Tissue Aitigens HistDcompatibility and Immunogenetics E D I T O R : F. K I S S M E Y E R - N I E L S E N , Aarhus, Denmark A S S O C I A T E E D I T O R S : W A L T E R B O D M E R , London, U K JOHN A. H A N S E N , Seattle, WA, USA E R I K T H O R S B Y , Oslo, Norway

Volume 33, 1989

M U N K S G A A R D , Copenhagen

© Copyright 1989 Munksgaard, International Publishers, Ltd., Copenhagen, Denmark

Unlversitäts-Bibüothek München

Printed in Denmark by AiO Print Ltd, Odense

Author Index Abbal, M . , 38 Abe, A. , 550 Adam, C , 501 Adrien, M . , 400 Agrewala, J. N . , 486 Alfinito, F., 457 Amsellem, S., 475 Awad, J., 30, 445

Balaguer, P., 475 Baur, M. P., 501 Benczur, M . , 491 Bender, K., 501 Bianchi, P. A . , 431 Bradley, B. A . , 437 Bratlie, A. , 520, 542 Betuel, H . , 475 Budkowska, A. , 495

Cambon-Thomsen, A. , 38

Carandente Giarrusso, P., 457

Carlsson, B., 531 Carthy, D., 30, 445 Cesana, M . , 431 Christiansen, B. S., 4 Clot, J., 475 Conte, D. , 431 Corbo, L. , 457 Cross, D., 41 Cutbush, S., 30, 445, 488

Debre, P., 495 Demeter, J., 491 Ditri, M. , 15 Doran, T. J., 41 Dos-Santos, A . , 445 duToit, E. D. , 367 du Toit, E. E., 41 Dunckley, H . , 418 Dupont, E., 400 Dwyer, E., 466 Dyer, P. A. , 21

Economicou, J., 488 Eliaou, J.-F, 475 Emtestam, L . , 531

Fargion, S., 431 Festenstein, H . , 30, 445,

488 Fielder, A . , 30

Fiorelli, G., 431 Forsberg, B., 415

Gatenby, P. A. , 418 Gebuhrer, L . , 475 Ghei, S. K., 486 Giordano, M . , 457 Girdhar, B. K., 486 Goetz, J., 501 Goldmann, S. F., 501 Graugaard, B. H. , 382 Grennan, D. M . , 21 Gruber, G., 389 Gualdieri, L . , 457 Gy, Petranyi G., 491 Gyoci, E., 491

Hajek-Rosemayer, A. , 389

Hancock, R. J. T., 437 Hannestad, K., 520, 542,

546 Hansen, T., 520 Hauptmann, G., 501 Hennessy, E., 15 Hirata, R., 421 Hitomi, Y., 421 Holthausen, H. , 375 Hu, S-A., 45 Huang, C.-C, 45, 425 Humbert, M. , 475

Ishimoto, A. L. , 550 Ito, I . , 550

Jaraquemada, D. , 445 Jazwinska, E. C , 418 Johansen, B., 520 Johnson, U . , 415 Joysey, V., 41 Jungl, L. , 389

Kaneko, T., 550 Kashiwagi, N . , 550 Kirnbauer, M. , 389 Kissmeyer-Nielsen, E ,

382 Kolstad, A. , 542, 546 Kuon, W., 511

Lee, S., 466 Lehoczky, D., 491 Leititis, J., 375

Lin, J.-L., 45 Litzenberger, J., 375

Maeda, H. , 421 Mandelli, C , 431 Marcusson, J. A. , 531 Mareenot, M. R., 495 martell, R. W., 41 Mauff, G., 501 May, R., 41 Meisel, C , 375 Mercuriali, F., 431 M0ller, B. K., 4 Möller, E., 531 Münchhoff, P., 375

Navarrete, C , 445 Neubert, U . , 375 Neugebauer, M . , 38, 501 Nicolas, J.-C, 475 Nunez-Roldan, A. , 466

Obata, R, 550 Ohkubo, M . , 550 Okoye, R., 30 Okoye, R. C , 445 Ollier, W., 15, 30, 445,

488 Oppolzer, E., 389 Oudshoorn, M . , 367

Pachoula-Papasteriadis, C , 488

Paloczi, K., 491 Panajotopoulcs, N . , 431 Petersen, C. M. , 4 Petzl-Erler, M. L. , 408 Pflüger, K.-H. , 375 Pillot, J., 495 Piperno, A. , 431 Pompeo, L. , 466 Pongratz, E., 375 Povlsen, J. V., 4, 382

Reekers, P., 400 Reimers, C .D. , 375 Riethmüller, G., 511

Sanders, P. A. , 21 Santamaria, J., 408 Sengupta, U . , 486 Serjeantson, S. W., 418 Silman, A. , 15, 30

Stammler, M . , 389 Steuer, M . , 501 Stordeur, P., 400 Sudhakar, K. S., 486

Taljaard, D. G., 367 Telek, B., 491 Thomsen, M . , 38 Thomson, W., 21 Thurau, S. R., 511 Tobngio, M. M . , 501 Tohyama, H . , 421 Trapp, B., 375 Turco, M. C , 457

Uring-Lambert, B., 501

Valentini, G., 457 van Rood, J. J., 1 Vandercruys, M . , 400 Vandvik, B., 546 Vartdal, F., 546 Venuta, S., 457 Völker, B., 375

Wallin, J., 531 Weiss, E. H . , 511 Wildner, G., 511 Winchester, R., 466 Wölpl, A . , 501 Wu, J .H . , 45

Yendle, J. E., 437

Contents:

Volume 33, no. 1 Editorial Obituary R. Ceppellini * 19.01.1917 t05.06.1988 Original Articles Cyclosporin A mediated immunosuppression in vitro: effect on high affinity interleukin-2 receptor expression and -turnover Co-segregation of HLA and rheumatoid arthritis in multicase families Haplotypes bearing HLA-A, -B, and -DR: Bf and C4 genes in rheumatoid arthritis families HLA-DR4 associated Dw types in rheumatoid arthritis Recombinations in the HLA system

Definition of the HLA-Aw43 antigen

Short Communication HLA and Chinese IgA nephropathy in Taiwan

1 J. J. van Rood

4 J. V. Povlsen, B. K. M0ller, B. S. Christiansen & C. M. Petersen

15 A. Silman, E. Hennessy, M. Ditri & W. Oilier

21 P. A. Sanders, W. Thomson, P. A. Dyer & D. M. Grennai

30 W. Ollier, D. Carthy, S. Cutbush, R. Okoye, J. Awad, A. Fielder, A. Silman & H. Festenstein

38 M. Thomsen, M. Abbal, M. Neugebauer & A. Cambon-Thomsen

41 R. W. Martell, E. E. du Toit, V. Joysey, T 7. Doran, D. Cross & R. May

45 C.-C. Huang, S.-A. Hu, J . - L . Lin & J.-H. Wu

Volume 33, no. 2

Abstracts from 49 Edited by Walter Knapp

4th International Conference on Human Leucocyte Differentiation Antigens Vienna, February 21-25, 1989

Volume 33, no. 3 Original Articles HLA-Dw RSH': a new HLA-Dw specificity associated with HLA-DRwl8(3) Lyme-Borreliosis and possible association with HLA-antigens

Lymphocytotoxic cross-matching performed on spleen cells: immunomagnetic technique versus current KN (Kissmeyer-Nielsen) technique Biochemical H L A typing: a population study in 112 Caucasians HLA-B SNA antigen: a BW6 associated B locus antigen belonging to the B5 CREG Are HLA class I I genes controlling susceptibility and resistance to Brazilian pemphigus foliaceus (fogo selvagem)? HLA-antigens in renal transplanted patients with varicella-zoster infection DNA-DR typing shows HLA-DRwll RFLPs are increased in frequency in both progressive systemic sclerosis and CREST variants of scleroderma A polymorphic monoclonal antibody, PLM10, that reacts with B-cell lines carrying HLA-DPwl, DPw5, and U P ^ o a "

Short Communication Strong association of HLA-DR3 in Chinese patients with idiopathic membranous nephropathy

Newsletter

Announcements

367 M. Oudshoorn, E. D. du Toit & D. G. Taljaard

375 K.-H. Pflüger, C.-D. Reimers, U. Neubert, C. Meisel, B. Trapp, J. Leititis, B. Völker, P. Münchhoff, J. Litzenberger, H. Holthausen & E. Pongratz

382 J. V. Povlsen, B. H. Graugaard & F. Kissmeyer-Nielsen

389 A. Hajek-Rosemayer, L . Jungl, E. Oppolzer, M. Stammler, G. Gruber & M. Kirnbauer

400 M. Adrien, P. Reekers, P. Stordeur, M. Vandercruys & E. Dupont

408 M. L . Petzl-Erler & J. Santamaria

415 B. Forsberg & U. Johnson

418 H. Dunckley, E. C. Jazwinska, PA. Gatenby & S. W. Serjeantson

421 H. Maeda, R. Hirata, Y. Hitomi & H. Tohyama

425 C-C. Huang

All

429

Volume 33, no. 4 Original Articles HLA typing in 67 Italian patients with 431 idiopathic hemochromatosis and their relatives MicroELISA assays of anti-HLA activity 437 and isotype of human monoclonal antibodies HLA-D region heterogeneity in a Niberian 445 population

Lymphocyte proliferative resonse to 457 mitogenic monoclonal antibodies in systemic sclerosis. Evidence for unresponsiveness to murine monoclonal antibodies of IgGl isotype Definition of DRwlO by restriction 466 fragment length polymorphism A method of HLA class I I typing using 475 non-radioactive labelled oligonucleotides

Short Communications HLA antigens and Erythema Nodosum 486 Leprosum (ENL) HLA antigens and haplotype frequencies 488 in Greeks Histocompatibility antigens in hairy cell 491 leukemia

N. Panajotopoulcs, A. Piperno, D. Conte, C. Mandelli M. Cesana, F. Mercuriali, G. Fiorelli, P. A. Bianchi & S. Fargion R. J. T. Hancock, J. E. Yendle & B.A. Bradley

R. C. Okoye, W. Ollier, D. Jaraquemada, J. Awad, C Navarrete, S. Cutbush, D. Carthy, A. Dos-Santos d H. Festenstein G. Valentini, P. Carandente Giarrusso, L . Gualdieri, L . Corbo, F Alfinito, M. C. Turco, S. Venuta & M. Giordano

S. Lee, A. Nunez-Roldan, E. Dwyer, L . Pompeo & R. Winchester J.-F Eliaou, M. Humbert, P. Balaguer, L . Gebuhrer, S. Amsellem, H. Betuel, J.-C. Nicolas & J. Clot

J. N. Agrewala, S. K. Ghei, K. S. Sudhakar, B. K. Girdhar & U. Sengupta C. Pachoula-Papasteriadis, W. Ollier, S. Cutbush, J. Economicou & H. Festenstein J. Demeter, E. Gyoci, K. Paloczi, B. Telek, D. Lehoczky, M. Benczur &. Petranyi G. Gy

Volume 33, no. 5 Original Articles HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination An estimate on the frequency of duplicated haplotypes and silent alleles of human C4 protein polymorphism. I . Investigations in healthy Caucasoid families

495 M. R. Marechot, A. Budkowska, J. Pillot & P. Debre

501 M. Steuer, G. Mauff, C. Adam, M. P. Baur, K. Bende J. Goetz, S. F Goldmann, G. Hauptmann, M. Neugebauer, M. M. Tongio, B. Uring-Lambert & A. Wölpl

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27 Two cytotoxic human-human hybridoma antibodies to HLA: TrAHIO (anti A3.1) and TrAG2 (anti B7,Bw42) Specificity of HLA restricting elements for human nickel reactive T cell clones A cytotoxic human-human hybridoma antibody (TrC7) specific for HLA-A29 Multiple sclerosis patients have a high frequency of an HLA-DQ8 epitope defined by a human-human hybridoma antibody Oligonucleotide-genotype as a method of detecting the HLA-DR2 (DRwl5)-Dw2, -DR2 (DRwl5)-Dwl2, -DR4-Dwl5, and -DR4-D"KT2" haplotypes in the Japanese population

Announcement

511 SR. Thurau, G. Wildner, W. Kuon, E. H. Weiss & G. Riethmüller

520 T. Hansen, A. Bratlie, B. Johansen & K. Hannestad

531 L . Emtestam, B. Carlsson, J. A. Marcusson, J. Wallin & E. Möller

542 A. Kolstad, A. Bratelie & K. Hannestad

546 A. Kolstad, K. Hannestad, B. Vandvik & E Vandal

550 F. Obata, I . Ito, T. Kaneko, M. Ohkubo, A. L . Ishimoto, A. Abe & N. Kashiwagi

559

Tissue Antigens (1989) 33, 511-519

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27 S. R . T H U R A U * , G . W I L D N E R § , W . K U O N , E . H . W E I S S and G . R I E T H M Ü L L E R

Institute for Immunology, University of Munich, Goethestr. 31, D-8000 München 2, Federal Republic of Germany * Present address: Eye Hospital of the University of Munich, Mathildenstr. 8, D-8000 München 2, Federal Republic of Germany § Present address: Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, D-8033 Martinsried, Federal Republic of Germany

The immunization of a (BALB/c x C57BL/6) Fi mouse with murine transfectants expressing the HLA-B27 antigen resulted in a panel of polymorphic monoclonal antibodies with specificity for HLA-B27 and some additional HLA-antigens. Speci­ficity of the antibodies was defined by cytofluorometric analysis on a panel of lymphoblastoid cell lines (LCL) derived from HLA typed individuals. Three of these antibodies are cytotoxic, and one of them inhibits B27-specific T cell cytotox­icity. Our results indicate that HLA-class I transfectants could be used to generate polymorphic antibodies, and that these antibodies may be helpful for HLA typing and for definition of epitopes recognized by T cells.

Received for publication 27 September, revised, accepted 2 December 1988

The transfection of isolated genes into recip­ient cells offers a new way to study the expres­sion and biological function of the gene prod­ucts isolated from their normal environment (Heyes et al. 1986, Barbosa et al. 1984, Ma-ryanski et al. 1985, Cowan et al. 1985, Go-mard et al. 1986, Herman et al. 1983, Mentzer et al. 1986, van de Rijn et al. 1984). Owing to its high association to rheumatoid diseases such as ankylosing spondylitis and Reiter's syndrome (Tiwari & Terasaki 1985, Terasaki 1980), high interest focuses on the HLA-B27 antigen. The gene has been isolated, se­quenced (Weiss et al. 1985, Szöts et al. 1986) and transfected into the mouse mastocytoma

line P815. Serological assays demonstrated that these transfectants express functional B27 antigens (W.K. , unpublished observa­tion).

These transfectants were used as immuno-gens to generate specific monoclonal antibod­ies. After immunization of mice with one high expressing transfectant clone, established with an in vitro mutated HLA-B27 gene, four monoclonal antibodies were isolated with specificity for B27 and additional H L A anti­gens (such as B8, B13, B15, B18, B37 and B44), according to indirect immunofluores­cence analysis. One monoclonal antibody rec­ognized all tested cells expressing HLA-class I

33 Tissue Antigens 33:5

512 THURAU E T AL.

antigens. Three of these antibodies show com­plement dependent cytolysis, and therefore may be useful reagents for H L A typing. One of the monoclonal antibodies also inhibits B27 specific T cell cytotoxicity.

Material and Methods

Transfection of the mutated and unmutated HLA-B27 genes

The cosmid clone cd2.6 encoding HLA-B27 W

(Weiss et al. 1985, Szöts et al. 1986) or a 6.5 kilobase Eco R I subclone (Kuon et al. 1986) were transfected into the mouse mastocytoma line P815 (Weiss et al. 1985, Van Pel et al. 1985). The Eco R I subclone contained a mu­tated B27 gene, generated in vitro by site di­rected mutagenesis, exchanging serine at posi­tion 131 to arginine. The transfection of the unmutated B27 w gene resulted in a transfec-tant clone designated B27.3, whereas trans-fectant clone B27.R3 expressed the mutated gene. After recloning, the transfectants were tested on a panel of monoclonal antibodies to control surface expression and specificity of the introduced H L A genes.

Immunization and fusion

A ( B A L B / c x C57BL/6) F i mouse was immu­nized with the P815 (DBA/2) transfectant B27.R3 expressing the mutated B27 antigen with an amino acid exchange at position 131 from serine to arginine. 7 x 106 B27.R3 cells were injected intra-peritoneally together with 108 Bordetella pertussis (Behring Institut, Marburg), followed by a second injection with 1.2 x 107 B27.R3 cells and 108 B . pert, on day 59. Three days later the spleen cells were fused with the mouse myeloma X63Ag8.653 (Kearney et al. 1979, Galfre et al. 1977).

Cells were grown in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS, Sebio, Boehringer) or 5% Clex (Falcon), 2 mM L-glutamine, 100 I U penicillin/ml, 100 u.g

streptomycin/ml, 20 pM 2-mercaptoethanol, 1 mM sodiumpyruvate and 1 mM non-essential amino acids at 37°C in a humidity-saturated atmosphere with 5% CO2.

Supernatants were screened in an indirect immunofluorescence assay on a homozygous B27 w L C L (LG-2 , Gatti & Leibold 1979) and the HLA-class I surface negative cell line Daudi (Ploegh et al. 1979). Positive hybrid-omas were cloned by limiting dilution before expansion.

Indirect immunofluorescence assay

Cells were pelleted in round bottom microti-terplates and incubated with antibody con­taining culture supernatant for 1 h. After washing with phosphate buffered saline (PBS), cells were stained with FITC-coupled goat anti mouse IgG & IgM (Dianova, Ham­burg) for 30 min, washed, fixed with 1% par­aformaldehyde and analyzed by fluorescence-microscopy or by cytofluorography ( E P I C S V , Coulter or FACScan, Becton and Dickin­son). All steps were carried out on ice.

Complement dependent microlymphocytotoxicity assay

Complement dependent cytotoxicity of mono­clonal antibodies was tested in a two-stage microlymphocytotoxicity assay on peripheral blood lymphocytes (PBL) according to Tera­saki & McClelland (1964). For cytolysis we used rabbit complement obtained from Behr­ing Institute, Marburg. Lympholysis in per­cent was calculated for a 1:10 dilution of su­pernatants. Lymphocytotoxicity was regarded as positive when at least 40% of cells were lysed.

Inhibition of cell mediated lymphocytotoxicity (CML)

P B L of donor MIWI (A2, B17, B51, C w l , Cw6) were stimulated with P B L of donor

GENERATION OF ANTIBODIES USING B27 TRANSFECTANTS 513

M E 1

B27.3

Figure 1. EPICS analysis: HLA-B27 surface antigen expression of P815 transfection clones (top: unmu­tated gene B27.3; bottom: mutated B27 gene B27.R3), determined by monoclonal antibody ME1 (specific for HLA-B27, -B7, -B22) (Ellis et al. 1982). Results are plotted as cell number (y-axis) versus log fluorescence intensity (x-axis).

H A S E (A2, A3, B27 w , B37, C w l ) as de­scribed elsewhere (Sehende! et al. 1978, Wildner et al. 1988). After 6 days, B27-specif-ic cytotoxicity was tested in a 4 h chromium release assay on 5 1 C r labeled L C L L G - 2 (A2, B27 w , Cwl ) in 200 pi of culture medium con­taining hybridoma supernatants of varying di­lutions. Chromium release was measured and calculated as described by Schendel et al. (1978).

For secondary response, cytotoxic T lym­phocytes ( C T L ) were maintained for 21 days with IL-2 but without further antigenic stim­

ulation. In this case the chromium release as­say was performed at the effector:target ratio of 5:1.

Results

Characterization of transfectants

Fluorescence profiles (Figure 1) show that both transfectants B27.3 and B27.R3 are rec­ognized by antibody ME1 (Ellis et al. 1982), which is specific for B7, B22 and B27. This pattern was confirmed by the monoclonal an­tibodies W6/32 (Barnstable et al. 1978), B27M1 (Grumet et al. 1981) and B27M2 (Grumet et al. 1982), whereas non-related an­tibodies did not bind to the transfectants (data not shown). Figure 1 also shows that the B27.R3 transfectant P815 clone with the mu­tated HLA-B27 gene expressed an increased amount of surface HLA-B27 antigen com­pared to the transfectant B27.3 containing the unmutated form. Transfectant B27.R3 was se­lected for immunization because of its clonal stability and increased expression of B27-sur-face antigen.

HLA specificity of generated antibodies

1010 hybridoma supernatants from two fusion experiments with the spleen cells of one single mouse were screened on a small informative panel of L C L with B27 positive and B27 nega­tive cells. Five of them were selected for fur­ther investigation and tested on a panel of 62 B-cell lines from unrelated, HLA-typed do­nors. The correlation between H L A antigens and monoclonal antibody reactivities was evaluated by the coefficient of correlation (r) computed from 2 x 2 tables, and significance (P) evaluated with a X 2 test (Simons & Tait 1984). The results are shown in Table 1.

The monoclonal antibodies TM-1, TM-4, TM-5 and TM-6 all recognize B27 + cells and show crossreactivity to other H L A - B , but not to H L A - A , - C or class II antigens. Antibody

33*

514 THURAU E T AL.

Table 1. Cytofluorometry. Reactivity and r values of TM-1, TM-3, TM-4, TM-5 and TM-6 on unrelated B cell lines.

Antibody Recognized antigens No. of cells No. of cells x° Pa Total No. tested reactive of cells

with anti­ tested with body antibody*

TM-1 B27 9 9 0.67 0.001 61 B44 17 13 0.60 0.001 61 B15 3 3 0.56 0.001 61 B27, B44, B15 28 24 0.68 0.001 61

TM-3 HLA-class I (monomorphic) 59 59 1.0 0.001 59

TM-4 B27 w 10 9 0.87 0.001 62 B15 1 1 1.00 n.s. 62 B27w, B15 11 10 0.84 0.001 62

TM-5 B13 3 3 0.54 0.001 62 B27 8 8 0.70 0.001 62 B37 2 2 0.43 0.001 62 B15 3 3 0.58 0.001 62 B13, B27, B15, B37 18 17 0.76 0.001 62

TM-6 B15 5 4 0.50 0.05 60 B44 14 13 0.69 0.001 60 B27 8 7 0.61 0.001 60 B8 6 5 0.55 0.025 60 B18 4 3 0.50 0.05 60 B15, B44, B27, B8, B18 38 32 0.66 0.001 60

a For calculation of r (coefficient of correlation) and P (probability), the other HLA antigens indicated in this table were excluded.

b Total number of tested cells, bearing 17 different HLA-A, 31 different HLA-B and eight different HLA-C antigens,

n.s. not significant.

TM-1 also bound to 76% of the B44 + and 37% of the B15 + cells. Exon shuffling experiments with hybrids of B27 and B7 indicate that the antigenic determinant for TM-1 is located on the al-domain of B27 involving amino acid residues 77 and 80 (Toubert et al. 1988).

TM-4 strongly bound to all B 2 7 w + cells, except for the subtype B27 K . Also, very few (two of 15) H L A - B 4 4 + and B15 + cells were stained with this antibody. In 1981 Grumet already described a monoclonal antibody, B27M1, dividing the B27 antigen into its sub­types B27 w and B27 K . TM-4 shows a similar

reactivity with respect to B27 subtypes, but the pattern of crossreactivity is different. The monoclonal antibody B27M1 crossreacts with B47, but not with B44 or B15 (Grumet et al. 1981).

Antibody TM-5 reacted with B27 and, to a lesser extent, B13, B15 and B37. This binding pattern is, except for B15, similar to that of the monoclonal antibody BD.7 (Bourel et al. 1987), supporting evidence of a common epi­tope on these antigens.

TM-6 recognized all B27-positive cells, and crossreacted with B44 and B15, whereas

GENERATION OF ANTIBODIES USING B27 TRANSFECTANTS 515

T M - 5

0.5 0.25 0.125 0.5 0.25 0.125 0.0625 Concent ra t ion of C o n c e n t r a t i o n of

S u p e r n a t a n t S u p e r n a t a n t

Figure 2. Inhibition of B27 specific cytotoxicity by monoclonal antibodies TM-5, W6/32 and HCIO.

A: Inhibition of CML by monoclonal antibodies TM-5, W6/32 and HCIO. Effector MIWI: HLA-A2, B17, B51, Cwl, Cw6 Stimulator HASE: HLA-A2, A3, B27w, B37, Cwl Target LG-2: HLA-A2, B27w, Cwl Effector: target ratio = 15:1 4000 targets per well Dotted horizontal line shows specific lysis without inhibitor (53%).

B: Inhibition of secondary CML of long term cultured CTL by monoclonal antibody TM-5. Effector MIWI: HLA-A2, B17, B51, Cwl, Cw6 stimulated with IL-2 Stimulator HASE: HLA-A2, A3, B27w, B37, Cwl Target LG-2: HLA-A2, B27w, Cwl Effector: target ratio = 5:1 3000 targets per well Dotted horizontal line shows specific lysis without inhibitor (40%).

TM-4 showed some crossreactivity with B8 and B18 bearing cells and recognized only some of these cells, although there are no serological splits of these antigens described so far (Terasaki 1988).

Antibody TM-3 bound to all HLA-positive cells. The binding-pattern was similar to that of W6/32 (Brodsky et ai. 1979, Barnstable et al. 1978). Nevertheless, the experiments could not distinguish between the recognition of a H L A - B specific or a monomorphic H L A -class I determinant.

The monoclonal antibodies TM-1, TM-3,

TM-4 and TM-5 did not show binding to the mouse mastocytoma P815, but, as expected, strongly reacted to a P815 transfectant ex­pressing the modified antigen B27.R3 (TM-6 was not tested on transfectant cells).

Inhibition of CTL

Various concentrations of antibody containing supernatants were tested for their ability to inhibit the lytic activity of B27 specific cyto­toxic T cells. Antibody TM-5 did inhibit cyto-

516 THURAU E T AL.

Table 2. IgG-class and complement dependent cytotoxicity of monoclonal antibodies determined at a 1:10 dilution of culture supernatant. Lympholysis was regarded as positive if 40% or more of cells were lysed. + and -indicate positive or negative lympholysis.

Antibody IgG-class % lysis of target cells

LEER WLGE B8, B27 B13,B44

FCS 5- 5 -S43 IgG2a 5 - 5 -W6/32 IgG2a 95+ 95+ ME-1 IgGl 20- 15-TM-1 IgG2b 95+ 10-TM-3 IgG2b 95+ 95 + TM-4 IgG2b 15- 5-TM-5 IgG2a 50+ 30-TM-6 IgM 5 - 5 -

toxicity of the primary response (Figure 2A) , whereas W6/32 and HCIO (Stam et al. 1986) did not show inhibition as supernatants, al­though inhibition is described for W6/32 when used as diluted ascites fluid (Aparicio et al. 1985). Inhibition by TM-5 was confirmed in a second assay using the same, but long-term cultured effector line (Figure 2B).

Complement dependent cytotoxicity

Monoclonal antibodies were tested for cyto­toxicity on B27 + and B27~ P B L (donor L E E R : A2, B8, B27 w , Cw2, Cw7; and donor W L G E : A2, A3, B13, B44, Cw6) as target cells. Most of our undiluted supernatants were directly cytotoxic. For this reason, the lysis obtained at a 1:10 dilution of antibody containing supernatants is shown in Table 2. Further dilution of supernatants abrogated specific cytotoxicity. Table 2 also shows the Ig-isotype of the antibodies as determined by immunodiffusion (Ouchterlony 1970).

Negative controls with F C S , the unrelated antibody S43 (IgG2a, Reth et al. 1978) and the non-complement fixing antibody ME-1

(IgGl) (Ellis et al. 1982) lysed 20% or less of both target cells, whereas W6/32 (IgG2a) and TM-3 (IgG2b) showed strong lysis of the tar­gets. TM-4 (IgG2b) and TM-6 (IgM) were not cytotoxic for these targets, although both anti­bodies recognized cells expressing the B27 or B44 antigen in indirect immunofluorescence. Antibodies TM-1 (IgG2b) and TM-5 (IgG2a) lysed the heterozygous B 8 + and B27 + cell, but failed to lyse the B13 + /B44 + P B L in this assay.

Discussion

Most commonly, P B L or Epstein-Barr virus transformed B cells are used as immunogens to generate monoclonal antibodies against H L A antigens. Since these cells present many foreign antigens to the mouse, the majority of obtained monoclonal antibodies is directed to unknown antigens on the B cell. Weak anti­gens hardly induce any antibody response. Murine transfectants carrying single H L A -genes offer the possibility of reducing the number of foreign antigens, especially in a syngeneic mouse system, and consequently may increase the yield of desired antibodies. Margulies et al. (1983) showed that syngeneic L-cells expressing a transfected alloantigen can induce antibodies specific for the trans­fected antigens in the serum of most mice. Heyes et al. (1986) described the generation of specific monoclonal antibodies by immuni­zation with murine L cells transfected with class II antigens.

In our experiments, the mouse was immu­nized with a xenogeneic HLA-class I antigen on an H-2 compatible background. A ( B A L B / c x C57BL/6) F i mouse with H2 d / b

was immunized with a P815 (derived from strain DBA/2 with H2 d ) transfectant express­ing the human MHC-antigen B27.

All antibodies presented in this paper are directed against the HLA-B27 antigen. In in­direct immunofluorescence assay they all crossreact with additional antigens, preferen-

GENERATION OF ANTIBODIES USING B27 TRANSFECTANTS 517

tially with B44 and B15. One antibody (TM-5) also recognizes to some extent B13 and B37, another (TM-6) B8 and B18. Antibody TM-4 did not bind to all cells carrying B44 or B15. Maybe it recognizes subtypes of these anti­gens, which are described elsewhere (Terasaki 1988).

Surprisingly, none of the common crossre-activities to H L A - B 7 , B22 or B47 were ob­served, although they are described for mono­clonal antibodies as well as for alloantisera (Darke 1983). Initially, we discussed a pos­sible change of immuno-dominant epitopes by the association with murine instead of human human ß2-microglobulin. Nevertheless, pres­ence of co-transfected human ß2-microglobu-lin did not significantly alter expression of the class I antigens on transfectants (preliminary observation), although antibodies are avail­able which differentiate human from murine ß2-microglobulin in association with human class I antigens. The described TM-antibodies bound to transfectants with murine ß2-micro-globulin as well as to B-cell-lines with human ß2-microglobulin. Moreover, different culture media supplemented with F C S or pooled hu­man sera did not influence recognition of transfectants by C T L or generation of C T L by transfectants (W. Kuon, unpublished obser­vation). Summing up, it may be said that the origin of ß2-microglobulin seems to play a minor role in this special system. The muta­tion from Ser to Arg cannot explain the differ­ent crossreactivities, as B7 encodes Arg in position 131. One monomorphic antibody, TM-3, was obtained, which bound to all cells expressing H L A (-B) on their surface.

Antibodies TM-1, TM-3 and TM-5 were active in complement dependent microlym­phocytotoxicity and thus may be useful for H L A typing. We observed some discrepancies between the reaction pattern when comparing the indirect immunofluorescence data with complement dependent cytotoxicity results. One explanation would be that L C L were

used for immunofluorescence, which, com­pared to P B L , expressed an increased amount of H L A antigens.

Moreover, TM-5 could inhibit B27 specifc cell mediated lympholysis even at very low antibody concentrations (diluted culture su­pernatant), at which the antibody W6/32 did not show inhibition any more. This antibody may be helpful to map antigenic epitopes for T cells.

Having obtained these antibodies, we could demonstrate that transfectants can induce a humoral response in a xenogeneic system and may be very useful for the generation of ser­ological reagents to cell-surface H L A class I-antigens. Owing to the mouse background of the transfectant cell, induction of antibod­ies to other human (MHC) antigens present on a normal H L A + human B cell can be avoided. Although in our case the mutation within the B27 gene did not alter the antigenic properties of the protein, transfectants may also be useful for generating serological re­agents to genetically modified surface anti­gens.

Abbreviations

C M L Cell mediated lymphocytotoxicity C T L Cytotoxic T lymphocyte F C S Fetal calf serum H L A Human leucocyte antigen L C L Lymphoblastoid cell line M H C Major histocompatibility complex F I T C Fluoresceinisothiocyanate P B L Peripheral blood lymphocytes PBS Phosphate buffered saline

Acknowledgments

The authors gratefully acknowledge the gen­erous gifts of typed L C L from Dr J . Johnson and Dr R. Wank. We also thank Dr J . John­son very much for the discussion of results and help during the preparation of this manuscript

518 THURAU E T AL.

as well as Mrs I . Contag for growing most of the tested B-cell lines. We are thankful to Mrs C. Dörner and Dr R. Mertz, who helped gen­erate the HLA-B27 mutant and Mr E . Le-derer and Mr T. Schlunck for E P I C S - abd FACScan-analysis.

References

Aparicio, P., Vega, M. A. & Lopez de Castro, J. A. (1985) One allogeneic cytolytic T lymphocyte clone distinguishes three different HLA-B27 sub­types: identification of amino acid residues influ­encing the specificity and avidity of recognition. / Immunol 135, 3074-3081.

Barbosa, J. A. , Mentzer, S. J., Minowada, G., Strominger, J. L . , Burakoff, S. J. & Biro, P. A. (1984) Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells. Proc Natl Acad Sei USA 81, 7549-7553.

Barnstable, C. J., Bodmer, B. F., Brown, G., Galfre, G., Milstein, C , Williams, A. F. & Zie-gler, A. (1978) Production of monoclonal anti­bodies to group A erythrocytes, HLA and other human cell surface antigens - new tools for ge­netic analysis. Cell 14, 9-20.

Bourel, D. , Fauchet, R., Dejour, G., Bouhallier, O., Merdrignac, G., Chales, G. & Genetet, B. (1987) A common epitope between HLA-B27, -B13 and -B37 alloantigens defined by a mono­clonal antibody. Tissue Antigens 30, 97-103.

Brodsky, F. M . , Parham, P., Barnstable, C. J., Crumpton, M. J. & Bodmer, W. F. (1979) Mono­clonal antibodies for analysis of the HLA system. Immunol Rev 47, 3-62.

Cowan, E. P., Coligan, J. E. & Biddison, W. E. (1985) Human cytotoxic T-lymphocyte recogni­tion of an HLA-A3 gene product expressed on murine L cells: the only human gene product required on the target cells for lysis is the class I heavy chain. Proc Natl Acad Sei USA 82, 4490-4494.

Darke, C. (1983) A reanalysis of the HLA-B7 cross-reactive group. Tissue Antigens 22, 326-334.

Ellis, S. A. , Taylor, C. & McMichael, A. (1982) Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum Immunol 5, 49-59.

Galfre, G., Howe, S. C , Milstein, C , Butcher, G. W. & Howard, J. C. (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550-552.

Gatti, R. A. & Leibold, W. (1979) HLA-D typing with lymphoblastoid cell lines. IV. Allelic rela­tionships. Tissue Antigens 13, 35-44.

Gomard, E., Begue, B., Sodoyer, S., Maryanski, J. L. , Jordan, B. R. & Levy J. P. (1986) Murine cells expressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T cells. Nature 319, 153-154.

Grumet, F. C , Fendly, B. M . & Engleman, E. G. (1981) Monoclonal anti-HLA-B27 antibody (B27M1): production and lack of detectable typ­ing difference between patients with ankylosing spondylitis, Reiter's syndrome and normal con­trols. Lancet ii, 174-176.

Grumet, F. C , Fendly, B. M . , Fish, L . , Foung, S. & Engleman, E. G. (1982) Monoclonal antibody (B27M2) subdividing HLA-B27. Hum Immunol 5, 61-72.

Herman, A . , Parham, P., Weissman, S. M . & En­gelhard, V. H. (1983) Recognition by xenogenic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer. Proc Natl Acad Sei USA 80, 5056-5060.

Heyes, J., Austin, P., Bodmer, J., Bodmer, W., Madrigal, A. , Mazzilli, M. C. & Trowsdale, J. (1986) Monoclonal antibodies to HLA-DP-trans-fected mouse L cells. Proc Natl Acad Sei USA 83, 3417-3421.

Kearney, J. F., Radbruch, A. , Liesegang, B. & Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123, 1548-1550.

Kuon, W., Riethmüller, G. & Weiss, E. H. (1986) Site specific mutagenesis as a tool to identify HLA-B27 unique determinants of a cloned HLA-B27 gene. Immunobiology 173, 270-271.

Margulies, D. H , Evans, G. A. , Ozato, K., Cam-erini-Otero, R. D. , Tanaka, K., Appella, E. & Seidman, J. G. (1983) Expression of H-2Dd and H-2L d mouse major histocompatibility antigen genes in L cells after DNA-mediated gene trans­fer. J Immunol 130, 463-470.

Maryanski, J. L . , Moretta, A. , Jordan, B., De Plaen, E., Van Pel, A. , Boon, T. & Cerottini, J.-C. (1985) Human T cell recognition of cloned HLA class I gene products expressed on DNA transfectants of mouse mastocytoma P815. Eur J Immunol 15, 1111-1117.

Mentzer, S. J., Barbosa, J. A. , Strominger, J. L. , Biro, P. A. & Burakoff, S. J. (1986) Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones. J Immunol 137, 408-413.

Ouchterlony, O. (1970) Handbook of Immunodif-

GENERATION OF ANTIBODIES USING B27 TRANSFECTANTS 519

fusion and Immunoelectrophoresis. Ann Arbor Science Publishers Inc., Ann Arbor.

Ploegh, H. L. , Cannon, L. E. & Strominger, J. L. (1979) Cell free translation of the mRNAs for the heavy and light chains of HLA-A and HLA-B antigens. Proc Natl Acad Sei USA 76, 2273-2277.

Reth, M . , Hämmerling, G. J. & Rajewsky, K. (1978) Analysis of the repertoire of anti-NP-anti-bodies in C57B1/6 mice by cell fusion. I . Charac­terization of antibody families in the primary and hyperimmune response. Eur J Immunol 8, 393-400.

Schendel, D. J., Wank, R. & Dupont, B. (1978) Cell-mediated lympholysis: examination of HLA genetic fine structure and complementation using cytotoxic lymphocytes. Eur J Immunol 8, 634-640.

Simons, M. J. & Tait, B. D. (1984). Detection of immune-associated genetic markers of human disease. In Practical Methods in Clinical Immu­nology Series 7, 82.

Stam, N. J., Spits, H . & Ploegh, H . L. (1986) Monoclonal antibodies raised against dena-turated HLA-B locus heavy chains permit bio­chemical characterisation of certain HLA-C locus products. / Immunol 137, 2299-2306.

Szöts, H. , Riethmüller, G., Weiss, E. & Meo, T. (1986). Complete sequence of HLA-B27 cDNA identified through the characterization of struc­tural markers unique to the HLA-A, -B and -C allelic series. Proc Natl Acad Sei USA 83, 1428-1432.

Terasaki, P. I . & McClelland, J. D. (1964) Micro-droplet assay of human serum cytotoxins. Nature 204, 998-1000.

Terasaki, P. I . (editor) 1980. Histocompatibility Testing 1980, UCLA Tissue Typing Laboratory, Los Angeles, California.

Terasaki, P. I . (editor) 1988. Histocompatibility

Testing 1988, UCLA Tissue Typing Laboratory, Los Angeles, California, (in preparation).

Tiwari, J. L. & Terasaki, P. I . (1985) Mechanisms of HLA and disease associations. In HLA and Dis­ease Associations, pp. 28-31, Springer Verlag, New York, Berlin, Heidelberg, Tokyo.

Toubert, A. , Raffoux, C , Boretto, J., Sire, J., So-doyer, R., Thurau, S. R., Amor, B., Colombani, J., Lemmonier, F. A. & Jordan, B. R. (1988) Epitope mapping of HLA-B27 and HLA-B7 anti­gens using intradomain recombinants. J Immunol (Paper submitted for publication).

Van De Rijn, M . , Bernabeu, C , Royer-Pokora, B., Weiss, J., Seidman, J. G., De Vries, J., Spits, H. & Terhorst, C. (1984) Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells. Science 226, 1083-1085.

Van Pel, A. , De Plaen, E. & Boon, T. (1985) Selection of highly transferable variant from mouse mastocytoma P815. Somatic Cell Mol Ge­net 11, 467-475.

Weiss, E. H . , Kuon, W., Dörner, C , Lang, M . & Riethmüller, G. (1985) Organization, sequence and expression of the HLA-B27 gene: a molec­ular approach to analyze HLA and disease associ­ations. Immunobiology 170, 367-380.

Wildner, G., Weiss, E. H . , Szöts, H . , Riethmüller, G. & Schendel, D. J. (1989) The use of fusion proteins to study HLA-B27-specific allorecogni-tion. Mol Immunol 26, 33-40.

Address: Dr Gerhild Wildner Max-Planck-Institute for Biochemistry Am Klopferspitz 18 D-8033 Martinsried F.R.G.