Negative trans-acting factors extinguish Ia expression in B cell-L 929 somatic cell hybrids

CELLULAR

IMMUNOLOGY

122,39 1-404 (1989)

Negative &w-Acting

Factors Extinguish la Expression 929 Somatic Cell Hybrids’

in B Cell-L

P. MICHAEL STUART,* JENNIFERL. YARCHOVER,AND JEROLD G. WOODWARD Department ofMicrobiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky 4OS36 Received April 4,1989; accepted May 20, 1989 Numerous studies have implicated trans-acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II geneswhile others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans-acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role transacting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk- cell line and normal B cells to determine if the existence of positive trans-acting factors from the B cell would lead to the expression of Ltk- class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk- class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggestthat Ltk- cells possessnegative trans-acting factors that appear to predominate over the positive transacting factors possessedby B cells. We have further extended these studies to test the MHC-inducing activity of IFN--y and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN--y with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II geneswere induced by IL-4. Taken together, these results indicate that Ltk- cells possessnegative trans-acting factors that not only maintain the Ia- phenotype of these cells, but also block the aCtiOn Of pOSitiVe trans-aCthg factOrS from B Cdk. 0 1989 Academic PESS, IIIC.

INTRODUCTION The major histocompatibility complex (MHC)3 of the mouse encodes two major classesof cell surface glycoproteins, termed class I and class II molecules. The class I genescode for a 45,000 D protein that is expressedon virtually all somatic cells. The classII genescode for dimeric proteins (Ia antigens) termed I-A and I-E, each of which consists of a 33,000 D cu-chain and a 27,000 D P-chain. These Ia antigens, unlike classI molecules, show a restricted tissue distribution, being limited to B cells, macrophages, and under certain conditions, particular nonhematopoietic-derived (NHD) ’ This work was supported by National Institutes of Health Grant CA39070. * Recipient of a National Institutes of Health Postdoctoral Fellowship A107344. [To whom all correspondence should be addressed.] 3 Abbreviations used: NHD, nonhematopoietic-derived, IFN-7, y interferon; IL-4, interleukin 4; MHC, major histocompatibility complex; PEG, polyethylene glycol; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; FCS, fetal calf serum; Ig, immunoglobulin; FACS, fluorescent-activated cell sorting. 391 OOOS-8749/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cells (1,2). These highly polymorphic Ia antigens have been shown to play an integral role in a wide variety of immune responses by their interaction with antigen and specific T cell receptors (3-5). Due to the importance of MHC antigens in the generation of MHC-restricted and alloreactive T cells and their association with disease (4, 6), considerable effort has gone into the identification of factors regulating MHC gene expression, Two such inducing agents have been well characterized to date, immune or y interferon (IFNy) and interleukin 4 (IL-4). These agents have both been shown to increase class I and class II MHC expression, though the cell types that respond to them are not the same (7-9). Mechanisms controlling Ia expression are not well established, but it is believed that truns-acting factors are involved in the regulation of classII genes.Positive transacting factors have been implicated in experiments involving the congenital severe combined immunodeficiency defect in man, which leads to a lack of class II MHC expression and has been shown to be due to a mutation in a gene located outside and unlinked to the MHC (10, 11). Recent evidence suggeststhis gene controls the expression of a class II-specific, DNA binding protein ( 12). The major source of evidence for the existence of positive trans-acting factors comes from work by several laboratories employing fusion technology to produce somatic cell hybrids. These hybrids have consisted of Ia-negative human B cell regulatory mutants fused to an assortment of cells including, Ia+ human B cells ( 13), Ia- human T cells ( 14), and mouse spleen cells ( 15) or fusions involving the Abelson leukemia virus-transformed murine Ia-negative pre-B cell line, R8205 and the human Ia-positive, Raji B cell line ( 16). In each of these experiments, the fusions resulted in the expression of class II genes of both fusion partners, presumably due to the presence of positive tram-acting factors donated by the Iaf partner of the fusion, or in the case of the fusion between Ia- B cells and Ia- T cell, factors from both partners. Accolla et al. have further demonstrated that one of these truns-acting factors, designated air- 1, was encoded by a locus on chromosome 16 ( 17). The existence of trans-acting factors has also been demonstrated in transfection experiments of two types. The first involved the transfer and expression of foreign class II genes into class II-expressing cells ( 18-22). The second type of experiment involved the transfer of genomic DNA, presumably containing genes encoding trarrs-acting factors, from an Ia-positive mouse lymphoma into the human class II-negative B cell line RJ 2.2.5, rescuing RJ 2.2.5’s class II gene expression (23). Some somatic cell hybridization studies have also suggestedthe existence of negative truns-acting factors in T cells (24) and plasma cells (25) that presumably block transcription of class II genesin these cells. This notion is supported by experiments in which negative, &-acting elements have been identified upstream of the class II genes(2 1,22). These observations raise the question whether positive trans-acting factors present in B cells can activate class II genes in NHD cells or whether NHD cells possess negative factors capable of suppressing Ia expression in B cells. To that end, we report here the results of fusion experiments between murine Ia-positive B cells and the Ianegative fibrosarcoma, Ltk- cell line. Our results indicate that not only do positive trans-acting factors from the B cell fail to activate Ltk- Ia expression, they also appear to be incapable of maintaining Ia expression of the B cell component of the resulting hybrid. We further show that Ia expression in the B cell-L cell hybrids cannot be

NEGATIVE

Ia tram-ACTING

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induced by IFN-7 or IL-4. This suggeststhat Ltk- cells possessnegative Ia trunsacting factors that have the capacity to block the activity of positive trans-acting factors that may be present within a cell. MATERIALS AND METHODS

Mice. Two to three-month-old female C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility were used for these experiments. Cell lines. A thymidine kinase deficient variant of the C3H-derived L929 fibrosarcoma (Ltk-) (26) was used. This cell line expressesKk and Dk antigens but not Ia antigens. The BALB/c B cell lymphoma, A20/2J (27) was used as an Ia-positive control in some experiments. Purijication ofB cells. B cells were purified from C57BL/6J mice as previously described (28,29). Briefly, spleens were removed from mice and a single-cell suspension made. The resulting cells were then treated with anti-Thy 1.2 and anti-L3T4 + complement followed by Ficoll-Hypaque gradient centrifugation to remove both dead T cells and red blood cells. The macrophages were then removed by passing the cells over a Sephadex GlO column. Cells were then counted and resuspended in DMEM and placed on ice until use in fusions. Purified B cells were shown to be greater than 95% Ia positive by FACS analysis. Somatic cellfusion. Hybrids between C57BL/6 B cells and Ltk- cells were obtained by fusion in polyethylene glycol (PEG) (Sigma, Mr 1000) as previously described (30). Briefly, 2 X 10’ Ltk- cells were mixed with lo* B cells and pelleted. To this mixture 1 ml of PEG was added and allowed to stand for 1 min at room temperature, followed by the dropwise addition of 7-10 ml of DMEM over a 5-min period. The cells were then gently washed several times and placed in DMEM supplemented with 10%FCS, 10 mM L-glutamine, 10 mM Hepes, 100 U/ml penicillin, 100 pg/ml streptomycin, 5 X 10e5M 2-ME, and 10 puMHypoxanthine + 1.6 PM thymidine (HT media) overnight. The following day this medium was removed and replaced with fresh HT medium to which 0.6 pA4 aminopterin was added (HAT media) as a selective agent for fusion hybrids. Cells were fed periodically with fresh HAT media for a period of 3 weeks. If, at that time, no colonies had appeared the cultures were terminated. When colonies did appear, these cells were maintained in HAT media. Cloning of one such fusion was performed using the limiting dilution technique by adding single cells to wells of a 96-well plate. Our cloning efficiency was about 40%. Antibodies. The mouse mAb MKD6 (anti I-Ak), 14-4-4 (anti I-Ek), 16-1-2 (antiKk), 34-5-3 (anti I-Ab), and 34- 1-2 (anti-Kb) were derived from supernatants of these cell lines (9). These mAb have been screened for Ia specificity on mouse spleen cells and for lack of nonspecific binding using the Ia- L-929 cell line. Lymphokines. We used 100 U/ml of recombinant interferon-y which was the kind gift of Schering-Plough Research (Bloomfield, NJ) and was distributed to us through the American Cancer Society Interferon Program. IL-4 supernatant was produced by stimulation of T cell hybridoma D9Cl. 12.17 (3 1) with 10 pg/ml of Con A for 24 hr in RPM1 1640 supplemented with 0.1% FCS. The Con A was neutralized by the addition of methyl-a-Dmannopyranoside. This IL-4 supernatant was used at a final concentration of 10% for all stimulation cultures performed. Fluorescenceassays.After the fusions were made the resultant hybrids or clones of hybrids were stained with mAb and FITC-labeled rabbit anti-mouse IgG as pre-

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viously described (32). The stained cells were analyzed by using a fluorescence-activated analyzer (FACS Analyzer; Becton-Dickinson, Mountain View, CA). Isolation ofgenomic DNA and Southern blot analysis. Genomic DNA was isolated from Ltk-, A20/2J, and the indicated clones from fusion III as previously described (33). DNA (10 pg) was digested overnight at 37°C with Eco RI restriction endonuclease.This DNA was then electrophoresed through a 0.8% agarosegel and blotted onto nitrocellulose according to Southern (34). Hybridization and wash conditions are as described elsewhere (33) with the only modification being that the final three washes were done at 61°C. The probes used were pJ1 I, a mouse J-heavy-chain probe (35) and pEbetaV, a 2-kb genomic subclone of pEbeta6 (36) including exons 4-6 of the E p gene. These probes were labeled with [32P]dCTP and sequentially hybridized to the blot by washing off the preceeding probe with two washes in 0.1 X SSC + 0.1% SDS at 100°C for 15 min each. Following hybridization and washing, the blot was exposed to Kodak XAR-5 X-ray film at -70°C using a DuPont Lightening Plus intensifying screen. RNA extraction and Northern blot analysis. Total cellular RNA was extracted from cells using the guanidine-thiocyanate procedure (37). For Northern blot analysis, 20 pg of RNA was electrophoresed on a 1%agarosegel containing 2.2 A4 formaldehyde (38). RNA was transferred to nitrocellulose filters (BA-85; Schleicher and Schuell, Keene, NH) with 20X SSCby blotting overnight. Hybridization analysis of RNA. Prehybridization and hybridization were done as previously described (9). The blots were sequentially probed with 32P-labeledgel-purified inserts derived from pAAC6 (A (YcDNA) (39), pEACl1 (E (YcDNA) (40), pA pk (41), pH2IIa (classI cDNA) (42), and pHF5 (human fi-actin cDNA) (43) by removing the preceeding probe as described above. Blots were exposed to X-ray film as described above. RESULTS Since previous reports had demonstrated that it was the probable presence of positive trans-acting factors in B cells that activate Ia expression in the Ia- partner of fusions between hematopoietic cells (17), we wished to determine if these positive trans-acting factors would be able to activate Ia expression in Ia- NHD cells as well. For these experiments we decided to use the thymidine kinase-deficient variant (Ltk-) of the L-929 C3H fibrosarcoma cell line as the Ia-negative NHD cell since it is well characterized as being devoid of Ia expression and is inducible for class I but not class II MHC expression with IFN-7 (J. G. Woodward, manuscript submitted). For our source of Ia-positive cells, we chose C57BL/6 (B6) B cells for two reasons. The first involved the ability to distinguish serologically the existence of both class I and class II expression of both Ltk- (H-2k) and B6 (H-2b) antigens. Second, since we were hoping to seeinduction of Ltk- Ia expression, we could utilize the fact that B6 does not produce an E (Ygene product. This would allow us to distinguish, on the basis of Northern blot analysis, whether there was Ia induction of Ltk- by measuring E (Ygene expression. We successfully produced four independent fusions between Ltk- and B6 B cells and characterized the MHC phenotypes of the uncloned populations by FACS analysis. The results in Table 1 demonstrate that no activation of Ia expression derived from the Ltk- fusion partner occurred, as evidenced by the lack of staining with the

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TABLE 1 MHC Cell-Surface Phenotype of Ltk- X C57BL/6 B Cell Hybrids” Monoclonal Ab (M.F.I.)b Cell type

No Ab

34-l-2 W’)

16-l-2 00

34-s-3 (I-Ab)

14-4-4 (1-E)

LtkC57BL/6 B cells

4.5 9.4

3.5 16.2

20.3 11.1

3.0 29.6

ND” ND

Fusion 3 Fusion 4 Fusion 6 Fusion 7

3.5 6.9 6.5 7.1

44. I 20.6 15.9 14.5

14.3 17.7 37.4 ND

3.8 8.9 8.0 4.5

3.6 7.0 6.0 9.7

a The cell lines indicated were stained with mAb and fluoresceinated rabbit anti-mouse IgG and analyzed by FACS. b M.F.I., Mean fluorescent intensity. ’ ND, Not determined.

mAb 14-4-4 (anti-I-E). Furthermore, Ia expression derived from the B6 B cell fusion partner was lost in all four hybrids as indicated by the lack of staining with 34-5-3 (anti-I-Ab). The histograms for the class II staining of these fusions revealed that these cells were uniformly negative for Ia expression with no minor population of Ia-positive cells present (see Fig. 3). The fact that the hybrids were derived from both Ltkand C57BL/6 B cells was confirmed by positive staining with 16-1-2 (anti-Kk) and 34-l-2 (anti-Kb). These data clearly demonstrated that somatic cell hybrids from all four fusions contained chromosome 17 derived from both fusion partners. In order to evaluate classII expression at the clonal level, we produced 50 independent clones from fusion III. These were first tested for class I surface expression to confirm the presence of both k and b haplotype alleles. Out of the 50 clones, all typed positive for both haplotypes (data not shown). We chose for further analysis those 17 clones that displayed the highest class I staining for both b and k class I alleles. These clones were tested for the expression of class II antigens and the results indicated that not one clone expressedIa of either haplotype (Table 2). The lack of Ia expression from either fusion partner on the surface of the hybrids was also reflected at the level of steady-state RNA expression. Total RNA was isolated from three hybrid lines and seven cloned lines and subjected to Northern blot analysis using an E Q!probe which would be specific for class II expression derived from the L cell partner. This was followed by probing these same blots with A cyto determine if the B cell component of the fusion hybrid retained its ability to transcribe class II genes. No class II mRNA expression was detected with either E a! or A CYprobing of any of the fusions or clones of fusion III (Fig. 1). Since the B cells used for fusion were purified by negative selection of T cells and macrophages, it is possible that the hybrids obtained were the result of the fusion of L cells with a non-B cell contaminant in our preparation. In order to evaluate this, we assessedthe presence of Ig heavy-chain gene rearrangements in the hybrids. From the 50 clones produced, we chose 10 which displayed strong class I staining of both k and b haplotypes to assessIg gene rearrangement. Southern blot analysis was per-

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TABLE 2 MHC Cell-Surface Phenotype of Clones from Fusion III Monoclonal Ab (M.F.I.)’ Clone number (1-E)

No Ab

34-l-2 W’)

16-1-2 W’)

34-5-3 (I-Ab)

14-4-4

8 9 10 11 12 13 16 31 36 39

5.8 4.4 4.7 3.9 4.5 4.4 4.4 1.4 3.6 3.4 2.4 2.6 2.6 2.2 1.2 3.1 2.1

11.9 11.4 18.1 11.3 18.7 18.1 42.5 25.5 22.9 57.5 13.0 11.4 14.1 15.3 29.4 24.7 17.3

ND’ ND ND ND ND 122.7 126.1 66.8 84.5 132.1 46.5 36.3 55.6 55.1 60.8 58.7 38.3

5.1 4.5 4.5 4.1 4.9 4.1 3.5 4.1 3.9 3.1 2.3 2.6 2.4 2.6 2.9 2.4 2.6

5.9 5.2 4.7 4.0 4.9 4.3 3.7 4.1 3.8 3.3 2.2 2.5 2.4 2.6 3.0 3.0 2.7

a M.F.I., Mean fluorescent intensity. b ND, Not determined.

formed on Eco RI digested genomic DNA from all 10 clones using a J-heavy-chain probe. This probe hybridizes to a 6.1-kb Eco RI fragment representing the germ line configuration of the heavy chain. Bands of any other size denote the presence of a heavy-chain rearrangement. As can be seenin Fig. 2, seven of the clones possessedIg gene rearrangement, indicating that they were the result of a fusion involving a B cell. In addition, they also possessedthe nonrearranged band at 6.1 kb, derived from the L cell partner and possibly the nonrearranged allele of the B cell. L cells had only the nonrearranged 6.1-Kb band, as did three of the hybrid clones. In order to further confirm the presence of I-region genesfrom both b and k haplotypes, we made use of a polymorphic restriction fragment at the 3’ end of the E p gene. The same Southern blot described above was boiled and reprobed with an E @ 3’ genomic Pst 1 fragment. As shown in Fig. 2, this probe hybridizes to two fragments of approximately 11.2 and 6.5 Kb sizes. The 11.2-Kb fragment is found in both L cells and all hybrid clones. However, the 6.5Kb fragment was only found in the hybrid clones and not in L cell DNA, indicating that these clones possessedI region genesfrom both fusion partners. Although no constitutive class II expression was found in the hybrids, it was possible that the class II genesof the hybrids would be responsive to the regulatory signals imposed by either IFN-7 or IL-4. In order to investigate this possibility, both cloned and uncloned lines were cultured for 72 hr in the presence of IFN--/ or IL-4 and tested for class I and class II expression by FACS analysis and Northern blotting. Our results show that 1FN-y failed to induce class II surface expression of either the b or k haplotype in uncloned fusion III cells (Fig. 3). We also tested fusion VI and

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Ia truns-ACTING FACTORS IN L-929 CELLS 12345676

397

91011

Ea

Aa

CLASS

I

FIG. 1.Northern blot analysis of fusions and clones to determine the presenceof MHC classII transcripts. Total RNA was isolated from class II negative Ltk- cells (lane 1) and from class II positive A20/2J cells (lane 11) and compared with RNA from fusion III (lane 2), fusion IV (lane 3), fusion VII (lane 4), and clones 7 (lane 5), 8 (lane 6) 10 (lane 7) 1I, (lane 8) 12 (lane 9) and 36 (lane 10) from fusion III. Each panel represents sequential hybridizations of the same blot with the probes for the indicated genes.

clone 11 of fusion III for their ability to respond to IFN-7 and found that these cells did not respond to IFN-7 by an increase in class II expression of either the b or k haplotype (Figs. 4C and 4D). In contrast, when these fusion cells were tested for class

1

2

3 4 5

6

7 8

9 101112

13

EP

FIG. 2. Southern hybridizations of 10 fig of EcoRI digested DNA to IgH and E p probes. DNA from Ltk- (lane 1) and the B cell lymphoma, A20/2J (lane 5), were compared with the following clones from fusion III: clone 10 (lane 2) clone 3 1 (lane 3), clone 36 (lane 4), clone 16 (lane 6) clone 11 (lane 7), clone 8 (lane 8), clone 13 (lane 9), clone 12 (lane lo), clone 7 (lane 1l), clone 9 (lane 12), and clone 39 (lane 13). The two panels represent sequential hybridizations for the same blot with the probes indicated.

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FIG. 3. FACS analysis of the induction of MHC class I and class II expression on fusion III following treatment for 72 hr with either 100 U/ml of IFIGy (. . . ) or 10% SN from the IL4-producing cell line D9C1.12.17(. . .) and compared with unstimulated control fusion III cells (-).

I MHC induction by IFN-y, we observed increased surface expression of both b and k alleles (Figs. 3,4C, and 4D). Since IIN- does not induce class I or II antigens on B cells (Fig. 4B), but does induce class I expression on Ltk- cells (Fig. 4A), these results indicate that the class I genes of the B cell partner are responding in tram to regulatory signals contributed by the L cell following IFN-7 stimulation. We next attempted to stimulate these same hybrids with IL-4, which is known to induce class II expression on B cells. The results in Figs. 3, 4C, and 4D demonstrate that there was no Ia expression of either the b or k haplotypes, indicating that the L cell’s negative influence on classII expression was also dominant over IL-4 responsive regulatory signals. As a control for the activity of IL-4, we isolated B cells from B6 mice and stimulated them with IL-4. As expected, IL-4 clearly increased class II expression on these cells as seen by an increase in mean fluorescent intensity of almost threefold over control levels (40.84 from 14.89) (Fig. 4B). In order to confirm the lack of Ia induction following stimulation with either IFNy or IL-4, we performed Northern blot analysis on fusion III clones 7, 8, 11, and 12 following stimulation with these reagents. The results shown in Fig. 5 indicate a lack of significant E LYor A /3 mRNA for either Ltk- or any of the hybrid clones tested, regardless of whether they were uninduced or had been induced with IFN-y or IL-4. Due to a hybridization artifact associated with the actin probing of the B cell RNA (lanes 4-6), and the fact that the UV shadow picture of this blot showed significant

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1-E”

Kk

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Ia truns-ACTING FACTORS IN L-929 CELLS

d

Kb

40

-0

20

1-d

I-Ek

0

Kk

FIG.4. Graphic representation of FACS analysis for MHC expression of Ltk- (A), C57BL/6 B cells (B), Fusion VI (C), and clone 11 of Fusion III (D) following induction with IL-4, IFN-7, or media alone. Bars represent the mean fluorescent intensity of the staining of the indicated cell with anti-Kb (34-l-2), anti-Kk (16-l-2) anti-I-Ab (34-5-3) or anti-I-Ed (14-4-4). Values displayed were corrected for nonspecific background staining of each cell type with FITC-labeled anti-mouse IG alone. These background values ranged from 6 to 12 mean fluorescent units.

underloading of the B cell control lane (lane 4), we cannot adequately determine the degree of IL-4 or IFN-7 induction of either A 6 or class I mRNA on this particular blot. Nevertheless, the response of normal B cells to these lymphokines is well established (8) and readily apparent in Figure 4B. In any case, these B cell lanes serve as controls for the specificity of our A /3 and E (Yprobes since C57BL/6 expressesA B but not E (Ygenes. On the other hand, both the hybrids and Ltk- cells did show induction of class I mRNA of roughly two to threefold above control levels when stimulated with IFN-y. As was seen with surface expression, IL-4 did not have any effect on class I MHC gene expression in Ltk- or any of the four clones tested. These results confirmed our surface expression data, which showed that neither IFN--/ or IL-4 had the capacity to induce class II gene activity, but that IFN-7 but not IL-4 was able to induce class I gene activity of both setsof genesin the hybrids. DISCUSSION In this study we have demonstrated, both by FACS analysis of surface expression and Northern blot analysis of steady-state RNA levels, that fusions between class II MHC-expressing B cells and the Ia-negative fibrosarcoma L-929 cell do not induce the expression of L-929 class II genes. Instead, we observed a loss of B cell Ia expression. This loss of B cell Ia expression was also observed for fusions between B cells and an Ia-negative squamous cell carcinoma, TDM4 (44) (data not shown). In fact,

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7 6

9 1011

1213

Ea

AP

CLASS

I

ACTIN

FIG. 5. Northern blot analyses of the induction of MHC class I and class II genes from uninduced cells (lanes 1, 4, 7, and 10) and compared with cells stimulated for 72 hr with either 10% SN from the IL-4producing cell line D9Cl. 12.17 (lanes 2, 5, 9, and 12) or 100 U/ml of 1FN-y (lanes 3,6, 8, and 11). Total RNA was isolated from Ltk- (lanes l-3), C57BL/6J B cells (lanes 4-6), clone 8 of fusion III (lanes 7-9), clone 12 of fusion III (lanes IO- 12), and A20/2J. Actin probing of blot was done in order to compare the relative amounts of RNA in each lane. Each pane1represents sequential hybridizations of the same blot with probes from the indicated genes.

we have not been able to demonstrate any induction of the class II genesfor any Ianegative NHD cell line fused to an Ia-positive hematopoietic cell, even when using transient fusion techniques (data not shown). Furthermore, we demonstrated that the resultant hybrid, formed from a L-929 and B cell fusion, appeared to respond to MHC regulatory agents (IFN-7 and IL-4) as if it were an L-929 cell and not a B cell. This was demonstrated first by the hybrid’s lack of responsivenessto the B cell-stimulating activity of IL-4, and second by the induction of class I expression when stimulated with IFN-7. These are both characteristics of the L-929 cell, whose MHC antigen expression is unaltered by IL-4 but whose class I expression is induced with EN-y. Our results are similar to a report by Aragnol et al. (45) who described a fusion between Ia positive C57BL/6 B cells and the Ia negative AKR T cell lymphoma, BW5 147. The hybrids derived from this fusion expressed the class I genes of both partners of the fusion but failed to expresseither partner’s classII genes.These results indicated that the fusion had, in some way, extinguished the Ia expression of the B

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cell partner to the fusion. The mechanism for this extinction of B cell Ia expression was presumably due to the presence of negative Ia truns-acting factors derived from the BW5 147 T cell. A similar study involving fusions between Ia-negative plasma cells and normal Ia-positive B cells also demonstrated that the resultant hybrids were devoid of Ia expression of either cell partner (25). This is another casewhere the loss of Ia expression was due to the presence of negative truns-acting factors, although in this instance the source for these factors was the plasma cell. Results demonstrating cell- or possibly species-specifictrans-acting factors showed that fusion of Ia-negative B cells and macrophages failed to rescue expression of class II genes from the B cell partner (46). Furthermore, when these hybrids were treated with EN-y, both classI and classII genesof the macrophage were induced. However, only class I but not class II genesof the B cell were induced. These results suggestthat IFN-7 mediated increases in macrophage class II expression do not employ the air- 1 gene, and that whatever truns-acting factors are involved they are not capable of activating class II expression in some class II-negative B cell mutants. Our results demonstrate a loss of Ia expression in fusions between B cells and Ltkcells. The mechanism for this effect is, as yet, undetermined, but could be due to either the presence of negative trans-acting factors originating from the L-929 cell or the lack of positive truns-acting factors, whose expression is lost as a result of fusion with L-929. We would argue that the first explanation is the most likely for several reasons. First, murine B cells are believed to produce positive trans-acting factors that are, at least in part, encoded by air- 1. Since fusion with human cells does not lead to the inactivation of this gene, one would expect that fusion with mouse cells should not, in and of itself, turn off the air- 1 gene or other genesinvolved in class II expression. It is also possible that the hybrids have lost chromosome 16, which codes for air- 1, or some other chromosome encoding positive regulatory factors from the B cell partner of the fusion and have therefore lost a positive truns-acting factor required for class II expression. We believe that it is improbable that those particular chromosomes would be preferentially lost in the vast majority of hybrids in all four fusions performed. In addition, we would have expected to see some Ia-expressing cells in the uncloned population early after fusion if this were the caseand we did not. Previous reports (47-49) describing expression of transfected genomic I-A genesin L cells provide evidence that L cells do have the capacity to expressIa gene products. However, these studies do not necessarily negate the possible existence of negative [runs-acting factors controlling Ia gene expression for several reasons. First, the transfected gene constructs contained viral sequencesthat had positive promotor and/or enhancer activity that could be overriding any potential negative factor possessedby the host L cell. Second, the expression of these transfected genescould have been the result of integration into transcriptionally active sites of the host genome not affected by host negative truns-acting factors. Finally, since not all transfectants expressed Ia genes, thereby necessitating selection of Ia-positive clones, some of these clones may have been the result of integration of multiple copies of the added genes.The presence of multiple copies of class II genes could competitively bind endogenous negative truns-acting factors leading to transcription of those transfected copies free of bound factor. Support for this explanation can be found in a recent report by Fukushima et al. (50). Their studies demonstrated that when A p genes were transfected to high copy numbers into Ia- B cell hybridomas, they not only observed expression of the transfected A p gene, but they also saw activation of the A (Ygene as well, leading to

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the expression of I-A molecules on the surface of these cells. This suggestedthe possibility that the increased number of extrachromosomal A /3genescompetitively bound a negative factor (repressor protein) resulting in the activation of the endogenous A (Ygene. This same hypothesis was also put forward by Nir et al. (5 1) to explain the regulation of insulin I gene expression in nonpancreatic cells. In similar experiments implicating the existence of negative trans-acting factors, Liou et a/. (24) describe three DNase I-hypersensitive sites 5’ of the A (Ygene, one of which was specific to cells that normally express class II molecules. This site, which they termed D2, was lost in Ia-negative hybrids between B cells and T cells. They concluded that the existence of negative trans-acting factors originating from the T cell prevents the appearance of the D2 site and that these factors can act in tram to eliminate this site in B cells. They believe that this site is critical to the ability of the A CY gene, and presumably other class II genesas well, to be transcribed and therefore expressed. It would be interesting to determine if the D2 site is altered in our hybrids as well. Thus, there is now a convincing body of evidence for a role of negative transacting factors in class II gene regulation. Therefore, it would seem to us that the most likely mechanism for the loss of B cell Ia expression in our studies is due to the presence of negative trans-acting factors originating from the Ltk- cell. The results described in this paper, along with other reports concerning expression of class II genes, suggestthere are three basic categories of cell types with respect to regulation of Ia expression. First are those cells, typified by B cells, which constitutively express class II genes. This expression is presumably due to the presence of positive trans-acting factors, that at least in one case (17) are not species specific. These factors appear to be somewhat labile since continuous protein synthesis is required to maintain transcription of the class II genes in B cells (J. G. Woodward et al., submitted). Second, there are those cells, typified by macrophages, which, in the presence of the appropriate inducing agent, will express class II genes. In the case of IFN-7 induction of class II in macrophages, it appears that the trans-acting factor pathway is somewhat distinct from that used in B cells (46 and J. G. Woodward et al., submitted). The third group consists of those cells that do not express class II genes under any known conditions. These cells presumably possessstrong negative trans-acting factors that can, even in the presence of positive trans-acting factors, block expression of class II genes. The question of why certain cells possesssuch strong negative regulatory signals is very intriguing, and we would like to propose a potential reason for their existence. It is known that many NHD cell types possessEN-y, IL-4, or both IFN-y and IL-4 receptors, yet they remain unresponsive to the class II-inducing activity of these agents, even though they retain responsiveness to other effects elicited by EN-7 or IL-4 (52,53). The existence of a dominant negative class II regulatory system in these cells may allow them to respond to these inducing agents in other ways while maintaining their Ia- phenotype. Such a mechanism would be beneficial to host immune function by preventing unnecessary and possibly harmful expression of Ia antigens, whose presence on these cells might activate nonspecific inflammatory immune responses, or immune responses that are specific for normally tolerated self antigens. In fact, recent reports have implicated inappropriate class II expression as potentially playing a role in the pathogenesis of collagen-induced arthritis (54), autoimmune nephritis (55), and diabetes (56). Whether negative trans-acting factors exist, at least in part, for this reason is not known and remains to be determined.

NEGATIVE

Ia truns-ACTING FACTORS IN L-929 CELLS

403

ACKNOWLEDGMENTS We thank Drs. D. Mathis, C. Benoist, L. Kedes, and R. Riblet for providing the plasmids used in these studies. We also thank Lisa Sloan, Kathy Omer, and Cathy Nowak for their excellent technical assistance.

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