Tissue (1972), 2, 78-83 LETTER TO THE EDITOR Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the authoris)

HL-A Model of the H-2 system?

JAN KLEIN*~AND DONALDC. SHREFFLER* *Department of Human , Medical School and ?Department of Oral Biology, Dental School, , .4nn Arbor, Michigan, U.S.A.

Received for publication 1 November, accepted 4 November 1971

In a recent article, Thorsby (1971a) pro- plex immunogenetic systems such as H-2 posed what he called an “HL-A inter- or HI,-A, and it therefore seems prema- pretation of the H-2 system”. According to ture to take a dogmatic position on thr this interpretation, each allele of the two matter. H-2 (H-2D and H-2K) controls In a reply to our criticisms, Thorsby only one “true” ; the rest of the (1971b) has reiterated the concept that a specificities presently listed in the H-2 “complex-simple’’ interpretation of sero- chart are assumed to be artifacts caused by logical data may be applied to the H-2 cross-reactive . In a review of system (a point which we do not contest), the H-2 system, we briefly criticized this but has, in our view, confused and con- view (Klein & Shreffler 1971) for the fol- founded the interpretation with the system lowing reasons : ( 1 ) The genvral idea that of notation used to represent serological at least some of the H-2 specificities may findings. We would like to further amplify be due to cross-reactive antibodies is not at and clarify our objections to Thorsby’s all new; (2) The specific model proposed H-2 model, and then to deal with the spe- by Thorsby is confusing and misleading, cific new points and criticisms of our review and contributes nothing to our understand- raised in his letter. ing of the H-2 system; (3) The traditional Ever since Landsteiner’s classical ex- interpretation of the H-2 system (several periments on cross-reactive antibodies specificities determined by each H-2 chro- against simple haptenic groups (Landstei- mosome) better suits present needs; (4) ner 1945), the concept, which much later There is no way to definitively prove or came to be called the “complex-simple” disprove, by serological methods, either of interpretation of serological systems the two alternative interpretations of com- (Hirschfeld 1965)>has existed in immuno-

Supported by U.S.P.H.S. Grants GM-15419, GM-18314, DE-02731, and K3-HE-24980. HI,-A MODEL OF THE H-2 SYSTEM? 79

Clearly, the interpretation and the notation Furthermore, we feel no temptation to applied fail to recognize substantial in- abandon the traditional notation for the compatibilities and have very little pre- H-2 system and replace it by such a rigid dictive value with respect to intra-H-2 complex-simple model for two additional recombination. This is hardly an improve- reasons. The first one is historical. The ment over the present interpretation. H-2 and HL-A systems have developed The primary basis for these difficulties under different circumstances. The H-2 is that the approach which was employed system was constructed on the basis of very in assigning a “single antigen” designation thorough serological analysis of a limited for each H-2D and H-2K allele was ar- number of H-2 chromosomes, extensive bitrary and artificial, and not even con- cross-immunization studies, and almost no sistent with the approach employed for population analysis. The HL-A system, on HL-A. In general, as we understand it, a the other hand, has been based, at least in number of narrow, mutually exclusive its first phase, almost exclusively on pop- HL-A antigens may be included in a ulation analysis, with very limited inten- “family”, cross-reactive with one or more tional immunization studies. Thus, there broadly reactive antisera. In cases in which has been almost no limitation on the com- a “piece” of a broadly reactive specificity plexity in the case of the H-2 system, while is not covered by a narrow specificity this in the case of the HL-A system some sim- is given a (*) designation, as IH* in Thors- plifications have been essential to bring by’s example ( 1971a). Thus, a better ap- order into the very complex data. To re- proach to an “HL-A interpretation of interpret the H-2 system and assign a new H-2” is the one taken by Snell et al. ( 197 1 ) notation at this stage of development just in assigning haplotypes composed of “pri- to make it look more like HL-A would be vate” H-2 specificities. The statement by most unwise. However, as we have sug- Thorsby (1971b) that “very few antisera gested earlier (Klein & Shreffler 1971), which clearly recognized private antigens further development of H-2 toward pop- of the H-2D and H-2K regions were pub- ulation studies and HL-A toward inten- lished at the time the tentative model was tional cross-immunization analysis, coupled developed,” is simply not correct. The fact with better chemical definition of the anti- is that most of the private H-2 antigens gens, might bring the two systems together have been recognized for more than a de- on the same platform quite naturally. cade and only three new private specifici- The second reason for maintaining the ties were added recently. A simple exami- traditional interpretation of the H-2 system nation of the past H-2 literature would is more pragmatic. One can, of course, trim have revealed this. For example, Amos the H-2 chart and leave in it only the pri- ( 1962), clearly identified certain of the vate antigens; but this will not stop in- private H-2 specificities. Beyond this ob- vestigators from using reagents made jection, even if the private specificities against public H-2 specificities. To pre- were not recognized, the usual HL-A in- serve some order in the system the speci- terpretation would have assigned a distinc- ficities should have symbols assigned to tive symbol to each antigen in the inclusion them and if the symbols are maintained, group. Thus the antigen of the D-series for there is no reason to exclude the public H-2 chromosomes f, g, 1 and n should at specificities from the H-2 chart. Even if least have been 6*, rather than 6, by HL-A the complex-simple interpretation is closer convention, etc. to reality, it would still be desirable to keep 80 KLEIN AND SHREFFLER genetics. H-2 investigators have been ascribed to cross-reactivity. In this way he aware of the two alternative explanations assigned 34 antigens to 17 H-2 chromo- of the H-2 system essentially from the time somes. In our opinion, of these 34 assign- of recognition of H-2 complexity. The pos- ments, 28 are entirely incorrect. Specifici- sibility of a complex-simple interpretation ties H-2.1,3,5,6,8,13 and 28, which Thors- of the H-2 system was most explicitly ex- by variously assigned to chromosomes b,d, pressed by Owen (1959a, b) and reiterated f,g,h,i,j,k,l,m,n,o,p,q,rand s, are all “pub- by one of us in 1966 (Shreffler 1966). The lic” antigens broadly distributed among “segregant series” in the H-2 system was different H-2 chromosomes. H-2.6, for first suggested by Gorer in 1956 (although, instance, is one of the broadest H-2 spe- at that time, they were given more prosaic cificities known, shared by all the known terms such as “alleles” and “antithetical H-2 chromosomes except one. None of antigens”). The bipartite structure of the these specificities fulfills the criterion on H-2 system has been a clearly established which Thorsby based his classification into fact for some time (Shreffler 1965). Under two segregant series, namely mutual ex- these circumstances the development of an clusiveness. If any, these specificities are “HL-A interpretation of the H-2 system” the most likely ones to be artifacts of cross- seems to us to reflect an unfamiliarity with reactivity. the history of immunogenetics and parti- As a consequence, Thorsby’s assignments cularly of the H-2 system. It is perhaps are full of internal inconsistencies which worthwhile to point out that immunogene- create paradoxical situations. We have al- tics as a scientific discipline did exist before ready noted that H-2 chromosomes f and the discovery of HL-A. g were assigned the same haplotype, even Our strongest objection is to the specific though they differ by 7 specificities. Thors- model and notation proposed by Thorsby. by’s “revised” H-2 chart also includes four In his model, Thorsby chose two of the haplotypes derived by recombination in- several specificities determined by each volving other haplotypes in the chart. In H-2 chromosome and designated them as every case the assignments are inconsistent, the “true” antigens with the rest being as shown below:

Parental Haplotype Haplotype Recombinant Parental haplotypes expected assigned chromosome chromosome D locus K locus D locus K locus D locus K locus

g -d 4r8 28 8 6 8 b 28 J 5

h 3 23

1 -a 4 5 4 1* b

0 -d 4r8 3** 8 3** 1 k 3** 1 23 HL-A MODEI. OF THE €IF2 SYSTEM? 81 the public specificities in the chart, because repeatedly stressed in the past by many this is a very useful mechanism for show- immunogeneticists. ing the cross-reactivities among the various In his reply to our criticism, Thorsby H-2 antigens. In fact, it has recently been ( 197 1b) argues that the complex-simple suggested by Ceppellini (1971) that it interpretation provides a better basis for would be of value to include in the HL-A explaining the work of Brondz & Gold- haplotype designations, the broad speci- berg (1969). The authors have shown ficities such as 4a and 4b. To take Thors- that immune do not kill third- by’s example ( 1971a), perhaps it would be party target cells in vitro even though the uspful to have an “HL-A chart” which third-party strain shares one public speci- shows the antigenic profile of haplotype ficity with the donor. According to Thors- HL-A2,5 as HL-A2, Da 2, HL-A5,4c, SL, by, the lymphocytes fail to do so because 4a, 4a1, 4a2, 4A*, because it could be that the cross-reactivity is not recognized at the factors Da2 or SL or 4a, etc. are more im- cellular level. This is certainly a posible portant in transplant survival or disease explanation. However, we fail to under- association than are HL-A2 or HL-A5. stand how this has any bearing on the One could still use such a chart under “code” used for the H-2 system. The fact whatever serological interpretation he is clearly established that there is serologi- might like. cal cross-reactivity between the H-2 anti- l’horsby suggests some experiments gens of the H-P and H-2b types. The which he thinks could test the complex- relevant specificity in this case is given the simple model of the H-2 system. For ex- designation H-2.5. The data of Brondz & ample, he would immunize H-2b mice with Goldberg indicate that this cross-reactivity H-2a cells and search for antibodies which is not recognized in their particular test would react with antigen H-2.4 and cross- system. This certainly requires an explan- react with antigens H-2.3 and 13. Such ation, but we do not understand the logic immunizations have been done many times in assigning, to the H-2a and H-2b anti- in several laboratories and have not proven gens, symbols which fail to show the cross- anything. Antisera obtained from such im- reactivity as a means for explaining the munizations react with all strains possess- data. Nothing is gained in the understand- ing H-2.4 as well as with strains possessing ing of the phenomenon by concealing the H-2.3 and 13. Thorsby would interpret cross-reactivity. If Thorsby’s notation were this as evidence that the antisera contain used for H-2, the phenomenon would not only one (anti-H-2.4) which even have been recognized; in fact, the ex- cross-reacts with H-2.3 and 13. It can also periment would probably not even have be postulated that the antiserum contains been done in this way. Whether the basis for at least three antibodies, anti-H-2.3, 4 and the effect lies in qualitative similarities and 13 and that these antibodies react dif- differences among single antigens or quan- ferentially with the corresponding antigens. titative differences in number of antigenic Thus the same data can be interpreted in sites, and whether the reactive lymphocytes two different ways. We see no way that are monovalent or polyvalent, are ques- this can be resolved by serology alone. One tions which must be answered at another interpretation may be more conuenient for level than that of serological interpretation. a particular system but that does not prove It is certainly possible to recognize these that it is correct. The ambiguity of such alternatives under either type of notation. serological data is not new. It has been One must be very careful not to confux 82 KI.EIN AND SHREFFLER notation with serological interpretation. In paration while the reactivity of the anti- any case, the distinction between the sero- H-2.5 antibodies remained unchanged. logical alternatives must come from a bio- Apparently, the treatment specifically in- chemical analysis of the antigens. Prelimin- activated the H-2.33 site and left the ary results from one such analysis seem to H-2.5 site intact. Therefore, there must be support the simple-complex rather than more than one antigenic site on the H-2Kb complex-simple interpretation of the H -2 molecule. Although this result is not com- system. pletely unambiguous, it speaks strongly in At the molecular level, the two models fauor of a multiple site interpretation of can be visualized in a simplified form as the H-2 system, at least with respect to follows : According to the complex-simple these two specificities. This result should model, each H-2 molecule carries only a serve as a caution that we must keep an single antigenic site which is recognized by open mind about interpretation of sero- a heterogeneous population of cross- logical data, and recognize the limitations reactive antibodies. According to the of the serological approach. It seems to us simple-complex model, each H-2 molecule quite possible that multiple H-2 specifici- carries more than one antigenic site and ties controlled by a single H-2D or H-2K the multiple sites are recognized by spe- allele will in some instances be found to be cific antibodies. If one could split the H-2 associated with a single antigenic site, in molecule into fragments and show either other instances with multiple sites on a that the antigenic sites are always on one single molecule. As we have stressed re- fragment or that different fragments carry peatedly (Shreffler 1966, Klein & Shreff- different sites, this would show which of ler I97 1 ) , this is primarily a chemical pro- the two models is more realistic. This has blem. not yet been achieved. However, Pancake Finally, we should emphasize that our & Nathenson (personal communication) purpose in the review (Klein & Shreffler used an indirect approach which tests the 1971) was not to advocate the simple- two models without the necessity of frag- complex over the complex-simple inter- menting the H-2 molecule. These investi- pretation, or vice versa, but to show that gators isolated the product of the H-2K many of the apparent serological differen- locus in the H-2b chromosome. According ces between H-2 and HL-A stemmed from to the complex-simple model, this molecule the differences in interpretation of the two should carry only one antigenic site systems. We indicated that a simple-com- (H-2.33) and the reaction of anti-H-2.5 plex interpretation of the HL-A system antibodies with the molecule should be due was possible, but recognizing our limited to cross-reactivity with the H-2.33 site. knowledge of the HL-A system, we did According to the simple-complex model, not feel it appropriate to offer a detailed the H-2Kb molecule could carry at least “H-2 interpretation of the HL-A system.” two antigenic sites, H-2.33 (private) and [Although we are not experts on the HL-A H-2.5 (public), Pancake & Nathenson system, we would like to note that our subjected the molecules to formaldehyde statements concerning the HL-A system treatment, followed by reductive alkyla- which were criticized by Thorsby as in- tion, and then tested the preparations with accurate are all based upon work of estab- anti-H-2.5 and anti-H-2.33 antisera. They lished HL-A investigators, uir. cross-reac- found that after treatment the anti-H-2.33 tivity between HL-A2 and Da15 (Walford antibodies no longer reacted with the pre- et al. 1970, Figure 2; Dausset et al. 1970), HI.-A MODEI. OF THE 11-2 SYSTEM? 83 formation of antibodies to specificities not Owen, R. D. (1959) Immunogenetics. Proc. Xth found on the immunizing cells (Amos 1970; int. Congr. Genet., p. 364-374. University of Toronto Press, Toronto. see also Ceppellini 1971).] We agree with Snell, G. D., Cherry, M. & DCniant, P. (1971) Thorsby that H-2 and HL-A are probably Evidence that the H-2 private specificities homologous and similarly organized - this can be arranged in two mutually exclusive is the point we tried to make. However, systems possibly homologous with the two sub- systems of HL-A. Transplant. Proc. 3, 183- we believe that the true relationship of the 186. two systems is more likely to be seen if one Shreffler, D. C. (1965) The Ss system of the keeps an open mind toward all possible mouse - a quantitative serum protein dif- interpretations. ference genetically controlled by the H-2 re- gion. Iroantigens and Interactions, ed. Palm, J., p. 11-17. (The Wistar Inst. Symp. 1LIonogr. No. 3) The Wistar Inst. Press. Phila- References delphia. Amos, D. B. (1962) Isoantigens of mouse red Shreffler, D. C. (1967) Genetic control of cel- cells. Ann. N.Y. Acad. Sci. 97, 69-82. lular antigens. Proc. 3rd int. Congr. Hum. Amos, D. B. (1970) Genetic aspects of human Genet., eds. Crow, J. F. & Neel, J. V., p. 217- HL-A transplantation antigens. Fed. Proc. 29, 231. The John Hopkins Press, Baltimore. 20 18-2025. Thorsby, E. (1971a) A tentative new model for Brondz, B. D. & Goldberg, N. E. (1969) Further organization of the mouse H-2 histocompati- in uitro evidence for polyvalent specificity of bility systems: Two segregant series of anti- immune lymphocytes. Folia biol. (Pralza) 16, gens. Europ. ]. Immunol. 1, 57-59. 20-28. Thorsby, E. (1971b) Homologies between the Ceppellini, R. ( 1971) Old and new facts and H-2 and HLA systems. Tissue Antigens 1, speculations about transplantation antigens of 294--297. man. Progress in , ed. Amos, M'alford, R. L., Waters, H. & Smith, G. S. D. B., p. 973-1010. Acad. Press, New York. ( 1970) Human transplantation antigens. Fed. Dausset, J., Colombani, J., Legrand, L., Fein- Proc. 29, 201 1-2017. gold, N. & Rapaport, F. T. (1970) Genetic and biological aspects of the HL-A system of human histocompatibility. 35, 591-612. Gorer, P. (1956) Some recent work on tumor 4, . Aduanc. Cancer Res. 149-186. Addresses : Hirschfeld, J. ( 1965) Serological codes: Inter- Dr. Jan Klein pretation of immunogenetic system5. Science Department of Human Genetics 148, 968-970. Medical School and Klein, J. & Shreffler, D. C. (1971) The H-2 Department of Oral Biology model for the major histocompatibility sys- Dental School, University of Michigan tems. Transplant. Rev. 6, 3-29. Ann Arbor, Michigan 48104 Landsteiner, K. (1945) The Specifirity of Sc- U.S.A. rological Reactions. Harvard Univ. Press, Cambridge. Dr. Donald C. Shreffler Owen, R. D. (1959) Discussion of a paper by Basel Institute for Immunology P. A. Gorer. Biological Problems of Grafting, 487, Grenzdcherstrasse eds. Albert, F. & Medawar, P. B., p. 30-33. CH 4058 Basel Blackwell Scientific Publications. Oxford. Switzerland