H-I, Atenth Allele at the Histocompatibility-2 Locus In

H-i, a Tenth Allele at the Histocompatibility-2 Locus in the Mouse as Determined by Tumor Transplantation*

SALLY LYMAN ALLENt

(Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, and Departmeni of Zoology, Universiiy of Chicago, CMcago, Ill.)

By crosses involving the A strain, Gorer demon rying H-Pp and H-p, but further genetic tests, on strated the existence of a histocompatibility gene the basis of reaction to transplantation, indicated which determines the presence of antigen II, found that the antigens produced by these alleles were in A strain erythrocytes as well as in other normal slightly different in nature. tissues and in tumors from this strain (5, 6). The The H-f@?locusis complicated by the presence of locus was later designated H4? (8). Thus, it is now composite alleles. Originally, the type allele char known that an allele at this locus not only deter acteristic of the A strain was designated simply mines the presence of a particular antigen or anti [email protected],by genetic crosses, it was found that gens, but, as a consequence, susceptibility or re animals of the heterozygous genotype H-@'/H-P@, sistance to certain transplantable tumors. irrespective of the strain from which the alleles The close linkage of H-@ with the gene for fused H-fi' and H@PJccame,grew strain A tumors (18, tail (Fu), producing distortion and fusion of tail 18). Mice bearing other alleles in various combina vertebrae, was found by Gorer, Lyman, and Snell tions did not grow strain A tumors. On the basis (8). Later, its linkage with two other loci on this of this evidence the type allele found in the A chromosome (IX), Ki and T, was reported (14). strain was presumed to be in some sense compound Kinky tail (Ki or Fu'@) is phenotypically similar and was redesignated H-@. That it actually de to fused tail (2., 11) and may be an allele of Fu (4). termines two antigens or two antigenic factors, D Brachyury (T) typically produces a shortened tail and IC, has been shown by Gorer and Mikulska (9) (3). All three or four of these loci are located close and becker, Counce, and Smith (10). It has not together on the ninth chromosome of the mouse in been determined whether two or more closely the order H-@ Fu (Ki) T (1). linked loci may be involved rather than a single The linkage of H-@ with these loci has been used locus with components. to identify new alleles at the H-@ locus and to de As is the case with the alleles determining the termine their distribution in 2.1inbred strains (18). M-N blood groups in man, each H-@ allele exerts Through extensive tests seven alleles and two sub an effect in the heterozygote. Furthermore, these group alleles have been found at locus H-i? (12., 18, alleles probably produce antigens of considerable 15, 16, 18). By far the greatest number of strains strength, since in many crosses the ratio of sus carry the alleles H-@, H-p, and H-p. The other ceptible to resistant animals reveals only the pres alleles (H-p, H-2â€•,H-Prn?â€•,andH-s) have so far ence of H-@t?,whereas earlier results with other tu been found in only one strain each. The two sub mors pointed to a multiplicity of histocompatibili group alleles are H-2â€•and H-p'. The strains con ty loci. Apparently, the H-fe alleles determine anti taining these alleles originally were classed as car gens which may â€œcrowdoutâ€•antigens of weaker strength produced by other histocompatibility S This investigation has been aided by a John M. Prather research fellowship from the University of Chicago and by a genes (7). However, some crosses do show other 2-year, 8-month predoctoral fellowship from the National loci segregating. Cancer Institute. Inbred mice and animal cages were provided Two other histocompatibility loci have been by,the Roscoe B. @1acksonMemorialLaboratory with the specifically identified. H-i is located on the first financial assistance of a grant-in-aid (EG-14) from the Amen can Cancer Society on recommendation of the Committee on chromosome in mice, crossing over with albinism Growth of the National Research Council. (c) being in the neighborhood of 2.0-2.5per cent t Present address: Department of Zoology,University of (17). H-,@ has been found closely linked with the Michigan, Ann Arbor, Mich. agouti locus.' Tentatively, it appears that these

Received for publication January 27, 1965. 1 Dr. George D. Snail, personal communication. 815

loci determine antigens â€œweakerâ€•ineffect than if they survived a small tumor which grew and H43. then regressed. The CA stock of mice is a â€œmultipledominantâ€• Here, M and A were known previously to carry stock of the genotype AA CaCa Fufu Ww de different H-2 alleles; therefore, (a) if linkage be signed for use in linkage tests. It was the stock tween H-fl and Fu is manifest, M and F' carry dif originally employed in establishing the H-2 locus ferent alleles; (b) if no linkage is manifest, it may in which Fu was found linked to â€œh-s,â€•agene for be presumed that M and F' carry the same allele, â€œresistanceâ€•to A-strain tumors. The H-L@allele although more extensive testing is necessary to present in this stock was introduced into strain A prove this conclusion beyond doubt. (genotype H-P@/H-P@) by successive backcrosses u@ing Fu as a chromosome marker. A line of the A RESULTS strain, which we may call F'@,was thus developed A summary of the results may be found in Table having the genotype H-9Fu/H-Pflu. Since the 1. In the first column are listed the crosses, fol Fh stock was to be used in setting up crosses de lowed by columns indicating the tumor inoculated, signed to determine the order of the loci Fu, T, and the results of tumor inoculation, the allelic group H-@ (1) it was of interest to identify the allele in under test, and the conclusions which may be this stock. Therefore, appropriate tests were set up drawn from the data. The statistic used in testing to determine if H-9 was an allele already estab for linkage of H43 with Fu was: lished or a new allele at the H-@ locus. .5â€”r 1=S.E. (q@â€”q@) MATERIALS AND METHODS The cross used in testing for new alleles at the where r = recombination value which is found by H-9@locus,described by Snell, Smith, and Gabriel combining the second and third classes. In every son (17), has the following form: (M X T) X N, cross except Case 15 linkage was found, and in this where M and N are any two inbred strains and T case there were too few positive animals. The con is one of the stocks carrying T, Fu, or Ki. Offspring elusion may be drawn that H-I?' is another new of the first cross which display the particular tail allele at the H-@ locus. anomaly segregating are chosen for crossing to N. Cases 3 and 9 were crosses set up to test whether Then, the offspring of the double cross are inocu H-il' produces either the D or I( antigenic factors. lated with a tumor native to the strain in the M An A-strain tumor was used in these cases. The position. Generally, the occurrence of linkage fact that linkage was manifest shows that H-9 proves that strains T and N carry an H-@ allele (or does not produce either D or K. Consequently, alleles) different from that in strain M. On the H-fY does not belong to either the H4â€•,H-9, or other hand, the absence of linkage establishes a H-@, H-P.?â€•groups. presumption but does not prove beyond question Analysis of the data contained in Table 1 re that strains M and T and/or M and N carry the vealed further information. One point of interest same H-@ allele. was the occurrence of ratios indicating the segre In the present crosses the F' stock was used in gation of the H-i locus in addition to H-i. It was the T position. All M X Fh mice resulting from the evident that in some of the crosses more than one first cross were initially checked to make sure that histocompatibility gene was segregating, since the a crossover had not arisen (H-@Fu), by mating number of negative animals observed was far more to a third strain, other than strain A, and by the than the theoretical 50 per cent expected for one inoculation of the offspring with A-strain tumor factor. In some of the crosses albinism (c) was in 15091a. Fu mice would all succumb to the tumor eluded, and, since H-i had been found by Snell and if crossing over had occurred; otherwise, all mice ICelton (17) to be linked with albinism, it was pos would be expected to be â€œnegativeâ€•tothe tumor. sible to determine if the increase in negative ani Then, the M X F'@mice known to be noncross mals in these cases was due to the additional seg overs were crossed to N. In most cases the A strain regation of the H-i locus. was used in the N position. Thus, the test cross Three of the crosses, Cases 4, 8, and 12., were could be rewritten: (M X F') X A. Offspring of found to show close approximation to four fac1@or the double cross were inoculated by trocar at 6â€”8 ratios for a backcross segregating for Fu, H4J weeks of age with a transplantable tumor native (withanestimatedSpercentcrossovervalue),c, to the strain in the M position. Mice were regarded and H-i (with an assumed 2.5 per cent crossing as â€œpositiveâ€•ifthey died as a result of a progres over). These crosses may be found in Table 2. re sively growing tumor. They were regarded as grouped into the eight possible phenotypes with @ â€œnegativeâ€•ifthey never bore a palpable tumor or observed and expected values for each cross. In

Case 4 the H-i allele determining resistance was The crossover value was 4.1 per cent among the on the same chromosome as full color (C), while in fused progeny produced by heterozygous males Cases 8 and 12.the allele for resistance entered the (11/2.68). For a smaller number of fused offspring cross with albinism (c). produced by females (78), this value was slightly From analysis of the data in Table 1 it was also higher (6.85 per cent). When tested by chi square possible to estimate the amount of recombination this sex difference in recombination was not sig between H-52and Fu (Table 3). The data could be nificant (P = 0.50). separated into that obtained from males as hetero zygous parent (Fufu) and that from females as DISCUSSION heterozygous parent. Two estimates of crossing The results show that H-s' is a tenth allele at over are given, one based on total animals, the the H-@ locus, since linkage was found in all other on fused-tail animals. The latter estimate is crosses testing for identity of this allele to one of probably more accurate, since it does away with the previously established alleles. Unfortunately, such variables as (a) â€œnormaloverlapsâ€•â€”animals the isogenic line carrying H43' used in the above phenotypically normal-tailed but genetically fused tests has been lost. The stock was heterozygous (11) ; (b) differences in number of histocompatibili for H-i? (H-@'Fu/H-@fu), and crossing over be ty loci segregating; and (c) differences in tumor tween H-L@and Fu resulted in elimination of H-2' virulence, all of which alter the total number of from it. Fortunately, H-9 has probably been pre negative animals. served in another line derived from the CA stock.

TABLE 1 ESTABLISHMENT OF H-@1 A5 A NEW ALLELE AT THE H4 Locus [email protected] RX5ULTI OP TUMOR INOCULATIONS Oaovp CASS NO. IP(AXFâ€•)XC67BL/6Cso.s Tu@oa+++â€”Fu+Fu CONCLUSI*N@ 1 .001(BALB/cXFâ€•)XA iSOOla8471256 .24< not H-Pg 2 5621 not H-Eq' 8 .001(CS7BR/aXFâ€•)XA(AXFâ€•)XCBA 15091a18 182 21 216 175.11 4.96<.001 < 4 5636 not H4@ 6 (C57BR/cdXFâ€•)XA, DBA/1 D1905 21 41 1 46 2.40 < .02 6 (CS7BR/cdXFâ€•)XDBA/l D1905 18 22 1 81 8.07 < .01 @ 7 (CBAxFâ€•)xA 32 1 9 4t) 15 4.92 <.001 8 (C3HXFâ€•)XA 6C3HED 20 19 2 it) 40 4.34 <.001 9 (AXP')XBALB/c.001(C57BL/6xF')XA 15091a14 1514 40 021 173.01 4.69<.01 < 10 H-2â€•(PXFâ€•)XA1824214.84<.001notfl-E'(DBA/IXFâ€•)XBALB/c510133.89<.001notfl4'(DBA/1XFâ€•)XA26091.21.30(RIIIXP')XAC1498 .90 .001 11 (CS7BL/ioxFâ€•)xA 8658 ii 2 0 14 4.44 <.001 12 (CS7LxFh)xA18 C954 92 161 114 324 3.14< <.01not 13 8637 14 dbrB 15 dbrB 16 5674 2.20 .05 H-@ @ atumor.trefers to normal-tailed, positive to tumor; the two â€œrecombinationdasses@@iiare:+â€”0and Fu+;19Fu refers to

TABLE 2 CROSSESFROMTABLE1 SHOWINGSEGREGATIONOFFu, H-2, c ANDH-i

CARN RESULTS OP TUMOR INOCULATION NO. CROSS Cfu+ C/uâ€” cfu+ Ã§fuâ€” CFu+ CFu cFu+cPux2P0110.4611.794.476.70 4 (CS7BR/aXFâ€•)XA 5 8 9 6 0 10 exp. H_ir@tC, 2.91 9.34 8.73 3.52 0.15 12.10 H@2r@Fu 8 (C3HxFh)xA 14 7 6 12 1 29 0 12@ exp. H-i@'c, 14.43 5.82 4.81 15.44 0.76 19.49 0.25 20.00 9.479 .30 H-2@Fu 12 (C57LXFâ€•)XA 4 6 5 10 0 16 1 16 dip. H-i@'c, 10.38 4.17 8.44 11.06 0.54 13.96 0.18 14.82 10.228 .20 H-@â€•Fu

@ S and â€” refer to results of tumor inoculation: + â€” died; â€” â€” survived. t .@â€”alleleofH-i orH.f @ej@g@inginresistantgenotypes. @ Includes one mouse probably genetically resistant to tumor on basis of long survivaL â€” In calculating expected classes 55 per cent crossing over was assumed for C or c and H-i; 5 per cent crossing over was assumed for Pu and H-I.

This is an JR line, A.CA, established by Snell. Evidence was also found for the additional seg Preliminary tests indicate that the genotype of the regation of the H-i locus in certain of the crosses. line is probably H-@'Fu/H-9Fu.' Recombination between H4 and Fu was esti Recently, another major allele at this locus, mated. Among fused progeny a crossover value of H-L?',has been found in the A.SW isogenic line' 4.1 per cent was obtained for heterozygous males (10). The results of preliminary tests with the and of 6.85 per cent for females. A.CA isogenic line indicate that the alleles in these ACKNOWLEDGMENTS two lines are different, so that we may presume I wish to thank Di,s. George D. Snell and Sewall Wright that H-53'is not H43'.' for their guidanceand criticismofthe workreported.

TABLE 8 ESTIMATION OF CROSSOVER PER CENT BETWEEN H-f AND Fu BY SEX FROM DATA IN TABLE 1 Cioesovaa PaR OVtT RXSVLT*or umos U(OCULLTION Per cent Per cent C*oss TUMOR ++ +â€” Fu+ Fu total Pu Males as Reterosygous Parent AX1Th)XCS7BL/6, CBA, BALB/c 56 9 2 55 ALB/cXF1')XA 5621 18 2 1 16 CS7BR/aXP')XA 5636 14 14 0 21 C67BRCdXF@)XA, DBA/1 Di905 21 41 i@ 46 CBAXF@')XA 5620 32 1 9(4* 15 6CSHEDCS7BL/6XP')XACSHXFâ€•)XAISOOla is 14 2(i@ 81 18 2 1 14 C57BL/10XF@')XA 5653 11 2 0 14 CS7LXFb)XA C954 9 16 1 82 dbrBTotal(AXFâ€•)XCS7BL/6BA/1XF@)XAC1498 2 4 0 7

194 105 11 257t 20.6 4.1 PemaI@s as Heterozygou. Parent 11 4 1 4 (CSHXFâ€•)XA 6@3HED 7 5 0 9 (PXF')XA 8637 18 2 4 21 (DBA/1XF')XA, BALB/c dbrB 5 8 0 15 (RIIIXP')XAiSOOla 5674 6 11 0 19 Total 46 25 5 68 20.8 6.85

* Genetically resistant to the tumor on the basis of long survivaL t IncludessixanimalsgeneticallyresistanttothetumoronthebasisoflongsurvivaL

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Sally Lyman Allen

Cancer Res 1955;15:315-319.

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