Animal Brood Groups and Biochemical Genetics 15 (1984) 117-122

Comparison of ELY-2.1 with blood group and ELY-1 markers in the horse*

Ernest Bailey and Pamela J. Henney

Department of Veterinary Science, University of Kentucky, Lexington, KY 40546- 0076, USA

Received 24 October 1983; accepted 15 December 1983

Keywords: lymphocyte antigen, alloantigen, non-MHS antigens, horse, blood

Summary

The distribution of ELY-2 was compared to the distribution of blood group factors Aa, Ab, Ac, Ae, Ca, Da, Db, Dc, Dd, De, Df, Dh, Dk, Ka, Pa, Pb, X, Qa, Qc, Ua, and W in 2465 American Standardbred horses and to ELY-1 in 193 American Standardbred horses. The distribution patterns were different in each case. The segregation of ELY-2.1 and factors at the A, C, 0, K, P, Q, U and T (W) blood group loci and at the ELA locus indicated that ELY-2.1 is not a product of any of those loci. No segregation data were available for the ELY-l locus. Family studies indicated that the gene for ELY-2.1 is not sex-linked.

Introduction

Antczak (1984) described a lymphocyte alloantigen which is not a product of the equine lymphocyte antigen (ELA) locus. Absorption studies demonstrated that ELY-2.1 is present on lymphocytes, platelets and erythrocytes. This specificity was also recognized at the Second International Workshop on Equine Lymphocyte Alloantigens and designated ELY-2.1 (Bailey et al., 1984).

Since ELY-2.1 is present on erythrocytes, it could be a product of one of the 8 known horse blood loci (reviewed in: Suzuki, 1978; Sandberg, 1979; Bell, 1983). Here we confirm that ELY-2.1 is present on red blood cells as well as on lym- phocytes, and describe studies undertaken to determine whether ELY-2.1 is a pro- duct of any of these blood group loci.

* The investigation reported in this paper (No. 83-4-205) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.

117

E. BAILEY AND PAMELA J . HENNEY

Materials and methods

Blood group and lymphocyte typing tests were done on blood samples from 2465 American Standardbred horses and 146 Thoroughbred mares. The group of Stan- dardbred horses included many sets of sire - dam - offspring. For reporting phenotypic frequencies, all mares on several farms were used to constitute a ron- domly selected sample.

Lymphocyte typing was done according to the methods of Terasaki & Park (1976) and as described previously (Bailey, 1983). Two reagents were used to test for ELY-2.1, namely R95 1 and R956. Both reagents were obtained from primiparous Thoroughbred mares and behaved as monospecific reagents in our tests. R956 was used in the Second International Workshop on Equine Lymphocyte Alloantigens as one of three reagents used to characterize ELY-2.1. A reagent for ELY-1 was kindly provided by Sandor Lbary. Horses were also tested for ELA lymphocyte specificities W1, W2, W3, W4, W5, W6, W7, W8, W9, W10, and LEX 8 by using reagents produced at this laboratory.

Blood group tests were done as described by Stormont, Suzuki & Rhode (1964) with few modifications. Horses were tested for blood group specificities Aa, Ab, Ac, Ae, Da, Db, Dc, Dd, De, Df, Dh, Ka, Pa, Pb, X, Qa, Qc, Ua, and W. Reagents for Ac, Ae, Dc, Dd and Qc were kindly provided by the Serology Laboratory at the University of California, Davis. Reagents for all other specificities were produced at this laboratory.

Erythrocytes for absorption studies were isolated free of platelets and leukocytes as follows: fresh blood was centrifuged across a ficoll-Hypaque gradient; erythrocytes were taken from the bottom of the tube and centrifuged a second time across a ficoll-Hypaque gradient; then the erythrocytes from the bottom of the tube were washed three times in RPMI media. At the conclusion of this process no platelets or leukocytes were visible at microscopic examination. Absorption were done by mixing equal volumes of reagent and packed erythrocytes 60 min. at 4 "C.

Results

Absorption The ELY-2.1 reagents, R951 and R956, were absorbed with erythrocytes from three horses possessing the ELY-2.1 specificity (Sw5, RP17 and Sw43) and one horse which did not possess that specificity (RP18). The results are shown in Table 1 for R956.

Unabsorbed R956 was cytotoxic for lymphocytes from Sw5, RP17 and Sw43 but not for lymphocytes from RP18:Absorption with erythrocytes from Sw5, RP17 or Sw43 removed all lymphocytotoxic activity. Absorption with erythrocytes from RP18 had no effect on the cytotoxicity of R956. The same results were obtained when absorbing reagent R951 (data not shown).

118 Animal Blood Groups and Biochemical Genetics I S (I984)

ELY-2. I AND RBC ANTIGENS

Table 1. Lymphocytotoxicity of an ELY-2.1 specific reagent (R956) before and after absorption with erythrocytes.

Lympho- Erythrocyte absorption

sw5 RP17 RP18 sw43 cytes none

dilution 0 4 16 0 4 16 0 4 16 0 4 16 0 4 16

s w s + ' + + - - - - - - + + i - - - RP17 + + + - - - - - - + + i - - - RP18 sw43 + + + - - - - - - + + + - - - * + and - represent cytotoxic and non-cytotoxic reactions, respectively, in lyrnphocytoxicity tests.

- - - - - - - - - - - - _ _ _

Blood group tests for EL Y-2.1 Since the specificity ELY-2.1 is present on erythrocytes, the next question is whether it could be detected on erythrocytes by using hemolytic or hemagglutination tests. We used the reagent R956 in standard blood grouping assays as described by Stor- mont, Suzuki & Rhode (1964). No hemolysis or agglutination occurred.

Distribution of EL Y-2. I and blood group factors The phenotypic frequency of ELY-2.1 is given in Table 2 for 146 Thoroughbred mares, 228 pacing Standardbred mares and 96 trotting Standardbred mares.

The distribution of ELY-2.1 and the distribution of blood group markers were compared among 2465 horses (Table 3). The distributions of ELY-2.1 and ELY-1 were also compared for 193 horses. The results indicated that ELY-2.1 is not iden- tical to any blood group factors for which we tested or for ELY-1. Furthermore, the distribution pattern of ELY-2.1 was not included within the pattern of any other factor, nor was it inclusive of the pattern of other factors. Therefore, ELY-2.1 does not appear to share a simple subtypic relationship with any of these serological fac- tors.

Segregation studies The critical test to determine whether ELY-2.1 is a product of an already identified locus is to compare the segregation of ELY-2.1 with that of other genetic markers. Sex linkage could be excluded for ELY-2.1 since stallions with that specificity had both male and female offspring without ELY-2.1 as well as male and female offspr- ing with ELY-2.1. ELY-2.1 was expressed on lymphocytes of newborn horses and its inheritance governed by an autosomal dominant gene. Seven Standardbred stallions were found which transmitted the allele coding for ELY-2.1 to some, but not all, offspring. For stallions heterozygous for markers at other loci, it was possi- ble to compare the segregation of ELY-2.1 with the segregation of those markers. The data are given in Table 4. If ELY-2.1 is a product of one of the blood group

Animal Blood Groups and Biochemical Genetics I5 ( I 984) 119

E. BAILEY AND PAMELA J. HENNEY

Table 2. Phenotypic frequency of ELY-2.1 in trotting and pacing American Standardbred mares and Thoroughbred mares.

n Frequency

Trotting Standardbred mare 96 0.0938 Pacing Standardbred mare 22 8 0.197 Thoroughbred mare 146 0.082

Table 3. Distribution of ELY-2.1 compared to the distribution of blood group and ELY-1 specificities in American Standardbred horses.

Specificities Distribution n*

I J I + J - I + J + I - J + I - J -

ELY-2.1 Aa Ab Ac Ae Ca K3 Pa Pb X Qa QC Ua W Da Db Dc Dd De Df Dh Dk

355 316 33 19

334 230 178 26

111 5

51 172 16 6

54 336 376 82

1 I5 37

275

117 156 439 439 133 232 290 446 361 467 412 296 449 466 417 121 90

389 340 429 192

1551 1154

61 140

1597 1004 1083 134 820

6 41 1 939 28 24

103 1429 1552 327 554 23 8

1015

442 839

1917 1822 386 945 900

1856 1169 1984 1568 1020 1946 1969 1882 520 402

1653 1432 1752 912

2465 2465 2456 2420 2450 241 1 245 1 2462 246 1 2462 2442 2427 2439 2465 2456 2406 2420 245 1 2441 2458 2454

ELY-I 16 23 80 74 193

* n = total number of horses tested for both specificities.

loci or ELA locus, then the gene for ELY-2.1 should always be transmitted with the allele ‘a’or ‘b’in each stallion. This did not occur. Therefore, ELY-2.1 is not a pro- duct of the ELA, A, C, 0, K, P, U or T (W) loci in horses.

The only family data for the Q blood group locus involved a single mare and her two offspring. The genotype of the mare was known based on her parents’ contribu- tion; her sire transmitted the genes Q- and ELY-2‘’ while her dam contributed the genes and ELY-2-. Based on her two offspring (Table 4), ELY-2.1 is not a product of the Q blood group locus.

No segregation data were available for ELY-1.

120 Animal Blood Groups and Biochemical Genetics 15 (1984)

ELY-2.1 AND RBC ANTIGENS

Table 4. Segregation of ELY-2.1 compared to ELA and blood group markers in the A, C, D, K, P, Q, U and T (W) systems.

1D Genotype Contribution to offspring

a /b 2 . I / - a - 02. I b - 62. I

22 1 EST 122051 l22lST 1307ST 2218ST I405ST 1220ST 1407ST 22 18ST 3 197ST 1407ST 22 I EST I22OST 22 18ST 3274ST 1407ST 221 8ST 122OST

A a / A b Aa/Abc Ca/C - Dc/Ddk Dcf/Ddh Dc/Ddk Dc/Ddk Ka /K- Ka/K - Pa/P - Ua/U- Ua/U- Ua /U- W/ - W/ - ELA- W4/WIO ELA- W3/ W4 ELA-Wl /Wl

2 .1 / - 2 .1 / - 2 . 1 / - 2 . 1 / - 2 . I / - 2. I / - 2 .1 / - 2 . 1 / - 2.11- 2.11- 2.11- 2 . 1 / - 2 . 1 / - 2. I/ - 2.11- 2 . 1 / - 2 . 1 / - .?.I/-

2 16 6 1 3 2 9 2 2 2 1 4

1 1 2 3 I 2

17

3 12 6 1 2 I

I 1 1 0 3 0 0

12 1 1 0 2

1 1

3 18 3 2 2 0

18 1 2 5 3 0

12 3 0 2 3

17

0 16 2 3 1 0

1 1 1 0 0 3 0 9 2 0 2 0

17

1863ST Q a d Q - 2.11- 1 1 0 0

* a and b represent allelic markers.

Discussion

The main emphasis of lymphocyte antigen studies in the horse has been to identify the major histocompatibility system (MNS). The MHS locus is highly polymorphic in most species and also includes genes controlling the immune system (reviewed by Snell, Dausset & Nathenson, 1976). The equine MHS has been well characterized with 10 specificities identified at International Workshops (Bailey et al., 1984). In- cidental to these studies, two non-ELA lymphocyte specificities were found, namely ELY-I and ELY-2.1.

Lazary et al. (1982) described a specificity which they designated ELY-1. This specificity is a product of a locus with 2 known alleles, one coding for the presence and the other coding for the absence of ELY-1. I t is not a product of the ELA, A , C, D or Q loci based on segregation studies. Absorption studies indicated that ELY-1 was present on T and B lymphocytes, but not on platelets or erythrocytes. ELY-I is particularly interesting because it had a higher frequency among horses with laminitis and chronic obstructive pulmonary disease than in the general population.

In this report, we demonstrate that ELY-1 is not identical to ELY-2.1 in its distribution. As reported by Antczak (1984) and Lazary et al. (1982), the tissue

121 Animal Blood Groups and Biochemical Generics 15 11984)

E. BAILEY AND PAMELA J. HENNEY

distribution of the two specificities is also different. Therefore they likely are pro- ducts of two different loci. However, informative family studies have not been reported.

With this report, we confirm the observations of Antczak (1984) that ELY-2.1 is not a product of the ELA locus and that it is present on erythrocytes as well as lym- phocytes. At the same time we demonstrated that ELY-2.1 is not a product of the A, C, 0, K, P, Q, U or T (W) blood group loci. No segregation data were available to determine whether it was a product of the ELY-Z locus. However, ELY-2.1 is not identical to the factor ELY-1.

Attempts to test for ELY-2.1 by using hemagglutination or hemolytic tests were unsuccessful. Our reagents may not be strong enough for conventional tests. Alter- nately, the distribution of ELY-I on erythrocytes may be too sparse to generate a reaction.

References

Antczak, D.F., 1984. Lymphocyte alloantigens of the horse. 111. ELY-2.1: a lymphocyte alloantigen not coded for by the MHC. Animal Blood Groups and Biochemical Generics IS: 103-115.

Bailey, E., 1983. Population studies on the ELA system in American Standard bred and Thoroughbred mares. Animal Blood Groups and Biochemical Generics 14: 201-21 I .

Bell, K., 1983. The blood groups of domestic animals. In: N.S. Agar & P.G. Board (Eds), Red blood cells of domestic animals. Elsevier, Amsterdam, p. 133-169.

Bailey, E., D.F. Antczak, D. Bernoco, R.W. Bull, R. Fister, G. Guerin, S. Liizary, S. Matthews, J . McClure, J. Meyer, V.D. Mottironi & J . Templeton, 1984. Joint Report of the Second International Workshop on Lymphocyte Alloantigens of the Horse. Animal Blood Groups and Biochemical Genetics IS: 123-132.

Lazary, S., H. Gerber, A.L. de Weck & P. Arnold, 1982. Equine leukocyte antigen system. 111. Non- MHC linked alloantigenic locus in horses. Journal of Irnrnunogenetics 9: 327-334.

Sandberg, K . , 1979. Studies on blood groups and genetic protein polymorphisms of the horse. Report 38. Swedish University of Agricultural Sciences, Uppsala.

Snell. G.D., J . Dausset & S. Nathenson, 1976. Immunogenetics, Academic, Press, New York, 401 p. Stormont, C., Y. Suzuki & E.A. Rhode. 1964. Serology of horse blood groups. Cornell Veterinarian 54:

439-452. Suzuki, Y., 1978. Studies on Blood Groups of Horses, Memoirs of the Tokyo University of Agriculture

20: 1-50. Terasaki, P.I. & M.S. Park, 1976. Microdroplet lymphocyte cytotoxicity test. In: J.G. Ray jr, D.B.

Hare, P.D. Pederson & D.I. Mullally (Eds), NIAID Manual of Tissue Typing Techniques. DHEW Publication No 76-545, pp. 69-80.

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