Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc.

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10T1/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10T1/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10T1/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.     

Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization.     

Transfer of ‘immortalizing’ oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes

Cell lines established after transfer into FR3T3 rat fibroblast cells of 'immortalizing' oncogenes (plt gene (large T protein) of polyoma virus, v-myc gene of MC29 virus, rearranged forms of c-myc) exhibited increased rates of sister chromatid exchange (SCE). This was observed neither in cells which expressed one of the oncogenes responsible for the terminal stages of tumorigenic transformation (polyoma virus pmt (middle T protein), mutated ras genes), nor in cell lines carrying oncogenes of both types. Abnormal chromosome numbers were observed in cell lines expressing plt or myc, but not after transformation by pmt or ras oncogenes.     

Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10Tl/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10Tl/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10Tl/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.     

Different regulation of vascular endothelial growth factor expression by the ERK and p38 kinase pathways in v-ras, v-raf, and v-myc transformed cells

Here we show that vascular endothelial growth factor (VEGF) mRNA expression is up-regulated in oncogene transformed rat liver epithelial (RLE) cell lines and that the extracellular signal-regulated kinase (ERK) and p38 kinase differentially regulate the oncogene-mediated stimulation of VEGF. The highest level of VEGF mRNA expression was observed in the v-H-ras transformed RLE cell line, followed by the v-raf and v-myc transformed lines. The PD98059 MEK inhibitor was used to block the ERK pathway and SB203580 inhibitor to block the p38 pathway. The parent and the v-H-ras transformed RLE cell lines showed up-regulation of VEGF RNA expression through the ERK pathway and down-regulation of VEGF through the p38 pathway. VEGF was regulated in a comparable manner in a human breast carcinoma cell line. In the v-raf and v-myc transformed RLE lines, positive regulation of VEGF was transduced through the p38 pathway. These findings suggest that (1) oncogenic ras differs from raf and myc in the recruitment of the MAPK signaling pathways for VEGF regulation; (2) that VEGF is regulated in ras transformed and human cancer cell lines in a positive and negative manner by the ERK and p38 signaling pathways.     

Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus

We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.     

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.     

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

An established rat cell line expressing chondrocyte properties

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.     

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes.     

Activation of ras and myc proto-oncogenes in human breast carcinoma and neuroblastoma

DNA from human breast carcinoma (SK-BR-3) and neuroblastoma (LA-N-1) cell lines are capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. The blot hybridization analysis of DNA from primary and secondary NIH 3T3 transformants identified additional sequences homologous to the c-Ha-ras 1 oncogene, and revealed amplification of nucleotide sequences homologous to the v-myc oncogene. Restriction fragments of the amplified myc-related sequences correspond to c-myc (SK-BR-3) and N-myc (LA-N-1) loci of the human genome. The results show that active Ha-ras oncogenes can coexist with altered myc oncogenes in breast carcinomas and neuroblastomas. This suggests that a multi-step mechanism involves both ras and myc genes and their cooperation in the development of these tumors.     

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.     

Partial transformation of mouse fibroblastic and epithelial cell lines with the v-myc oncogene

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v-myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic.     

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.     

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.     

Comparison of antigen specificity, class II major histocompatibility complex restriction, and in vivo behavior of myelin basic protein-specific T cell lines and clones derived from (BALB/c x SJL/J) mice

Immunization with myelin basic protein (BP) causes experimental allergic encephalomyelitis (EAE) in certain strains of mice. SJL/J (H-2s) is the prototype sensitive strain. Although BALB/c (H-2d) is resistant to EAE through use of an identical immunization protocol, (BALB/c x SJL/J)F1 hybrid mice develop EAE after immunization with BP. T cell clones specific for BP have been isolated from a highly encephalitogenic line of (BALB/c x SJL/J)F1 hybrid T cells raised against bovine BP. The clones were examined for their H-2 restriction and specificity for heterologous forms of BP (mouse, rat, and bovine BP). The results revealed the clones cross-reacting with mouse (self) BP were almost always restricted to F1 hybrid class II major histocompatibility complex (MHC) elements. In contrast, mouse cross-reactive clones derived from a nonencephalitogenic (BALB/c x SJL/J) T cell line raised against rat BP were largely restricted to H-2d elements. These clones did not cross-react with bovine BP. Four additional lines were generated by carrying the original rat and bovine F1 T cell lines on parental antigen-presenting cells thus generating lines biased toward homozygous (SJL/J, H-2s, or BALB/c, H-2d) restriction elements. These "parentally restricted" T cell lines did not induce EAE when injected in vivo. These results suggest that in this F1 strain sensitivity to T cell-induced EAE is associated with epitopes on murine BP that associate with F1 class II MHC restricting elements. In contrast, nonencephalitogenic T cell lines contain a high proportion of murine cross-reactive clones restricted to H-2d, the haplotype of the classically resistant BALB/c mouse. This work illustrates the use of T cell lines and clones in a model system to further analyze the role of MHC restriction elements in autoimmune disease occurring in heterozygous individuals.