WO1994008454A1 - Methods, compositions and screening assays relating to autoimmune disease - Google Patents

Abstract

Disclosed herein is the discovery that the insertion of a retroviral transposon of the ETn family into the fas apoptosis gene coding region underlies the 1pr/1pr rodent model of systemic autoimmune disease. These results establish a link between endogenous retrovirus expression and autoimmune disease. The present invention embodies both novel nucleic acid probes and new screening assays for use in the identification of agents for the treatment of autoimmune and lymphoproliferative diseases. The invention also contemplates new therapeutic strategies for autoimmune disease involving modulating the expression of retroviruses associated with tolerance-related or lymphocyte activation genes.

Description

DESCRIPTION

METHODS, COMPOSITIONS AND SCREENING ASSAYS RELATING TO AUTOIMMUNE DISEASE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to immunology and autoimmunity. Disclosed is the discovery that the faε apoptosis gene, a gene of importance in tolerance induction, is rendered dysfunctional by a retroviral insert. The invention describes novel nucleic acid probes and screening assays for use in the identification of candidate substances for the treatment of autoimmune and lymphoproliferative diseases.

2. Description of Related Art

The phenomenon of autoimmunity underlies the development of several common diseases, including many arthritis-linked disorders such as rheumatoid arthritis and systemic lupus erythematosus. Rheumatoid arthritis is a chronic, immune-mediated inflammatory disease that primarily affects the joints and their supporting structures, although the disease sometimes involves other organs and tissues such as the eyes, lungs, heart and skin (Harris, 1990) . This common disease produces profound morbidity and excess mortality in an estimated 1% of the population (Lawrence et al . , 1989).

Despite progress in understanding how the immune system works, the pathogenesis of autoimmune diseases still remains obscure. As a consequence, bar er treatment of these diseases remains elusive Although life expectancy has improved, due principally to ancillary services such as antibiotics, blood banks and control of disease complications, treatment remains non-specific relying upon corticosteroids and immunosuppressive drugs which have dangerous and life threatening side-effects. Significant progress clearly needs to be made in understanding the etiology and pathogenesis of autoimmune disease.

It is important to distinguish between autoimmunity and autoimmune disease. The former is often benign whereas the latter is potentially fatal. Autoimmunity is the presence of serum autoantibodies and is a normal consequence of aging, it is readily inducible by drugs or infectious agents, and is potentially reversible in that it disappears when the offending drug or agent is removed or eradicated. Autoimmune disease results from activation of self-reactive T and B cells which, following stimulation by genetic or environmental triggers, cause actual tissue damage.

The mechanism of autoimmune disease is not well understood, but such diseases may arise because of a defect in removing immune cells that react against the organism itself. The generation of self-reactive lymphocytes is a consequence of the immune system designed for immunorssponses against foreign antigens. However, in a normal immune system, the self-reactive cells generated are usually removed, either by clonal deletion or inactivation on interaction with body antigens, thereby triggering cell death.

Autoimmune disease is dependent upon at least four factors. Two of the major factors are genetic and viral. A third factor is endocrine, based on the ability of estrogen to promote autoimmune disease, whereas androgen acts as a natural immunosuppressive agent. These are physiological modulatory effects of sex hormones acting on normal immune responses, and explain the marked female predominance of autoimmune disease. The fourth factor is psychoneuroimmunologic (i.e. the influence of stress and neurochemicals on the immune response) . A common feature of these four factors is the ability to affect gene expression. Genes influence autoimmune disease through their classic role as immune response regulators acting primarily through the major histoco patibility complex (MHC) , but perhaps also through immunoglobulin (lg) and T cell receptors (TCR) .

Attempts to develop effective pharmacological agents for use in treating autoimmune diseases have not met with significant success, partly because the few animal models used for autoimmune studies are equally poorly understood. Some success has been achieved in studying autoreactive immunoglobulins which have been studied in transgenic mice expressing an antibody to murine red blood cells (Oka oto et al . , 1992). These mice, unlike normal mice, have large numbers of self reactive B lymphocytes so that it is relatively easy to follow the selection of these cells in vivo .

Another model system is the MRL-lpr/lpr mouse, a model of systemic autoimmune disease in which intrinsic defects of intrathymic T cell development have been noted (Cohen & Eisenberg, 1991; Zhou et al . , 1991; 1992). MRL- lpr/lpr mice have been reported to express endogenous retroviruses (Kreig et al . , 1991), but the relationship between such expression and the development of autoimmune disease remained to be elucidated prior to the present invention.

Programmed cell death or apoptosis is a fundamental mechanism in the development of the organism and occurs from e bryogenesis throughout life. Unfortunately, little is known about the process on the molecular level. Of the two mechanisms thought to be important, the more classical type of programmed death is thought to require activation of a set of genes that lead to DNA fragmentation and subsequent apoptotic morphological changes. One gene thought to be involved is PD-1 which is specifically expressed after induction by a cell death signal. PD-1 is a member of the lg supergene family that expresses a membrane receptor protein thought to associate with a tyrosine kinase.

A second membrane receptor-like protein, Fas, has also been reported to be involved in programmed cell death. A mutation in the faε gene has been implicated in the cause of the lymphoproliferative disorder seen in MRL-lpr/lpr autoimmune mice, however, the nature of the mutation has not yet been defined. Determining the nature of the faε defect which occurs in MRL-lpr/lpr mice would be a significant advance in that it would lead to an understanding of autoimmune disease such that effective treatment strategies could be designed.

An increased understanding of autoimmune processes will likely provide a significant insight into immune regulation in both health and disease and contribute to a broader understanding of the interaction of host and external factors in -the immune system. Furthermore, elucidating the molecular basis of autoimmune diseases is of great importance as, without such knowledge, screening for effective therapeutic agents for use in treating many relatively common disorders, including rheumatoid arthritis, will continue to be severely limited. Additionally, without an indepth knowledge of the molecular mechanisms underlying autoimmunity, the rational design of therapeutics will remain virtually impossible. SUMMARY OF THE INVENTION

By establishing the first link between endogenous retrovirus expression, defective apoptosis and autoimmune disease, the present invention seeks to overcome several drawbacks inherent in the art of autoimmune disease diagnosis and treatment. The discoveries disclosed herein concerning the inactivation of the faε apoptosis gene by an ETn retroviral insert have opened the way for the development of new molecular-based strategies for diagnosing and treating autoimmune and ly phoproliferative diseases.

This invention first provides novel nucleic acid segments and probes for use in molecular biological embodiments including diagnostic and screening assays. The inventors discovered that an ETn retroviral insert into the faε apoptosis gene is the molecular mechanism underlying the symptoms of an autoimmune mouse model. Accordingly, the nucleic acids of the present invention are generally purified nucleic acid segments, isolated free from total genomic DNA, which include a Fas cell surface protein-encoding sequence in combination with an ETn gene sequence, or the complements of such sequences.

— In certain embodiments, nucleic acid segments are provided which will encode an apoptosis-defective Fas cell surface protein (also termed Fas antigen) with an ETn amino acid sequence insert, such as a Fas protein including an ETn sequence insert within an extracellular domain. In one exemplary embodiment, the ETn gene sequence will be inserted at position 232 of the faε gene, in another embodiment the coding sequence for the Fas protein may also include an additional triplet at position 240. Nucleic acid segments including an ETn insert may be exemplified by segments which have a sequence in accordance with the sequence of seq id no:3 or seq id no:4, however, the present invention is intended to encompass all ETn sequence insertions.

It will be understood that the nucleic acid segments may contain the entire faε gene, as described by

Watanabe-Fukunaga et al . (1992a), or may contain smaller sections, as represented by, e.g., seq id no:7 and seq id no:8, so long as such faε sequences have ETn sequences inserted therein. The nucleic acid segments of the present invention may encode an apoptosis-defective Fas cell surface protein. Such a protein may include within its sequence an amino acid sequence in accordance with seq id no:2, and may be encoded by a DNA sequence which includes a sequence in accordance with seq id no:l, which may also include extended faε sequences so that the entire Fas antigen in encoded.

Alternatively, the nucleic acid segments of the present invention may be smaller so that they do not encode an entire protein. Purified nucleic acid segments with sequences in accordance with the nucleic acid sequences set forth in seq id no:l, seq id no:21 or seq id no:22, or the complements of such sequences, are contemplated to be particularly useful. Small nucleic acid segments which span the junction of the faε and ETn sequences, such as those represented by the nucleic acid sequences of seq id no:21 or seq id no:22, or sequences which comprise at least a ten nucleotide long stretch which corresponds to seq id no:21 or seq id no:22, are contemplated to be particularly useful.

As used herein, the term nucleic acid segment is intended to refer to DNA and RNA molecules which have been isolated free from total genomic or total cellular nucleic acids. Therefore, a nucleic acid segment of the present invention most often refers to a nucleic acid segment which is isolated away from total T cell nucleic acids. Included within the term "nucleic acid segment" are segments which may be employed in the preparation of vectors, as well as the vectors themselves, including, for example, plasmids, cosmids, phage, viruses, and the like. It will be understood that the present invention also encompasses sequences which are complementary to the sequences listed herein, along with biological functional equivalents thereof, including naturally occurring variants and genetically engineered mutants.

The DNA segments and recombinant vectors of the present invention may variously include the DNA coding regions set forth herein, coding region bearing selected alterations or modifications in the basic coding region, or may encode larger polypeptides which nevertheless include disclosed sequences, particularly from seq id nos:l, 21 or 22. Nucleic acid molecules having stretches of 10 or 12 nucleotides or so, complementary to seq id nos:21 and 22 will have utility, for example, as hybridization probes. However, the total size of fragment, as well as the size of the complementary stretch(es) , may be varied depending on the intended use or application of the particular nucleic acid segment. Nucleic acid fragments of up to 50 or 100 basepairs in length which include a sequence in accordance with, or complementary to, the sequences of seq id no:21 or seq id no:22 are particularly contemplated.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

It will also be understood that this invention is not limited to the exact nucleic acid and amino acid sequences described herein. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acid sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.

The nucleic acid segments and probes of the present invention may be used in detecting apoptosis-defective T cells from animals or humans and may also be employed in molecular biological embodiments such as screening assays. However, they are not limited to such uses and also have utility in a variety of other embodiments, for example, as probes or primers in nucleic acid hybridization embodiments and in the expression of peptides and polypeptides for antibody generation.

The ability of these nucleic acid segments to specifically hybridize to faε and ETn-like sequences will enable them to be of use in various ways, e.g., as primers for the cloning of further portions of genomic DNA, and particularly, for the preparation of mutant species primers. The specific mutagenesis and subsequent analysis of various Fas proteins which cause apoptosis defects in T cells would be invaluable as a tool in more precisely defining the apoptotic pathways in these cells and their interaction with disease processes. This would allow more effective drugs and therapeutic strategies to be designed to combat a variety of disorders.

Fas proteins (antigens) and Fas peptides and polypeptides, purified relative to their natural state, are also encompassed by the present invention. The invention particularly concerns Fas proteins and polypeptides which also include ETn amino acid sequences, as exemplified by an apoptosis-defective mutant Fas protein which includes within its sequence an amino acid sequence in accordance with seq id no:2. Fas proteins and peptides may contain only such sequences themselves or may be linked to other protein sequences, such as, e.g., ^λnatural' sequences derived from other T cell proteins or portions of engineered' proteins such as, e.g., glutathione-S-transferase (GST), ubiquitin, β-galactosidase and the like. Antibodies having binding affinity for Fas/ETn combination proteins are also contemplated by the present inventors.

This invention also provides a method for identifying an apoptosis-defective T cell, which method generally comprises obtaining a DNA or RNA sample from a population of T cells suspected of containing apoptosis- defective T cells and probing said sample with a nucleic acid probe capable of differentiating between normal faε gene transcripts and aberrant faε gene transcripts. To "differentiate" in this manner generally requires the use of a nucleic acid probe, such as those described herein, which allows faε DNA or RNA from normal T cells to be identified and differentiated from faε DNA or RNA from apoptosis-defective T cells. This may be achieved using criteria such as, e.g., number, pattern and size of faε nucleic acids, and more particularly, by the presence of an Etn sequence insert within the faε sequence. The method for identifying apoptosis-defective T cells described herein may be employed as a method for, or part of a method for, identifying an individual at risk for developing systemic autoimmune disease. Therefore in addition to cells from animal models, and cultured cells, the T cells analyzed in such methods may obtained from an individual suspected of being at risk and for developing systemic autoimmune disease, or from a patient known to be presently or previously suffering from such a disorder. In such a method, the identification of apoptosis-defective T cells would be indicative of a disease state or the propensity to develop such a disease state.

To identify an apoptosis-defective T cell in accordance with the present invention one may employ Northern blotting technology, as described in the text and Figures 3 and 4 herein, and known to those of skill in the art (e.g., see Sambrook et al . , 1989). In this method, one would obtain RNA, and preferably, mRNA, from a population of T cells suspected of containing apoptosis-defective T cells, and probe the RNA with a nucleic acid probe capable of identifying normal faε gene transcripts and aberrant faε gene transcripts, preferably those including an ETn gene sequence insert, wherein a reduction in the amount of a normal faε gene transcript or the presence of an aberrant faε gene transcript is indicative of an apoptosis-defective T cell. Nucleic acid probes for use in such embodiments may include faε gene sequences, ETn gene sequences or both such sequences.

In certain embodiments concerning mouse cells, a reduction in the amount of faε gene transcripts estimated to be of about 2.2 kb in length, as compared to the level of such transcripts in normal mouse cells, is indicative of T cells which are apoptosis-defective. In lpr/lpr mouse cells, multiple aberrant faε mRNA transcripts ranging from about 2 kb to about 9.5 and 10.5 kb were detected, with a prominent _ETn-containing insert of 10.5 kb being particularly noticeable. Accordingly, an elevation in the levels of such transcripts is indicative of an apoptosis-defective T cell.

It is important to note that the size of normal and aberrant faε transcripts may vary between different species and cell lines, however, in light of the present invention, aberrant faε species will always be distinguishable from faε species in normal cells of the same species. It is therefore clear that using the technology described herein one may differentiate between normal and mutant faε transcripts, and thus identify apoptosis-defective T cells in an assay or screening protocol, regardless of the actual size and pattern of the aberrant transcripts themselves.

Another method which may be employed to identify an apoptosis-defective T cell and, therefore, to identify an individual at risk for developing systemic autoimmune disease, is based upon Southern blotting. In this method, the nucleic acids obtained for analysis would be DNA, and preferably, genomic DNA, which would be digested with one or more restriction enzymes and probed with a nucleic acid probe capable of hybridizing to normal-sized faε DNA bands and aberrant-sized faε DNA bands, preferably those which include an ETn gene sequence insert. A reduction in the amount of normal-sized faε DNA, or the presence of aberrant-sized faε DNA, e.g., JETn-including faε DNA, would be indicative of an apoptosis-defective T cell.

A large battery of restriction enzymes are commercially available and the conditions for Southern blotting are described hereinbelow, suitable odifications of which will be known to those of skill in the art (see e.g., Sambrook et al . , 1989, incorporated herein by reference) . The utility of Southern blotting is exemplified herein in embodiments using mouse cells (see, e.g., Figure la) where an additional 5.3 kb of DNA within the extracellular domain of the genomic faε gene, as compared to normal mouse cells, was found to be indicative of T cells which are apoptosis-defective.

Kits for use in Southern and Northern blotting to identify apoptosis-defective T cells, or individuals at risk for developing systemic autoimmune disease, are also contemplated to fall within the scope of the present invention. Such kits will generally comprise a first container including faε or ETn nucleic acid probes, and preferably both; a second container including unrelated probes for use as controls; and optionally, a third container which includes one or more restriction enzymes.

In still further embodiments, the present invention concerns a method for identifying agents capable of promoting normal apoptosis in apoptosis-defective T cells, which agents are herein termed "candidate substances." It is contemplated that this screening technique will prove useful in the general identification of -any compound that-will serve the purpose of promoting or restoring the normal apoptotic mechanisms in such cells. As such, candidate substances which have activity in such assays would be good potential agents for use in the treatment of systemic autoimmune diseases. It will be understood that positive candidate substances, i.e., T cell apoptosis-promoting substances, and pharmaceutical compositions thereof, identified by the methods disclosed herein are encompassed by the present invention.

The screening methods of the invention generally include obtaining a composition containing apoptosis- defective T cells, preferably those which express a Fas cell surface protein including an ETn gene sequence insert, and admixing or contacting this cell composition with a candidate substance. One would then determine the ability of the candidate substance to decrease the expression of aberrant faε gene transcripts, particularly those which include an ETn gene sequence insert; to decrease the expression of ETn gene transcripts; or to increase the expression of normal faε gene transcripts.

Any suitable method may generally be employed to identify normal and aberrant {ETn insert-including) faε gene transcripts and ETn gene transcripts. Preferred methods are those described hereinabove, particularly those utilizing faε and ETn probes in Northern blotting studies. The CD2 -faε transgenic mouse studies presented herein demonstrate that faε transcription is less disrupted under conditions that suppress expression of the ETn retrotransposon. Therefore, in general, a candidate substance that produces a Northern blotting pattern positive for normal faε transcripts and negative for ETn transcripts (i.e., similar to that shown on the left hand side of Figure 4, as opposed to that shown on the right) would be indicative of a useful candidate substance.

Such candidate screening assays are relatively simple to set up and perform, and may be conducted in cell culture or by using an animal model such as the lpr/lpr mouse model which contains apoptosis-defective T cells. After contacting the cells with the candidate substance for an appropriate period of time, as may be achieved by administering the candidate substance to an animal, one would then perform an assay, preferably a Northern blot, to determine the levels of faε and ETn transcripts. A potentially useful substance would promote a more normal Northern blot pattern as opposed to a typical defective pattern, as exemplified by the data in Figure 4.

It is proposed that chemical compositions, man-made compounds and compounds isolated from natural sources, such as plants, animals or even sources such as marine, forest or soil samples, may be assayed for the presence of potentially useful candidate substances. However, it will be understood that the candidate substances to be screened could also be derived from or comprise known pharmaceutical agents, including cytokines, which are not currently used in conjunction with cell apoptosis or autoimmune diseases. The ^λcandidate substances' may be also be genes, oligonucleotides or anti-sense oligos. The suitability of the technique for use in such embodiments is exemplified by the CD2-_fas transgenic mouse studies described herein.

In still further embodiments, the discoveries of the present invention are contemplated for use in designing new treatment strategies for autoimmune diseases. For example, drugs may be identified or designed to normalize transcription of genes important in tolerance induction and apoptosis, such as the faε gene and other apoptosis genes known to those of skill in the art, and to restore normal gene function-despite the presence of a mutation. The inventors thus envision methods and compositions to modulate the expression of a retrovirus, either upward or downward depending on its mode of action and its association with tolerance related or lymphocyte activation genes, as part of the treatment for autoimmune diseases.

The invention thus concerns a method for treating systemic autoimmune disease comprising administering to a patient with such a disease an immunologically effective amount of a pharmaceutical composition capable of promoting normal apoptosis in apoptosis-defective T cells. More specifically, this may be achieved by administering a pharmaceutical composition comprising a positive candidate substance identified by the screening assays of the invention, or by administering a composition comprising an anti-sense oligonucleotide specific for a nucleic acid segment which includes a Fas cell surface protein-encoding sequence in combination with an ETn gene sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Southern blot analysis of the faε gene in MRL-lpr/lpr and wild-type MRL-+/+ mice and 5' Genomic map of Murine Faε Gene. Figure 1 consists of Figure la and Figure lb.

Figure la. DNA was purified from thymuses of MRL-lpr/lpr (L) and wild-type MRL-+/+(+) mice. High molecular weight DNA was digested with the indicated restriction enzyme d probed with a Hindi cDNA fragment corresponding to tne extracellular domain fragment extending from 219-569 bp of the rurine sequence.

Figure lb. Restriction map derived by single and multiple double enzyme digestions, and probing with a cDNA fragment corresponding to either a 170 bp Pstl- Hincll cDNA fragment (+49 bp to +219 bp) which is entirely 5' of the ETn insertion, a probe corresponding to the remainder of the extracellular domain which was a 345 bp Hindi cDNA fragment (+219 bp to +569 bp) , or the full-length faε cDNA probe (+49 to +1033 bp) . The location of the 5.3 kb inserted DNA and the approximate location of Exl, Ex2 and Ex3 was derived using the different probes on multiple identical blots. Additional enzyme sites present in the 5.3 kb insert are indicated. R=EcoRI, B=BamHI, H=HindIII, P=PvuII.

Figure 2. An abnormal faε RNA containing ETn is expressed in MRL-lpr/lpr mice but not in wild type mice. Figure 2 consists of Figure 2a, Figure 2b and Figure 2c.

Figure 2a. The position of the ETn sequence inserted within the faε gene as determined by sequence analysis of cDNA prepared from the thymus of MRL-lpr/lpr and wild- type mice. The PCR primers used and their relative locations with the faε gene are indicated as PI, P2, P3 and P4. Also shown is the location of the 170 bp 5' probe which is the Pstl-HincII fragment, and the 345 bp extracellular domain probe which is the Hindi fragment derived from the normal faε cDNA clone.

Figure 2b. PCR products using primers P2-P3 subjected to agarose gel electrophoresis and visualized by ultraviolet illumination in the presence of ethidium bromide. A unique larger PCR product was observed using thymic RNA from six different MRL-lpr/lpr mice.

Figure 2c. The insertion of the ETn sequence found within the otherwise normal extracellular coding region of -the wild-type xas_gene. The faε gene sequence (early portion, seq id no:7; late portion, seq id no:8) is numbered according to the numbering in the Watanabe- Fukunaga et al . (1992a), the position of which numbers are indicated as •. The nucleotide sequence of the Etn- derived insert (seq id nos:5 and 6) is numbered from position 1 of the insert, the position of which numbers are as indicated as T. The length of the inserted portion including the additional G residue (seq id no:4) is 168 bp. The ETn insert in the faε gene of MRL-lpr/lpr mice results in a novel sequence (seq id no:l) and an in-frame amino acid sequence (seq id no:2) shown below the cDNA sequence.

Sequences in Figure 2c: The Etn nucleotide sequence corresponding to the nucleotide sequence of the insert from position 1 through position 45 (prior to the addition of a G at the corresponding position of the MRL-lpr sequence) is represented by seq id no:5. The Etn nucleotide sequence after the addition of a G at the corresponding position of the MRL-lpr sequence through position 168 is represented by seq id no:6. A continuous nucleotide sequence of 168 residues comprising the sequence of seq id no:5 linked directly to the sequence of seq id no:6 by a single additional nucleotide (A, C, T or G) is represented by seq id no:3. A continuous nucleotide sequence of 168 residues comprising the sequence of seq id no:5 linked directly to the sequence of seq id no:6 by a single additional G residue, as was found to occur in one particular instance, is represented by seq id no:4. The sections of the wild-type faε nucleotide sequence prior to, and after, the insert are represented by seq id no:7 and seq id no:8, respectively. The nucleotide sequence of the resultant MRL-lpr coding segment, which includes the faε gene segments and an ETn- derived insert, is represented by seq id no:l; and the corresponding amino acid sequence resulting from the altered MRL-lpr coding segment is represented by seq id no:2.

Figure 3. Northern blot analysis of faε RNAs from the thymus of wild-type MRL-+/+ and MRL-lpr/lpr mice. Figure 3 consists of Figure 3a, Figure 3b, Figure 3c and Figure 3d.

Thymus poly-A⁺ RNA from the indicated mouse strains were analyzed by probing four identical blots with (a) a full length faε cDNA probe, (b) a 5' Pst-I/Hincll faε cDNA probe corresponding to position 49-219, (c) a faε cDNA probe corresponding to the 345 bp Hindi fragment of extracellular domain, and (d) a 168 bp ETn probe derived from ETn sequences within the abnormal sized faε transcript obtained by PCR amplification of the extracellular domain of faε cDNA from lpr/lpr mice. MRL- +/+, BXSB male and NZB female mice were 2 mo of age. MRL-lpr/lpr mice were 1 mo old (lanes 4,6) and 3 mo old (lane 5) . The upper arrows in panels a,b and c indicate the abnormal faε transcripts in MRL-lpr/lpr mice which correspond in size to a unique transcript which also hybridizes to the ETn probe used in panel d.

Figure 4. Decreased ETn expression in CD2-fas transgenic MRL-lpr/lpr mice. Poly-A RNA from thymus, lymph node (LN) and brain of 4 wk old CD2-fas transgenic and non-transgenic MRL-lpr/lpr mice was blotted as described in Figure 3. Figure 4 consists of Figure 4A, Figure 4B and Figure 4C.

Figure 4A. ETn expression is decreased in the thymus of 4 wk old CD2-fas transgenic MRL-lpr/lpr mice but not in age-matched non-transgenic littermate control mice.

Figure 4B. CD2-fas transgenic MRL-lpr/lpr mice have high levels of fas RNA in the thymus and LN but not in non-T cell sites including-the brain. Non-transgenic litter mate control mice do not express fas.

Figure 4C. The blot was stripped and hybridized with a jS-actin probe to ensure that nearly equal amounts of RNA was present in all samples. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The MRL-lpr/lpr mouse strain is a model of systemic autoimmune disease. The homozygouε expression of the lpr/lpr gene leads to autoimmunity and lymphadenopathy in different strains of mice including MRL, C57BL/6, C3H, AKR, and Balb/c mice (Cohen & Eisenberg, 1991) . In such mice, intrinsic defects of intrathymic T cell development exist, including defective deletion of self-reactive T cells (Zhou et al . , 1991; 1992) and expression of endogenous retroviruses (Kreig et al . , 1991). Expression of the 3' long terminal repeat (ltr) of the endogenous mouse mammary tumor virus (MMTV) as a superantigen has been reported to influence the shaping of the repertoire of the normal immune response (Choi, et al . , 1991;

Frankel et al . , 1991; Woodland et al . , 1991; Pullen et al . , 1992). However, the relationship between the expression of modified endogenous retroviruses in the thymus of autoimmune mice and the development of autoimmune disease was not defined prior the present invention.

A defect in Fas expression, a cell surface antigen which mediates apoptosis, has been described in MRL- lpr/lpr mice. A germline mutation in the faε gene which leads to abnormal Fas expression has been proposed to cause defective deletion of self-reactive T cells in the thymus of these mice (Itoh et al . , 1991; Watanabe- Fukunaga et al . , 1992b). One report identified a point mutation in the intracellular region of the faε gene in CBA/J- pr*⁸ mice which is believed to be functionally significant (Watanabe-Fukunaga et al . , 1992b) .

The faε gene also has been found to be abnormal in MRL-lpr/lpr mice in which Southern blot analysis indicated altered restriction enzyme digestion and faε RNA expression was not detectable in the thymus (Watanabe-Fukunaga et al . , 1992b). These results led to the conclusion that the faε mutation in MRL-lpr/lpr mice was different from the mutation in CBA/J-lpr⁰⁸ mice, and that in MRL-lpr/lpr mice the mutation leads to disruption of normal transcription of the faε gene.

Different strains of lpr/lpr mice develop different types of lymphoproliferative autoimmune disease (Cohen & Eisenberg, 1991) . Genetic differences between the different strains of lpr/lpr mice play a role in determining the levels in autoantibody production, the type and severity of autoimmune disease and extent of lymphoproliferation (Cohen & Eisenberg, 1991) . Genes determining the severity of renal disease in mice expressing the lpr/lpr gene have been mapped to chromosome 7 and chromosome 12, whereas genes associated with arthritis, although known to exist, have not yet been mapped. The heterozygous expression of the lpr gene also leads to a less severe form of lymphoproliferative autoimmune disease. It is not yet known if these disease differences are related to differences in expression of the faε gene, or to the influence of other genes in the immune response.

It is apparent that, despite intensive efforts in the—field, the molecular mechanisms underlying the Fas apoptosis defect in MRL-lpr/lpr mice had not been elucidated prior to the present invention. This lack of understanding thus prevented the development of the kind of useful screening assays which the art so clearly needs. The present inventors surprisingly found that the mutation of the faε gene is due to the insertion of a retroviral transposon of the ETn family (Shell et al . , 1987; 1990), resulting in inclusion of an ETn sequence within the coding region of the mature faε mRNA in the thymus. Using different fragments of a faε cDNA probe, the inventors determined that the lpr/lpr mutation was a 5.3 kb insertion of DNA within the second intron of the faε gene. cDNA corresponding to this region was derived from thymic RNA from MRL-lpr/lpr and MRL-+/+ mice using the polymerase chain reaction. All thymic RNA samples from MRL-lpr/lpr mice yielded a unique product that vn. 168 bp larger than that of MRL-+/+ mice. Complete sequence analysis indicated that this inserted sequence had 98% homology with a sequence from the 3' LTR of the ETn transposon. RNA analysis indicated higher expression of ETn RNA in the thymus of MRL-lpr/lpr than MRL-+/+ mice. This mutation leads to abnormal transcription and splicing of the faε gene in MRL-lpr/lpr mice, resulting in l/50th reduced amounts of normally spliced faε mRNA. The expression of ETn was found to be increased in the thymus of younger mice, but to decrease with age.

The inventors next analyzed the interdependence of ETn expression and abnormal faε expression in a CD2 -faε transgenic mouse model in which a full length murine faε cDNA under the regulation of the CD2 promoter and enhancer was used to correct defective faε expression in T cells of MRL-lpr/lpr mice. In CD2-fas transgenic MRL- lpr/lpr mice, increased expression of faε mRNA results in decreased expression-of ETn in the thymus. This indicates that the extent of interruption of the faε transcription by ETn is not constant, and that faε transcription is less disrupted under conditions that suppress expression of the ETn retrotransposon.

The results from these studies suggest that autoimmunity may result from the combination of the ability of predisposing background genes to facilitate the transcription of endogenous retroviruses, and the integration of a retrovirus or transposon near, or within, a gene of importance in tolerance induction. In this type of genetic mechanism, endogenous retroviral sequences (ERS) act as autogenes to adversely modulate immune cell function creating a state of immunologic dysregulation. Herein, an autogene is defined as an ERS integrated into the mammalian chromosome in such a way that it promotes autoimmune disease, in a similar manner to oncogenes promoting cancer.

Since the predisposition to autoimmune disease dependent upon classical immune response genes is relatively weak, additional causative factors have long been suspected. In recent years, the role of infectious viruses has received much attention. Epstein-Barr, the agent of infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma in southeastern China, can function as a polyclonal B cell activator and it may be a co-factor in some human autoimmune diseases. Numerous autoimmune, arthritic, and dermatologic manifestations as well as a diffuse lymphoproliferative disorder can occur as a consequence of human immunodeficiency virus (HIV) infection. Some patients with systemic lupus erythematosus (SLE) , Sjogren's Syndrome (SS) and scleroderma make antibodies which are reactive with the p24 gragr-protein of HIV-1.

— The defective function of the faε protein in the MRL/ pr thymus, shown herein to be due to the insertion of a retroviral transposon of the ETn family, results in defective apoptosis. Failure or suppression of physiologic apoptosis is also important in cancer, where it is regulated by oncogenes. Cell death is a well-modulated and active process that may be blocked in some lymphomas and leukemias. Bc -2 protein, an oncogene found in mitochondria, suppresses apoptosis in neurons as well as in lymphocytes. T lymphocytes that overexpress Jc -2 are resistant to apoptotic killing. Certain types of oncogenes are mammalian genes which are captured by tumor viruses and reinserted into the host genome in a way that leads to carcinogenesis. Oncogenes and autogenes belong to the twin worlds of both virus and host. Just as the clinical consequence of oncogene activation is cancer, the clinical consequence of autogene activation is autoimmune disease. These pathologic effects may actually be quite similar since autoimmune disease is often accompanied by lymphoproliferation and may predispose to malignant lymphoma.

Autogene products may act as controlling elements regulating the immune response directly (e.g. as antigens) or indirectly (e.g. by controlling the lymphocyte growth cycle, particularly through apoptosis) . Autogenes, like oncogenes, may not be a fixed genetic defect but rather inducible by endogenous triggers which increase the predisposition for development of disease. These triggers might be other viruses or genes, sex hormones, or neurotransmitters. As in the case of oncogenes, the genetic basis behind increased autogene expression may be a mutation or translocation leading to abnormal transcription or altered genetic coding regions, which in turn leads to altered function of the autogene. Possibly ERS evolved-originally to augment the immune response, thereby imparting a selective advantage and leading gradually by natural selection to a human population better adapted to survive in a world full of infectious organisms but also more prone to autoimmune reactions. In any event certain ERS acquire the ability to become expressed in a way that promotes autoimmune disease.

Almost all endogenous retroviruses are non-infectious. Perhaps 5% of the mammalian genome arises through reverse transcriptase (RT) of retroviral sequences. There are many mechanisms by which retroviruses can induce autoimmunity including polyclonal B cell activation, cytokine dysregulation, immune suppression, molecular mimicry, etc.

The ability of ERS to act as superantigens lends further evidence to their possible role as autogenes. Minor lymphocyte stimulating (Mis) antigens can act as endogenous superantigens that activate or induce the deletion of large portions of the T cell repertoire.

They are encoded by mouse mammary tumor viruses (MMTV) that have integrated into the germ line as DNA proviruses. Hence, T cells bearing TCR VjS-specific for the superantigen Mls-la (encoded in the open reading frame of the 3' long terminal repeat of endogenous MMTV) can lead to deletion of T cells expressing Mis reactive V3 regions. A murine leukemia virus (MuLV) with similar superantigen properties has been discovered. A rapid activation and proliferation of CD4' T cells is associated with the development of an immunodeficiency syndrome of mice caused by a replication-defective MuLV.

Retroviruses can also alter immune function by integration and disruption of either the structure or regulation of the host gene. Retrotransposons are retroviral sequences that are especially capable of this type of immune disfunction. Mtv retroviral sequences can also effect transcription of adjacent genes. Mtv-8 is an endogenous retrovirus located 4.6-kb upstream of a VK region gene (called VK9M) within the K-Ig locus. The proximity of these two genes results in reciprocal transcriptional activation. Mtv-8 transcription can be detected after juxtaposition of the K-enhancers to the normally silent provirus. Reciprocally, the frequency of VJT9M rearrangement is 5- to 10-fold higher in spleens from Mtv-8-positive mice compared to spleens from mice that lacked the Mtv-8 provirus.

ETn elements are 5.7-kb retrotransposons found in the murine genome. The ETn family of long-repeated sequences is abundantly transcribed in early mouse embryos from retroviral-like long terminal repeats (LTR) . Nucleotide sequencing of two ETn elements did not reveal any long open reading frame or significant homology with other retroviral proteins. The genetic polymorphism, monitored by Southern blotting within and across mouse species, reflects a conserved mode of evolution for the ETn sequences.

The ETn retrotransposon has been identified in several autosomal recessive disease states in mice and man. The ETn transposon has inserted into the major mammalian-skeletal-muscle-chloride-channel (CIC-1) in myotonia mice, destroying its coding potential for several membrane-spanning domains (Steinmeyer et al . ,

1991) . In some cases, as in faε , the ETn is incorporated into the cDNA of genes that are mutated by this transposon. Genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line contain sequences for the remnants of a mouse ETn (Baughman et al _*-, 1991).

The transposon is strongly expressed not only in embryonic cells but also in plasmacytomas, B lymphomas and T cell lines. ETn integration within a gene was first observed upstream of Vλ2 in the P3X63Ag8 cell line. A unique 2.2-kb mRNA is transcribed from Q6 and Q8 genes of the mouse MHC. The 3' portion of Q8 contains extensive homology with the ETn transposon.

The ability of a retrovirus to disrupt the regulation of host genes, as is the case for ETn in the faε gene of the pr mice, may be related to common control regions for expression of retroviruses and developmental genes in lymphocytes. Early development of B and T cells are also associated with a number of enhancer binding proteins which regulate T and B cell development. One transcription factor, etε , is found in the enhancer of TCRα, TCR/3, TCR5, CD3Σ, CD3<S and IL2, and is also found in the enhancers of retroviruses including HIV-1, MuLV and ETn .

High levels of expression of ets during T cell development might also lead to increased transcription of ETn and disruption of faε transcription. This model was tested by the present inventors in a TCR/3 transgenic MRL-lpr/lpr mouse. The TCR/3 transgene results in a complete loss of lymphoproliferative disease and production of the unusual CD4 CD8 B220+ T cell in MRL- lpr/lpr mice. Examination of ETn expression in T cells of TCR_/8 transgenic lpr/lpr mice indicated that faε expression increased relative to non-transgenic lpr/lpr mice. Thus, the ETn mutation within faε is not a fixed mutation but is a site of active transcription which might disrupt normal transcription during early T cell development.

— The results obtained during the present studies give rise to the following conclusions. First, the entire complex of autoimmune and lymphoproliferative disease that occurs spontaneously in lpr/lpr mice is intimately linked to a defect in apoptosis. Second, the molecular basis of this defect is traceable to an ETn integrated into the faε apoptosis gene. Also, the inventors found that the major features of the disease do not develop when this aberrant faε gene is accompanied by a TCR/3 transgene, suggesting that the apoptosis defect is compensated for, or otherwise bypassed by, the action of this normal TCR gene. It is now clear that endogenous retroviruses, apoptosis and autogenes are linked to autoimmune diseases in a fundamental way which may be exploited in the development of new strategies to combat autoimmune diseases. For example, it is envisioned that the present invention may be utilized to screen for, or design, drugs to normalize autogene transcription which may restore normal gene function despite the presence of an autogene mutation. In addition, the invention may be employed to gain further information regarding autogene regulation which will allow a better definition of the precise triggers (both endogenous and exogenous) that cause abnormal autogene expression and disease. Since most cytokine and immunosuppressive drugs in current usage induce apoptosis in susceptible cells, it is contemplated that this invention will allow the development of drug therapy which will restore normal apoptosis in autoimmune patients.

The present invention has opened the way for new therapeutic strategies for autoimmune disease. Thus, modulation of the expression of a retrovirus, either upward or downward depending on its mode of action and its association with tolerance related or lymphocyte activation genes, is envisioned to be of use in inhibiting an autoimmune response even in the presence of an uncorrected genetic defect. More particularly, in the present case, the use of an agent which downregulates ETn expression and thus promotes normal faε gene expression is contemplated. Such ^λagents' may be genetic constructs or pharmacological agents identified by the screening assays of the invention.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE I CHARACTERIZATION OF THE MRL-lpr/lpr .fas MUTATION

A. MATERIALS AND METHODS

1. Normal Mice . The original breeding pairs of MRL-lpr/lpr and MRL-+/+ mice used for all the studies described herein were obtained from the Jackson Laboratory (Bar Harbor, ME) .

2 . Southern Blot Analyεiε . MRL-lpr/lpr and MRL-+/+ mice were obtained from the Jackson Laboratory, Bar Harbor, ME. DNA was prepared from the thymus and digested with the indicated restriction enzymes. Approximately 10 μg of the digested DNA was separated on a 0-7% agarose gel, -blotted to a nylon membrane and hybridized with ³²P-labelled cDNA probes.

3 . Northern Blot Analyεiε . Five micrograms of poly (A)⁺ RNA was denatured at 65°C for 5 min in electrophoresis buffer (0.4 M 3-morpholinopropanesulfonic acid, 0.1 M sodium acetate, 2 mM EDTA pH=7.0) containing 6% formaldehyde and 50% formamide, and size fractionated by electrophoresis through 1% agarose gels containing O.S% formaldehyde. Gels were stained with ethidium bromide to assure integrity of the loaded RNA. RNA was transferred to nylon membranes (Nitroplus 2000, M.S.I. Inc. , Eastboro, MA) and baked for 2 h at 80°C in a vacuum oven. Membranes were prehybridized and then hybridized with 1 to 3 x 10⁶ cpm/ml of different DNA probes that had been labeled with ³²P by random priming to a specific activity of 10⁹ cpm/μg. Filters were then washed with 2x SSC + 0.1% SDS at 42°C for 30 min and then with 0.1 x SSC + 0.1% SDS at 60°C for 30 min; they were then exposed to Kodak XAR-2 film (Eastman Kodak, Rochester, NY) at -70°C with intensifying screens.

4 . PCR Analyεiε . Thymuses of mice were homogenized and total RNA was extracted from the homogenates by the guanidinium-CsCl method. Total RNA (2-4 μg) from each tissue was used for cDNA synthesis followed by PCR amplification using the Perkin-Elmer RNA-PCR kit

(Perkin-Elmer, Norwalk, CT) . Reaction conditions were as specified by the manufacturer. An oligo (dT) primer was used to initiate cDNA synthesis. Thirty PCR cycles (1 min at 95°C; 1.5 min at 55 °C; 2.5 min at 72°C) were run followed by extension for 10 min, and the amplification products visualized after electrophoresis on agarose gels (1.0%) under ultraviolet illumination in the presence of ethidium bromide. Gels were blotted and hybridized to a labelled internal faε probe to verify that the bands were faε specific. The full length faε cDNA was obtained by PCR- amplification of- cDNA made from thymus mRNA from MRL-lpr/lpr or MRL-+/+ mice.

The full length cDNA PCR primers used were as described below (in all the following, the sequence positions are referenced to the published murine faε sequence of Watanabe-Fukunaga et al. (1992a)). PCR primers PI and P4, the sequences of which are shown below (seq id no:9 and seq id no:12, respectively), were employed to obtain the full length cDNA.

PI = 5'-GGC-CGC-CCG-CTG-TTT-TCC-CTT-GCT-GCA-GAG-3', position +20 P4 = 5'-ATT-GAC-ATT-GGC-AAC-TCC-TGG-TCT-3', position 1110

Internal PCR primers P2 and P3, the sequences of which are shown below (seq id no:10 and seq id no:11, respectively) , were used to obtain the extracellular domain cDNA which was sequenced.

P2 = 5'-CA-CAG-TTA-AGA-GTT-CAT-ACT-CAA-GGT-ACT-AAT-3' position+93

P3 = 5'-AA-AGT-CCC-AGA-AAT-CGC-CTA-TGG-TTG-TTG-3' , position 540

Also used in the sequencing reactions were the universal vector 5' primer, 5'-CTG-TGG-ATC-TGG-GCT-3', position 53 (seq id no:13); and the following 5' primers:

5'-TGT-CAA-CCA-TGC-CAA-CCT-3' , position 215 (seq id no:14) 5'-CGA-AAG-TAC-CGG-AAA-AGA-3', position 608) — (seq id no:15) - 5'-CGA-GAA-AAT-AAC-ATC-AAG-3', position 773 (seq id no:16)

The 3' universal vector primer, 5'-GAA-TCT-AGA-ACC- TCC-AGT-3', position 656 (seq id no:17); and the following 3' primers were also used in the sequencing reactions:

5'-TGT-GTT-CGC-TGC-GCC-TCG-3' , position 464 (seq id no:18) 5'-ACA-GAA-GGG-AAG-GAG-TAC-3', position 293 (seq id no:19) 5'-GTT-GAG-GAC-TGC-AAA-ATG-3', position 245 (seq id no:20)

5. Genomic Cloning . High molecular weight DNA from the thymus of MRL-lpr/lpr and MRL-+/+ mice was digested with PstI and Hindi. The digested sample was divided into several wells and electrophoresed through 0.8% agarose. After electrophoresis, a portion of the gel was blotted and probed with the 345 bp Hindi fragment of faε and the 168 bp ETn fragment and another portion of the gel was sliced into thin sections representing different,. molecular weights. The gel slices correspond in size to bands hybridizing with the faε or ETn probes were extracted from the gel using Geneclean II (Bio 101, La

Jolla, CA) . DNA fragments ranging from 3.8 kb to 5.6 kb were amplified using the appropriate 3' or 5' faε cDNA primers in combination with ETn primers from the 168 bp sequence (Fig. 2) and published ETn sequences (Shell et al . , 1990). PCR products were cloned into the PCR 2000 vector (Invitrogen, San Diego, CA) for sequence analysis.

6. Seguence Analyεiε . Sequence analysis was carried out on double stranded DNA derived from PCR amplification and cloning into the PCR 2000 (Invitrogen, San Diego, CA -. Sequence analysis was carried out in both directions using 5' and 3' universal primers and faε specific primers. For genomic sequencing, universal primers were used to determine the sequence of both the 3' end of the second exon and the 5' end of the third exon and flanking intronic sequences.

7. DNA Probe ε . The full-length murine faε cDNA probe (49 bp-1033 bp) was derived by PCR amplifica ion of cDNA prepared from MRL-+/+ thymus mRNA as described above and using previously described faε primers (Watanabe-Fukunaga et al . (1992a). A probe corresponding to the first and second exons of faε , which are 5' of the normal ETn insertion, was the 170 bp Pstl-HincII fragment (49 bp-219 bp) derived from the full length faε cDNA clone (Fig. 2a) . A probe corresponding to the remainder of the extracellular domain was the 345 bp Hindi fragment (219 bp-569 bp) derived from the full length faε cDNA clone. The ETn probe was the 168 bp ETn sequence that was isolated by PCR amplification of cDNA prepared from MRL- lpr/lpr thymus mRNA and reamplified using primers specific for the 5' and 3' sequences of ETn . The jS-actin probe was a gift from Dr. K. Gordon (GenZyme Corp., Framingham, MA) (13) and may be produced as described by Roberts et al . (1992).

B. RESULTS

1. The lpr Mutation Results from a 5.3 kb Insertion of DNA

High molecular weight DNA from the kidney and thymus of MRL-lpr/lpr and MRL-+/+ mice was digested with various restricted enzymes and probed with a 345 base pair sequence corresponding to the extracellular domain of the faε cDNA (Fig. la) . There was no difference in restriction fragment lengths between high molecular weight DNA from the-kidney and the thymus. There was an additional 5.3 kb of DNA within the extracellular domain of the genomic faε gene from MRL-lpr/lpr mice as determined by restriction fragment length analysis of Southern blots prepared using multiple single and double enzyme digestions and hybridization with a 170 base pair cDNA probe corresponding to the first and second exons of faε cDNA (49 bp-219 bp) , an extracellular domain probe (219 bp-569 bp) , and a full length fas cDNA probe (49 bp- 1033 bp) . The inserted DNA contained additional digestion sites for EcoRI, Hindlll and PvuII but not for BamHl (Fig. lb) . Using probes corresponding to exon 1 and exon 2, or a Hindi cDNA fragment corresponding to the extracellular domain, the insert was localized to the region of the faε gene corresponding to the second intron (Fig. lb).

Insertion of ETn in the Extracellular Domain of fas

To determine if the lpr/lpr mutation in the extracellular domain of the fas gene results in abnormal faε RNA, cDNA corresponding to the extracellular domain was derived from thymus RNA from several MRL-lpr/lpr and MRL-+/+ mice using the polymerase chain reaction and primers P2-P3 (Fig. 2a) . All RNA samples from the thymus of different MRL-lpr/lpr mice yielded a unique polymerase chain reaction product that was 168 bp larger than that of wild-type MRL-+/+ mice (Fig. 2b) .

The mutation of the faε gene in MRL-lpr/lpr mice was confirmed to be in the extracellular domain by sequencing, using full length primers P1-P4 (Figure 2a) and using the extracellular faε cDNA clones from MRL-lpr/lpr and MRL-+/+ mice. The sequence of the transmembrane and cy oplasmic domains were identical in MRL-lpr/lpr and MRL-+/+ mice. Complete sequence analysis of cDNA corresponding to the extracellular domain of the faε gene was carried out using two different MRL-lpr/lpr mice and indicated that there was a 168 bp insert into the fas cDNA sequence at position 232 of an otherwise normally encoded extracellular domain (Fig. 2c) .

This 168 bp sequence was analyzed by a GenBank search and found to exhibit an extremely high degree of homology with a sequence from the 3' LTR of the ETn retroviral transposon (portions of which are represented by seq id no:5 and seq id no:6, Figure 2c). The inserted sequence within the faε cDNA is 98%-99% homologous to portions of ETn sequences previously found to be integrated into the lg locus of mice, namely the sequences MUS ETn Xi (bp 1120-1285; Shell et al . , 1987;1990) and Mus ETn IgM (bp 270-435; Weiss & Johansson, 1989) .

The differences between the previously identified ETn sequences and that found within the faε gene are the exchange of A for C at ETn position 22, the exchange of G for T at ETn position 66, and the insertion of an additional G residue at position 43 of the MRL-lpr/lpr ETn combined sequence. The nucleotide sequence of the MRL-lpr coding segment which has the i?Tn-derived insert in the faε gene is represented by seq id no:l, and the corresponding amino acid encoded by this altered, or mutated, MRL-lpr sequence is represented by seq id no:2 (Figure 2C) . One MRL-lpr/lpr mice was found to have an additional AAA triplet at position 240 of the normal murine faε cDNA (Watanabe-Fukunaga et al . (1992a), making a total of four AAA codons in this region of the sequence.

3. Aberrant faε Gene Expression

A full length faε cDNA was used to probe northern blots of poly-A RNA prepared from the thymus of MRL-+/+, MRL-lpr/lpr, MRL-lpr/+, BXSB male and NZB mice and from the BW5147 cell line. In MRL-+/+ mice there was a 2.2 kb normal sized faε cDNA (Fig. 3a, lane 1-3) . In contrast, in 1 mo old MRL-lpr/lpr mice there were multiple bands ranging from 2 kb to 10.5 kb (Fig. 3a, lane 4,6). Fas expression was highest in the thymus of 1 mo old MRL- lpr/lpr mice, and decreased in 3 mo old mice (Fig. 3a, lane 5) . When identical blots were hybridized with a 170 bp Pstl/HincII faε cDNA fragment corresponding to the first and second exons, faε expression in MRL-lpr/lpr mice was very low compared to faε expression in MRL-+/+ mice (Fig. 3b, lane 4-6) . A faint abnormal high molecular weight species of 10.5 kb was present using this 5' probe (Fig. 3b, lane 4, arrow). When blots were probed with a 345 bp Hindi faε cDNA fragment corresponding to the extract lular domain of faε , the primary species of RNA expressed in the thymus of young MRL-lpr/lpr mice was a high molecular weight 10.5 kb and 9.5 kb transcript (Fig. 3c) .

These results indicate t.v.l the faε mutation leads to production of abnormal high molecular weight faε transcripts in the thymus. There was high expression of the 2.2 kb faε transcript in (MRL-lpr/lpr x MRL-+/+)F_X mice, and also in BXSB male and NZB autoimmune mice (Fig. 3a,b,c; lanes 7-9). Expression of normal levels of Faε RNA in BXSB and NZB mice indicates that autoimmune disease in these mice is not related to defective expression RNA.

The 168 bp ETn probe, derived from within the faε cDNA prepared from thymus RNA of MRL-lpr/lpr mice, strongly hybridized to a 5.7 kb full-length ETn transcript which was expressed in the thymus of younger MRL-lpr/lpr mice (Fig. 3d, lane 4,6), but not strongly expressed in the thymus of older MRL-lpr/lpr mice (Fig. 3d, lane 5) or in the thymus of MRL-+/+ mice (Fig. 3d, lanes 1-3). RNA corresponding to the full-length 5.7 kb ETn transcripts was also abundant in the thymus of MRL-lpr/+ and BXSB mice, and also in the BW5147 cell line. The largest faε transcript corresponds in size to an abnormal 10.5 kb ETn transcript in MRL-lpr/lpr mice

(arrows, lanes 4; Fig. 3a,b,c,d) suggesting the presence of the ETn sequence within one of the abnormal sized high molecular weight fas transcripts of MRL-lpr/lpr mice. A higher approximately 10.5 kb molecular weight fas transcript was also present in the thymus of MRL-lpr/+ and BXSB mice, but not NZB mice or the BW5147 cell line (Fig. 3a, lanes 7-10) .

4. Germline Organization of the Mutated fas Gene in lpr Mice

Using PCR primer pairs to the 3' end of fas exon 2 and the 5' end of the 168 bp ETn found in the faε transcript or the 5' end of faε exon 3 and the 3' end of ETn , the sequence of cloned genomic fragments isolated using the 345 bp Hindi fragment of faε and the 168 bp ETn fragment were determined. In MRL-lpr/lpr mice the ETn sequence began at the 5' terminal of exon 2 and continued for an additional 5.3 kb. There was a conserved splice consensus nucleotide sequence on the 3' end of exon 2-intron 2 (G/A) and on the 5' end of the 168 bp ETn (A/G) which was found to be spliced into the faε transcript. Also, there was a conserved splice consensus at the 3' end of the 168 bp ETn (A/G) and on the 5' end of the faε exon 3 (A/G) . These splice consensus sequences allow for splicing of the 168 bp ETn into the faε cDNA. Additional ETn sequence was present in the

MRL=1 pr/lpr mice directly adjacent to the 3' terminus of the second exon. In MRL-+/+ and MRL-lpr/lpr mice there was an additional 3.5 kb of intron sequence consistent with the restriction map (Fig. 1) . EXAMPLE II

DOWNREGULATION OF ETn IN CD2-fas TRANSGENIC MRL-lpr/lpr MICE

A. METHODS

1. Production of CD2-faε Tranεgenic MRL-lpr/lpr Mice . The 1.1 kb full length faε cDNA was obtained by PCR amplification of cDNA made from thymus mRNA from MRL-+/+ mice as described herein. This was cloned into an EcoRI site in front of exon 1 of a human CD2 minigene consisting of 5.5 kb of 5' flanking sequence, exon 1, the first intron, fused exons 2 to 5 and 2.1 kb of the 3' flanking sequence. The 3' sequence has been shown to be sufficient to allow T cell specific, copy-dependent, integration-independent expression in transgenic mice (Lang et al . , 1988; Greaves et al . , 1989).

MRL-lpr/lpr male and female mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Single cell MRL-lpr/lpr embryos were produced, injected with approximately 100 copies of the CD2- faε transgene, and then placed into the distal oviduct of CDl pseudopregnant female mice. Tail DNA prepared from offspring was digested with EcoRI and probed with a ³²P labeled full length faε cDNA to identify CD2 -faε transgenic mice.

B. RESULTS

To determine if high ETn expression was dependent on abnormal faε expression, CD2 -faε transgenic mice were produced that utilized a full-length murine faε cDNA under the regulation of the CD2 promoter and enhancer (8- 9) to correct defective faε expression in T cells of MRL-lpr/lpr mice. The presence of the faε transgene resulted in reduction of expression of ETn in the thymus, suggesting that high ETn expression is related to abnormal faε expression (Fig. 4a) . Northern blot analysis indicated that there was high expression of the fas transgene in the thymus and lymph node, but not in the brain of 4 wk old CD2 -faε transgenic MRL-lpr/lpr mice (Fig. 4b) .

C. DISCUSSION

ETn retrotransposon transcription occurs during early embryonic development in mice (Wang et al . , 1992). Increased expression of endogenous retroviruses in the thymus of autoimmune strains has been proposed to be related to development of autoimmune disease (Gourley et al . , 1992). Inhibition of translation of retroviral transcripts by antisense RNA has been reported to result in increased proliferation of lymphocytes leading to the speculation that full-length retroviral transcripts and protein products are a compensatory mechanism for increased lymphocyte proliferation in autoimmune mice

(Krieg et al . , 1989). The present data suggests a second mechanism of association of retroviruses with autoimmunity, in that increased retroviral expression is be related to defective faε expression in MRL-lpr/lpr mice.

The ETn retrotransposon in the second intron of the faε gene might interfere with faε expression by promoting abnormal transcription initiation and interfering with abnormal splicing. High expression of ETn correlates with high expression of an abnormal large sized faε transcript with a molecular weight of approximately 10.5 kb (Fig. 3; lane 4,6). The largest faε transcript corresponds in size with an abnormal ETn transcript of the same size. This transcript contains both 5' and 3' faε cDNA sequences because it is detected by both the 5' 170 bp Pst-1/HincII faε probe, and the 3' 345 bp Hindi faε probe. A lower unusual faε transcript with an approximate molecular weight of 7.5 kb which retains 5' and 3' faε sequences (Fig. 3, lane 4) does not hybridize with the 168 bp ETn probe indicating that the 168 bp portion of ETn is spliced out of this transcript. Other aberrant splicing events can lead to faε transcripts which contain only 168 bp of ETn sequences (Fig. 2) . These results suggest that thymic developmental factors which lead to high ETn expression also promote production of an abnormally large faε transcript in MRL-lpr/lpr mice due to the integration of ETn within the second intron of the faε gene.

In MRL-lpr/+ heterozygous mice, the inventors found an increased expression of ETn and abnormal faε , despite the presence of apparently normal levels of faε transcription from the unmutated allele. It is possible that abnormal faε and high ETn are expressed in a subpopulation of thymocytes that express low levels of normal faε and exhibit abnormal thymic development. This was investigated in CD2 -faε transgenic MRL-lpr/lpr mice. In these mice, faε expression is regulated by the CD2 promoter/enhancer which results in high expression of faε in all thymocytes, and elimination of ETn expression (Fig. 4) . These results suggest that faε expression and ETn-expression are functionally related.

The inventors also observed that ETn expression is decreased and faε expression is partially normalized in TCR_/3 transgenic mice. They previously demonstrated that in TCRjS transgenic mice, there is nearly total elimination of the CD4"CD8'B220⁺ subpopulation of T cells and lymphoproliferation, but not elimination of autoimmunity (Mountz et al . , 1990; Zhou et al . , 1993). The inventors recently demonstrated that there is decreased apoptosis of thymocytes of MRL-lpr/lpr mice and an increase of a large, proliferating CD4⁺CD8⁺ subpopulation of thymocytes. The TCR0 transgene was found to reduce these large, proliferating CD4⁺CD8⁺ thymocytes and there was no difference between this population in TCR0 transgenic MRL-lpr/lpr mice the same population in MRL-+/+ mice. These results suggested that the presence of the TCRβ transgene corrected the defect in early T cell development related to lymphoproliferation despite the presence of a germline mutation of the faε apoptosis gene.

Rearrangement of the TCRjS chain gene has been proposed to play a critical role in early T cell development in the thymus (Teh et al . , 1992). The TCR3 transgene suppresses rearrangement of the endogenous TCR/3 gene (Ue atsu et al . , 1988). Suppression of rearrangement of the endogenous TCR/3 gene might accelerate T cell maturation resulting in decreased levels of retroviral LTR and eukaryotic gene enhancer binding proteins associated with T cell development (Thompson et al . , 1992). Prevention of aberrant transcription initiation at the site of the ETn integration within the second intron of the murine faε gene could result in normal transcription initiation from the 5' end of the faε gene. This would lead to the observed increased levels of faε expression in the thymus of the TCR/3 transgenic MRL-lpr/lpr mice. This interpretation is consistent with the concept that ETn expression and abnormal faε expression are functionally related in lpr mice.

Abnormal fas expression and T cell development in the thymus of autoimmune mice might lead to continued high expression of retroviruses. Abnormal populations of T cells, or B cells found in the periphery of autoimmune mice exhibit common features of developmental defects and retrovirus expression. In the case of the lpr/lpr gene the present data suggest that retrovirus expression is intimately related to abnormal faε gene transcription and abnormal lymphocyte development.

EXAMPLE III

Fas EXPRESSION IN DIFFERENT T CELL SUBSETS

LN T cells from MRL-lpr/lpr and MRL-+/+ mice were sorted (50,000 cells/sample) by flow cytometry into either normal CD4⁺CD8^* or CD4"CD8⁺ T cells, abnormal

Thyl⁺B220⁺ T cells, or ThylΕ220⁺ cells. Quantitative PCR analysis indicated that there was no Fas RNA expression by the Thyl⁺B220⁺ subpopulation of LN T cells from MRL- lpr/lpr mice. In contrast, nearly normal levels of Fas RNA was expressed on the CD4⁺ or CD8⁺B220" T cells.

Quantitative PCR analysis of FACS sorted thymocytes indicated that Faε RNA expression is highly regulated during normal thymocytes development. Faε RNA was not expressed on normal immature CD4^*CD8" or large proliferating CD4⁺CD8⁺ thymocytes, but was present on small, non-proliferating CD4⁺CD8⁺ thymocytes. However, Faε expression was not evident in mature single positive CD4⁺CD8^" or CD4"CD8⁺ thymocytes. Absence of Faε on mature thymocytes might be due to down-regulation of Faε expression, or due to- apoptosis of thymocytes that express Faε . In the thymus MRL-lpr/lpr mice, Fas expression was abnormally expressed on the large proliferating CD4⁺CD8⁺ population as well as in the mature CF4⁺ or CD8⁺ (single positive) population.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: UAB Research Foundation

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SCREENING ASSAYS RELATING TO AUTOIMMUNE DISEASE

(Ui) NUMBER OF SEQUENCES: 22

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I oo

TGCTGTCAAC CATGCCAACC TGCTCGAGCG GCCTTCTCAG TCGAACCGTT CACGTTGCGA 60 I

15 GCTGCTGGCG GCCGCAACAT TTTGGCGCCT GAACAGGGAC CTGAAGAATG GCAGAGAGAT 120

GCTAAGAGGA ACGCTGCATT GGAGCTCCAC AGGAAAGGAT CTTCGTATCG GACATCGGAG 180

CAACGGACAG GTAAAAAAAA AGTTGAGGAC TGCAAA 216

20

( 3 ) INFORMATION FOR SEQ ID NO : 2 :

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I 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

I

KD

Cys Cys Gin Pro Cys Gin Pro Ala Arg Ala Ala Phe Ser Val Glu Pro I 1 5 10 15

15

Phe Thr Leu Arg Ala Ala Gly Gly Arg Asn lie Leu Ala Pro Glu Gin 20 25 30

Gly Pro Glu Glu Trp Gin Arg Asp Ala Lys Arg Asn Ala Ala Leu Glu

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15

GCTCGAGCGG CCTTCTCAGT CGAACCGTTC ACGTTGCGAG CTGCTXGCGG CCGCAACATT 60

TTGGCGCCGG AACAGGGACC TGAAGAATGG CAGAGAGATG CTAAGAGGAA CGCTGCATTG 120

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GAGCTCCACA GGAAAGGATC TTCGTATCGG ACATCGGAGC AACGGACA 168

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GCTCGAGCGG CCTTCTCAGT CGAACCGTTC ACGTTGCGAG CTGCTGGCGG CCGCAACATT 60

15 TTGGCGCCGG AACAGGGACC TGAAGAATGG CAGAGAGATG CTAAGAGGAA CGCTGCATTG 120

GAGCTCCACA GGAAAGGATC TTCGTATCGG ACATCGGAGC AACGGACA 168

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GCTCGAGCGG CCTTCTCAGT CGAACCGTTC ACGTTGCGAG CTGCT 45

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20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GCGGCCGCAA CATTTTGGCG CCGGAACAGG GACCTGAAGA ATGGCAGAGA GATGCTAAGA 60

GGAACGCTGC ATTGGAGCTC CACAGGAAAG GATCTTCGTA TCGGACATCG GAGCAACGGA 120

5 CA 122

I

Ul ) I

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TGCTGTCAAC CATGCCAACC T 21

(9) INFORMATION FOR SEQ ID NO:8:

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_(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GGTAAAAAAA AAGTTGAGGA CTGCAAA 27

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GGCCGCCCGC TGTTTTCCCT TGCTGCAGAG 30

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

CACAGTTAAG AGTTCATACT CAAGGTACTA AT 32

( 12 JL INFORMATION FOR _SEQ ID NO : 11 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid

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(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AAAGTCCCAG AAATCGCCTA TGGTTGTTG 29

(13) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

ATTGACATTG GCAACTCCTG G 21

(14) INFORMATION FOR SEQ ID NO: 13:

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(A) LENGTH: 15 base pairs

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( i) SEQUENCE DESCRIPTION: SEQ ID NO:13

CTGTGGATCT GGGCT 15

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

TGTCAACCAT GCCAACCT 18

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(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CGAAAGTACC GGAAAAGA 18

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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGAGAAAATA ACATCAAG 18

(18) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

GAATCTAGAA CCTCCAGT 18

(19) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TGTGTTCGCT GCGCCTCG 18

(20) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

ACAGAAGGGA AGGAGTAC 18

(21) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

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(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20;

GTTGAGGACT GCAAAATG 18

(22) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

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(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CAACCTGCTC GA 12

(23) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

CGGACAGGTA AA 12

Claims

1. A purified nucleic acid segment, isolated free from total genomic DNA, which includes a Fas cell surface protein-encoding sequence in combination with an ETn gene sequence.

2. The nucleic acid segment of claim 1, further defined as encoding an apoptosis-defective Fas cell surface protein wherein the ETn gene sequence is inserted within the coding sequence for the Fas protein.

3. The nucleic acid segment of claim 2, wherein the ETn gene sequence is inserted at position 232 of the fas gene.

4. The nucleic acid segment of claim 3, wherein the ETn gene sequence insert includes within its sequence a DNA sequence in accordance with seq id no:3.

5. The nucleic acid segment of claim 4, wherein the ETn gene sequence insert includes within its sequence a DNA sequence in accordance with seq id no:4.

6. The nucleic acid segment of claim 5, wherein the ETn gene sequence insert has the DNA sequence of seq id no:4.

7. The nucleic acid segment of claim 2, wherein the apoptosis-defective Fas cell surface protein includes within its sequence an amino acid sequence in accordance with seq id no:2.

8. The nucleic acid segment of claim 7, wherein the coding sequence for the Fas protein includes within its sequence a DNA sequence in accordance with seq id no:l.

9. The nucleic acid segment of claim 2, wherein the coding sequence for the Fas protein includes an additional triplet at position 240.

10. The nucleic acid segment of claim 1, further defined as comprising at least a ten nucleotide long stretch which corresponds to the nucleic acid sequence of seq id no:21 or seq id no:22.

11. The nucleic acid segment of claim 10, further defined as comprising at least a twelve nucleotide long stretch which corresponds to the nucleic acid sequence of seq id no:21 or seq id no:22.

12. The nucleic acid segment of claim 10, further defined as comprising a nucleic acid fragment of up to 100 basepairs in length.

13. The nucleic acid segment of claim 12, further defined as comprising a nucleic acid fragment of up to 50 basepairs in length.

14. A purified nucleic acid segment with a sequence in accordance with the nucleic acid sequence of seq id no:l, seq id no:21 or seq id no:22 or the complement of such a sequence.

15. An apoptosis-defective mutant Fas protein which includes within its sequence an amino acid sequence in accordance with seq id no:2.

16. A method of identifying an apoptosis-defective T cell, comprising:

obtaining mRNA from a population of T cells suspected of containing apoptosis-defective T cells; and

probing said mRNA with a nucleic acid probe capable of identifying faε gene transcripts or ETn gene transcripts, wherein a reduction in the amount of a normal faε gene transcript or the presence of an aberrant faε gene transcript including an ETn gene sequence is indicative of an apoptosis-defective T cell.

17. The method of claim 16, wherein the nucleic acid probe includes a faε gene sequence.

18. The method of claim 16, wherein the nucleic acid probe includes an ETn gene sequence.

19. The method of claim 16, wherein the nucleic acid probe includes a faε gene sequence in combination with an ETn gene sequence.

20. The method of claim 16, wherein the population of T cells is obtained from an individual suspected of being at risk for developing systemic autoimmune disease and wherein the identification of apoptosis-defective T cells is a positive indication of such a risk.

21. A method of identifying an apoptosis-defective T cell, comprising:

obtaining genomic DNA from a population of T cells suspected of containing apoptosis-defective T cells;

digesting said DNA with one or more restriction enzymes; and

probing said digested DNA with a nucleic acid probe capable of hybridizing to normal-sized faε DNA bands and aberrant-sized faε DNA bands which include an ETn gene sequence insert, wherein a reduction in the amount of normal-sized faε DNA bands or the presence of aberrant-sized faε DNA bands including an ETn gene sequence is indicative of an apoptosis-defective T cell.

22. A method for identifying a candidate substance capable of promoting normal apoptosis in apoptosis- defective T cells, comprising: containing apoptosis-defective T cells which express a Fas cell surface protein including an ETn gene sequence insert with a candidate substance; and

determining the ability of the candidate substance to decrease the expression of aberrant faε gene transcripts which include an ETn gene sequence insert, to decrease the expression of ETn gene transcripts or to increase the expression of normal faε gene transcripts.

23. The method of claim 22, wherein the apoptosis- defective T cells are located within an experimental animal and the candidate substance is administered to the animal.

24. A T cell apoptosis-promoting substance identified by the method of claim 22.

25. A pharmaceutical composition for use in treating sysjemic autoimmune disease comprising a substance identified by the method of claim 22 dispersed in a pharmacologically acceptable vehicle.

26. A method for treating systemic autoimmune disease comprising administering to a patient with autoimmune disease an effective amount of a composition capable of promoting normal apoptosis in apoptosis-defective T cells.

27. The method of claim 26, wherein said composition comprises an anti-sense oligonucleotide specific for a nucleic acid segment which includes a Fas cell surface protein-encoding sequence in combination with an ETn gene sequence.

28. The method of claim 26, wherein said composition is a composition in accordance with claim 25.