Phylogenetically conserved epitopes of leukocyte antigens

Veterinary Immunology and Immunopathology 52 (1996) 415-426

ELSEVIER

Veterinary immunology and immunopathology

Phylogenetically conserved epitopes of leukocyte antigens P. Vilmos aT*, 6. Kurucz a, I. Ocsovszki b, G. Keresztes a, I. And6 a a Institute

of Genetics,

Biobgicul

Szeged, H-6701, b Department

HungarianAcademy

Research Center ofthe

of Biochemistry,

Albert

of Sciences, PO Box 521.

Hungary

Szent-Gyijrgyi

Medical

Uniuersity,

Szeged, Hungary

Abstract The occurrence of conserved epitopes in the immune system was investigated on the leukocytes of cattle, river buffalo, sheep, camel, swine and humans by indirect immunofluorescence and flow cytometry. The distribution of the most conservative epitopes on leukocyte sub-populations suggests that the expression pattern of the proteins is similar. Western blotting experiments indicate that the recognized antigens are structural homologues. Keywords:

Phylogenetic conservation; Leukocyte antigens; Ruminant; Monoclonal antibody

1. Introduction The identification of phylogenetically conserved structures in the immune system has many theoretical and practical aspects. First, it helps to understand analogies and homologies in the defence mechanisms of various phyla (Naessens, 1991; Hein and Mackay, 1991). Second, the mapping of conserved structures may be important in revealing the cause and possible therapy of infectious and autoimmune diseases caused by superantigens (Conrad et al., 1994; Shafer and Sheil, 1995). Third, the description of conserved epitopes will open a way to develop serological reagents for identification of

Abbreviations: CD, cluster of differentiation; EDTA, ethylendiamine tetmacetic acid; FACS, fluorescence Activated Cell Sorter; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; MHC, major histocompatibility complex; PBM cells, peripheral blood mononuclear cells; PBS, phosphate buffered saline; TBS, tris buffered saline * Corresponding author. 0165-2427/96/$15.00 PII

Published by Elsevier Science B.V.

SOl65-2427(96)05595-X

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analogous leukocyte sub-populations as well as for diagnosis, in phylogenetically distant species. One method to detect conserved structures is based on serological cross-reactions between leukocytes representing different steps of phylogeny. Although monoclonal antibodies (mAbs) against leukocytes generally show little cross-reactivity between species, bovine or ovine antigens show a degree of similarity within the ruminant family (Naessens, 1993). A few, highly conserved structures have also been found: the p2-microglobulin detected in lower animal phyla (Shalev et al., 1981; Roth et al., 1983); the MHC molecules (Hashimoto et al., 1992); a sequence in the cytoplasmic region of the E subunit of the T-cell receptor-CD3 complex (Kurucz et al., 1992; Kurucz et al., 1993; Jones et al., 1993); and extracellular epitopes of cellular adhesion molecules (KumagaiBraesch et al., 1995) are good examples of the highly conservative evolution of elements in the immune system. In this work we performed a detailed flow cytometry and western blotting analysis of leukocyte antigens in cattle, river buffalo, sheep, camel, swine and humans with the aim of identifying highly conserved structures in the immune system. We found that there is a substantial level of cross-reactivity between the leukocytes of the species tested, which suggests a conservative evolution of the mammalian immune system.

2. Materials and methods 2.1. Antibodies and cells Mouse monoclonal antibodies were provided by the Third International Workshop on Ruminant Leukocyte Antigens and diluted according to recommendations. Positive controls were: anti-CD14 (MA5, And6 et al., 1984); anti-CDllb (CC1.7, And6 et al., 1984); anti-MHC Class II (JD2, Takacs et al., 1983); and anti-CD3 (UCHTl, Beverley and Callard, 1981) kindly provided by Professor Peter C.L. Beverley, ICRF, London. Workshop antibodies known to react with leukocyte sub-populations [IL-A109 (monocytes), IL-Al45 (granulocytes), IL-Al 15 (all T and monocytes) and IL-Al54 (B cells)] were used as positive controls in the cattle. Peripheral blood mononuclear (PBM) cells derived from three ruminant even-toed ungulates (cattle: Bos faurus; river buffalo: Bubalus bubalis; sheep: Ouis aries); two non ruminant even-toed ungulates (camel: Camelus bactrianus; pig: Sus domesticus); and from humans. In each experiment, except for sheep, cells from different animals were examined. 2.2. Preparation of PBM cells Heparinized blood samples were diluted with cool PBS (1: 1) and layered on Ficoll-hypaque solution (r = 1.077). After centrifugation the cells from the interface

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were collected and washed three times with cool RPM1 1640 tissue culture medium (RPMI). Red blood cells were lyzed by hypotonic NH&l solution. 2.3. Indirect immunojluorescence 2 X lo5 cells were incubated with mAbs for 60 min and washed three times in cold RPM1 supplemented with 10% fetal calf serum. After washing, cells were incubated for 60 min with FITC-conjugated rabbit anti-mouse Ig (Sigma-Aldrich, Budapest, Hungary) diluted l:lOO, washed as before and analyzed with a fluorescence microscope (cattle, sheep, swine) or with a FACSTAR-PLUS instrument (Becton-Dickinson, Mountain View, CA) (river buffalo, camel). Anti-bovine and anti-human antibodies were used to identify major leukocyte sub-populations and dot-plots were analyzed using the PaintA-Gate software (Becton Dickinson) to further characterize leukocyte sub-populations. Propidium iodide (0.01 mg ml-‘) was used to exclude dead cells. 2.4. Western blotting 2 x 10’ PBM cells were incubated in 1 ml swelling buffer (10 mM Tris-HCl, 0.5 mM MgCl,, 1 mM phenylmethylsulphonyl fluoride, 10 mM iodoacetamide, 10 mg ml-’ leupeptin, 10 mg ml- ’ aprotinin, 25 mM para-nitrophenyl p’-guanidino-benzoate) for 20 min on ice. After homogenization the NaCl concentration was adjusted to 0.15 M and the suspension was centrifuged twice to remove the nuclei. After adjusting the EDTA concentration to 5 mM, the sample was ultracentrifuged and the pellet was solubilized in Laemmli sample buffer. The proteins were separated on a 5-15% gradient SDS-polyacrylamide gel and transferred to Immobilon-PVDF membrane (Millipore Co., Bedford, MA). The membrane was saturated with washing buffer (TBS, 0.1% Tween-20) supplemented with 3% non fat milk powder and cut into slices. The slices were incubated with different antibodies for one hour and washed five times. Anti-CD3e (DAKO, Copenhagen, Denmark) (Kurucz et al., 1992; Kurucz et al., 1993; Jones et al., 1993) was used as a positive control. The membranes were incubated with biotinylated anti-mouse Ig F(ab’), (DAKO) for another 60 min, washed as before, than incubated with Horseradish peroxidase conjugated streptavidin (DAKO, Copenhagen, Denmark, 1:500) for 60 min and washed again. The detected proteins were visualized using DAB as a chromogen.

3. Results 3. I. Cross-reaction

of the antibodies

Table 1 summarizes the reactions of the individual antibodies detecting leukocyte antigens in all species tested. Out of the 159 antibodies (100%) reacting with bovine leukocytes 110 mAbs (69%) reacted with cells of river buffalo, 51 mAbs (32%) with

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Table 1 Positive reactions of the antibodies on PBM cells mAB No.

Name

004 005 006 009 020 022 023 026 028 029 030

Co-IC6E3 IL-Al36 17-3 IAH-CC199 DM7 IL-A 123 IL-A98 CACTB22 CACTI00 MUC2 TH17

032 035 036 040 042 044 046 049 0.52 053

MM12 Co-33B3 4G10 IL-A 109 DIl2-104 BAQ44 IL-AI 19 TD14 IAH-CC49 CACTB17

mAb No.

Name

054 056 0.58 062 065 066 068 069 080 083 084 085 089 090 092 094 096 098 099 202 204 205 206

Bf5 IVA84 TH18 IL-AI 15 GB25 HI-25 Buf34 IVA30 Fw3-47- 11 Du2-74 2G8 20-27 IVAl14 LCTB28 lF7 NAM4 IVA12 17-15 72-87 Co-3DID4 IAH-CC57 IAH-CC76 MM13

C

BU

Sh

0 0 0

0

0

0 0 0 0

0 0

0

0 0 0 a

0

0

0

S

S

HU

Ca

SW

HU

0

0 S

0

0 0 0 0 0 0 a a 0 0

??

0 0

0

0

C

Bu

Sh

Ca

SW

0

0

0 0

S

0

0 0 0

0 0

?? 0

0

0 0 0 0

: 0

0

51

0

: 0

0 0 0 0

?? 0 0

0 0 0

a

S

419

P. Vilmos et al./ Veterinary Immunology and Immunopathology 52 (1996) 415-426 Table

1 (continued)

mAB No.

Name

208 220 224

IL-A63 CACT65 K-17

mAb No.

Name

225 226 230 232 233 234 235 238 239 240 242 243 245 246 248 249 250 254 255 256 258 259 260 262 263 264

IVAll2 IL-Al66 Bf20 CAPP2 MM20 BAQ78 IL-Al45 GB22 Fw4-101 VPM54 IL-A65 38-38 IL-Al54 IVA20 IL-A 137 Fw l -86D-11 MM10 7G8 DH59 Co&D5 BUfl BAQl50 IAH-CC45 IAH-CC43 DM6 VPM67

mAb

C

Bu

0

0 0 0

C

0

Name

265 266 269 280 282 283 284 285 286 289 290 295 296

PI0 IL-A62 IAH-CC63 IL-A77 BAGB20 IL-Al41 GB127 LCTB32 CACTB 10 CACTB81 IVA198 GC65 CACT60

Ca

SW

Hu

BU

Sh

Ca

SW

Hu

0 0

0

0

0

S

S

??

??

48

30

:

S

SW

Hu

0 0 0

0

0 0 0 0

0

0 0

0

a

0

0 0

0 : 36

C

No.

Sh

0

0

0

0 0

0 0 0

0

Bu

Sh

0

??

0

0 0 0 0 0 0 0 0 0 0

0 0

0 0 0

0

Ca

420

P. Vilmos et al, / Veterinary lmmurwlogy and Immumpathology

52 (1996) 415-426

Table 1 (continued) C

mAB No.

Name

302 303 304 306 309 322 323 324 325 326 328 329 330

BT3/8.12 IL-i162 WA369 IL-A24 IAH-CC58 CACTI35 1 IVA120 CACTl16 GB21 IVA17 CH138 WA37 IAH-CC 189

mAb No.

Name

332 333 335 336 338 339 340 342 343 344 345 346 348 349 350 352 353 354 355 356 358 360 362 363 364 365 mAb No.

CACTI8 MM1 Buf42 BAQ89 LCTB2 FW- l-2-4 IVA35 CH127 MM41 IL-AI 22 CACTI9 Fwl-136 BAGB27 IVA38 MF14B4 TD22 218 IL-Al30 IL-A47 BAQl51 CACT26 IAH-CC32 ANA8 CACT77 VPM66 CACTB16

382 383

CA17 25-32

385 386

GBllO Co-20A 1

Bu

Sh

Ca

SW

Hu

?? 0

0 0

26

24

SW

Hu

0

0

s it 0 0 0 0 0

0 0 0 0 0 0

0

0

0

0 0 0

0 0

0 0

C

BU

Sh

Ca

0

0

0 0

s

0 0

0

??

s 0 0 ?? 0 ?? 0 ?? 0 42

0

0 ??

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0

0

21

91

0 0 0

0 0 ??

0

41 0

24

0

C

Bu

Sh

??

0 0

0

Ca

SW

Hu

S

s

Name S

?? 0

421

P. Vilmos et al./ Veterinary Immunology and Immurwpathology 52 (1996) 415-426 Table 1 (continued) mAB No.

Name

389 390 392 393 395 399 402 403 404 405 406 408 409 420 422 423 424 425 426 428 429 430 434

But38 4G9 Buf46 WA373 MD7C5 46-91 IAH-CC125 VPM72 Hl-198 IL-Al35 IAH-CC17 B18 Buf24 LCTB19 IAH-CC20 Fw3-181 VPM65 GB84 J5 MCBO LCTBl7 IL-Al64 HI-50

mAb No.

NEUlle

436 438 440 442 443 445 446 448 452 453 454 456 458 459 462 463 466 468 469 482 483 484

IL-Al65 CACTB44 CACTl 11 IL-A87 GB20 IL-Al43 VPM36 IL-Al38 Buf44 MM11 IAH-CC84 4C7 P5 LCT2 IL-Al47 7ElO CACTB14 IVA31 20-76 GB16 LCTB39 But32

C

BU

Sh

0

0

??

0 0 0

0 0

0 0

0 ?? 0 0 0 0 0 0 0 0 0

0

Ca

SW

Hu

0

0 0 0 0 0 0

0

s

0

0

S

0 0

S

0

0 0

C

Bu

Sh

30

Ca

SW

Hu

0

S

0

S

0 0

24 62 0 0

0 0 0 0 0 0 0 0

0 0

0 0 0 0 0 0 0 0 0 0

0 0 0

??20

23 42

28

422 Table

P. Vilmos et al./ Veterinary immunology and hnmunopathology 52 (1996) 415-426

1 (continued)

mAb No.

Name

485 486 488 492

BAQ92 Bt21 CAC’IX IVA313

mAb No.

Name

493 495

IVA352 IL-A75 8Ell Co-38D3 46-66 GB26 TD26 Bufl3 WA40 VPM61 IVA77 IL-A% IL-A97 ILAl25 DH16 CACI7 IVA197 BUf2 IVA103 CACTB32 IVA50 IB12 IL-A55 MM29 IL-Al61 FC8-15

4% 498 502 504 50.5 506 520 522 524 525 526 528 529 530 533 534 535 536 538 539 540 542 543 544 mAb No.

Name

545 554 555 556 559 560 562 563 564 565 582 583

LCT-27 5F8 60-2 CACTB6 TH57 BAQ90 LCTB16 IVA94 LCTBSO IAH-CC 187 TD4 IL-A25

C

BU

0 0

0

Sh

Ca

SW

Hu

Ca

SW

Hu

0 0 C

BU

Sh

0

0 0 0

0

0

0 ?? 0

0 ?? 0

0 0 0 0 0

0 0

0

0

0

0

0

49

0 0 0 43 0 0 0 0

0

0 0 0 0

0

30

s

0 0 0

S

0

0

M

0 0

C

Bu

0 0

0 0 0

Sh

Ca

0 0

0 0 0 0 0

??

?? 0 0 0 0 0

0 0

SW

Hu

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Table 1 (continued) mAb No.

Name

58.5 586 589 590 592

Bo42 Bol IL-Al 14 IL-Al50 IL-A59

C

BU

Sh

0 0 0

0 0 0 0

0 0

SW

Ca

HU

0

C: Cattle PBM; Bu: River buffalo PBM; Sh: Sheep PBM; Ca: Camel PBM; SW: Swine PBM; Hu: Human PBM; S: Positive peak shifted; 0: Positive reaction; Bold percentage of positive cells.

34 mAbs

sheep,

(21%) with camel, 23 mAbs (15%) with swine and 20 mAbs (13%)

with human. 3.2. Expression pattern of the most conserved epitopes Thirteen antibodies (TH17, IVA30, Fw4-101, DH59, Bufl, LA24, IVA35, 25-32, B18, Fw3-181, VPM36, Buf44, DH16), which reacted with cattle, swine and human PBM, were further tested to determine the expression pattern using individual populations as assessed by the forward and side scatter profiles. As shown in Table 2, antibodies Th17, Fw4-101, Bufl, 25-32 and Fw3-181 reacted with most leukocytes including monocytes, granulocytes and lymphocytes in these three species, Fw4-101 being CD29 specific, 25-32 is an anti-CD44, B18 is anti-TAPA- and Fw3-181 reacts

Table 2 The results of the ‘paint-a-gate’ Species: Antibody No.

Name

030 069 239 255 258 306 340 383 408 423 446 452 529

TH17 IVA30 Fw4-101 DH59 Bufl IL-A24 IVA35 25-32 B18 Fw3-181 VPM36 Buf44 DH16

FACS

and western blotting analysis Cattle

Swine

WB

FACS

WB

all: M, G, L maj.: M, G, sp. L maj.: M, G, L H sub.: M, G all: M, G, L H sub.: M, G H sub.:? all: M, G, L all: M. G, L all: M, G, L H min.: M, sp. L Hmin.: L H sub.: L

-

all: M, G, L H maj.:? maj.: M, G, L 124 kD H sub.: M, G maj.: M, G, L H sub.: M, G 113kD * all: M, G, L 90kD all: M, G, L 86kD * maj.: M, G, L n.d. all: M,G,L H sub.: M, sp. L 35 kD H maj.: M, G, sp. L H sub.: G, sp. L 214kD ’

Human FACS

maj.: M, G, L maj.: M, G, sp. L 117 kD maj.: M, G, L H maj.: M, G all: M, G, L 101 kD H maj.: M, G, sp. L H all: M, G, sp. L 86 kD all: M, G, L n.d. min.: G, sp. L all: M, G, L 37 kD min.:sp.L min.: L 236 kD * H sub.: G, sp. L ??

WB

116kD 83 kD n.d. 38 kD -

H: heterogeneity (distinct subpopulation); sp: subpopulation; M: monocytes; G: granulocytes; L: lymphocytes; all: > 90% positive; maj.: 5090% positive; sub.: 25-50% positive; min.: < 25% positive; WB: Western blotting; -: negative reaction; * positive in one experiment; n.d.: not determined.

~

116

-

-

1

45

29

1:

124(Fw4-101)

-

35 (VPM36)

113 (IL-A24)

t

._ -

214 (DH16)

-

kDa (mAb)

-

-

-

-~

45

29

-

66

97.4 -

116

205

kDa

23 (CD~E)

37 (VPM36)

117 (Fw4-101) 101 (IL-A24) 66 (25-32)

236 (DH16)

kDa (mAb)

18 14

kDa

-

-

-

23 (CD3e)

38 (VPM36)

kDa (mAb)

Fig. I. Western blotting experiments with the antibodies reacting with the most conserved epitopes. Cell membranes of bovine (A), swine (B) and human CC> PBM were run in a 5515% gradient SDS-PAG. Standard molecular weights of the molecular weight markers are shown on the left side of each panel while right side arrows indicate the relative molecular mass of the detected proteins. The designations of the antibodies are in parentheses.

mAb

~

66

97.4

~

205

kDa

A

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with MHC Class I. Antibodies IVA30, DH59, IL-A24, IVA35, VPM36, Buf44 and DH16 marked distinct sub-populations of the PBM. Antibodies DH59 and IL-A24 marked monocytes and granulocytes in the three species tested while antibodies VPM36, Buf44 and DH16 recognized lymphocytes in all species. 3.3. Conservation

of the antigens at the molecular

level

Western blotting experiments were performed to examine the identity of the most conserved antigens. Table 2 shows that six out of the 12 antibodies showed reactivity in western blotting experiments. Three antibodies (Fw4-101, 25-32, VPM36) detected proteins in cattle, swine and human with similar molecular weights suggesting that the recognized homologues. The antibody Fw4-101, 25-32 and VPM36 detected proteins of M, 116-124000,83-86000 and 35-38000 respectively. Antibodies IL-A24 and DH16 showed reactivity with cattle and swine (M, 101-113000 and 214-236000 respectively); antibody IVA35 detected a protein of M, 90000 (Fig. 1).

4. Discussion

Previous studies (Naessens, 1993) have demonstrated that bovine or ovine antigens show a degree of similarity within the ruminant family and we have expanded these studies to include non-ruminants. By measuring cross-reactivity of the antibodies with cattle, buffalo, sheep, camel, swine and human antigens, we investigated the occurrence of highly conserved structures on leukocyte surface molecules. Our results indicate that 13 out of the 298 antibodies examined recognize phylogenetically conserved epitopes and that the expression patterns and molecular weights of some of these thirteen antigens are also conserved. Conservation of intracellular epitopes on leukocyte cell surface antigens is not surprising, since such domains are often responsible for conserved biochemical and transmembrane signaling processes. On the other hand, conservation of extracellular structures participating in recognition and cell-cell cooperation is a less common phenomenon, because these determinants are forced to adapt to a changing environment. However, a high degree of phylogenetic conservation was revealed by the MHC Class I (Hashimoto et al., 1992); B,-microglobulin (Shalev et al., 1981) and cellular adhesion (Kumagai-Braesch et al., 1995) molecules. We found that 13 mAbs detected cross-reactive, extracellular structures which may be involved in cell-cell communication or adhesion. Indeed, five of them were shown to be antibodies to CD29 (pl-integrin chain), CD44, CD81 (TAPA-I), MHC Class I and MHC Class II, while others clustered with molecules involved in adhesion (CD1 1a, CD41 and CD62L).

Acknowledgements

We thank Levente Toszegi and L&z16 T&h of the Slaughter-house of Szeged, Lkzl6 Mezasy of the Budapest Zoo, Endre Bajmocy of the Veterinary Institute, Debrecen and

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Antal Huszenyicza and Jakab Bal&zs of the Hortobagy National Park for providing the blood samples, and Peter C.L. Beverley for providing the UCHT-1 antibody. We are grateful to Jan Naessens and John Hopkins for critical revision of the manuscript. The technical assistance of Gabriella Bogdan is acknowledged. This work was supported by OTKA T016527, EEC grant RBCI PACT 92 3003.

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