Scand. J. fmmitnol. 7, 447 452, 1978
Identification of H-2 and la-Antigen Analogues in Several Species by Immunological Crossreactions of Xenoantisera S. KVIST. L. KLARESKOG & P. A. PETERSON Inslitute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Uppsala, Sweden
Kvist, S., Klareskog, L. & Peterson. P.A. Ideniificalion of H-2 and Ia-Anligen Analogues in Several Species by Imniunological Crossreactions of Xenoantisera. Scand. J. Immunol. 7, 447-452, 1978. Rabbit aniisera against H-2K and D aniigens react with molecules compo.sed ol' 12,000 and 45,000 dalton subunits derived from human, monkey, rat. guinea-pig, cow and pig lymphocytes. The antisera failed, however, to react with similar type molecules from chicken. A rabbit antihuman fJ^-microglobulin serum reacted with chicken molecules comprising 12.000 and 45,000 dalton polypepnde chains. An anliserum against HLA-DR antigens reacted with la-aniigen-likc molecules from monkey, ral. mouse, guinea-pig, eow, pig and chicken lymphocyies. All laantigen-like molecules displayed two dissimilar subuniis. The present daia suggest that xenoanliscra directed against highly purified MHC antigens from one species may lie useful tools in elucidating the structure of similar antigens in other species where alloantisera are nol available. Sune Kvi.st, Institute of Medical nnd Physiological Chemistry. The Biomedical Center, Box S75 S-751 23 Uppsala. Sweden.
Work on the structure of the MHC-antigens.* which requires large amounts of tissue as starting material to obtain reasonable quantities of highly purified antigens, is hampered by the fact that most inbred animals belong to species with smal! individuals. Recently, we have raised xenoantisera directed against highly purified MHC antigens from mouse [S. Kvist, L. Ostberg & P. A. Peterson, submitted] and man [8] and this communication shows that such antisera. due to the conservative structure of the MHCantigens, are suitable reagents to detect MHCantigens from several species where alloantisera are not available. •Abbreviations used are: MHC, major histocompatibilily complex; SAC, Staphylococcus aureus Cowan 1; SDS, sodium dodecylsulphate; NRS, normal rabbit serum.
MATERIALS AND METHODS CW/.V. Splenocytes were obtained from Vervet monkeys, Sprague-Dawley rats, BIO.A mice, guineapigs and chickens. All animals were maintained al the Animal House, Biomedical Cenire. Spleens from cows and pigs were obtained fresh from a local abattoir. Human peripheral blood lymphocytes were isolated from blood samples drawn from healthy volunteers [I]. Antisera. A rabbit anliserum against H-2K and D antigens was raised by repeated lymph node and footpad injections with a sample of highly purified H-2 antigens. The characterization and specificity of the antiserum is outlined elsewhere [S. Kvist et al.. submilled]. Tbe rabbit anti-HLA-DR antigen serum was raised by the same immunization schedule. The reactivity of this antiserum has been the subject of a previous publication (8]. The rabbit antiserum against human ^^-microglobulin was the same as earlier used [151. Radioactive labelling and indirect immunoprecipitation. The splenocytes were labelled for 14 h with ^H-tyrosine
0300-9475/78/0600-0447 $02.00 © !978 Blackwell Scientific Publications
447
448
S. Kvist. L. Klareskog & P. A. Peter.son
H-2K and D antigen analogues on lymphocytes derived from various species. Internally labelled glycoproteins were isolated by means of lens culinaris haemagglutinin afllinity chromatography and molecu]es reactive with the antiserum were recovered by indirect immunoprecipitation. Fig. 1 shows that the precipitated antigens gave rise to two radioactive peaks on SDSpoly aery I amide gel eiectrophoresis for all species examined except for the chicken. In the latter case the rabbit anti-H-2 antigen scrum did not react measurably with any molecules, as shown in Fig. 1 (h). The SDS-polyacryiamide gel patterns for molecules derived from monkey, human, mouse, rat, guinea-pig., swine and cow lymphocytes were very similar inasmuch as the smaller ofthe two precipitated chains displayed a molecular weight of about 12.000 whereas the larger chain exhibited an eiectrophoretic tnobility consistent with an apparent molecular weight of about 45,000. The larger chain varied slightly in molecular weight for the difTerent species. Thus, the chain from guinea-pigs had an apparent molecular weight of about 42,000 RESULTS whereas those of mice and rats were consisIdentification of H-2K and D antigen analogues tently larger with apparent molecular weights of about 47.000. With use of the rabbit anti-H-2 antigen-serutn Since the rabbit anti-H-2 antigen serum attempts were made to establish the presence of
(Amersham, England) and soUibilized with 2"'o (v/v> Triton X-100. Soluble glycoproteins were isolated by alRnity chromatography on Sepharose-4B columns containing covalently bound lens culinaris haemaggluiinin. Hound glycoproleins were desorbed by including 10% (w/v) a-methylmannoside in ihe cluent. After dialysis the glycoprotein fractions were incubaled with NRS overnight and radioactive components binding un specifically to IgG were removed by ihe precipilalion of the IgG wilh SAC. The supernatants were divided in Uvo equal fractions to which 50 |xl of NRS and anliserum, respectively, were added. After about 10 h of incubation immune complexes were precipitated by the addition of SAC. The bacteria were washed three limes and the immune complexes were dissolved by the addition of 200[xl of a solution containing 2% SDS. 8 M urea and 3"/,, fJ-mercaptoelhanoL Further details of this procedure have been published 114]. SDS-polyacrylamtde gel electrophoresis and isoelectric focusing. SDS-poIyacrylamide gel electrophoresis was carried oul essentially as described hy Laemmli [10]. The technique employed for isoeleciric focusing was that of O'Farrell [13]. When samples were to be subjected lo isoelectric focusing SDS was omitted from the solution used to dissolve the immune precipitates.
1,0
40 80 MIGRATION
40 DISTANCE
80
80
(mm)
FIG. I. SDS-polyacrylamide gel electropboreses of "H-tyrosinc-!abelled membrane glycoproteins precipitated wilb a rabbit anliserum against highly purilied H-2 antigens {•—•) and NRS ( ). respectively. The membrane protein.s were derived from lymphocytes obtained from monkey (a), human (b), mouse (c), rat (d), guinea-pig (e). pig (f), cow (g) and chicken (h). The arrows denote the migration positions of marker IgG heavy (HJ and light (L) chains and Pa-microglobulin (|3a|i).
MHC-Atitigen Analogites in Several Species
449
molecules which on SDS-polyacrylamide gel electrophoresis displayed the typical profile, i.e. two polypeptide chains with the apparent molecular weights 12,000 and 45.000, respectively. 2 •
Molecular weight determination of la-antigen analogttes
1 •
20 iO 60 80 100 MIGRATION DISTANCE (mm) FIG. 2. SDS-polyacryiamide gel etectrophoresis of chicken lymphocyte membrane glycoproleins, internally labelled wilb ^H-tyrosine, precipitated by a rahhil anti-human p.-microglobulin serum (•—•) and NRS ( ), respectively. The arrows have the same meaning as in Fig. I.
available did not react wilh any lymphocytederived molecules from chicken it appeared of interest to examine if antisera against fi..microglobulin might crossreact with the chicken homoiogue. To this end a rabbil anti-human;io-microglobulin serum was used for indirect immunoprecipitations of ^H-tyrosine-labelled lymphocyte glycoproteins from the chicken. Fig. 2 shows that the antiserum reacted with
a
The human analogues of the murine Ia antigens have recently been identified [7], These antigen are the products of the HLA-D locus. In a series of experiments similar to the preceding ones attempts were made to isolate faantigen analogues from several species by means of indirect immunoprecipitation with use of a rabbit anti-HLA-DR antigen serum. Fig. 3 shows that the glycoprotein fractions derived from lymphocytes of the dilTerent species examined all contained molecules that reacted with the antiserum. The SDS-polyacrylamide gel electrophoreses of the precipitated antigen revealed the typical la-antigen-like two-chain pattern for all species except the pig. In this case only a single peak of radioactivity could be discerned (Fig. 3f). The molecular weights estimated from the SDS-polyacrylamide gel electrophoresis of the two types of chains from the
H
tl. Q
< o Q
80
^0 80 MIGRATION
40 DISTANCE
80 {mm)
80
FIG. _1. SDS-polyacrylamide gel electrophoreses of ''H-tyrosine-labelled glycoproteins precipitated with a rabbit anti-HLA-DR antigen serum (•—•) and NRS ( ), respectively. The precipitated lymphocyte antigens were derived from monkey (a), human (b), mouse (c), rat (d), guinea-pig (e), pig (f), cow (g) and chicken (hj. The arrows have ihe same meaning as in Fig. 1.
450
S. Kvist. L. Kliire.skog & P. A. Peterson
I. Apparent molecular weights of the laantigen analogue sutiunits from dilTerent species*
Molecular weight Species Monkey Human Mouse Rat Guinea-pig Pig Cow Chicken
a-Chain
p-Chain
.14.000 34.000 35.000 35,000 33,000 27,000 28,000 .^3,000
28,000 28,000 27,000 27,000 25,000 27,000 25,000 28,000
* The determinaiions were made by SDS-polyacrylamide gel electrophoresis.
various species are summarized in Table J. It can be seen in the Tabie that the molecular weights of the smaller ({i) chain ranged from 25,000 to 28,000 daltons and those of the larger {yi) chain from 27,000 to 35.000 daltons. It is noteworthy that the rabbit anti-human HLA-DR antigen serum reacted with the laantigen analogues of the chicken and that the chicken displays la-antigens with the typical subunit structure. The SDS-polyacrylamide gel electrophoresis did not allow the conclusion to be drawn that the la-antigen analogues ofthe pig are composed of two types of polypeptide chains. Therefore, pig lymphocyte glycoproteins recognized by Ihe rabbit anti-HLA-DR antigen serum were subjected to isoelectric focusing. By this type of separation technique the pig Ia-antlgen analogues resolved into two broad radioactive peaks, as can be seen in Fig. 4. Interestingly, the isoelectric focusing profile of the pig la-antigen analogues and those of human and chicken were very similar (Fig. 4), thereby further strengthening Ihe notion that they represent homologous proteins. Material in the fraciions denoted by the arrows in Fig. 4 was subjected to SDS polyacrylamide gel electrophoresis. It can be seen in Fig. 5 that the more acidic component comprised the a-chain whereas the more basic material represented the [i-chain. Therefore it seems reasonable to conclude that all the species examined produce la-antigen-like glycoproteins that are composed of two types of dissimilar polypeptide chains.
20 DISTANCE
40 FROM
60
80
THE ANODE
100 (mm)
FIG. 4. Isoelectric focusing of *H-lyrosine labelled laantigen analogues derived from pig (A), human (Bl. and chicken (C) lymphocyies. The aniigens wore isolated by indirect immunoprecipitalion with use of a rahhil anti-Ht.A-DR antigen serum. Material in the fractions denoted hy the arrows was subjected to SDSpolyacrylamide gel electrophoreses (see Fig. 5). A denotes Ihe pH.
DISCUSSION It is well established that the MHC displays a very conservative genetic organization since narrow chromosomal segments controlling various aspects of several immunobiological phenomena have been identified in a number of species [6, 9, 18]. The conservative nature at the DNA-level seems to be paralleled by the similarity in structure between the MHC antigens of several species. The present report demonstrates that antisera raised against highly purified MHC-antigens crossreact exlensively with the analogous antigens from other species. Thus, the antiserum raised against highly puri-
MHC-Antlgen Analogues in Several Species
80 MIGRATION DISTANCE ( m m )
451
80
FIG. S. SDS-polyucrylamidc gel electrophoresis of la-aniigcn analogues separated by isoeleclric focusing. The analysed materials were obtained from the fractions ilcnoicd by the arrows in Fig. 4, Pig (a-d), human (e-h) anti chicken (i-l) la antigen analogues were analysed. The arrows have the same meaning as in Fig. I.
fied H-2K- and D antigens combines with H-2 antigen-like molecules from monkey, human, rat. guinea-pig, pig and cow. The antiserum employed did not recognize the chicken anligens, which, however, were precipitated with an anliserum against human pj-microglobulin. In all cases the H-2 antigen-like molecules were shown to be composed of two subunits dissimilar in size. This confirms earlier findings obtained wilh alloanlisera in some specie.s [2, 3, II. 12, 17. 19] and it seems reasonable lo conclude thai the struciural similarity between the MHC-anligens of the various species is also reflected by the occurrence of common, interspecies anligenic determinants. The anliserum against the human la-antigen analogues (HLA-DR) reacted wiih la-antigenlike molecules from all the species examined, i.e. also with the chicken antigens. Also in this case were Ihe isolated molecules very similar in their gross structure. However, ihe molecular weights of the separated chains varied considerably more between the species than did the molecular weights for the H-2 aniigen-like molecules. The significance of this observation
cannot be fully realized until it is ascertained whether the dilTerences are accounted for by ihe polypeptide poriions or by the carbohydrate moieties of the subunits. It is. thus, possible that the immunotogical crossreactiviiy observed is restricted to one or several of the l-rcgion subloci in the ditTerent species assuming that alt species, like the mouse [18], display several genes controlling the expression of la-antigen-Uke molecules. However, separation of the Taaniigcn-like subunits from pig, human and chicken by isoelectric focusing revealed striking similarities in charge regardless of the difterences in the measured molecular weights. Although Ihis finding does not argue againsi the possibility thai the isolated pig and chicken [a-antigen-tike molecules arc derived from a lesser number of genes Ihan are their human counterparts it suggests that, if so, Ihe products of the ditTerent 1-region genes have Io be very similar. In any event, Ihis observation makes it likely that isoelectric focusing can be used advantageously to distinguish the two types of la-antigen-like subunits when ihe resolution obtained by SDSpolyacrylamide gel electrophoresis is insufficient.
452
S. Kvist, L. Klareskog & P. A. Peterson
It is somewhat surprising that the antisera used exhibit the wide cross-reactivity observed. It would be expected that the antisera only recognize structures that are dilTerent between the immunogen and the rabbit counterpart of the immunogen. In that case it is remarkable that human HLA-DR antigens, used as the immunogen. should exhibit structures that are dissimilar from those of the rabbit la-antigens but shared with la-antigens from the chicken. It seems, however, that the immune system of most if not all animals may preferentially be be geared towards dealing with MHC-antigen [5] and antigenie complexes involving the MHC antigens [4]. Thus, monkey antibodies raised against free human fia-microglobulin are not easily obtained but [i.-microglobulin, as part of the HLA-A, B, and C antigens, gives rise to a good antibody production [16]. Therefore, it is possible that also HLA-DR antigens may give rise to an anomalously vigorous antibody response which would account for the high degree of interspecies cross-reactivity observed. Another explanation for the crossreactiviiy would be that the antiserum against the HLADR antigens recognizes carbohydrate structures that may be similar despite vast differences in tbe protein moieties of the la-antigens from dilTerent species. This would not be unreasonable to assume since the present studies were restricted to analyses of MHC antigens bound to the Lens CuHnaris haemagglutinin. Thus, a subfractionation of the MHC antigens from the various species may have occurred so that only la-antigens with similar carbohydrate moieties were isolated. However, HLA-DR antigens that have been separated by means of their dilTerent afTinity for the Lens Culinaris haemagglutinin react identically with the antiserum although they most probably display differences in their prosthetic groups [B. Curman, L. Rask, L. Ostberg & P. A. Peterson, in preparation].
REFERENCES
ACKNOWLEDGMENT
t Boyum, A. Separation of leucocytes from blood ami bone marrow, Scand. J. clin. Lab. Invest. 21, Suppl. 97, 31. 1968. 2 Callahan, N.G, & Dewilt, C.W. Ral cell surface antigens, I. Isolation and partial characterization of an Ag-B antigen, J. Immunol. 114, 776. 1975. I Cramor. D.V., Shonnard, J.W. & Gill, T.J. Genetic siudies in inbred rals. II. Rclationsbip between the [iiajor histocompatibilily complex and mixed, lymphocyte reactivily.y./HimHHrt^rnc/u,V.I. 421.1974, 4 Doherty, P.A.. Blanden. R.V, & ZinkcrnaBel. R.MSpecificity of virus-immune efleclor T cells Tor H-2K or H-2D compatible inferattions: Implications for H-antigen diversity. Transplant. Rev. 29, 89, 197C>. 5 Jerne, N.K, The somatic generation of immune recognition, Eur.J. IniDiuiiol. 1, 1, 1971, 6 Kal7. D.H, Lymphocyte Oi^Hi-nwuiation, Rfcofuji/;()«,fl;f(/i?ci'M/a//on. Academic Press. New York. 1977. 7 Klareskog, L., Sandberg-Trag^rdh. L., Rask. L,, Lindblom, J.B., Curman, B. & Peterson. P.A. Chemical properties of human la anligens. Nature (Lorn/.), 265, 248. 1977. 8 Klareskog, L., TragArdh, L.. Lindhlom. J.B, & Peterson. P.A. Reactivity of a rabbil aniiserum against highly purilied HLA-DR anligens. Scaml. J. Immunol. 7, 199, 1978, 9 Klein. J. Tfw Biology of the Mou.sc tiistocompaiihility 2 Comph:x. Springer, New York. 1975. 10 Laemmli, V.K, Cleavage of strucliiral proteins during the assembly of the head of hacleriophage T4, Nanire {Lond.), 227, 680, 1970, II Leight. G.S.. Sachs. D.H., Williams, G.M. & Rosenberg. S.A. Transplantation in minialurc swine. Transplant. Proc. 9, 575. 1977, 12 Moller. G. (ed.) [ij-Microglobulin and HL-A aniigen. Transplant. Rcw 21, 1974. 13 O'Farrell, P.H. High resolution two-dimensional electrophoresis of proieins. J. biol. Chem. 250, 4007. 1975, 14 Ostberg, L.. Scge. K.. Rask, L. & Peterson, P.A. Isolation of radiolabelled H-2 antigens. Folia Not.. Praha. 22, 372, 1976. 15 Peterson, P.A.. Rask, L. & Lindblom. J.B, Highly purilied papain-solubilized HL-A antigens contain P^-microglobulin. Proc. nat. Acad. Sci. U.S.A. 7t, 35, 1974, 16 Sanderson, A.R. HLA "help" for human p^-microglobulin across species barriers. Nature (Lond.), 269,414. 1977, 17 Schwartz. B.D,. Kask. A.M., Patii, W.E. & Shevach, E.M. Structural characteristics of the alloantigens determined by ibe major histocompatibility complex of tbe guinea pig. J. e.xp. Med. 143, 541. 1976, 18 ShrelHcr. D.C, & David. C.S, The H-2 major histocompatibility complex and the 1 immune response region; genetic variety, funclion, and organization. AJv. Immunol. 20, 125, 1975. 19 Ziegler, A. & Pink, R, Chemical propenies or iwo antigens conirolleti by ihe major histocompatibility complex of the chicken. J. biol. Chem. 251, 5391. 1976,
This work was supported by the Swedish Cancer Society.
Received 5 December 1977 Received in revised form 13 January 1978
Whatever the reason is for the immunological cross-reactivity it seems reasonable to conclude that xenoantisera raised against highly purified MHC-antigens from one species may be useful tools in monitoring the purification of MHC antigens from olher species where alloantibodies are not available.
Identification of H-2 and la-Antigen Analogues in Several Species by Immunological Crossreactions of Xenoantisera S. KVIST. L. KLARESKOG & P. A. PETERSON Inslitute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Uppsala, Sweden
Kvist, S., Klareskog, L. & Peterson. P.A. Ideniificalion of H-2 and Ia-Anligen Analogues in Several Species by Imniunological Crossreactions of Xenoantisera. Scand. J. Immunol. 7, 447-452, 1978. Rabbit aniisera against H-2K and D aniigens react with molecules compo.sed ol' 12,000 and 45,000 dalton subunits derived from human, monkey, rat. guinea-pig, cow and pig lymphocytes. The antisera failed, however, to react with similar type molecules from chicken. A rabbit antihuman fJ^-microglobulin serum reacted with chicken molecules comprising 12.000 and 45,000 dalton polypepnde chains. An anliserum against HLA-DR antigens reacted with la-aniigen-likc molecules from monkey, ral. mouse, guinea-pig, eow, pig and chicken lymphocyies. All laantigen-like molecules displayed two dissimilar subuniis. The present daia suggest that xenoanliscra directed against highly purified MHC antigens from one species may lie useful tools in elucidating the structure of similar antigens in other species where alloantisera are nol available. Sune Kvi.st, Institute of Medical nnd Physiological Chemistry. The Biomedical Center, Box S75 S-751 23 Uppsala. Sweden.
Work on the structure of the MHC-antigens.* which requires large amounts of tissue as starting material to obtain reasonable quantities of highly purified antigens, is hampered by the fact that most inbred animals belong to species with smal! individuals. Recently, we have raised xenoantisera directed against highly purified MHC antigens from mouse [S. Kvist, L. Ostberg & P. A. Peterson, submitted] and man [8] and this communication shows that such antisera. due to the conservative structure of the MHCantigens, are suitable reagents to detect MHCantigens from several species where alloantisera are not available. •Abbreviations used are: MHC, major histocompatibilily complex; SAC, Staphylococcus aureus Cowan 1; SDS, sodium dodecylsulphate; NRS, normal rabbit serum.
MATERIALS AND METHODS CW/.V. Splenocytes were obtained from Vervet monkeys, Sprague-Dawley rats, BIO.A mice, guineapigs and chickens. All animals were maintained al the Animal House, Biomedical Cenire. Spleens from cows and pigs were obtained fresh from a local abattoir. Human peripheral blood lymphocytes were isolated from blood samples drawn from healthy volunteers [I]. Antisera. A rabbit anliserum against H-2K and D antigens was raised by repeated lymph node and footpad injections with a sample of highly purified H-2 antigens. The characterization and specificity of the antiserum is outlined elsewhere [S. Kvist et al.. submilled]. Tbe rabbit anti-HLA-DR antigen serum was raised by the same immunization schedule. The reactivity of this antiserum has been the subject of a previous publication (8]. The rabbit antiserum against human ^^-microglobulin was the same as earlier used [151. Radioactive labelling and indirect immunoprecipitation. The splenocytes were labelled for 14 h with ^H-tyrosine
0300-9475/78/0600-0447 $02.00 © !978 Blackwell Scientific Publications
447
448
S. Kvist. L. Klareskog & P. A. Peter.son
H-2K and D antigen analogues on lymphocytes derived from various species. Internally labelled glycoproteins were isolated by means of lens culinaris haemagglutinin afllinity chromatography and molecu]es reactive with the antiserum were recovered by indirect immunoprecipitation. Fig. 1 shows that the precipitated antigens gave rise to two radioactive peaks on SDSpoly aery I amide gel eiectrophoresis for all species examined except for the chicken. In the latter case the rabbit anti-H-2 antigen scrum did not react measurably with any molecules, as shown in Fig. 1 (h). The SDS-polyacryiamide gel patterns for molecules derived from monkey, human, mouse, rat, guinea-pig., swine and cow lymphocytes were very similar inasmuch as the smaller ofthe two precipitated chains displayed a molecular weight of about 12.000 whereas the larger chain exhibited an eiectrophoretic tnobility consistent with an apparent molecular weight of about 45,000. The larger chain varied slightly in molecular weight for the difTerent species. Thus, the chain from guinea-pigs had an apparent molecular weight of about 42,000 RESULTS whereas those of mice and rats were consisIdentification of H-2K and D antigen analogues tently larger with apparent molecular weights of about 47.000. With use of the rabbit anti-H-2 antigen-serutn Since the rabbit anti-H-2 antigen serum attempts were made to establish the presence of
(Amersham, England) and soUibilized with 2"'o (v/v> Triton X-100. Soluble glycoproteins were isolated by alRnity chromatography on Sepharose-4B columns containing covalently bound lens culinaris haemaggluiinin. Hound glycoproleins were desorbed by including 10% (w/v) a-methylmannoside in ihe cluent. After dialysis the glycoprotein fractions were incubaled with NRS overnight and radioactive components binding un specifically to IgG were removed by ihe precipilalion of the IgG wilh SAC. The supernatants were divided in Uvo equal fractions to which 50 |xl of NRS and anliserum, respectively, were added. After about 10 h of incubation immune complexes were precipitated by the addition of SAC. The bacteria were washed three limes and the immune complexes were dissolved by the addition of 200[xl of a solution containing 2% SDS. 8 M urea and 3"/,, fJ-mercaptoelhanoL Further details of this procedure have been published 114]. SDS-polyacrylamtde gel electrophoresis and isoelectric focusing. SDS-poIyacrylamide gel electrophoresis was carried oul essentially as described hy Laemmli [10]. The technique employed for isoeleciric focusing was that of O'Farrell [13]. When samples were to be subjected lo isoelectric focusing SDS was omitted from the solution used to dissolve the immune precipitates.
1,0
40 80 MIGRATION
40 DISTANCE
80
80
(mm)
FIG. I. SDS-polyacrylamide gel electropboreses of "H-tyrosinc-!abelled membrane glycoproteins precipitated wilb a rabbit anliserum against highly purilied H-2 antigens {•—•) and NRS ( ). respectively. The membrane protein.s were derived from lymphocytes obtained from monkey (a), human (b), mouse (c), rat (d), guinea-pig (e). pig (f), cow (g) and chicken (h). The arrows denote the migration positions of marker IgG heavy (HJ and light (L) chains and Pa-microglobulin (|3a|i).
MHC-Atitigen Analogites in Several Species
449
molecules which on SDS-polyacrylamide gel electrophoresis displayed the typical profile, i.e. two polypeptide chains with the apparent molecular weights 12,000 and 45.000, respectively. 2 •
Molecular weight determination of la-antigen analogttes
1 •
20 iO 60 80 100 MIGRATION DISTANCE (mm) FIG. 2. SDS-polyacryiamide gel etectrophoresis of chicken lymphocyte membrane glycoproleins, internally labelled wilb ^H-tyrosine, precipitated by a rahhil anti-human p.-microglobulin serum (•—•) and NRS ( ), respectively. The arrows have the same meaning as in Fig. I.
available did not react wilh any lymphocytederived molecules from chicken it appeared of interest to examine if antisera against fi..microglobulin might crossreact with the chicken homoiogue. To this end a rabbil anti-human;io-microglobulin serum was used for indirect immunoprecipitations of ^H-tyrosine-labelled lymphocyte glycoproteins from the chicken. Fig. 2 shows that the antiserum reacted with
a
The human analogues of the murine Ia antigens have recently been identified [7], These antigen are the products of the HLA-D locus. In a series of experiments similar to the preceding ones attempts were made to isolate faantigen analogues from several species by means of indirect immunoprecipitation with use of a rabbit anti-HLA-DR antigen serum. Fig. 3 shows that the glycoprotein fractions derived from lymphocytes of the dilTerent species examined all contained molecules that reacted with the antiserum. The SDS-polyacrylamide gel electrophoreses of the precipitated antigen revealed the typical la-antigen-like two-chain pattern for all species except the pig. In this case only a single peak of radioactivity could be discerned (Fig. 3f). The molecular weights estimated from the SDS-polyacrylamide gel electrophoresis of the two types of chains from the
H
tl. Q
< o Q
80
^0 80 MIGRATION
40 DISTANCE
80 {mm)
80
FIG. _1. SDS-polyacrylamide gel electrophoreses of ''H-tyrosine-labelled glycoproteins precipitated with a rabbit anti-HLA-DR antigen serum (•—•) and NRS ( ), respectively. The precipitated lymphocyte antigens were derived from monkey (a), human (b), mouse (c), rat (d), guinea-pig (e), pig (f), cow (g) and chicken (hj. The arrows have ihe same meaning as in Fig. 1.
450
S. Kvist. L. Kliire.skog & P. A. Peterson
I. Apparent molecular weights of the laantigen analogue sutiunits from dilTerent species*
Molecular weight Species Monkey Human Mouse Rat Guinea-pig Pig Cow Chicken
a-Chain
p-Chain
.14.000 34.000 35.000 35,000 33,000 27,000 28,000 .^3,000
28,000 28,000 27,000 27,000 25,000 27,000 25,000 28,000
* The determinaiions were made by SDS-polyacrylamide gel electrophoresis.
various species are summarized in Table J. It can be seen in the Tabie that the molecular weights of the smaller ({i) chain ranged from 25,000 to 28,000 daltons and those of the larger {yi) chain from 27,000 to 35.000 daltons. It is noteworthy that the rabbit anti-human HLA-DR antigen serum reacted with the laantigen analogues of the chicken and that the chicken displays la-antigens with the typical subunit structure. The SDS-polyacrylamide gel electrophoresis did not allow the conclusion to be drawn that the la-antigen analogues ofthe pig are composed of two types of polypeptide chains. Therefore, pig lymphocyte glycoproteins recognized by Ihe rabbit anti-HLA-DR antigen serum were subjected to isoelectric focusing. By this type of separation technique the pig Ia-antlgen analogues resolved into two broad radioactive peaks, as can be seen in Fig. 4. Interestingly, the isoelectric focusing profile of the pig la-antigen analogues and those of human and chicken were very similar (Fig. 4), thereby further strengthening Ihe notion that they represent homologous proteins. Material in the fraciions denoted by the arrows in Fig. 4 was subjected to SDS polyacrylamide gel electrophoresis. It can be seen in Fig. 5 that the more acidic component comprised the a-chain whereas the more basic material represented the [i-chain. Therefore it seems reasonable to conclude that all the species examined produce la-antigen-like glycoproteins that are composed of two types of dissimilar polypeptide chains.
20 DISTANCE
40 FROM
60
80
THE ANODE
100 (mm)
FIG. 4. Isoelectric focusing of *H-lyrosine labelled laantigen analogues derived from pig (A), human (Bl. and chicken (C) lymphocyies. The aniigens wore isolated by indirect immunoprecipitalion with use of a rahhil anti-Ht.A-DR antigen serum. Material in the fractions denoted hy the arrows was subjected to SDSpolyacrylamide gel electrophoreses (see Fig. 5). A denotes Ihe pH.
DISCUSSION It is well established that the MHC displays a very conservative genetic organization since narrow chromosomal segments controlling various aspects of several immunobiological phenomena have been identified in a number of species [6, 9, 18]. The conservative nature at the DNA-level seems to be paralleled by the similarity in structure between the MHC antigens of several species. The present report demonstrates that antisera raised against highly purified MHC-antigens crossreact exlensively with the analogous antigens from other species. Thus, the antiserum raised against highly puri-
MHC-Antlgen Analogues in Several Species
80 MIGRATION DISTANCE ( m m )
451
80
FIG. S. SDS-polyucrylamidc gel electrophoresis of la-aniigcn analogues separated by isoeleclric focusing. The analysed materials were obtained from the fractions ilcnoicd by the arrows in Fig. 4, Pig (a-d), human (e-h) anti chicken (i-l) la antigen analogues were analysed. The arrows have the same meaning as in Fig. I.
fied H-2K- and D antigens combines with H-2 antigen-like molecules from monkey, human, rat. guinea-pig, pig and cow. The antiserum employed did not recognize the chicken anligens, which, however, were precipitated with an anliserum against human pj-microglobulin. In all cases the H-2 antigen-like molecules were shown to be composed of two subunits dissimilar in size. This confirms earlier findings obtained wilh alloanlisera in some specie.s [2, 3, II. 12, 17. 19] and it seems reasonable lo conclude thai the struciural similarity between the MHC-anligens of the various species is also reflected by the occurrence of common, interspecies anligenic determinants. The anliserum against the human la-antigen analogues (HLA-DR) reacted wiih la-antigenlike molecules from all the species examined, i.e. also with the chicken antigens. Also in this case were Ihe isolated molecules very similar in their gross structure. However, ihe molecular weights of the separated chains varied considerably more between the species than did the molecular weights for the H-2 aniigen-like molecules. The significance of this observation
cannot be fully realized until it is ascertained whether the dilTerences are accounted for by ihe polypeptide poriions or by the carbohydrate moieties of the subunits. It is. thus, possible that the immunotogical crossreactiviiy observed is restricted to one or several of the l-rcgion subloci in the ditTerent species assuming that alt species, like the mouse [18], display several genes controlling the expression of la-antigen-Uke molecules. However, separation of the Taaniigcn-like subunits from pig, human and chicken by isoelectric focusing revealed striking similarities in charge regardless of the difterences in the measured molecular weights. Although Ihis finding does not argue againsi the possibility thai the isolated pig and chicken [a-antigen-tike molecules arc derived from a lesser number of genes Ihan are their human counterparts it suggests that, if so, Ihe products of the ditTerent 1-region genes have Io be very similar. In any event, Ihis observation makes it likely that isoelectric focusing can be used advantageously to distinguish the two types of la-antigen-like subunits when ihe resolution obtained by SDSpolyacrylamide gel electrophoresis is insufficient.
452
S. Kvist, L. Klareskog & P. A. Peterson
It is somewhat surprising that the antisera used exhibit the wide cross-reactivity observed. It would be expected that the antisera only recognize structures that are dilTerent between the immunogen and the rabbit counterpart of the immunogen. In that case it is remarkable that human HLA-DR antigens, used as the immunogen. should exhibit structures that are dissimilar from those of the rabbit la-antigens but shared with la-antigens from the chicken. It seems, however, that the immune system of most if not all animals may preferentially be be geared towards dealing with MHC-antigen [5] and antigenie complexes involving the MHC antigens [4]. Thus, monkey antibodies raised against free human fia-microglobulin are not easily obtained but [i.-microglobulin, as part of the HLA-A, B, and C antigens, gives rise to a good antibody production [16]. Therefore, it is possible that also HLA-DR antigens may give rise to an anomalously vigorous antibody response which would account for the high degree of interspecies cross-reactivity observed. Another explanation for the crossreactiviiy would be that the antiserum against the HLADR antigens recognizes carbohydrate structures that may be similar despite vast differences in tbe protein moieties of the la-antigens from dilTerent species. This would not be unreasonable to assume since the present studies were restricted to analyses of MHC antigens bound to the Lens CuHnaris haemagglutinin. Thus, a subfractionation of the MHC antigens from the various species may have occurred so that only la-antigens with similar carbohydrate moieties were isolated. However, HLA-DR antigens that have been separated by means of their dilTerent afTinity for the Lens Culinaris haemagglutinin react identically with the antiserum although they most probably display differences in their prosthetic groups [B. Curman, L. Rask, L. Ostberg & P. A. Peterson, in preparation].
REFERENCES
ACKNOWLEDGMENT
t Boyum, A. Separation of leucocytes from blood ami bone marrow, Scand. J. clin. Lab. Invest. 21, Suppl. 97, 31. 1968. 2 Callahan, N.G, & Dewilt, C.W. Ral cell surface antigens, I. Isolation and partial characterization of an Ag-B antigen, J. Immunol. 114, 776. 1975. I Cramor. D.V., Shonnard, J.W. & Gill, T.J. Genetic siudies in inbred rals. II. Rclationsbip between the [iiajor histocompatibilily complex and mixed, lymphocyte reactivily.y./HimHHrt^rnc/u,V.I. 421.1974, 4 Doherty, P.A.. Blanden. R.V, & ZinkcrnaBel. R.MSpecificity of virus-immune efleclor T cells Tor H-2K or H-2D compatible inferattions: Implications for H-antigen diversity. Transplant. Rev. 29, 89, 197C>. 5 Jerne, N.K, The somatic generation of immune recognition, Eur.J. IniDiuiiol. 1, 1, 1971, 6 Kal7. D.H, Lymphocyte Oi^Hi-nwuiation, Rfcofuji/;()«,fl;f(/i?ci'M/a//on. Academic Press. New York. 1977. 7 Klareskog, L., Sandberg-Trag^rdh. L., Rask. L,, Lindblom, J.B., Curman, B. & Peterson. P.A. Chemical properties of human la anligens. Nature (Lorn/.), 265, 248. 1977. 8 Klareskog, L., TragArdh, L.. Lindhlom. J.B, & Peterson. P.A. Reactivity of a rabbil aniiserum against highly purilied HLA-DR anligens. Scaml. J. Immunol. 7, 199, 1978, 9 Klein. J. Tfw Biology of the Mou.sc tiistocompaiihility 2 Comph:x. Springer, New York. 1975. 10 Laemmli, V.K, Cleavage of strucliiral proteins during the assembly of the head of hacleriophage T4, Nanire {Lond.), 227, 680, 1970, II Leight. G.S.. Sachs. D.H., Williams, G.M. & Rosenberg. S.A. Transplantation in minialurc swine. Transplant. Proc. 9, 575. 1977, 12 Moller. G. (ed.) [ij-Microglobulin and HL-A aniigen. Transplant. Rcw 21, 1974. 13 O'Farrell, P.H. High resolution two-dimensional electrophoresis of proieins. J. biol. Chem. 250, 4007. 1975, 14 Ostberg, L.. Scge. K.. Rask, L. & Peterson, P.A. Isolation of radiolabelled H-2 antigens. Folia Not.. Praha. 22, 372, 1976. 15 Peterson, P.A.. Rask, L. & Lindblom. J.B, Highly purilied papain-solubilized HL-A antigens contain P^-microglobulin. Proc. nat. Acad. Sci. U.S.A. 7t, 35, 1974, 16 Sanderson, A.R. HLA "help" for human p^-microglobulin across species barriers. Nature (Lond.), 269,414. 1977, 17 Schwartz. B.D,. Kask. A.M., Patii, W.E. & Shevach, E.M. Structural characteristics of the alloantigens determined by ibe major histocompatibility complex of tbe guinea pig. J. e.xp. Med. 143, 541. 1976, 18 ShrelHcr. D.C, & David. C.S, The H-2 major histocompatibility complex and the 1 immune response region; genetic variety, funclion, and organization. AJv. Immunol. 20, 125, 1975. 19 Ziegler, A. & Pink, R, Chemical propenies or iwo antigens conirolleti by ihe major histocompatibility complex of the chicken. J. biol. Chem. 251, 5391. 1976,
This work was supported by the Swedish Cancer Society.
Received 5 December 1977 Received in revised form 13 January 1978
Whatever the reason is for the immunological cross-reactivity it seems reasonable to conclude that xenoantisera raised against highly purified MHC-antigens from one species may be useful tools in monitoring the purification of MHC antigens from olher species where alloantibodies are not available.
