Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta

Acha-Orbea, H.

Held, W.

MacDonald, H.R.

Scarpellino, L.

Shakhov, A.N.

Serveur académique lausannois

Br ie f  De~nitive Report Iphibition of Mouse Manunary Tumor Virus-induced T Cell Responses In Vivo by Antibodies to an Open Reading Frame Protein By Hans Acha-Orbea, Leonardo Scarpdlino, Alexander N. Shakhov, Werner Hdd, and H. Robson MacDonald From the Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland Summary Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) ~ chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (off) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism ofaction of the off gene product is unknown. Whereas predicted off amino acid sequences show strong overall homology, the 20-30 COOH- terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR Va specificity. We have generated monoclonal ntibodies to a synthetic peptide ncompassing the 19 COOH-terminal amino acid residues of Mtv-7 off, which encodes the Mls-1 ' determinant. We show here that these antibodies block Mls responses invitro and can interfere specifically with thymic clonal deletion of Mls-P reactive Vt~6 + T cells in neonatal mice. Furthermore, the antibodies can inhibit V~6 + T cell responses in vivo to an infectious MMTV that shares off sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the off COOH terminus and imply that the off proteins of both endogenous and exogenous MMTV interact directly with TCR V~. E xpression of minor lymphocyte stimulating (Mls) antigens in mice leads to thymic lonal deletion of Mls-reactive T cells. In contrast to conventional antigen recognition where both the TCR ce and fl chains contribute, T cells interacting with a particular Mls antigen are characterized bythe TCR Vfl chain they express on the call surface (1, 2). Thus, to as- sure stir-tolerance in mice expressing MIs-P, the majority of T cells expressing TCR Vfl6, 7, 8.1, and 9 are deleted from the mature CD4 + 8- and CD4-8 + thymocytes a well as from peripheral T cells (1-4). Encounter of Mls antigens later in life can lead to an initial activation of the reactive T ceils followed by induction of unresponsiveness and dele- tion (5-9). Mls phenotypes are encoded by open reading flames (or 0 in the 3' LTRs of endogenous mouse mammary tumor virus (MMTV) (10-14). Transfection studies howed that ex- pression of Mtv-7 off leads to stimulation of TCR Vfl6 § T ceils (14). Additionally, we have recently described infectious MMTV(SW) with properties of Mls-P such as activation and deletion of the same subsets of T cells and strong off sequence homology (9). Amino acid sequence comparisons of MMTV orf mole- cules suggest acorrelation between the highly polymorphic COOH-terminal 20-30 amino acids and the TCR Vfl specificity (10-12). In vitro translation studies uggested that off molecules are type II membrane proteins (15-17). Thus, the COOH terminus would be accessible tothe TCR. There is no experimental evidence, however, that MMTV off mol- ecules directly interact with the V~ dement of the TCR. Herein we describe the characterization of five mAbs specific for an Mn,-7 off Peptide ncompassing the 19 COOH-terminal amino acids. These mAbs specifically block anti-Mls-1 are- sponses in vitro, rescue V~6 + T cells from donal deletion in vivo, and strongly inhibit the Vr + T cell response in- duced by MMTV(SW) infection in vitro. These results ug- gest that the viral superantigen directly interacts with the V~ chain of the TCR. Materials and Methods Production ofAnti-M~7 orfmAbs. For production ofthe mAbs described in this study, 8-wk-old BALB/c mice were immunized intraperitoneally with 50/~g of the COOH-terminal Mw-7 orf pep- tide KILYNMKYTHGGKVGFDPF (9) coupled to albumin i  CFA and boosted 4 wk later in IFA. The mouse having the strongest titer in serum, as judged by inhibition of an anti-Mls-l" response, was reimmunized intravenously and fusions with the myeloma X63Ag8 were performed 3 d later. Five mAbs were selected that exhibited strong binding to the peptide used for immunization as 1769 J. Exp. Med. 9 The Rockefeller University Press * 0022-1007/92/12/1769/04 $2.00 Volume 176 December 1992 1769-1772 determined in solid phase ELISA: 6B9.4 (IgG1), 2E5.6 (IgG1), 4D1.3 a (IgG1), 6E1.8 (IgG2b), and 1G12.7 (IgM). Inhibition of Mls Response In Vitro Mls-l" LBB cells (5-500 x 103, irradiated with 103 tad using a cobalt source) were cultured with 2 x 104 V06 § T hybridoma (RG17) cells for 24-36 h (18). Supernatants were tested at different dilutions for IL-2 production using CTLL cells as indicator cells. Different doses of protein G-Sepharose (Pharmacia, Uppsala, Sweden) -purified antibodies and Mtv-7 off peptide were added from the onset of culture. All five anti-Mw-7 mAbs were similarly efficient in inhibiting an anti- Mls-l" response in vitro (data not shown). Expression of M~ 7 orf in Baculovirus. Mtv-7 orfDNA was ob- tained by PCR amplification from DNA of BALB.D2 mice using the 5' oligonucleotide GATCGTCGACATGCCGCGCCTGCAG- CAGA and the 3' oligonucleotide GTGTCGACCCAAACCAAGT- CAGGAAACCACTTG (9). The start codon is underlined. The b product was cut with the restriction enzyme SalI and ligated into the SalI-digested vector pGem3ZF + (Promega Biotec, Madison, WI). The insert was excised using the restriction enzymes BamHI and HindlII and ligated in the BgllI-EcoRI-digested vector pCSD92 (obtained from F. Godeau). After 10-h ligation and blunting the ends with Klenow fragment the vector was recircularized by liga- tion and transfected together with wild-type baculovirus DNA into SF9 cells using standard methods. Colonies containing recombinant baculovirus were plaque purified twice. SF9 cells were infected with a multiplicity of infection of 5 and analyzed 36 h later. FACS| FACS | (Becton Dickinson & Co., Mountain View, CA) analysis of infected SF9 cells was performed using an amplification system consisting of 6 #g of biotinylated antibody 6E1.8 followed by PE-streptavidin (Caltag Laboratories Inc., Burlin- game, CA), 1.25/~g/ml biotinylated antistreptavidin (Vector Labora- tories Inc., Burlingame" CA), and PE-labeled streptavidin (19). Dead cells were excluded using forward scatter and side scatter analysis. For three-color analysis, thymocytes were stained with FITC- conjugated anti-Vo6 (44-22-1; 20) or anti-V08.2 (F23.2; 21) anti- bodies, PE-conjugated anti-CD4 (GK1.5; Becton Dickinson & Co.), and biotinylated anti-CD8 followed by avidin-tandem (Southern Biotechnologies, Birmingham, AL). Two-color analysis of thymo- cytes (after CD8 depletion with antibody 3.168.1 and rabbit com- plement) (22) or lymph node cells was carried out with FITC- conjugated anti-Ve antibodies and PE-conjugated anti-CD4. Dead cells were gated out using forward scatter and side scatter analysis on a FACScan | flow cytometer. Virus Isolation and Purification. Virus was purified from milk of BALB/c mice carrying MMTV(SW) as described (9). The virus was titered using a sandwich ELISA and independently b injec- = tion into the foot pads of BALB/c mice and measuring the lymph node Vo6+CD4 + T cell response. Limiting virus doses (~10 r virus particles) giving an increase of CD4 § V06 + T cells from 10 to 25% were used in these experiments. Results and Discussion Based on the off sequences of Mtv-7 (Mls-1 ~) and the related infectious MMTV(SW) (9), mouse anti-off mAbs specific for a peptide corresponding to the unique 19 COOH-  terminal amino acid residues were produced. Five mAbs were selected based on their strong binding to the Mtv.7 orf pep- tide. As shown for the representative antibody 6E1.8 in Fig. 1 a, all five mAbs inhibited the in vitro anti-Mls-l" response of the Va6 + T cell hybrid RG-17. This inhibition was specific since addition of the Mr  7 off IX*pride restored a normal no antibody 2oooo I 10000t 30 10 3 1 0.3 Amount of 6E1.8 mAb in culture (pg/ml) 20000 E 0,. 0 10000 no antibody , . , 9 , . , . , . , , , , 6 2 0.7 0.2 07 .03 0 Amount of Mtv-7 orf peptide added (1.1g) Figure 1. Inhibition of Mls-l= response by anti-Mw-7 orf mAbs. (a) The Mls-l*-reactive T cell hybrid (KG17) was cultured with Mls-l*-expressing LBB cells (18) in the absence orpresence ofdifferent doses of mouse anti- orf mAb 6E1.8. After 36 h of coincubation, IL-2 production was assessed in the supematants by measuring proliferation ofIb2-dependent CTLL cells. (b) Same as a, except using 10 #g/m1 ofmAb 6E1.8 and graded oses of Mtv-7 off peptide. a lb t0'0 t 060 - Relative fluorescence intensity Figure 2. Flow microfluorometric analysis of baculovirus-infected SF9 cells expressing Mtv-7 off. SF9 cells infected with either wild-type baculovirus or recombinant baculovirus were stained with biotinyhted anti-Mw-7 orf mAb 6E1.8. The signal was amplified with PE-streptavidin, biotinylated anti-streptavidin, andPE-streptavidin as described in Materials and Methods. (a) Wild-type baculovirus-infected SF9 cells, (b) Mtv-7 off-~pressing SF9 cells, and (c) same as b, but in the presence of10 #g of the peptide used for immunization. 1770 Inhibition by Antibodies to an Open Reading Frame Protein Vl36 day 6 day 10 12 10- >~ 4- 2- 0 . , V[[],14 day 6 or 10 B 13 9 '- ' -  ~ _a ~ g 0 0 Figure 3. Inhibition of thymic clonal deletion by anti-orf mAbs. V~6 and Ve8.2 expression was measured in CD4+CD8 - tby- mocytes 6 or 10 d after birth in anti-orf-treated or control mAb- treated BALB.D2 (Mls-l~) mice or in untreated BALB/c (Mls-1 b) mice. All the Ig, G anti-orf mAbs gave comparable r sults and are pooled in this figure. As control IgG1, antibody B8-24-3 (anti-H-2 K b) (25) was used. + D 0 r > + r~ 0 t-. > 30"  mm 20 him 10-  ml t o co oq < + et~ + o > # Figure 4. Inhibition of anti- MMTV(SW) T cell response by anti-orf mAbs. Adult (5-8 wk old) BALB/c mice were injected with 10 ~ purified MMTV(SW) virus particles as described (9) into the left hind footpad. After 12 h and every 12 h thereafter, the mice were injected in the same footpad r with 50 #g of either isotype- - matched control antibody (SJL2- o 4) (26) or anti-orf mAb 6E1.8 E 8 (IgG2b). After 4 d the draining + lymph nodes were removed and single cell suspensions were ana- > ~- lyzed for Vo6+CD4 + or V~14 + CD4 § T cells (27). response (Fig. 1 b). Furthermore, since RG-17 cells express a second TCR V~ chain (Val, provided by the BW fusion partner), the response could be restored in the presence of staphylococcal enterotoxin A (SEA), which crosslinks Val with MHC class II molecules (data not shown). Flow microfluorometric analysis of SF9 cells overexpressing the Mtv-7 orf molecule 36 h after infection with recombinant baculovirus showed strong and specific staining with all the anti-orf mAbs. This staining could be completely blocked by addition of the Mtv-7 orf peptide (Fig. 2). The orf mole- cule in SF9 insect cells might therefore be expressed atthe cell surface confirming in vitro translation studies (15-17). Similar analysis of Mls-l' LPS induced B cell blasts showed very weak to undetectable staining (data not shown). Immu- noprecipitation fMtv-7 off molecules from 3SS-methionine- labeled infected SF9 cells gave a very similar band pattern after SDS-PAGE to that observed by Brandt-Carlson a d Butel (23). SF9 cells infected with recombinant baculovirus ex- pressing the control orf molecule of MMTV (GK) did not yield any detectable bands (data not shown), consistent with the fact that MMTV(GR) orf has a completely different COOH-terminal sequence. In vivo, most Mls-l' mouse strains intrathymicaUy delete Mls-P-reactive mature Va6 + T cells within the first 10 d after birth (24). This deletion occurs at the transition from CD4+8 + to either CD4+8 - or CD4-8 + mature thymo- cytes (1). To see whether anti-orf mAbs are able to rescue Va6 + T cells from deletion, we injected neonatal Mls-1 ~ mice with the different anti-Mtv-7 orf peptide-specific mAbs. As shown in Fig. 3, all four IgG antibodies tested were able to rescue aconsiderable proportion (40-60%) ofVa6 + ma- ture thymocytes from clonal deletion after either two in- traperitoneal injections of 70-320 #g of antibody on days 0 and 4 or after nine daily injections with 100 #g starting within the first 24 h after birth. The percentage ofT cells expressing TCR molecules not interacting with Mls-1 ~ (VaS.2, Va14) did not change, and isotype-matched control antibody did not interfere with the clonal deletion patterns (Fig. 3). We recently described the characterization f infectious MMTV(SW) that has the same TCR specificity as Mls-1 ~ (Mtv-7 off) and displays avery similar COOH-terminal orf amino acid sequence (9). Thus, we attempted using the anti- off mAbs to interfere with a proliferative Va6 + CD4 + re- sponse induced after MMTV(SW) infection in vivo. Prelim- inary experiments determined the minimal virus dose (~107 particles) required for a significant and specific (2.5-fold) in- crease in Va6 +CD4 + T cells in the draining lymph nodes. We injected 50 #g of anti-orf mAb 6E1.8 (IgG2b) 12 h after virus challenge into the hind footpad and repeated the anti- body injections every 12 h until day 4, when the lymph node cells were isolated. As can be seen in Fig. 4, a strong and specific inhibition of the local increase of Va6 + CD4 + T cells is observed in anti-off-treated but not control IgG2b-treated mice. This effect likely represents blocking of orf presenta- tion to V~ + T cells by MMTV(SW)-infected B cells, since the antibody should not interfere with MMTV infection per se. In conclusion, the results described here indicate that MMTV orf molecules are expressed on the cell surface and that the off COOH terminus i accessible toantibodies, thus confirming predictions made from sequence omparisons and in vitro translation studies. Furthermore, the in vivo blocking data indicate that MMTV-induced T cell stimulation and de- letion both depend upon direct interaction of TCR Va with the off protein. We thank F. Godeau for the vector pCS1392, G. P. Corradin for the mAb SJL2-4, and J. A. Lop6z for communication of results before publication. H. Acha-Orbea was supported by a START fellowship and a grant (31-32271.91) from the Swiss National Science Foundation. 1771 Acha-Orbea et al. Brief Definitive Report Address correspondence to Hans Acha-Orbea,. Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland. Received for publication 24 August 1992. P~ef~l~nces 1. MacDonald, H.R., K. Schneider, R.L. Lees, R.K. Howe, H. Acha-Orbea, H. 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Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

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