Proc. Natl. Acad. Sci. USA

Vol. 76, No. 9, pp. 4636-4640, September 1979

Immunology

Monoclonal antibody directed to a B-cell antigen present in rats, mice, and humans (hybridomas/immune response-associated antigens/major histocompatibility complex)

DAVID L. GASSER*, B. ALLEEN WINTERS*, JEAN B3. HAAS*, THOMAS J. MCKEARNt, AND ROGER H. KENNETT* Departments of *Human Genetics and tPathology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Communicated by Peter C. Nowell, June 25, 1979

ABSTRACT Spleen cells from a LEW.AVN rat immunized with cells from an MNR r41 were fused with mouse myeloma cells to produce hybrid ell lines. One of these hybridomas produced a monoclonal antibody that was cytotoxic, for bo'ne marrow-derived (B)'but not thymus-derived-() cells. The antigen defined by this antibody is determined by a gene linked to the major histocompatibility complex MHC). e antigen is also present on B cells of most mouse strains and is determined by an MHC-linked gene in this species as well. In both rats and mice, the gene dete'rmining the antigen maps within the immune response region of the MHC. All human B-cell lines, but not T-cell lines, and B but'not T cells of all human donors tested so far are also positive for this antigen. Among human-mouse somatic cell lines that have lost-various human chromosomes, this B-cell antigen is present on all lines that are positive for HLA antigen but -is a4isent from all lines that.'have lost HLA. A class of antigenic specificities present on bone marrow-derived (B) cells are known to be coded for by genes mapping within the immune response region of the major histocompatibility complex (MHC) of the'-mouse, and are referred'to as immune response-associated (Ia) antigens (1-3). Similar B-cell antigens controlled by MHC-linked genes have also been described in humans (4) aintd rats (5, 6) as well as various other species. In order to study the Ta antigen specificities of the rat, we have utilized the techniques of somatic cell fusion to generate cell lines that produce monoclonal antibodies and have isolated a cell line that produces an antibody that is cytotoxic not only for B cells of many rat strains but also for B cells of many mouse strains and of many humans as well. The rat MHC was discovered in 1960 by Bogden and Aptekman (7), who designated this genetic locus R-1. Soon after this discovery was published, the locus'was referred to as AgB or H-1 by other investigators, and a considerable amount of nomenclature confusion ensued. It has now been agreed' that this locus be designated.RTJ (8), and this terminology will- be utilized in this report. At the present time, only two genetic regions of this complex have been defined by-laboratory recombinants, Putcher and Howard*(9) first reported a'riombination between the RT1 a (AgB4) haplotype of the OTA strain and the RT1 C (AgB5) haplotype of HO. This recombinant, designated iR, possessed RTJ a serological specifcities of the class I transplantation antigens'but was compatible with RT1 c cells in the mixed lymphocyte culture (MLC) test and was positive for class II B-cell antigens characteristic of RT1 c rats. These regions are now designated RTL.A and RT1.B, respectively, and the genotype of IR is therefore RTI.Aa, RTJ.Rc. The MNR rat strain has an interesting'MHC haplotype The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

which, although not a laboratory recombinant, has many of the RTI.Aa and RTI.Bc specificities (10-12). We have therefore immunized LEW.AVN (RTl a) rats with MNR cells and, from the immune cells, have obtained a hybridoma line that produces an anti-Ia antibody. MATERIALS AND METHODS Animals. The Lewis, Al, A2, BN.B2, BN, F.BN, DA, and August rats were obtained from stocks maintained at the University of Pennsylvania or Wistar Institute. Fischer, Wistar/ Furth, and Buffalo rats were obtained from Microbiological Associates (Walkersville, MD). The wild rat used in the development of the partially congeneic line DA. 1931 was trapped by the Philadelphia Department of Public Health near Philadelphia International Airport. LEW.AVN and AVN rats were kindly provided by Jane Schultz (Veterans Administration Hospital, Ann Arbor, MI). Donald Cramer (University of Pittsburgh) provided the KGH strain, and Carl Hansen (National Institutes of Health) provided the MNR rats. (Lewis X 1R)Fj hybrids were kindly provided by Jonathan Howard (Institute of Animal Physiology, Babraham, Cambridge, U.K. ). All mice were obtained from strains maintained at the University of Pennsylvania. Cell Lines. The P3/X63-Ag8 mouse plasmacytoma cell line was originally obtained from C. Milstein. All human cell lines were obtained from the Cell Center of the University of Pennsylvania. Hybridoma Development. LEW.AVN recipients received multiple injections of spleen and lymph node cells from MNR donors.- Four days after the last injection, a recipient was killed and a suspension of 108 spleen cells was mixed with 107 P3/ X63-Ag8 mouse plasmacytoma cells in the presence of polyethylene glycol. After fusion,- the hybrids were maintained in HY medium as described (13). Hypoxanthine and guanine were present in the HY medium,'and aminopterin was added 1 day after fusion. The cells were maintained in 96-well microtiter plates and fed weekly until hybrid clones appeared. At this time, supernatants were drawn from the wells and tested in the cytotoxicity assay, as described below. Positive lines were then cloned in semisolid agarose and retested after cloning. The concentration of rat immunoglobulin was measured by using an inhibition of passive hemagglutination assay as described by- Gutman and Weissman (14). This assay utilizes an anti-K alloantiserum (kindly provided by I. L. Weissman) that reacts with Kla allotype of the rat but does not react with the Abbreviations: B, bone marrow-derived; T, thymus-derived; MHC, major histocompatibility complex; MLC, mixed lymphocyte culture; Ia, immune response-associated.

4636

Immunology:

Gasser et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

P3/X63-Ag8 proteins. By comparison with the inhibitory activity of a known concentration of a second antibody, a pool of H76 culture supernatants was found to contain 10 ,ug of rat

Table 1. Strain distribution of susceptibility to H76 monoclonal antibody

RTl-

antibody per ml. Cytotoxicity Assay. Mineral oil was poured into each well of a microtiter plate, and 103 target cells in 1 Atl were injected under the mineral oil along with 1 Ail of culture supernatant containing the hybridoma antibody. The plates were incubated at 370C for 30 min, after which 5 ,l of guinea pig or rabbit complement was added to each well. After an additional 90-min incubation at 370C, 1 Al of 2.5% trypan blue was added to each well. The plates were examined in an inverted phase microscope and viabilities were recorded. Cytotoxicity index was defined as (A - B)/(100 - B), in which A = percentage of cells lysed by antibody and complement, and B = percentage of cells lysed by complement alone. MLC Test. Peripheral blood lymphocytes were obtained by cardiac puncture, sedimented in citrate/dextran, washed, and counted. In each well of a microtiter plate, 2 X 105 responding cells were mixed with 1 X 105 irradiated target cells; each combination was set up in quadruplicate. The cultures were grown, in minimal essential medium with glutamine, penicillin, streptomycin, and 5% rat serum, at 37°C in 5% CO2/95% air. On the fifth day of culture, 0.25 ACi (1 Ci = 3.7 X 1010 becquerels) of [3H]thymidine was added to each well. Four hours later, the cells were harvested in an automated sample harvester and counted in a Searle Delta 300 scintillation counter. Absorption. Hybridoma cell culture supernatants diluted 1:50 were incubated at 4°C with an equal volume of absorbing cells. The cells were changed several times during a 20-hr incubation period, after which the supernatants were tested for antibody activity. Mouse-Human Hybrids. The hybrids were produced by fusion of P3/X63-Ag8 cells with peripheral blood cells from a patient with chronic lymphocytic leukemia of the B-cell type (kindly provided by Peter Nowell and David Rowlands). The fusion and maintenance of cultures was as reported (13). 80

60

x

40 0

0

U

20

10°

10-'

10-2

10-3

Dilution

FIG. 1. Cytotoxic activity of H76 monoclonal antibody against lymph node cells of KGH rats. Nylon wool-adherent cells (0) are predominantly B cells; nylon wool-nonadherent cells (A) are predominantly T cells. *, Unfractionated lymph node cells.

4637

*

Cyto-

Strain haplotype toxicity, %* 45 Lewis 45 Black Hooded I 40 Fischer I v 45 Wistar/Furth v 35 Al v 50 A2 v 40 BN.B2 n 20 BN n 50 F.BN a DA 2 a AVN 1 a LEW.AVN 1 c 60 August b 45 Buffalo 70 KGH g m MNR 80 Values shown represent the approximate number of nylon wooladherent cells killed at the optimal concentration of hybridoma antibody. The percentage killed in the complement control has been subtracted in each case.

Immunoperoxidase Assay. Antigens were detected on mouse-human hybrids by using a modification of the method described by Bross et al. (15). Cells were treated in suspension with hybridoma supernatant, washed, and then treated with peroxidase-linked rabbit anti-mouse IgG. The cells were attached to poly(L-lysine)-coated slides and then treated with diaminobenzidine and osmium tetroxide. HLA antigens were detected by using the monoclonal antibody W6/32 reported by Barnstable et al. (16).

RESULTS Among the 576 wells in which the fused cells were distributed, 340 hybrid cell lines developed. Supernatants from all of these were tested, and 23 were cytotoxic for MNR lymph node cells. One line, designated H76, gave especially good activity and was cloned in semisolid agarose. In most experiments, only 20-40% of the best target lymph node cells (MNR or KGH) were lysed by this antibody, so its activity was tested against cells that adhere and cells that do not adhere to a nylon wool column (17). Nylon wool-nonadherent cells were almost completely resistant to killing by this antibody, and nylon wool-adherent cells were very susceptible (Fig. 1). The specificity of the antibody was then tested against a panel of rats with different MHC haplotypes (Table 1). Significant cytotoxicity was observed with lymph node target cells from all strains tested except those with the RTJ a haplotype. Although there was considerable variation from strain to strain in the number of cells killed by this antibody, the percentage of cells killed among rats of the same strain was fairly reproducible. For example, not one of the three BN rats tested Table 2. Cytotoxic activity of H76 monoclonal antibody after absorption by rat nylon wool-adherent lymphocytes Target Unabsorbed Cytotoxicity after absorption, % cells H76, % DA KGH Fischer BN F.BN DA 0 0 2 0 0 0 KGH 60 70 0 0 0 0 Fischer 45 50 2 0 0 0 BN 30 30 2 0 0 0 F.BN 60 40 0 0 0 0

4638

Immunology:

Gasser et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

Table 3. Susceptibility of mouse strains to cytotoxic activity of H76 antibody H-2 genotype* CytoStrain K A B J E C S G D toxicity, %t b b b b b b b b b 65 B10 BALB d d d d d d d d d 40 C3H k k k k k k k k k 50 DBA/1 q q q q q q q q q 2 B1O.Q q q q q q q q q q 2 AQR q k k k k d d d d 70 BlO.T(6R) q q q q q q q - d 2 q q q q q q q q s 5 B1O.DA B1O.S s s s s s s s s s 30 * H-2 genotypes of recombinant haplotypes are listed in ref. 18. t Values shown represent the approximate number of nylon wooladherent cells killed at optimal concentration of hybridoma antibody, with the amount killed in the complement control subtracted.

showed greater than-30% killing and not one of five KGH rats tested showed less than 60% killing. To determine whether there might be differences in the specificity of the antigen possessed by various strains, the absorption experiment shown in Table 2 was done. These results show that KGH, Fischer, BN, and F.BN all are capable of absorbing out all of the cytotoxic activity of this antibody. The negative strain DA did not remove any significant amount of the activity. In order to establish if the antigen defined by this antibody is determined by a gene linked to the MHC, the partially congenic line DA. 1931 was tested. About half of the progeny in each generation are homozygous for the RTJ a haplotype of the inbred partner strain (DA) and the other half are heterozygous for a haplotype obtained in the wild that is similar if not identical to RT1g. Individual rats were tested in the MLC test as well as for susceptibility to the H76 hybridoma antibody. All rats were either positive in both tests (15 animals) or negative in both tests (10 animals), so the antigen defined by H76 is determined by a gene that is closely linked to the MHC. Further evidence for this linkage was obtained by the observation that the LEW.AVN congeneic strain is negative for the antigen and thus corresponds to the phenotype of the MHC donor (AVN) rather than that of the genetic background (Lewis). To map this gene as precisely as possible within the MHC, descendants of the 1R strain (9) were then studied. (Lewis X 1R)Fj hybrids, kindly provided by J. Howard, were crossed with LEW.AVN partners, and the progeny were tested for susceptibility to the H76 antibody as well as MHC haplotype (by the MLC test). All progeny of both heterozygous types (RTI '/RTJ a and RTI 'IRT1 lr) were strongly positive for the antigen defined by H76 antibody. RT1 lr/RT1l r homozygotes which were subsequently derived also were strongly positive for this antigen. Because the RT1 lr haplotype has the RTJ.Aa, RTJ.Bc composition and RT1a rats are negative, the antigen is determined by a gene either within or closely linked to the RTI.B region. The possibility that this antigen might be present in mice was then explored. Several strains (B1O, BALB, and C3H) were

Target cells KGH MOLT-4 SCBM

Table 5. Expression of HLA and 2 B-cell antigen detected by H76 monoclonal antibody on mouse-human hybrid cell lines

H76+ H76W6/32+ 3 0 W6/320 9 Numbers shown represent the numbers of mouse-human hybrid clones that were positive or negative for HLA or the B cell antigen. Binding of the monoclonal antibodies was detected by peroxidaselinked anti-mouse IgG, reaction with diaminobenzidine and osmium tetroxide, and observation of a black precipitate around the surface of cells expressing the antigens. All cells tested were confirmed to be hybrids by the demonstration that both mouse and human glucose phosphate dehydrogenase were present.

clearly positive for the antigen, and the DBA/1 strain was negative (Table 3). The congenic strain B1O.Q was also negative, which suggested that the gene determining this antigen was linked to H-2. The AQR strain was strongly positive and the BlO.T(6R) and BlO.DA strains were negative, so the gene determining the antigen does not map in the H-2K or H-2D region. Various human cell lines were then tested. Very strong reactions were observed with the B-cell lines MWF (85%), IDF (80%), LBF (70%), BJAB (100%), AS (80%), 8866 (90%), and MST (80%). The DAUDI B-cell line, which lacks 132-microglobulin as well as HLA A, B, and C antigens, was fairly susceptible to the antibody (40-80% of the cells were killed). The T-cell lines MOLT-4 and RPMI 8402 were negative or only weakly positive, with cytotoxicities of 0-20% in various tests with MOLT-4 and 5% with RPMI 8402. The null cell line REH was strongly positive, with a cytotoxicity of 80% above background. In order to determine whether the human antigen was the same as that present in rats, the absorptions shown in Table 4 were done. These results show that absorption by BJAB or SCBM removed almost all of the activity not only for human cells but also for rats cells as well. The T-cell line MOLT-4 removed very little of the activity for either human or rat cells. Susan Radka and Chester Zmijewski (Hospital of the University of Pennsylvania) then tested this antibody for cytotoxicity against B and T cells obtained from 14 normal donors. The B cells of all 14 of these donors were highly susceptible to the antibody; all of the T cells were resistant. To determine whether the antigen detected by the H76 antibody is syntenic with HLA, a series of independent clones of mouse-human hybrids were assayed for the expression of HLA by using monoclonal antibody W6/32, which reacts with HLA A, B, and C molecules (16), and H76. All of the hybrid lines expressed either both HLA and the B-cell antigen defined by H76 or neither of these antigens (Table 5). This supports the conclusion that the gene for the B-cell antigen is on the same chromosome (no. 6) as the human MHC. The class of antibody produced by the H76 cell line was studied by several methods. Supernatant from H76 cells grown overnight in the presence of [35S]methionine was run on a sodium dodecyl sulfate/polyacrylamide gel along with labeled

Table 4. Cytotoxic activity of H76 monoclonal antibody for human cell lines Cytotoxic index, % Absorbed by Absorbed by Unabsorbed H76 BJAB cells MOLT-4 cells Cell type antibody Rat B cells Human T cells Human B cells

50 0 100

30 10 90

5

0 0

Absorbed by SCBM cells 0 0 0

Immunology:

I...

....

Gasser et al.

%.

a

.!- millilimmomakw....

Proc. Natl. Acad. Sci. USA 76 (1979)

4639

are conserved among such species as mice, rats, guinea pigs, and humans (19). In the present communication, we report evidence for the occurrence of an antigenic specificity that is shared by rats, mice, and humans. In all three species, the antigen is present on B but not T cells. The possibility that this antibody is directed against a surface immunoglobulin is ruled out by its strongly positive reaction with the human REH cell line, which is known to lack surface immunoglobulins (20). In both mice and rats, the antigen has been shown to be determined by a gene linked to the MHC, and in humans, the gene determining this antigen maps on the same chromosome as HLA. In rats, the gene maps in a region that is known to code for Ia antigens. In mice, the gene is linked to H-2 but does not map in H-2K or H-2D, suggesting that it most probably maps in one of the I-region loci between K and D. The antigen is present on the human cell line DAUDI, which lacks HLA A, B, and C antigens, so it is likely that the antigen is an HLA.Drelated antigen. This would be consistent with its occurrence on B cells and absence from T cells. It is not clear how large a genetic segment has been conserved among the various species studied. It is known that most antigenic determinants consist of no more than five or six amino acids (21) and can involve as small a difference as one amino acid substitution, as in the case of the Oz immunoglobulin allotypes (22). The possibility that the same antigenic specificity could appear among various species by chance alone does not seem likely, because this specificity was not detectable on the T cells of any of the three species examined. Finding an antigenic specificity shared by rats, mice, and humans is in itself not surprising because oligopeptide sequences of many proteins have been shown to be highly conserved. Presumably such sequences are maintained by strong selective forces, but the specificity described in this report is not present in all rats and mice and is therefore not essential for survival. Why allelic variation can occur within a segment of genetic material that is kept invariant among fairly unrelated orders of mammals is an important question for future research. We are grateful to Drs. Susan Radka and Chester Zmijewski for testing the activity of the hybridoma antibody against B and T cells of normal human donors. This work was supported by Grants 15146 and 24263 from the National Cancer Institute and PCM-82997 from the National Science Foundation.

FIG. 2. Autoradiography of polyacrylamide gel comparing 35Slabeled H76 antibody (Left) with 35S-labeled myeloma protein produced by the mouse P3/X63Ag8 cells (Right).

supernatant from the mouse P3 cells used for fusion. The P3 heavy and light chains were clearly visible in the H76 supernatant, as well as an additional light chain and a larger molecule with the mobility expected for a ,u chain (Fig. 2). It was also observed that the antibody activity could be destroyed by 2mercaptoethanol and that a positive reaction between H76 supernatant and a rabbit anti-(rat IgM) antiserum (Miles Laboratories) could be demonstrated in both agar diffusion and antigen binding assays. DISCUSSION It has been well established that a MHC coding for transplantation antigens, immune response genes, and complement components is present in most, if not all, vertebrate species. What has not been well defined is the degree of similarity of the products of the same locus among various species, although amino acid sequence data clearly indicate that many residues

1. Hammerling, G. J., Deak, B. D., Mauve, G., Hammerling, U. & McDevitt, H. 0. (1974) Immunogenetics 1, 68-81. 2. Shreffler, D. C. & David, C. S. (1975) Adv. Immun. 20, 125195. 3. McDevitt, H. O., Delovitch, T. L., Press, J. L. & Murphy, D. B. (1976) Transplant. Rev. 30, 197-235. 4. Snell, G. D., Dausset, J. & Nathanson, S. (1977) Histocompatibility (Academic, New York). 5. Shinohara, N., Cullen, S. E. & Sachs, D. H. (1977) J. Immunol.

118,2083-2097. 6. Shinohara, N., Lunney, J. K. & Sachs, D. H. (1978) J. Immunol.

121,637-640. 7. Bogden, A. & Aptekman, P. A. (1960) Cancer Res. 20, 13721382. 8. Report of the First International Workshop on Alloantigenic Systems in the Rat (1978) Transplant. Proc. 10, 271-285. 9. Butcher, G. W. & Howard, J. C. (1977) Nature (London) 266, 362-364. 10. Cramer, D. V., Davis, B. K., Shonnard, J. W. & Gill, T. J. (1977)

Transplantation 23, 498-503. 11. Radka, S. F., Cramer, D. V. & Gill, T. J. (1977) J. Immunol. 119,

2037-2044.

4640

Immunology: Gasser et al.

12. Gotze, D. (1978) Immunogenetics 7,491-506. 13. Kennett, R. H., Denis, K. A., Tung, A. S. & Klinman, N. R. (1978) Curr. Top. Microbiol. Immunol. 81,77-91. 14. Gutman, G. A. & Weissman, I. L. (1971) J. Immunol. 107, 1390-1393. 15. Bross, K. J., Pangolis, G. A., Staatz, C. G. & Blume, K. G. (1978) Transplantation 25, 331-334. 16. Barnstable, C., Bodmer, W. F., Brown, G., Galfre, G., Milstein, C. & Williams, A. F. (1978) Cell 14,9-20. 17. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J. Immunol. 3, 645-649.

Proc. Nati. Acad. Sci. USA 76 (1979) 18. Klein, J., Flaherty, L., Vande Berg, J. L. & Shreffler, D. C. (1978) Immunogenetics 6, 489-512. 19. Blankenhorn, E. P., Cecka, J. M., Goetze, D. & Hood, L. (1978) Nature (London) 274,90-92. 20. Rosenfeld, C., Goutner, A., Choquet, C., Venuat, A. M., Kayibanda, B., Pico, J. L. & Greaves, M. F. (1977) Nature (London) 267,841-843. 21. Van Vunakis, H., Kaplan, J., Lehrer, H. & Levine, L. (1966) Immunochemistry 3,393-402. 22. Appella, E. & Ein, D. (1967) Proc. NatW. Acad. Sci USA 57, 1449-1454.

Monoclonal antibody directed to a B-cell antigen present in rats, mice, and humans.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4636-4640, September 1979 Immunology Monoclonal antibody directed to a B-cell antigen present in rat...
1MB Sizes 0 Downloads 0 Views