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Complementation of Major Histocompatibility Haplotypes in Regression of Rous Sarcoma Virus-Induced Tumors in Noninbred Chickens1,2
BREEDING AND GENETICS Complementation of Major Histocompatibility Haplotypes in Regression of Rous Sarcoma Virus-Induced Tumors in Noninbred Chickens1'2 D. W. BROWN, W. M. COLLINS, and P. H. WARD Department of Animal Sciences, University of New Hampshire, Durham. New Hampshire 03824 W. E. BRILES Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 (Received for publication May 26, 1981)
1982 Poultry Science61:409-413 INTRODUCTION T h e B alloantigen locus, as originally described by Briles et al. ( 1 9 5 0 ) , is a m a r k e r for t h e major histocompatibility c o m p l e x (MHC) of t h e chicken (Schierman and Nordskog, 1961) and has a decisive effect o n t h e o u t c o m e of R o u s sarcoma virus-induced t u m o r s (Collins et al, 1 9 7 7 ; Schierman et al, 1977). Utilizing highly inbred lines, a r e c o m b i n a n t B h a p l o t y p e , and a variety of t e c h n i q u e s , Pink et al. ( 1 9 7 7 ) and Hala et al. ( 1 9 7 7 ) have subdivided t h e MHC into t h r e e regions and characterized three classes of antigens t h e synthesis of which are u n d e r control of genes of the B c o m p l e x . C o m p l e m e n t a t i o n , as used in this paper, is characterized by an e n h a n c e d specific response in heterozygotes. One h a p l o t y p e " c o m p l e m e n t s " t h e o t h e r such t h a t t h e immunological response t o a particular antigen, or set of
1 Scientific Contribution Number 1076 from the New Hampshire Agricultural Experiment Station. 2 This investigation was supported in part by Grants CA 17680 (UNH) and CA 12796 (NIU), awarded by the National Cancer Institute, Department of Health and Human Services, and by the New Hampshire Agricultural Experiment Station.
antigens, is greater t h a n t h a t observed in h o m o z y g o t e s for either c o m p l e m e n t i n g haplot y p e . In a preliminary study concerning regression of R o u s sarcoma virus (RSV)-induced t u m o r s involving nonpedigreed p r o g e n y of flock-mated parents of a n o n i n b r e d line of chickens (Collins et al, 1979), c o m p l e m e n t a tion of genes within t h e MHC was suggested. A m o n g six B g e n o t y p e s investigated, t h e greatest a n t i - t u m o r response, as measured b y a t u m o r profile index (TPI), was observed in h e t e r o zygous B23/B26 hosts and the least response in B^/B24 hosts. T h e m e a n response of B23 IB26 chickens, however, did n o t differ significantly from t h a t of g e n o t y p e s B23 IB23 and B26/B26. In t h e present study t h e relative responses of these same six B g e n o t y p e s were m o r e clearly characterized. T h e B23 IB26 a n t i - t u m o r response was found t o be significantly greater t h a n t h a t of either B23 IB23 or B26 /B26 hosts, implicating gene c o m p l e m e n t a t i o n .
MATERIALS AND METHODS Stock. UNH 105, a n o n i n b r e d line of N e w Hampshire chickens originally o b t a i n e d from a commercial breeder, was used. Chickens were
409
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ABSTRACT Relative responses to Rous sarcoma virus (RSV)-induced tumors were studied in UNH 105 chickens, a noninbred line of New Hampshires. A total of 799 chickens blood typed for B alloantigens were classified into six genotypes: B23 IB13, B1* IB™, B26 /B26, B"IB2", B23 IB26, and B 24 IB26. Chickens were inoculated with subgroup A Rous sarcoma virus in the left wingweb at 6 weeks of age. The in vivo response was evaluated and given a tumor profile index (TPI) based on the change in tumor size over a 10-week period postinoculation. The TPI's ranged from 1 (regressor) to 3 (progressor). The mean TPI of B23 IB26 hosts (1.7) was significantly smaller than that of all other genotypes studied. The mean TPI's of the homozygous genotypes B23IB23 , B26 IB26, and B 2 4 /B 2 4 , were 2.0, 2.3, and 2.9, respectively, and differed significantly in all comparisons. These results suggest complementation of haplotypes influencing the anti-Rous sarcoma response. (Key words: major histocompatibility complex complementation, Rous sarcoma, tumor regression, B genotype)
410
BROWN ET AL.
TPI 1
2
3
Criterion Complete, or nearly complete, regression (tumor not exceeding 1.2 cm diameter at 70 days PI). Intermediate response (tumor exceeding 1.2 cm diameter but not completely filling the wingweb at 70 days PI). Terminal tumor by 70 days, or massive tumor completely filling or extending
TABLE 1. Analysis of variance of tumor profile indices (TPI's)
beyond the wingweb at 70 days PI). Statistical Analyses. Data were subjected to analysis of variance and to a posteriori contrasts of means using SPSS subprogram ANOVA and the modified least significant difference (LSDMOD) of subprogram ONEWAY, respectively. The LSDMOD was used since it is exact for groups of unequal sizes (Nie et al., 1975). Statistical significance was determined at P<.05. RESULTS Table 1 gives the analysis of variance of TPI's of 795 chickens which developed tumors. Since the two and three-way interaction effects were not statistically significant, variances attributed to them were pooled with the residual. Differences among genotypes in response to tumor were statistically significant. Table 2 gives the percentage distribution of chickens to TPI's according to B genotype. Of B23 IB26 chickens 54% were regressors (TPI of 1) compared to 34% of B23 IB23 and 26 % for B26/B26. B^IB24 chickens had the greatest incidence of tumor progression, 87% (TPI of 3), in contrast to 23% for B23IB26. Table 3 gives the mean TPI of each genotype and comparisons of the means based upon the one way analysis of variance and modified LSD of subprogram ONEWAY. The homozygous genotypes - B23 IB23, B26/B26, and B24 IB24 gave mean TPI's of 2.0, 2.3, and 2.9, respectively, which differed significantly in all comparisons, B 3 IB23 having the greatest response and B24 IB24 the least. The greatest anti-tumor response was observed when the B 3 and B 6 haplotypes were combined. However, when the B 24 haplotype combined with either the B23 or B26 haplotype, anti-tumor response appeared
TABLE 2. Percentage distribution of chickens to tumor profile indices, by B genotype Tumor profile index
Source of variation
df
Genotype Sex Hatch Residual
5 1 5 783
Probability of associated P<.05.
Mean square 17.8 .6 .8 .6
B genotype
1
2
3
23/26 23/23 23/24 26/26 24/26 24/24
54 34 17 26 14 0
23 32 39 15 21 13
23 34 44 59 65 87
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pedigreed by sire only in the first hatch and by sire and dam in five subsequent hatches. A total of 28 sires and 112 dams contributed progeny. Blood Typing and Blood Group Genotypes. Chickens were blood typed for B alloantigens at 4 weeks of age using a panel of alloantisera and classified according to the patterns of reactivity obtained (Briles et al, 1950, 1980). Three homozygous genotypes — B23/B23, 24 26 26 B^/B , and B IB - and three hetero3 23 / D 2 zygous genotypes - B" /BM4, B" IBM and 24 26 _ died. A fourth allele, B22, fi /£ w e r e stu was found to exist in very low frequency. Due to the rarity of genotypes having B22, however, no regression data are reported for this haplotype. Virus. A highly purified pseudotype of Bryan high-titer Rous sarcoma virus designated BH RSV(RSV-l) was provided by L. B. Crittenden of the Regional Poultry Research Laboratory in East Lansing, MI, Chicks were inoculated with .1 ml of virus (approximately 20 pock-forming units) into the left wingweb at 6 weeks of age. Evaluation of Tumor Development and Progression. A tumor profile index (TPI), modified from that of Collins et al. (1977) to account for the different distribution of responses observed with line UNH 105, was assigned to each chicken according to the following criteria:
REGRESSION OF RSV-INDUCED TUMORS
411
TABLE 3. Mean tumor profile index (TPI), by B genotype together with a mean separation test B genotype
Mean TPI Number/genotype
23/26
23/23
23/24
26/26
24/26
24/24
1.7^ 145
2.0 b 176
2.3bc 88
2.3 C 181
2.5 C 133
2.9 d 76
' ' ' Means having no superscripts in common are significantly different, P<.05.
implicated by enhanced anti-tumor response in B23IB26 hosts compared to that of B23 IB23 and B26/B26 chickens. Gene complementation has been observed with H-2 complex immune response (Ir) genes of mice for the response to certain synthetic antigens (Dorf and Benacerraf, 1975). The MHC complementation observed in UNH line 105, however, may represent a phenomenon distinct from the responses observed against highly purified synthetic antigens. Conceivably, a hybrid molecule produced by the MHC of B23 IB26 chickens (possibly a T-cell receptor or antigen-presenting structure on the cell surface) could elicit a relatively greater immune response against a highly immunogenic determinant than that produced by either B23 IB23 or B26 IB26 chickens. However, RSV-transformed avian cells have been shown to express a variety of antigens relevant to the immune response (Ignjatovic et al, 1978; Hall et al, 1979). Therefore, response against tumor cells could involve interactions with a wide spectrum of antigenic
DISCUSSION The MHC haplotype complementation was
TABLE 4. Number and mean tumor profile index (TPI) of progeny from various kinds of matings of Line 105 parents, according to B genotype B genotype of progeny Mating no.
B genotype of parents
1 2 3
B23/B26 X B23/B26 B23/B26 X B26/B26 B23/B26 X B23/B23 B"/B" X B"/B26 Above mating types combined All mating types'
4 5
23/26
23/23 hatches
26/26
No.
XTPI
No.
XTPI
No.
XTPI
31
1.90 a
2.75 b 2.71b
2.00 a
1.80 a 1.39 a 2.04 a
24 17
25
49 23 28
56 176
1.95 a 2.0 a
100 145
1.77 a 1.7°
41 181
2.73 b 2.3 C
3 1 2 1
a,b,c;Means in the same row with the same superscript do not differ significantly (P<.05). 'Includes matings of B"IB23 X B23/B23, to matings 1 through 3 (see Table 3).
B26/B26
X B26/B26
and B23IB23
X B26/B26
in addition
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smaller than that observed for homozygotes of the respective better responding B haplotype, B23 or B26. In Table 4, results from Mating 1 (an F 2 type mating) and Matings 2 and 3 (backcross type matings) were compared with the results from all matings. Data from only three genotypes - B23/B23, B23/B26 and B26 IB26 - are summarized here. The differences between B23IB23 and B23 IB26 in anti-tumor response were in favor of the B23 IB26 genotype in Mating 1 (F 2 type mating) and Mating 4 (matings 1, 2, and 3 pooled). The differences between genotypes B26 IB26 and B23 IB26 were consistently in favor of B23 IB26 in Matings 1, 2, and 4. When data from all mating types were combined, the anti-tumor response of B23 IB26 chickens was greater than that of the other two genotypes and the differences were statistically significant.
412
BROWN ET AL. TABLE 5. Observed frequencies and frequencies expected under Hardy-Weinberg equilibrium for B genotypes from a sample of 442 New Hampshire chickens B genotype
Frequency
23/23
23/24
23/26
24/24
24/26
26/26
Observed Expected
.004 .007
.08 .09
.08 .06
.27 .28
.44 .41
.12 .15
Calculated x 2 with 5 df = 5.51; P>.05.
Heterozygotes carrying the B haplotype appeared to have a lower anti-tumor response than homozygotes for the higher responding B haplotype (either B23 or B26). This could be explained by an Ir gene dose effect similar to that seen with certain hybrid mice in their response to the synthetic terpolymer GL0 (Dorf et al, 1979). A B23IB23 homozygote, having two doses of the higher responding B23 haplotype, would, thus, give a greater response than a B23 /B24 heterozygote, carrying only one dose. An alternative explanation could involve cross-reactivity of B24 alloantigens with tumor antigen(s) responsible for stimulating an anti-tumor response. Tolerance to the B 4 alloantigens could result in a decreased response to the crossreacting tumor antigens and lead to tumor progression. Several studies using various tumor systems in rats and mice have demonstrated such cross-reactivity (Bear et al, 1977; Parmiani et al, 1979; Russell et al, 1979; Paciucci et al, 1980). Heinzelmann et al, (1981) investigated the role of tolerance to blood alloantigens on Rous sarcoma development in chickens. The anti-tumor response of B2B2 (high responder) White Leghorns made
partially tolerant to blood alloantigens from Bs B5 (low responder) animals was significantly smaller than that of B2B2 controls. In earlier work, 461 chickens of a strain of New Hampshires had been obtained from 25 individual sire matings of a commercial breeder. These chickens were tested with a broad panel of B system reagents in an effort to determine haplotype frequencies. In 442 chickens of designated genotypes, the frequency of the B24 allele was .53 while that of B 2 3 was .08. Since the commercial breeder had not used blood type as a criterion of selection, this higher frequency suggests a selective advantage for B24. Whether or not such advantage resulted from artificial selection for higher numbers of eggs or from natural or artificial selection for greater resistance to certain pathogens is unknown. Chickens of this line are quite robust, and no apparent increase in susceptibility to other particular pathogens was observed during the study. Furthermore, the observed frequencies of the 6 genotypes are in agreement with those expected on the basis of Hardy-Weinberg equilibrium (Table 5). Thus, the low anti-tumor response of B24 IB24 chickens appears to be specific and not the result of a generalized immunological weakness. Complementation of genes controlling regression of RSV-induced tumors in inbred lines was recently reported (Watanabe et al, 1980). F t chickens of a cross between two related inbred lines had a higher incidence of tumor regression than either parental line. The authors attributed such complementation to genes within or closely linked to the MHC. Knowing the relative frequency of genetic complementation within the MHC in the avian sarcoma system may provide insight into die nature of the immune response against these tumor cells. If tumor regression in chickens resulted from immune interactions with only a few antigenic determinants, frequency of the complementa-
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determinants, each contributing in varying degrees toward the observed anti-tumor response. The B26 haplotype, although providing a smaller response against determinants of high immunogenicity than the B23 haplotype, could conceivably complement B23 by providing a greater response against determinants of lesser immunogenicity. For complementation to occur at least some of the high responses would have to be dominant in nature, allowing high responses to sum across the two haplotypes. Some responses could be conditioned by gene dose effects, however, and these would be less likely to prevent complementation from occurring if both haplotypes had similar frequencies of gene dose dependent high responses.
REGRESSION OF RSV-INDUCED TUMORS
REFERENCES Bear, R. H., O. A. Roholt, and D. Pressman, 1977. Protection against syngeneic tumor grafts induced by inoculation with normal allogeneic tissues. Immunol. Commun. 6:547—558. Briles, W. E., R. W. Briles, W. H. McGibbon, and H. A. Stone, 1980. Identification of B alloalleles associated with resistance to Marek's disease. Pages 395—413 Resistance and immunity to Marek's disease. P. M. Biggs, ed. CEC Publ. EUR 6470, Luxembourg. Briles, W. E., W. H. McGibbon, and M. R. Irwin, 1950. On multiple alleles affecting antigens in the chicken. Genetics 35:633—652. Collins, W. M., W. E. Briles, A. C. Corbett, K. K. Clark, R. M. Zsigray, and W. R. Dunlop, 1979. B locus (MHC) effect upon regression of RSV-induced tumors in noninbred chickens. Immunogenetics 9:97-100. Collins, W. M. W. E. Briles, R. M. Zsigray, W. R. Dun-op, A. C. Corbett, K. K. Clark, J. L.Marks, and T. P. McGrail, 1977. The B locus (MHC) in the chicken: association with the fate of RSVinduced tumors. Immunogenetics 5:333—343. Dorf, M. E., and B. Benacerraf, 1975. Complementation of H-2 linked Ir genes in the mouse. Proc. Nat. Acad. Sci. 72: 3671-3675. Dorf, M. E., J. H. Stimpfling, and B. Benacerraf, 1979. Gene dose effects in Ir gene-controlled systems. J. Immunol. 123:269-271. Hala, K., M Vilhelmova, and J. Hartmanova, 1977.
The structure of the major histocompatibility complex of die chicken. Pages 227—232 in Avian Immunology. A. A. Benedict, ed. Plenum Press, New York, NY. Hall, M. R., L. F. Qualtiere, and P. Meyers, 1979. Cellular and humoral immune reactivity to tumor-associated antigens in chickens infected with Rous sarcoma virus. J. Immunol. 123: 1097-1105. Heinzelmann, E. W., R. M. Zsigray, and W. M. Collins, 1981. Increased growth of RSV-induced tumors in chickens partially tolerant to MHC alloantigens. Immunogenetics 12:275—284. Ignjatovic, J., H. Rubsamen, M. Hayami, and H. Bauer, 1978. Rous sarcoma virus-transformed avian cells express four different cell surface antigens that are distinguishable by a cell-mediated cytotoxicity-blocking test. J. Immunol. 120:1663— 1668. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H. Bent, 1975. Statistical package for the social sciences. 2nd ed. McGraw-Hill Book Company, New York, NY. Paciucci, P. A., S. Macphail, J. M. Zarling, and F. H. Bach, 1980. Lysis of syngeneic solid tumor cells by alloanrigen stimulated mouse T and non-T cells. J. Immunol. 124:370-375. Parmiani, G., G. Carbone, G. Ivernizzi, M. A. Pierotti, M. L. Sensi, M. J. Rogers, and E. Apella, 1979. Alien histocompatibility antigens on tumor cells. Immunogenetics 9:1—24. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and A. Zeigler, 1977. A three-locus model for the chicken major histocompatibility complex. Immunogenetics 5:203-216. Russell, J. H., L. C. Ginns, G.Terres, and H. Eisen, 1979. Tumor antigens as inappropriately expressed normal alloantigens. J. Immunol. 122: 912-919. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134:1008-1009. Schierman, L. W., D. H. Watanabe, and R. A. McBride, 1977. Genetic control of Rous sarcoma regression in chickens: linkage with the major histocompatibility complex. Immunogenetics 5: 325-332. Watanabe, D. H., J. A. Cutting, F. S. Strebel, and R. A. McBride, 1980. Genetic complementation of MHC genes which control regression of avian sarcoma virus-induced tumors in inbred lines of chickens. Fed. Proc. (3, part 2): 1154.
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tion p h e n o m e n o n likely would n o t greatly exceed t h a t observed with inbred mice in their response t o highly purified synthetic antigens. However, if a n t i - t u m o r response were t h e result of a spectrum of interactions with n u m e r o u s different antigenic d e t e r m i n a n t s , c o m p l e m e n t a t i o n conceivably could occur m u c h m o r e frequently. T h e spectrum of m e a n responses to t u m o r associated with t h e six g e n o t y p e s tested in this investigation provides insight into the subtle differences which m a y occur b e t w e e n genotypes. T h a t t h e MHC has a decisive influence o n t u m o r regression, in spite of varying background genes f o u n d in n o n i n b r e d chickens, is clearly relevant t o o t h e r r a n d o m breeding populations, including m a n .
413
1982 Poultry Science61:409-413 INTRODUCTION T h e B alloantigen locus, as originally described by Briles et al. ( 1 9 5 0 ) , is a m a r k e r for t h e major histocompatibility c o m p l e x (MHC) of t h e chicken (Schierman and Nordskog, 1961) and has a decisive effect o n t h e o u t c o m e of R o u s sarcoma virus-induced t u m o r s (Collins et al, 1 9 7 7 ; Schierman et al, 1977). Utilizing highly inbred lines, a r e c o m b i n a n t B h a p l o t y p e , and a variety of t e c h n i q u e s , Pink et al. ( 1 9 7 7 ) and Hala et al. ( 1 9 7 7 ) have subdivided t h e MHC into t h r e e regions and characterized three classes of antigens t h e synthesis of which are u n d e r control of genes of the B c o m p l e x . C o m p l e m e n t a t i o n , as used in this paper, is characterized by an e n h a n c e d specific response in heterozygotes. One h a p l o t y p e " c o m p l e m e n t s " t h e o t h e r such t h a t t h e immunological response t o a particular antigen, or set of
1 Scientific Contribution Number 1076 from the New Hampshire Agricultural Experiment Station. 2 This investigation was supported in part by Grants CA 17680 (UNH) and CA 12796 (NIU), awarded by the National Cancer Institute, Department of Health and Human Services, and by the New Hampshire Agricultural Experiment Station.
antigens, is greater t h a n t h a t observed in h o m o z y g o t e s for either c o m p l e m e n t i n g haplot y p e . In a preliminary study concerning regression of R o u s sarcoma virus (RSV)-induced t u m o r s involving nonpedigreed p r o g e n y of flock-mated parents of a n o n i n b r e d line of chickens (Collins et al, 1979), c o m p l e m e n t a tion of genes within t h e MHC was suggested. A m o n g six B g e n o t y p e s investigated, t h e greatest a n t i - t u m o r response, as measured b y a t u m o r profile index (TPI), was observed in h e t e r o zygous B23/B26 hosts and the least response in B^/B24 hosts. T h e m e a n response of B23 IB26 chickens, however, did n o t differ significantly from t h a t of g e n o t y p e s B23 IB23 and B26/B26. In t h e present study t h e relative responses of these same six B g e n o t y p e s were m o r e clearly characterized. T h e B23 IB26 a n t i - t u m o r response was found t o be significantly greater t h a n t h a t of either B23 IB23 or B26 /B26 hosts, implicating gene c o m p l e m e n t a t i o n .
MATERIALS AND METHODS Stock. UNH 105, a n o n i n b r e d line of N e w Hampshire chickens originally o b t a i n e d from a commercial breeder, was used. Chickens were
409
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ABSTRACT Relative responses to Rous sarcoma virus (RSV)-induced tumors were studied in UNH 105 chickens, a noninbred line of New Hampshires. A total of 799 chickens blood typed for B alloantigens were classified into six genotypes: B23 IB13, B1* IB™, B26 /B26, B"IB2", B23 IB26, and B 24 IB26. Chickens were inoculated with subgroup A Rous sarcoma virus in the left wingweb at 6 weeks of age. The in vivo response was evaluated and given a tumor profile index (TPI) based on the change in tumor size over a 10-week period postinoculation. The TPI's ranged from 1 (regressor) to 3 (progressor). The mean TPI of B23 IB26 hosts (1.7) was significantly smaller than that of all other genotypes studied. The mean TPI's of the homozygous genotypes B23IB23 , B26 IB26, and B 2 4 /B 2 4 , were 2.0, 2.3, and 2.9, respectively, and differed significantly in all comparisons. These results suggest complementation of haplotypes influencing the anti-Rous sarcoma response. (Key words: major histocompatibility complex complementation, Rous sarcoma, tumor regression, B genotype)
410
BROWN ET AL.
TPI 1
2
3
Criterion Complete, or nearly complete, regression (tumor not exceeding 1.2 cm diameter at 70 days PI). Intermediate response (tumor exceeding 1.2 cm diameter but not completely filling the wingweb at 70 days PI). Terminal tumor by 70 days, or massive tumor completely filling or extending
TABLE 1. Analysis of variance of tumor profile indices (TPI's)
beyond the wingweb at 70 days PI). Statistical Analyses. Data were subjected to analysis of variance and to a posteriori contrasts of means using SPSS subprogram ANOVA and the modified least significant difference (LSDMOD) of subprogram ONEWAY, respectively. The LSDMOD was used since it is exact for groups of unequal sizes (Nie et al., 1975). Statistical significance was determined at P<.05. RESULTS Table 1 gives the analysis of variance of TPI's of 795 chickens which developed tumors. Since the two and three-way interaction effects were not statistically significant, variances attributed to them were pooled with the residual. Differences among genotypes in response to tumor were statistically significant. Table 2 gives the percentage distribution of chickens to TPI's according to B genotype. Of B23 IB26 chickens 54% were regressors (TPI of 1) compared to 34% of B23 IB23 and 26 % for B26/B26. B^IB24 chickens had the greatest incidence of tumor progression, 87% (TPI of 3), in contrast to 23% for B23IB26. Table 3 gives the mean TPI of each genotype and comparisons of the means based upon the one way analysis of variance and modified LSD of subprogram ONEWAY. The homozygous genotypes - B23 IB23, B26/B26, and B24 IB24 gave mean TPI's of 2.0, 2.3, and 2.9, respectively, which differed significantly in all comparisons, B 3 IB23 having the greatest response and B24 IB24 the least. The greatest anti-tumor response was observed when the B 3 and B 6 haplotypes were combined. However, when the B 24 haplotype combined with either the B23 or B26 haplotype, anti-tumor response appeared
TABLE 2. Percentage distribution of chickens to tumor profile indices, by B genotype Tumor profile index
Source of variation
df
Genotype Sex Hatch Residual
5 1 5 783
Probability of associated P<.05.
Mean square 17.8 .6 .8 .6
B genotype
1
2
3
23/26 23/23 23/24 26/26 24/26 24/24
54 34 17 26 14 0
23 32 39 15 21 13
23 34 44 59 65 87
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pedigreed by sire only in the first hatch and by sire and dam in five subsequent hatches. A total of 28 sires and 112 dams contributed progeny. Blood Typing and Blood Group Genotypes. Chickens were blood typed for B alloantigens at 4 weeks of age using a panel of alloantisera and classified according to the patterns of reactivity obtained (Briles et al, 1950, 1980). Three homozygous genotypes — B23/B23, 24 26 26 B^/B , and B IB - and three hetero3 23 / D 2 zygous genotypes - B" /BM4, B" IBM and 24 26 _ died. A fourth allele, B22, fi /£ w e r e stu was found to exist in very low frequency. Due to the rarity of genotypes having B22, however, no regression data are reported for this haplotype. Virus. A highly purified pseudotype of Bryan high-titer Rous sarcoma virus designated BH RSV(RSV-l) was provided by L. B. Crittenden of the Regional Poultry Research Laboratory in East Lansing, MI, Chicks were inoculated with .1 ml of virus (approximately 20 pock-forming units) into the left wingweb at 6 weeks of age. Evaluation of Tumor Development and Progression. A tumor profile index (TPI), modified from that of Collins et al. (1977) to account for the different distribution of responses observed with line UNH 105, was assigned to each chicken according to the following criteria:
REGRESSION OF RSV-INDUCED TUMORS
411
TABLE 3. Mean tumor profile index (TPI), by B genotype together with a mean separation test B genotype
Mean TPI Number/genotype
23/26
23/23
23/24
26/26
24/26
24/24
1.7^ 145
2.0 b 176
2.3bc 88
2.3 C 181
2.5 C 133
2.9 d 76
' ' ' Means having no superscripts in common are significantly different, P<.05.
implicated by enhanced anti-tumor response in B23IB26 hosts compared to that of B23 IB23 and B26/B26 chickens. Gene complementation has been observed with H-2 complex immune response (Ir) genes of mice for the response to certain synthetic antigens (Dorf and Benacerraf, 1975). The MHC complementation observed in UNH line 105, however, may represent a phenomenon distinct from the responses observed against highly purified synthetic antigens. Conceivably, a hybrid molecule produced by the MHC of B23 IB26 chickens (possibly a T-cell receptor or antigen-presenting structure on the cell surface) could elicit a relatively greater immune response against a highly immunogenic determinant than that produced by either B23 IB23 or B26 IB26 chickens. However, RSV-transformed avian cells have been shown to express a variety of antigens relevant to the immune response (Ignjatovic et al, 1978; Hall et al, 1979). Therefore, response against tumor cells could involve interactions with a wide spectrum of antigenic
DISCUSSION The MHC haplotype complementation was
TABLE 4. Number and mean tumor profile index (TPI) of progeny from various kinds of matings of Line 105 parents, according to B genotype B genotype of progeny Mating no.
B genotype of parents
1 2 3
B23/B26 X B23/B26 B23/B26 X B26/B26 B23/B26 X B23/B23 B"/B" X B"/B26 Above mating types combined All mating types'
4 5
23/26
23/23 hatches
26/26
No.
XTPI
No.
XTPI
No.
XTPI
31
1.90 a
2.75 b 2.71b
2.00 a
1.80 a 1.39 a 2.04 a
24 17
25
49 23 28
56 176
1.95 a 2.0 a
100 145
1.77 a 1.7°
41 181
2.73 b 2.3 C
3 1 2 1
a,b,c;Means in the same row with the same superscript do not differ significantly (P<.05). 'Includes matings of B"IB23 X B23/B23, to matings 1 through 3 (see Table 3).
B26/B26
X B26/B26
and B23IB23
X B26/B26
in addition
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smaller than that observed for homozygotes of the respective better responding B haplotype, B23 or B26. In Table 4, results from Mating 1 (an F 2 type mating) and Matings 2 and 3 (backcross type matings) were compared with the results from all matings. Data from only three genotypes - B23/B23, B23/B26 and B26 IB26 - are summarized here. The differences between B23IB23 and B23 IB26 in anti-tumor response were in favor of the B23 IB26 genotype in Mating 1 (F 2 type mating) and Mating 4 (matings 1, 2, and 3 pooled). The differences between genotypes B26 IB26 and B23 IB26 were consistently in favor of B23 IB26 in Matings 1, 2, and 4. When data from all mating types were combined, the anti-tumor response of B23 IB26 chickens was greater than that of the other two genotypes and the differences were statistically significant.
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BROWN ET AL. TABLE 5. Observed frequencies and frequencies expected under Hardy-Weinberg equilibrium for B genotypes from a sample of 442 New Hampshire chickens B genotype
Frequency
23/23
23/24
23/26
24/24
24/26
26/26
Observed Expected
.004 .007
.08 .09
.08 .06
.27 .28
.44 .41
.12 .15
Calculated x 2 with 5 df = 5.51; P>.05.
Heterozygotes carrying the B haplotype appeared to have a lower anti-tumor response than homozygotes for the higher responding B haplotype (either B23 or B26). This could be explained by an Ir gene dose effect similar to that seen with certain hybrid mice in their response to the synthetic terpolymer GL0 (Dorf et al, 1979). A B23IB23 homozygote, having two doses of the higher responding B23 haplotype, would, thus, give a greater response than a B23 /B24 heterozygote, carrying only one dose. An alternative explanation could involve cross-reactivity of B24 alloantigens with tumor antigen(s) responsible for stimulating an anti-tumor response. Tolerance to the B 4 alloantigens could result in a decreased response to the crossreacting tumor antigens and lead to tumor progression. Several studies using various tumor systems in rats and mice have demonstrated such cross-reactivity (Bear et al, 1977; Parmiani et al, 1979; Russell et al, 1979; Paciucci et al, 1980). Heinzelmann et al, (1981) investigated the role of tolerance to blood alloantigens on Rous sarcoma development in chickens. The anti-tumor response of B2B2 (high responder) White Leghorns made
partially tolerant to blood alloantigens from Bs B5 (low responder) animals was significantly smaller than that of B2B2 controls. In earlier work, 461 chickens of a strain of New Hampshires had been obtained from 25 individual sire matings of a commercial breeder. These chickens were tested with a broad panel of B system reagents in an effort to determine haplotype frequencies. In 442 chickens of designated genotypes, the frequency of the B24 allele was .53 while that of B 2 3 was .08. Since the commercial breeder had not used blood type as a criterion of selection, this higher frequency suggests a selective advantage for B24. Whether or not such advantage resulted from artificial selection for higher numbers of eggs or from natural or artificial selection for greater resistance to certain pathogens is unknown. Chickens of this line are quite robust, and no apparent increase in susceptibility to other particular pathogens was observed during the study. Furthermore, the observed frequencies of the 6 genotypes are in agreement with those expected on the basis of Hardy-Weinberg equilibrium (Table 5). Thus, the low anti-tumor response of B24 IB24 chickens appears to be specific and not the result of a generalized immunological weakness. Complementation of genes controlling regression of RSV-induced tumors in inbred lines was recently reported (Watanabe et al, 1980). F t chickens of a cross between two related inbred lines had a higher incidence of tumor regression than either parental line. The authors attributed such complementation to genes within or closely linked to the MHC. Knowing the relative frequency of genetic complementation within the MHC in the avian sarcoma system may provide insight into die nature of the immune response against these tumor cells. If tumor regression in chickens resulted from immune interactions with only a few antigenic determinants, frequency of the complementa-
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determinants, each contributing in varying degrees toward the observed anti-tumor response. The B26 haplotype, although providing a smaller response against determinants of high immunogenicity than the B23 haplotype, could conceivably complement B23 by providing a greater response against determinants of lesser immunogenicity. For complementation to occur at least some of the high responses would have to be dominant in nature, allowing high responses to sum across the two haplotypes. Some responses could be conditioned by gene dose effects, however, and these would be less likely to prevent complementation from occurring if both haplotypes had similar frequencies of gene dose dependent high responses.
REGRESSION OF RSV-INDUCED TUMORS
REFERENCES Bear, R. H., O. A. Roholt, and D. Pressman, 1977. Protection against syngeneic tumor grafts induced by inoculation with normal allogeneic tissues. Immunol. Commun. 6:547—558. Briles, W. E., R. W. Briles, W. H. McGibbon, and H. A. Stone, 1980. Identification of B alloalleles associated with resistance to Marek's disease. Pages 395—413 Resistance and immunity to Marek's disease. P. M. Biggs, ed. CEC Publ. EUR 6470, Luxembourg. Briles, W. E., W. H. McGibbon, and M. R. Irwin, 1950. On multiple alleles affecting antigens in the chicken. Genetics 35:633—652. Collins, W. M., W. E. Briles, A. C. Corbett, K. K. Clark, R. M. Zsigray, and W. R. Dunlop, 1979. B locus (MHC) effect upon regression of RSV-induced tumors in noninbred chickens. Immunogenetics 9:97-100. Collins, W. M. W. E. Briles, R. M. Zsigray, W. R. Dun-op, A. C. Corbett, K. K. Clark, J. L.Marks, and T. P. McGrail, 1977. The B locus (MHC) in the chicken: association with the fate of RSVinduced tumors. Immunogenetics 5:333—343. Dorf, M. E., and B. Benacerraf, 1975. Complementation of H-2 linked Ir genes in the mouse. Proc. Nat. Acad. Sci. 72: 3671-3675. Dorf, M. E., J. H. Stimpfling, and B. Benacerraf, 1979. Gene dose effects in Ir gene-controlled systems. J. Immunol. 123:269-271. Hala, K., M Vilhelmova, and J. Hartmanova, 1977.
The structure of the major histocompatibility complex of die chicken. Pages 227—232 in Avian Immunology. A. A. Benedict, ed. Plenum Press, New York, NY. Hall, M. R., L. F. Qualtiere, and P. Meyers, 1979. Cellular and humoral immune reactivity to tumor-associated antigens in chickens infected with Rous sarcoma virus. J. Immunol. 123: 1097-1105. Heinzelmann, E. W., R. M. Zsigray, and W. M. Collins, 1981. Increased growth of RSV-induced tumors in chickens partially tolerant to MHC alloantigens. Immunogenetics 12:275—284. Ignjatovic, J., H. Rubsamen, M. Hayami, and H. Bauer, 1978. Rous sarcoma virus-transformed avian cells express four different cell surface antigens that are distinguishable by a cell-mediated cytotoxicity-blocking test. J. Immunol. 120:1663— 1668. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H. Bent, 1975. Statistical package for the social sciences. 2nd ed. McGraw-Hill Book Company, New York, NY. Paciucci, P. A., S. Macphail, J. M. Zarling, and F. H. Bach, 1980. Lysis of syngeneic solid tumor cells by alloanrigen stimulated mouse T and non-T cells. J. Immunol. 124:370-375. Parmiani, G., G. Carbone, G. Ivernizzi, M. A. Pierotti, M. L. Sensi, M. J. Rogers, and E. Apella, 1979. Alien histocompatibility antigens on tumor cells. Immunogenetics 9:1—24. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and A. Zeigler, 1977. A three-locus model for the chicken major histocompatibility complex. Immunogenetics 5:203-216. Russell, J. H., L. C. Ginns, G.Terres, and H. Eisen, 1979. Tumor antigens as inappropriately expressed normal alloantigens. J. Immunol. 122: 912-919. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134:1008-1009. Schierman, L. W., D. H. Watanabe, and R. A. McBride, 1977. Genetic control of Rous sarcoma regression in chickens: linkage with the major histocompatibility complex. Immunogenetics 5: 325-332. Watanabe, D. H., J. A. Cutting, F. S. Strebel, and R. A. McBride, 1980. Genetic complementation of MHC genes which control regression of avian sarcoma virus-induced tumors in inbred lines of chickens. Fed. Proc. (3, part 2): 1154.
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tion p h e n o m e n o n likely would n o t greatly exceed t h a t observed with inbred mice in their response t o highly purified synthetic antigens. However, if a n t i - t u m o r response were t h e result of a spectrum of interactions with n u m e r o u s different antigenic d e t e r m i n a n t s , c o m p l e m e n t a t i o n conceivably could occur m u c h m o r e frequently. T h e spectrum of m e a n responses to t u m o r associated with t h e six g e n o t y p e s tested in this investigation provides insight into the subtle differences which m a y occur b e t w e e n genotypes. T h a t t h e MHC has a decisive influence o n t u m o r regression, in spite of varying background genes f o u n d in n o n i n b r e d chickens, is clearly relevant t o o t h e r r a n d o m breeding populations, including m a n .
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