An antiserum reacts with an evolutionary conserved region in the ε subunit of the T-cell receptor—CD3 complex in phylogenetically distant species

Immunology Letters, 38 (1993) 167-170 0165 2478 / 93 / $ 6.00 © 1993 ElsevierSciencePublishers B.V. All rights reserved

1MLET 02035

Letter to the E d i t o r

An antiserum reacts with an evolutionary conserved region in the e subunit of the T-cell receptor-CD3 complex in phylogenetically distant species E. Kurucz a, R. Glavits b, L. Krenacs c, T. Krenacs c, I. Ocsovszky d, G. Keresztes a, E. M o n o s t o r i a and I. Ando a'* aInstitute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary; bCentral Veterinary Institute, Budapest, Hungary, Departments of CPathology and dBiochemistry, Albert Szent-Gyorgyi Medical University, Szeged, Hungary

(Received 26 July 1993; revision receivedand accepted 15 September 1993)

CD3 proteins are first expressed in the cytoplasm of thymocytes at the earliest stages of intrathymic T-cell maturation [1]. It has been suggested that they are directly involved in transmembrane signaling events: the subunits have large cytoplasmic domains [2,3] the y, ~ and r/ chains undergo phosphorylation upon T-cell stimulation [4-6]. The CD3 e chain on human T cells is a 20 kDa non-glycosylated peptide [7] that is essential for cell surface expression of the T-cell receptor (TCR)-CD3 complex [1]. Monoclonal antibodies to the e chain mimic the antigen interaction with T C R in that they trigger early and late events of T-cell activation: rapid increase in phosphatidylinositol turnover and intracellular calcium level, lymphokine secretion and cell proliferation [8]. Functional conservation of the molecules involved in signal transduction has been described previously [9-11]; however, little is known about the phylogenic conservation of the CD3 e chain, although it can be deducted from the D N A sequences for human, mouse, and dog e chains [12-14] that the D N A sequence for residues 156-168 is conserved. In this work we have studied the phylogenic conservation of the CD3 e

"Corresponding author: Dr. lstvan Ando, Institute of Genetics,

Biological Research Centre of the Hungarian Academy of Sciences, H-6701, P.O. Box 521, Szeged, Hungary.

chain in secondary lymphoid tissues of various mammalian and avian species with a polyclonal antibody [15] to the intracytoplasmic sequence of the human CD3 chain comprising residues 156-168 (NH2-Glu-Arg-Pro-Pro-Pro-Val-Pro-AsnPro-Asp-Tyr-Glu-Pro-COOH). On the tissue sections of human, bovine, water buffalo and swine lymph nodes (Fig. 1A,B,C,D, respectively) extensive staining of the T-cell area is observed in contrast to that of the B-cell follicles in which several scattered cells are stained, presumably helper T cells. The T-cell areas show selective reactivity in spleen sections also obtained from chicken (Fig. 1E) where the unreactive follicles are surrounded by strongly labeled cells in the extrafollicular region. However, in cryostat sections of Bursa of Fabricius (a specialized tissue of birds for B-cell development) almost all the follicular lymphocytes both in the medulla and in the cortex are unreactive (Fig. 1F). The antigen recognised by CD3 e antibody in different species was further characterized by immunoblotting. Fig. 1G shows that the antibody reacts with a band varying from 19 to 22 kDa in human (lane a), hen (lane b), buffalo (line c) and bovine (lane d) lymphocytes. Lanes a',b',c' and d' show the lack of reaction of immunoglobulins prepared from an immune serum to an irrelevant antigen. Lysates prepared from non-lymphoid organs of the mammalian species and from the bur167

kDa 97-66-45-29 w 18-14 w

G 168

a

bcd

a' b' c' d'

sa o f the hen also d i d n o t react in d o t - b l o t a n a l y sis ( d a t a n o t shown). The immunohistological and biochemical analysis on l y m p h o i d tissues f r o m different verteb r a t e s p r e s e n t e d in this s t u d y d e m o n s t r a t e s a sequence o f the e chain o f the C D 3 c o m p l e x t h a t is c o n s e r v e d between h u m a n , cow, buffalo, swine a n d hen. A s t r o n g i n d i c a t i o n for the c o n s e r v a t i o n o f this region o f the C D 3 in all m a m m a l i a n a n d a v i a n species is given b y analysis o f several t a x o n o m i c a l l y u n r e l a t e d species. T h e following species have been tested: Xenopus, s a n d lizard, E u r o p e a n green frog, hen, d u c k , t u r k e y , mole, h e d g e h o g , mouse, rat, r a b b i t , nerts, ferret, cat, dog, cattle, sheep, goat, w a t e r buffalo, d o m e s t i c horse, chimp a n z e e a n d h u m a n . T h e tissues o f all a v i a n a n d m a m m a l i a n species stained with the C D 3 antib o d y while the a m p h i b i a n a n d reptile tissues did n o t react. Birds a n d m a m m a l s evolved f r o m their reptilian a n c e s t o r s over 200 million years ago. C o n s e r v a t i o n o f a sequence o f the c y t o p l a s m i c tail o v e r a significant p h y l o g e n i c d i s t a n c e suggests a s t r o n g selective pressure p e r h a p s a s s o c i a t e d with a general cell biological function. These d a t a with o t h e r e x a m p l e s [9-11] s h o w t h a t r e c e p t o r s i n v o l v e d in cell signaling c o u l d be c o n s e r v e d between p h y l o genetically d i s t a n t species in their c y t o p l a s m i c d o mains. The d a t a also s h o w t h a t a n t i b o d i e s to conservative regions m a y be used in the v e t e r i n a r y p r a x i s for d i a g n o s t i c p u r p o s e s .

Acknowledgements W e t h a n k P r o f e s s o r Peter C.L. Beverley, I C R F T u m o u r I m m u n o l o g y Unit, L o n d o n , U K , for cri-

tical r e a d i n g a n d c o m m e n t s on the m a n u s c r i p t . This w o r k was s u p p o r t e d by O T K A G r a n t s 179, 928 a n d 5257 a n d the B a s t y a i - H o l c z e r M e m o r i a l F o u n d a t i o n . T h e C D 3 e a n t i b o d y was a gift f r o m D a k o p a t t s . T h e technical assistance o f Mrs. G a briella B o g d a n , Ms. M a r i a L a b d y a n d Ms. A g n e s D u d a s is a c k n o w l e d g e d .

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Fig. 1. Reactivity of the CD3 e chain antibody. Cryostat sections were obtained from human (A), bovine (B), buffalo (C) and swine (D) lymph nodes (original magnification: x 130), and from chicken (E) spleen and Bursa of Fabricius (F) (original magnification: x 325). Immunohistochemical staining was performed by an affinity purified rabbit polyclonal antibody, raised to a peptide sequence of the cytoplasmic domain 156-168 of the human CD3 chain [15] and by the ABC streptavidin-biotin peroxidase (Dakopatts) procedure according to the manufacturer's instructions. Peroxidase reaction was developed with 0.05% 3,9-amino-ethyl-carbazole (Sigma) in an acetate buffer, pH 4.6, containing 0.05% H202 for 15 min. Slides were counter-stained with Mayer's hematoxylin. Fig. 1G is a Western blot analysis on human lymph node (lanes a and a'), chicken spleen (lanes b and b'), buffalo (lanes c and c') and bovine (lanes d and d') lymph node cells. NP-40-solubilized cells were analyzed with SDS-PAGE (10% gel) followed by electroblotting onto a nitrocellulose filter. The blots were probed with CD3 antibody (a,b,c,d) and an irrelevant antibody (a',b',c',d') as a negative control, developed by biotinylated sheep anti-rabbit Ig followed by streptavidin-peroxidase (Dakopatts) and chromogenic substrate 3,3'-diaminobenzidine. The position of the molecular-weight markers (kDa) is marked on the left. 169

Wileman, T. and Terhorst, C. (1988) Proc. Natl. Acad. Sci. USA 85, 8623. [14] Nash, A.R., Scherrf, U. and Storb, R. (1991) Immunogenetics 33, 396.

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