Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing

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Miriam A. OssevoorP, Alice J. A. M. Sijts., Karin J. H. van Veen., Frank Momburg., Giinter J. Hammerling., Angela Seelig., Geoffrey W. Butcher', Jonathan C. Howard., W. Martin Kast." and Cornelis J. M. MelieP Division of Immunohaematology and Blood Bank., University Hospital, Leiden, German Institute for Immunology and Genetics., Heidelberg and Department of Immunology., The AFRC Babraham Institute, Cambridge

Eur. J. Immunol. 1993. 23: 3082-3088

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing* The protein products of the Tap (Transporter associated with antigen processing) 1and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line M A - S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of M A - S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced 1evels.Transfectionof aTap 2 gene of mouse (BALB/c, H-2d)or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

1 Introduction Cytotoxic T lymphocytes (CTL) recognize intracellular antigen as 8-10 amino acid long peptides presented in the groove of MHC class I molecules [l-31. These peptides are thought to arise from cleavage of proteins in the cytosol[4]. The products of the MHC-encoded peptide transporter genesTap 1and Tap 2 together form a heterodimer thought to be involved in the transport of peptides into the endoplasmic reticulum (ER) where they can assemble with MHC class I molecules [5-lo]. The mouse mutant T lymphoma cell line RMA-S contains a mutated Tap 2 gene. The putative aberrant Tap 2 gene product is very short and so would not be expected to form a functional heterodimer with the Tap 1 gene product, which probably results in failure of peptide delivery into the ER. Phenotypically, this

leads to a reduced cell surface expression of MHC class I molecules which can partly be restored by culture at reduced temperature or by incubation with MHC class I binding peptides [ l l , 121. Restoration of MHC class I cell surface expression is accomplished by transfection of a rat or a mouse Tap 2 gene [13, 141.

We here describe the effect of Tap 2 gene transfection into RMA-S on the ability to present viral and allogeneic antigens. Moloney murine leukemia virus (MuLV)-specific CTL which cross-react with Rauscher viral antigens recognize RMA-S albeit at a strongly reduced level in comparison with the parental RMA tumor cell line [15]. Our results show that transfection of a Tap 2 gene of rat or allogeneic (BALBk) origin into RMA-S does not result in an increase in presentation of peptides derived from Rauscher MuLV. RMA-S cells also process and present allogeneic and Sendai viral antigens at reduced levels, but the presentation of these antigens, in contrast to Rauscher MuLV antigen presentation, is increased after transfection with a Tap 2 [I 117361 gene of rat or mouse (BALB/c) origin.

*

This work was supported by the Netherlands Organization for Scientific Research (NWO) (Grant 900-509-151) and the Dutch Cancer Society (Grant IKW 89-09). Fellow of the Royal Netherlands Academy of Arts and Sciences (KNAW).

These data confirm that the defect of RMA-S for the presentation of H-2b-restricted antigens is leaky and demonstrate that transfection of a Tap 2 gene of rat or allogeneic (BALB/c, H- 2d) origin into RMA-S has differential effects on the processing and presentation of viral antigens.

Correspondence: Cornelis J. M. Melief, Division of Immunohaematology and Blood Bank, University Hospital, Building 1,E3-Q, PO. Box 9600, NL-2300 RC Leiden, The Netherlands (Fax: 31 71 21 6751)

2 Materials and methods

Abbreviations: Tap: Transporter associated with antigen processing MuLV: Murine leukemia virus

2.1 Mice

Key wonls. RMA-S cells / Transporter gene / Sendai virus / Rauscher virus / Cytotoxic T lymphocyte

C57BL/6 (B6, Kb, Db) mice were obtained from IFFA Credo (UArbresle, France). The spontaneously derived

0014-2980/93/1212-3082$10.00+ .25/0

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1993

Eur. J. Immunol. 1993.23: 3082-3088 B6.CH-2bm1(bml, Kbml, Dh) and the spontaneously derived B6.CH-2bm13(bm13, Kb, Dbm13)mutant mice were obtained from the Central Animal Facility of the Netherlands Cancer Institute (Amsterdam,TheNetherlands).The Kb mutations in bml resulted in amino acid changes from Glu to Ala at position 152, Arg to Tyr at position 155 and Leu to Tyr at position 156 [16]. The Db mutations in bm13 resulted in amino acid changes from Leu to Tyr at position 114, Phe to Tyr at position 116 and Glu t o Asp at position 119 [17]. All mice were maintained at the Central Animal Facility of the University Hospital Leiden under pat hogen-free conditions.

Viral antigen presentation by Tap 2-transfected RMA-S

3083

84K-

5OK-

2.2 Peptide and virus Peptide was synthesized on a Biosearch 9500 peptide synthesizer (Milli GedBiosearch, Middlesex, GB) according to Merrifield [18] and was dissolved in 0.9% NaCl and stored at - 20 "C. The Sendai virus peptide encompasses the immunodominant epitope of Sendai virus nucleoprotein for H-2Kb-restricted CTL (S9; amino acid residue 324-332: FAPGNYPAL) [ 19,201. Live non-virulent Sendai virus lot 403440089 was obtained from Flow Laboratories (McLean, VA, USA) and stored at - 70 "C.

Figure I. Expression of Tap 1and Tap 2 in RMA, RMA-S cells and RMA-S cells transfected with mTap 1,2 genes measured by Western blot analysis. Cell lysates were probed with rabbit antisera against synthetic peptides based on the carboxy termini of rTap 1 and rTap 2 (see Sect. 2.3).

2.3 Cell lines

and were incubated with the second Ab goat anti-mouse F(ab')2-FITC (Tago, Burlingame, CA) for 30 min at 4 "C. FCM analyses were performed using a FACScan flow cytometer with consort 30 software (Becton Dickinson, Immunocytometric Systems, Mountain View, CA).

RMA, RMA-S cells (H-2b) were obtained from Dr. K. Karre, Karolinska Institute, Stockholm, Sweden. RMA-S cells used in this study were transfected with rTap 1 cDNA (mtpla, rat) [8] or rTap 2 cDNA ( m t ~ 2rat) ~ , [13] or with the BALB/c derived cosmid I1 5.9 [21], kindly provided by Drs.Y. Uematsu and M. Steinmetz (Basel, Switzerland), which encompasses the mouse Tap 1 and Tap 2 genes [14,22]. The protein level of Tap 1 and Tap 2 in RMA-S cells transfected with mTap 1,2 genes was measured by Western blot analysis as described [ 131. Each track contains an equal amount of cell extracts. Antisera recognizing rat Tap 1 [13] was used at a l/700 dilution and antisera recognizing rat Tap 2 [23] was used at a 'hooo dilution (Fig. 1). Western blots using sera raised against Tap2 showed that the RMA-S cells transfected with mTap 1,2 genes expressed the Tap 2 polypeptides in amounts comparable to RMA cells. I7MA-S cells did not express the Tap 2 polypeptides. As a control, Western blots using sera raised against Tap 1 showed that all cell lines expressed Tap 1 polypeptides (Fig. 1). All cell lines were cultured in Iscove's modified Dulbecco medium (Seromed, Biochrom KG, Berlin, FRG) supplemented with 8% heat-inactivated FCS, penicillin (100 IU/ml) , kanamycin (100 pg/ml) , Lglutamine (2 mM) and 2-ME (20 pM) at 37 "C. 2.4 Indirect peptide binding assay Cell lines (RMA, M A - S or RMA-S cells transfected with a Tap gene) were washed with serum-free medium and incubated with 1 pglml S9 peptide at 37 "C.After 1 , 2 or 4 h cells were washed with PBS supplemented with 0.02% NaN3 and 0.5% BSA and incubated with saturating amounts of anti H-2Kb Ab (B8-24-3 [24], 28-13-3 [25], K10.56[26]) orantiH-2DbAb(28-14-8s [27], H141.31[28], B22.2491 [28]) for 30 min at 4 "C. Cells were washed twice

anti-Tap1

anti-Tap2

2.5 CTL clones The Moloney MuLV-specific H-2Kb-restricted CTL clone 10B6 was derived by limiting dilution from a Moloney MuLV-specific B6.CH-2bm13CTL bulk culture as described [29]. The alloreactive anti-Kb specific CTL clone 30D6 was derived by limiting dilution from a bml (Kbm*) anti B6 (Kb) specific CTL bulk culture. Cells were stimulated weekly with irradiated (25 Gy) Moloney MuLV-infected syngeneic LPS blasts or spleen cells of B6 origin, respectively, and culture supernatant from PMA/ConA-stimulated rat spleen cells.

2.6 Induction of secondary CTL responses Moloney MuLV-specific B6 CTL bulk cultures were generated as described [29] and weekly stimulated with irradiated Moloney MuLV-infected syngeneic LPS blasts. Allo-H-2Kb specific CTL bulk cultures were generated by culturing 5 x 106 spleen cells of b m l origin with 2.5 x 106 irradiated spleen cells of B6 origin. For the induction of a secondary Sendai virus-specific CTL response B6 mice were primed by one i.p. injection of 102 hemagglutinating units (HAU) of non-virulent Sendai virus [30] at the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service Amsterdam, The Netherlands. At 2-6 weeks after the immunization, spleen cells (5 x lo6) were co-cultured in vitro with 2.5 x 106irradiated (25 Gy) non-virulent Sendai virus infected syngeneic spleen cells. For the induction of S9 peptide-specific CTL response B6 mice were immunized twice with a 2-week interval with 100 pg S9 peptide dissolved in IFA (Difco Laboratories,

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Detroit, MI): 0.9% NaCl = 1: 1 (v/v) supplemented with 0.5% BSA (Sigma Chemicals Co., St. Louis, MO). Two weeks after the second immunization spleen cells ( 5 x lo6) were stimulated in virro with 2.5 x 106 irradiated S9 peptide sensitized syngeneic spleen cells. Spleen cells were sensitized with 50pg/ml S9 peptide for 4 h at room temperature.

2.7 CTL assay After 5 days of culture the effector cells were harvested on a Lympholyte M gradient (Cedarlane Laboratories, Hornby, Ontario, Canada). Various numbers of effector cells were incubated with 2000 %r-labeled target cells for 6 h at 37 "C.Target cells were RMA, RMA-S and RMA-S cells transfected with one or two Tap genes. S9 peptide-loaded target cells were incubated for 10 min at room temperature with 3 pg/ml S9 peptide after labeling and before adding them 1:2 (v/v) to the effector cells. To generate Sendai virus-infected target cells, 2 x lo6 cells were incubated with 200,20,2 or 0.2 HAU non-virulent Sendai virus during labeling with W r . The percentage specific %r release was calculated by the formula: 100 x [(cpm experimental well background W r release) : (cpm 2% Triton X-100 release background 51Cr release)]. All assays were carried out in triplicate. No substantial difference in background release was observed between the different cell lines and the cell lines sensitized with S9 or infected with non-virulent Sendai virus.

Eur. J. Immunol. 1993. 23: 3082-3088

fected with a rTap 2 gene or mTap 1,2 genes compared to RMA-S and RMA. Alloreactive CTL bulk cultures recognize RMA-S transfected with a flap 2 or mTap 1,2 genes, but the percentage W r release was 56% and 52%, respectively, versus 81% for RMA and 21% for RMA-S at an E:Tratio of 3:l (Fig. 2). RMA-S cells transfected with a flap 1 gene were lysed to an even lesser extent than were RMA-S cells (Fig. 2). Three types of H-2Kb-specificCTL clones can be distinguished by their ability to lyse RMA-S cells [31,33]. Some CTL clones lyse RMA-S efficiently, whereas other CTL clones show an intermediate lysis. The third type of CTL clone fails to lyse RMA-S. Our H2Kb-specificCTL clone 30D6 fails to lyse both RMA-S cells and RMA-S cells transfected with a rTap 1 gene (Fig. 3). RMA-S cells transfected with a 'Tap 2 gene are specifically lysed but to a lesser extent than RMA (Fig. 3). Thus, although the cell surface expression of Kb is repaired on RMA-S cells transfected with a rTap 2 gene or the mTap 1,2 genes, the presentation of this particular allogeneic antigen is partly restored.

-

76 specific lysis IW

-RMA 80-

W - S + rTap2

* RMA-S+ mTap1,Z

60-

3 Results

RMA-s RMA-S +[Tap 1

40-

3.1 Presentation of allogeneic antigens by RMA-Scells is partly restored by Tap 2 gene transfection The cell surface MHC class I expression on RMA-S cells is partly restored after transfection of a rTap2 gene and completely restored after transfection of the mTap 1and 2 genes into RMA-S and is not restored on RMA-S transfected with a rTap 1 gene (Table 1,[13, 141). Allo-H-2Kbspecific CTL bulk cultures recognize RMA-S less well than they do RMA, the parental cell line [31-33].We determined the lytic activity of H-2Kb-specificCTL bulk cultures and an H-2Kb-specificCTL clone 30D6 on RMA-S cells trans-

- 0

1

10

100

EiT ratio

Figure 2. Lytic activity of the allo-H-2Kb-specificbulk CI'L, generated from bml mice against Wr-labeled RMA, RMA-S, RMA-S cells transfected with a rTap 1, r n p 2 or mTap 1,2 gene. Values shown represent specific lysis and standard deviation of three determinations.Thisfigure is representative of the results obtained in three independent experiments.

Table 1. MHC class I Kb (B8-24-3,28-13-3, K10.56) and Db (28-14-8S,H141.31, B22.249.1) expression on RMA, RMA-S and RMA-S cells transfected with a transporter (Tap) gene measured by FCM analyses MHC expression H-2Kb) B8-24-3 28-13-3 K10.56 H-2Db) 28-14-83 H141.31 B22.249.1

Cell lines

RMA

RMA-S

+

RMA-S+ fmP 1

RMA-S + fip2

RMA-S mlkp1,2

518) 34 61

14 10 11

12 8 9

35 22 33

61 38 47

66 67 92

9 18 16

6 9 15

21 23 34

59 46 82 a) Mean fluorescence channel.

Viral antigen presentation by Tap 2-transfected RMA-S

Eur. J. Immunol. 1993.23: 3082-3088

-

% specific lysis

7 -

50-

--C

*

3085

(Fig. 4B). Thus, transfection of a rat or allogeneic mouse Tap 2 gene into RMA-S cells which does restore the MHC class I cell surface expression, did not augment the presentation of Rauscher viral peptides.

RMA RMA-s RMA-S + rTap 1 RMA-S+rTap2

40-

3.3 Sendai virus- and S9 peptide-specitk CTL cultures recognize Sendai virashfected RMA-S cells to a lesser extent than Sendai virus-infected RMA cells and Tap 2 gene-transfected RMA-S cells

30 -

RMA-S cells have been reported to be incapable of presenting several endogenous antigens, including influenza viral antigens [12]. Transfection of a rat or mouse Tap 2 1 10 100 gene into RMA-S results in a recovery of the ability to EJT ratio process and present influenza viral antigens [13, 141. DeFigure 3. Lytic activity of the allo-H-2Kb-specificCTL clone 30D6 spite the above, RMA-S cells are permissive for presentagenerated from bml mice against 51Cr-labeledM A , RMA-S and RMA-S cells transfected with a r'hp 1or rTap 2 gene.Values shown tion of vesicular stomatitis virus (VSV) antigens and to represent specific lysis and standard deviation of three determina- some extent Rauscher viral antigens [15, 36,371. To chartions. Similar results were obtained in three independent experi- acterize further the RMA-S defect, we investigated the presentation of Sendai viral antigens by RMA-S cells, ments. RMA cells and RMA-S cells transfected with aThp 1and or a Thp2 gene. Sendai virus-infected RMA-S cells were specifically lysed by Sendai virus-specific bulk CTL (Ta3.2 Presentation of Rauscher viral antigens by RMA-S ble 2). However, Sendai virus-infected RMA-S cells as well is not restored after transfection of a Tap 2 gene as RMA-S cells transfected with a rQp 1gene were lysed to RMA and RMA-S were derived from RBL-5, which is a a lesser extent than Sendai virus infected RMA and RMA-S Rauscher-induced T cell lymphoma [34,35]. Moloney transfected with a rTap 2 or the mTap 1,2 genes by Sendai MuLV-specific CTL cross-react with Rauscher viral anti- virus-specificbulk CTL (Table 2). S9 peptide-specific bulk gens presented by RMA cells. RMA-S cells are also lysed CTL specifically lysed Sendai virus-infected RMA cells and specifically,but to a greatly reduced extent. The Rauscher RMA-S cells transfected with a rTap 2 or the mTap 1,2 MuLVexpression on RMA and RMA-S cells is similar [15]. genes (Table 2). All cell lines were sensitized for lysis by S9 FCM analyses with virus-specific antibodies against the peptide and Sendai virus-specific bulk CTL in the presence viral core and envelope protein on intact cells demonstrated of exogenous antigenic S9 peptide (Table 2). Uninfected that in RMA-S cells Rauscher viral expression does not cell lines were not lysed by these CTL (data not shown).The differ from that in RMA-S cells transfected with a rTap 1or lysis of Sendai virus-infected RMA-S cells could not be rTap 2 gene or the mTap 1,2 genes (data not shown). 10B6is attributed to the presence of S9 peptide in the Sendai virus, a class I Kb-restricted Moloney-specific CTL clone of because after 4 h of infection with Sendai virus the MHC B6.CH-2bm13 origin [15].We demonstrate that the transfec- class I Kb cell surface expression on RMA-S cells did not tion of either allogeneic mThp 1,2 genes (BALB/c) or a increase (Table3). Such an increase in expression is r n p 2 gene into RMA-S cells did not result in an increased observed if exogenous Sendai virus peptide has bound to susceptibility to CTL clone 10B6 (Fig. 4A). All cell lines Kb. Thus, the defect of RMA-S cells to present Hwere sensitized for lysis by addition of the, Moloney 2b-restricted antigens is leaky for Sendai viral antigens. MuLV-encoded,10B6CTL epitope (Sijts et al., manuscript Transfection of a Thp 2 gene of rat or allogeneic (BALB/c) in preparation). Moloney MuLV-specificCTL bulk cultures origin into RMA-S increases the presentation of Sendai of B6 origin behave similarly to CTL clone 10B6 with viral antigens. respect to recognition of RMA, RMA-S and RMA-S cells transfected with a rTap 2 gene or the mTap 1,2 genes

-

% specific lysis

-RMA

30j

2.5

5

EJT ratio

I

2.5

T

5

10

20

EJT ratio

RMA-s RMA-S + ffap 1 RMA-S+ffapZ Rh4.4-S + mTapl.2

Figure 4. Lytic activity of the Kb-restricted Moloney MuLV virus-specific bm13 CTL Clone 10B6 (A) and the Moloney MuLV-specific B6 CTL bulk culture (B) against 51Cr labeled RMA, RMA-S, RMA-Scells transfected with a rTap 1, i m p 2 or the mTap 1,2 genes. Values shown represent means and standard deviation of three independent experiments.

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Table 2. Lytic activity of Sendai virus-specific bulk CTL and S9 peptide-specific bulk CTL against RMA, RMA-S, RMA-S cells transfected with a rTapl, rTap2 or mTapl,2 genea) Cell lines

SV-specific S9P

sv

S9p-specific CTL bulkh) S9P sv

42cJ 57 33 51

24 23 23 15

57 43 37 23

11 8 4 -3

52 56

56 63 58 48

49 35 30 15

38 26 18 14

21 16

56 19

6 3 1 0

52 39 26 31

58 55 44 23

31 21 7 1

52 61 48 42

44 36

37 35 27

CTL bulkb,

Eur. J. Immunol. 1993. 23: 3082-3088

-

% specific lysis

-m

I"

60 50

RMA-s RMA-S+rTap 1 RMA-S+rTapZ

40 30

RMA-S

RMA

46 42

RMA-S rTapl

+

41

48 40 37 49

RMA-S +

54 40 34 27 26 32 31

+

RMA-s mTapl, 2

48 38

13 10

33 35

14

a) %get cells were sensitized with S9 peptide (S9p) or infected with 200 HAU Sendai virus (Sv). The lysis of uninfected cells was less than 10%. SD were less than 8%. Data are representative of the results obtained in three independent experiments. b) Emratio = 50:1, 25:1, 12.5:1, 6.2:l. c) YO specific lysis.

Table 3. Kb (B8-24-3) expression on RMA-S cells after 1,2 or 4 h of incubation with S9 peptide (1 pg/ml) or non-virulent Sendai virus (25 HAU) measured by FCM analyses

Cell lines

Time (h) -

RMA-S

1 2 4

5a)

7 9

Kbexpression S9 peptide Sendai virus

31 54 94

6

8 10

a) Mean fluorescence channel.

3.4 M A - S cells require a higher virus dose to become sensitized for CTL recognition than either RMA cells or R M A - S cells transfected with a Tap 2 gene

RMA, RMA-S and RMA-S cells transfected with a Tap gene were infected with different doses of Sendai virus to determine the minimal concentration of Sendai virus needed to sensitize these cells for lysis by Sendai virusspecific bulk CTL. RMA-S cells and RMA-S cells transfected with a rTap 1 gene required approximately tenfold more Sendai virus to become targets for recognition by Sendai virus-specific bulk CTL than RMA cells or M A - S

20 10

0 0

0.2

2 20 200 Sendai virus (HAW

Figure 5. Lytic activity of Sendai virus-specific B6 bulk CTL against Sendai virus-infected M A , RMA-S and RMA-S cells transfected with a rTap 1or rTap 2 gene. Target cells were infected with different concentrations (0, 0.2, 2, 20 or 200 HAU) of non-virulent Sendai virus during W r labeling. Points represent percent specific lysis (% f S.D.) of these cell lines by CTL bulk at an E/T ratio of 50: 1. This figure is representative of the results obtained in three independent experiments.

cells transfected with a 'Tap 2 gene (Fig. 5). Thus, RMA cells and RMA-S cells transfected with a rTap 2 gene do not differ with respect to processing and presentation of Sendai virus.

4 Discussion Treatment of RMA cells with anti-H-2 class I alloantisera and complement resulted in the selection of an H-2 variant cell line RMA-S [34, 35].The strongly reduced MHC class I cell surface expression of M A - S cells is restored after transfection of a rTap 2 or the allogeneic mTap 1,2 genes into RMA-S (Table 1) [13, 14,221. Several experimental results have been reported regarding the ability of RMA-S cells to present viral antigens. RMA-S cells can present VSV antigens and to some extent Rauscher viral antigens but are incompetent to present influenza viral antigens [12, 14, 151. In addition, we report that RMA-S cells can process and present Sendai viral antigens, but to a lesser extent than RMA cells (Table 2, Fig. 5). Transfection of an allogeneic mTap 2 gene into RMA-S cells restores the ability to process and present influenza viral antigens [14]. We have demonstrated that M A - S cells transfected with a rTap 2 gene or allogeneic mTap 2 gene and RMA cells do process and present Sendai viral antigens equally well (Table 2, Fig. 5). The presentation of Rauscher viral antigens is not increased after transfection of allogeneic mTap 1,2 genes or a rTap 2 gene into M A - S cells (Fig. 4A, 4B). Several explanations can be postulated. First, during the anti-MHC class1 selection procedure [34] mutations in the endogenous Rauscher viral DNA could have taken place. This may result in the presentation of mutated viral epitopes by RMA-S cells which therefore could act only poorly as targets for Moloney MuLV-specific H-2Kb-restricted CTL of bm13 origin as well as the Moloney MuLV-specific CTL bulk cultures of B6 origin. However, these CTL each

Eur. J. Immunol. 1993. 23: 3082-3088

recognize different epitopes derived from the Moloney virus (unpublished observations). We know that RMA and RMA-S cells do not differ in the RNA expression of Rauscher virus and cell surface expression of the Rauscher viral core end envelope protein [15]. Furthermore, an increase in the presentation of Rauscher viral antigens to T cells by RMA-S cells is observed when cultured at reduced temperature [15]. Together, this implies that RMA-S cells do present the proper Rauscher MuLV peptides, although at a strongly reduced level. Second, the class I-associated epitopes from Rauscher MuLV could be generated and transported to the area where they can assemble with MHC class I molecules by an other unknown mechanism independent of the MHCencoded Tap gene products. This unknown mechanism might be a second defect in RMA-S versus RMA and obviously can not be restored by a Tap 2 gene transfection. Third, the transporter genes of rat origin display a complex polymorphism which affects the identity of peptide found in association with a given MHC class I molecule [38,39]. These data imply that the transporter plays a role in the selection of peptides that are available for binding to MHC class I molecules. Interestingly, both chains of the human transporter are also polymorphic [40-42]. In this study, the Tap 2 genes transfected into RMA-S (H-2b) were of rat origin or of mouse origin of a different haplotype (BALBk, H-2*) [8, 13, 14,211. It is possible that the set of peptides which were transported into the ER by the rTap 2 gene or allogeneic mTap 2 gene did not contain the peptides recognized by the Moloney MuLV virus-specific CTL. The last two explanations can be put forward with respect to the data obtained with the allogeneic H-2b-restricted CTL which demonstrated that transfection of a Tap 2 gene into M A - S only partly restored the recognition (Figs. 2 and 3). Future studies with RMA-S cells transfected with theTap 2 gene of syngeneic origin (H-2b) will test this hypothesis. We thank Simon J. Powis for providing the RMA-S cells transfected with a rTap2 ( m t ~ 2 gene ~ ) and Evelyn Tredget for technical assistance. We thank Drs. E Koning and E. Goulmy for critically reading of the manuscript.

Received April 8, 1993; in revised form July 30, 1993; accepted August 18, 1993.

5 References 1 Rotzschke, 0. and Falk, K., Immunol. Today 1991. 12: 447. 2 Van Bleek, G. M. and Nathenson, S. G., Trends Cell Biol. 1992. 2: 202. 3 Bjorkman, €? J., Saper, M. A., Samraoui, B., Bennet, W. S., Strominger, J. L. and Wiley, D. C., Nature 1987. 329: 506. 4 Moore, M. W., Carbone, F. R. and Bevan, M. J., Cell 1988.54: 777. 5 Braciale, T. J. and Braciale, V. L., Immunol. Today 1992. 12: 124. 6 Monaco, J. J., Curr. Opin. Immunol. 1993. 5: 17. 7 Monaco, J. J., Cho, S. and Attaya, M., Science 1990. 250: 1723.

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Eur. J. Immunol. 1993. 23: 3082-3088 Note added in proof: Recently, Zhou et al. (Eur. J. Immunol. 1993.23: 1796) also described that RMA-S cells are capable of presenting low levels of Sendai viral antigens and found this presentation to be restored to the RMAlevel by introductionof the ' h p 2 gene.

Received September 20, 1993.