes from CD7 deficient mice were found to be able to lyse YAC targets to the same degree as age- matched control mice, demonstrating that functional NK cells were present in CD7 deficient mice. Antigen-specific MHC Class I-restricted CTL activity. Next, the ability of CD7 deficient mice to generate CTL activity to the EG.7ova cell line expressing chicken ovalbumin was assessed, after immunization of CD7 deficient mice with an ovalbumin peptide in a system that has previously been demonstrated to generate CD8+, MHC Class I restricted CTL in MHC H2D b C57BL/6 mice (Porgador et al, / Immunol 158:834-41 (1997)). The CTL lytic activity in CD7 deficient mouse splenocytes was significantly decreased compared to control splenocytes at multiple effector:target ratios (Figures 3 A and 3B). Interestingly, supernatants of splenocytes after 7 days of in vitro culture with irradiated ovalbumin expressing cells showed equal g-IFN levels in splenocytes from CD7 deficient and control mice. Thus, deficiencies in g-IFN production in ovalbumin stimulated splenocyte bulk cultures cannot explain the decrease in ova-specific CTL activity in CD7- deficient spenocytes versus normal +/+ splenocytes. EXAMPLE II Susceptibility to LPS-induced Shock Two model systems have been used for the study of susceptibility to LPS-induced shock, a high dose LPS-induced shock model (LPS lOOmg/kg of body weight given IP), and a low dose LPS induced shock and liver necrosis model (LPS lμg + 8mg D-galactosamine IP) (Gutierrez-Ramos et al, Immunol. Today 18:329-334 (1997)). The high dose LPS-induced shock model is known to be mediated by IFN-γ and TNF-α as is the low dose LPS- induced shock model (Gutierrez-Ramos et al, Immunol. Today 18:329-334 (1997), Fantuzzi et al, J. Immunol. 157:291-296 (1996)). Moreover, the liver necrosis in the low dose LPS-induced shock model is dependent on IFNγ induced nitric oxide and FAS mediated liver cell necrosis (Gutierrez-Ramos et al, Immunol. Today 18:329-334 (1997), Fantuzzi et al, J. Immunol. 157:291- 296 (1996), (Galanos et al, Proc. Natl. Acad. Sci. USA 76:5939-5943 (1979)). Following challenge with high dose LPS, wild type C5BL/6 mice had an 80% mortality after 7 days, CD7 deficient mice only had a 20% mortality (p< 0.001) (Figure 5). In the low dose LPS challenge model, in which D- galatosamine sensitizes the liver to damage by low dose LPS (Fantuzzi et al, J. Immunol. 157:291-296 (1996) (Galanos et al, Proc. Natl. Acad. Sci. USA 76:5939-5943 (1979)), whereas C57BL/6 mice had 80% mortality at 72 hours after injection of LPS and D-galactosamine, CD7 deficient mice had 0% mortality ' (Figure 6). In this latter model, measurement of serum TNFα production demonstrated that TNFα production in response to LPS and D- galatosamine was decreased (Figure 7) throughout the first 6 hours after injection of LPS, and IFNγ was totally absent (Figure 8). Thus, CD7 deficient mice have defective release of IFN-γ and TNFα that protects them from the pathogenic effects of LPS with regard to shock. In addition, in the low dose LPS model, liver damage is markedly attenuated in CD7 deficient mice, suggesting that CD7 deficient mice also have deficient nitric oxide generation in response to LPS and D-galactosamine. Taken together, these data demonstrate that the CD7 molecule is important for mediation of LPS induced issue damage and shock, and is important for mediation of IFNγ, TNFα and likely nitric oxide triggered tissue damage. There is increasing evidence that inhalation of bacterial endotoxin (LPS) causes and/ or exacerbates hyperreactive lung disease or asthma (Michel et al, Clin. Exp. Allergy 21:441 (1991); Michel et al, Am. J. Respir. Crit. Care Med. 154:1641 (1996); Jagielo et al, Chest 110:263 (1996); Schwartz et al, Am. J. Resp. Crit. Care Med. 152:603 (1995)). Alterations in the CD7 gene may protect in this setting as well, or other alterations in the CD7 gene may predispose to enhanced CD7 activation and lung TNFα and IFN-γ release leading to more severe LPS-induced asthma. Thus, inhibition of CD7 function can be therapeutic for asthma via inhibition of lung macrophage and T cell TNFα and IFN-γ. All documents cited above are hereby incorporated in their entirety by reference. One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. Table 3 Sensitivity or Resistance to Death in the High Dose or Low Dose LPS-induced Shock Model Low Dose LPS + Mouse Strain High Dose LPS Model D-galactosarnine Model C57BL/6 Normal Sensitive Sensitive ICAM-1 Deficient Resistant Sensitive ICE Deficient Resistant Sensitive TNFRII Deficient Partially Resistant Sensitive TNFRI Deficient Sensitive Resistant IFN-γR Deficient Sensitive Resistant CD7 Deficient Partially Resistant Resistant

Claims: WHAT IS CLAIMED IS: 1. A method of identifying an individual predisposed to autoiirurtune disease comprising isolating a DNA-containing sample from said individual and screening said DNA in said sample for a mutation in the CD7 gene that results in said predisposition. 2. The method according to claim 1 wherein said autoimmune disease is rheumatoid arthritis. 3. The method according to claim 1 wherein said screening is effected using a method selected from the group consisting of direct sequencing, mismatch detection, single strand polymorphism analysis, restriction fragment length polymorphism analysis, amplification refractory mutation system analysis and in situ hybridization. 4. A method of treating a patient suffering from an autoimmune disease comprising administering to said patient an agent that inhibits CD7 expression or function in an amount sufficient to effect said treatment. 5. A method of identifying an individual predisposed to a condition mediated by TNFα or IFNγ comprising isolating a DNA- containing sample from said individual and screening said DNA in said sample for a mutation in the CD7 gene that results in said predisposition. 6. The method according to claim 5 wherein said condition is lipopolysaccharide (LPS)-induced shock. 7. A method of identifying an individual predisposed to an inflammatory condition or an infection comprising isolating a DNA- containing sample from said individual and screening said DNA in said sample for a mutation in the CD7 gene that results in said predisposition. 8. A method of treating or preventing a condition mediated by TNFα or IFNγ comprising administering to a patient in need thereof an amount of an agent that inhibits CD7 expression or function sufficient to effect said treatment or prevention. 9. The method according to claim 8 wherein said condition is LPS-induced shock. 10. A method of treating or preventing an inflammatory condition or an infection comprising administering to a patient in need thereof an amount of an agent that inhibits CD7 expression or function sufficient to effect said treatment or prevention. 11. A non-human animal the genome of which comprises a disrupted CD7 allele. 12. The anirnal according to claim 11 wherein said animal is a rodent. 13. A method of testing a compound for its ability to relieve symptoms associated with LPS-induced shock comprising contacting said compound with stimulated peripheral blood mononuclear cells (PBMCs) and deterrnining whether said compound inhibits IFNγ or TNFα production by said PBMCs, wherein a compound that effects said inhibition is capable of effecting said relief. 14. The method according to claim 13 wherein said PBMCs are human PBMCs stimulated by tetanus toxoid. 15. A method of screening a test compound for it ability to inhibit CD7 mediated functions comprising assessing the ability of said compound to inhibit death, liver injury, IFNγ production or TNFα production in a high-dose LPS-induced or low-dose LPS + D-galactosamine model of shock. 16. A method of screening a test compound for it ability to inhibit CD7 mediated functions comprising assessing the ability of said compound to inhibit IFNγ production triggered by CD3 monoclonal antibodies in C57BL/6 wild-type thymocytes.

Inventors: HAYNES BARTON F, LEE DAVID, PATEL DHAVALKUMAR D, HAYNES, BARTON, F., LEE, DAVID, PATEL, DHAVALKUMAR, D.
Applicants: HAYNES BARTON F, LEE DAVID, PATEL DHAVALKUMAR D, UNIV DUKE, DUKE UNIVERSITY, HAYNES, BARTON, F., LEE, DAVID, PATEL, DHAVALKUMAR, D.
Application year: 1999
Main classification: G01N33
IPC codes: C12Q1