I "eterinary Immunology and lmmunopathologl'. 27 ( 1991 ) 7 !-76

71

Elsevier Science Publishers B.V., Amsterdam

4.7 Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes

(WC1, WC2) W.I. Morrison a and W.C. Davis" alAH, Complotl, Neu'bur.l', RGI6 0NN, L'K t, ll'ashington State University, Pullman, II Z4 99164- 7040, ~"S.-I

INTRODUCTION

In 1986, MacKay et al. described a monoclonal antibody (mAb), 19-19, which recognised an antigen expressed on a discrete population of sheep lymphocytes; these cells were negative for B cell markers and for the T cell subpopulation markers CD4 and CD8, but were positive for CD5. As they were present as a small population in the thymus, it was suggested that they represent a novel subpopulation ofT lymphocytes. Shortly thereafter, a number of antibodies which recognise a similar antigen in cattle were produced (Davis et al., 1987; Morrison et al., 1988; Howard et al., 1989). Recent studies in both sheep and cattle indicate that the populations of cells recognised by these mAbs represent T lymphocytes that synthesise the gamma/delta form of the T cell receptor (Hein et al., 1989; MacKay et al., 1989; Clevers et al., 1990 ). Nineteen mAbs considered to recognise populations of CD4- CD8- T lymphocytes were submitted to the workshop. Two of these reagents ( 19-19 and 197) had been produced against sheep lymphocytes and the remainder against bovine lymphocytes. Based on the workshop findings, 14 of the antibodies could be clustered into one group all of which recognised an antigen of 215/300 kDa. A second cluster of two mAbs, which reacted with an antigen of 37/47 kDa, was also established. Three mAbs (BAQ 136A, CACTB35 and BAQ53A) which fa~led to immunoprecipitate could not be clustered. PROPERTIES O F MABS IN W O R K S H O P CLUSTER ! (WC I )

The fourteen mAbs listed in the first part of Table l precipitated molecules of the same relative molecular weights from PBM. Analyses of the immunoprecipitates in SDS polyacrylamide gels run under reducing conditions revealed two bands of approximately 215 and 300 kDa (Fig. l ). In separate studies, immunoprecipitates obtained with mAbs CCI 5 and IL-A29, when 0165-2427/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

72

W.l. MORRISONAND W.C. DAVIS

TABLE 1

List of monoclonai antibodies forming workshop clusters 1 and 2 and the percentages of cells recognised in peripheral blood mononuclear cells from two cattle Workshop cluster

WC 1

WC2

Monoclonal antibody

Laboratory of origin

CC 15 11 F5 197 CACTB31A IL-A29 CC39 19-19 BAQ4A CACTB32A BAQ 128A BAQ89A BAq 159A BAQ90A B7AI CACTB81A CACTB6A

lsotype

IAH-Compton Namur Basel Pullman ILRAD IAH-Compton Melbourne Pullman Pullman Pullman Pullman Pullman Pullman Pullman Pullman Pullman

Percentage cells recognised in PBM

G2a G2a G2b G2b GI GI G1 G1 G1 G1 GI G1 G3 M G1 M

Animal 1

Animal 2

32 36 33 36 28 35 26 34 11 19 20 21 16 31 19 25

11 10 6 14 7 10 11 9 5 9 7 10 6 16 10 11

run under non-reducing conditions, also resolved as two bands but with lower apparent molecular weights (Clevers et al., 1990). Thus, the 215 and 300 kDa entities are not covalently linked. vv

~j

W~,IAAAq~A A~

~AAV~A~

~J~

~JL

qt~A

tJ~0.~]~,7~AI~A~,711tJ

fJ4,JLl~

JtlJLl--

munohistology indicated that the phenotype and tissue distribution of the cells reactive with the 14 mAbs are similar. However, it is clear that the numbers of cells with which the different mAbs react varies considerably. Thus for example, in two cattle the percentages of cells in PBM recognised by the mAbs varied from 6% to 16% in one animal and from I 1% to 36% in the other (Table l ). Differences in the numbers of positive cells were also apparent in tissue sections. The maximum percentage of cells recognised by the mAbs also varies between individual animals. Separate studies with two of the mAbs (CCI 5 and lL-A29) have shown that the population of cells reactive with these mAbs is much larger in young calves than in adults but also varies markedly among young animals of t~e same age (Clevers et al., 1990). Double staining experiments have shov:n that the cells reacting with these 14 mAbs are negative for CD2, CD6, CD4 and CD8. The positive cells show a characteristic pattern of tissue distribution. In the lymph nodes, they are concentrated in the outer areas of the cortex adjacent to the subcapsular and trabecular sinuses (Fig. 2a). A few cells are also found in the paracortex and medullary cords. In the spleen, they are localised mainly

DIFFERENTIATION ANTIGENS ON CD4-CD8- T LYMPHOCYTES

1 2 3 4 5

6

7

8

73

9

-200

-92 -69

-46

i I

-30

Fig. 1. Autoradiographs of immunoprecipitates analysed by SDS-PAGE under reducing conditions. Immunoprecipitation was carried out on detergent-solubilised lysates of peripheral blood mononuclear cells surface-labelled with 12Sl.Lanes l, 2 and 3 contain precipitates obtained with IgM, IgGl and IgG2a mouse myeloma proteins. Lane 4, MAB I I FS; lane 5, MAb 197; lane 6, MA~ BAQ89A; lane 7, MABQ 128A; lane 8, MAb CACTB81 A; lane 9, CACTB6A.

in the marginal zones. In the thymus, where they constitute 2-3% of thymocytes, they are concentrated mainly in the medulla with a few also scattered through the cortex (Fig. 2b). Independent studies with mAbs CC 15 and ILA29 have shown that there are substantial populations of positive cells in the intestines, mainly in the lamina propria, and in the skin where they are found in the dermis and basal layers of the epidermis (Howard et al., 1989; Clevers et al., 1990). Only occasional positive cells are found in the Peyer's patches. Despite the fact that the 14 mAbs recognise different numbers of cells, the similarity in tissue distribution and phenotype of the positive cells, together with the finding that they all immunoprecipitate molecules of the same molecular weight, suggests that they recognise different epitopes on the same surface antigen complex. Such differences might arise as a result of different types of post-translational modification of the molecule. Statistical analyses of the results of flow cytometry have enabled the 14 antibodies to be clustered. Since the antigen does not have an obvious homologue in man, the groups of antibodies have been designated workshop cluster I (WCI). Although there was some evidence from the clustering data and from other studies (W.C. Davis, unpublished data; Sopp et al., pp. 163-168) that the mAbs could be subdivided into two or more subgroups, this subgrouping was considered not to be

74

W.I. MORRISON AND W.C. DAVIS

sufficiently precise to define subclusters. Clearly, further studies of the biochemical structure of the antigen recognised by these 14 mAbs are required to define the basi ~, of the difference in the numbers of cells recognised by the mAbs.

PROPERTIES OF MABS IN WORKSHOP CLUSTER 2 (WC2)

Clustering analyses revealed a second group of two mAbs, CACTB81A and CACTB6A, which could be distinguished from the W C I antibodies (Table l ). Both of these mAbs identified molecules of 37 and 47 kDa in immunoprecipitates subjected to S D S - P A G E under reducing conditions (Fig. l ). The tissue distribution of the cells identified by these mAbs was zimilar to that of those reactive with the WC 1 mAbs. The numbers of cells detected in PBM by

®

® d

,~

Wl't

&~ili~

•

•

t/k

'~

• ~"~ll, " Wmd~Lq ~ d

0

•

. . . . . .

~ •"

Q

•

~"..

+%

~~~i I

~'"'. .

+.

""

~

..'

111,

'

,

~,

.

•

.

~.'

~.~

,.

%,I+ "+' ,,"" ,~" ,

"°

.

. +,

*

,,

"~'o ~'

'

,:~L~ -.,~ , a w m .,j+

,",",

,+ , ~ ~ ~

".",.

. "~

+~

~i

+~

•

•

+~

•

•.

O

:P ;+

'

~

,"

¢¢

41

4~

•

~

~

I'~.."" h'"" "

. j~.,+~ ~¢.,,. .

.

:., '..,+, ,,, ~ . . • ' . . ! . ,. .-.

.. ,

-.

. •

+.

.

Fig. 2. Sections of bovine lymphoid tissues stained by immunoperoxidase with MAb IL-A29. (a) Lymph node: positive cells are found adjacent to the subcapsular sinus (arrows). F - follicular region: P - paracortex ( × 100). (b) Thymus: positive cells are present mainly in the medulla, shown in the lower part of the figure ( × 95 ).

DIFFERENTIATION ANTIGENS ON C D 4 - C D 8 - T LYMPHOCYTES

75

these mAbs was within the range of, or in some instances less than, those detected by the WC 1 mAbs. The population of cells with which the two mAbs reacted overlapped to a large extent with the cells recognised by WC 1 mAbs. However, an important difference was that, in at least some animals, the two mAbs reacted with small populations of cells which were negative with the WCI mAbs; most of these cells expressed CD2 and some CD8 (Sopp et al., this volume). The two mAbs did not react with the same population of cells; in the animals tested, there was considerable overlap, but CACTB6A ÷ CACTB81 - and CACTB6A- CACTB8 ÷ cells were identified. In view of the molecular weight of the molecule recognised by these mAbs, it is possible that they are specific for T cell receptor molecules. However, until the precise identity of the antigen has been resolved, the two mAbs have been designated WC2. SUMMARY

The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric, molecule expressed in a subpopulation of the WC 1+ cells and on, an additional small population ofT lymphocytes. The cell populatiun~ rccognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor ma!ecules. REFERENCES Clevers, H., MacHugh, N.D., Bensaid, A., Dunlap, S., Baldwin, C.L., Kaushal, A., lams, K., Howard, C.J. and Morrison, W.I., 1990. Identification of a bovine surface antigen uniquely expressed on CD4/CD8-negative, T cell receptor gamma/delta-positive T lymphocytes. Eur. J. lmmunol. 20: 809-815. Davis, W.C., Marusic, S., Lewin, H.A., Splitter, G.A., Perryman, L.E., McGuire, T.C. and Gotham, J.R., 1987. The development and analysis of species specific and cross-reactive mmioclonai antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species. Vet. lmmunol. Immunopathol., 15: 337-376. Hein, W.R., Dudler, L., Beya, M.-F., Marcuz, A. and Grossberger, D, 1989. T cell receptor gene expression in sheep: differential usage of TCRI in the periphery and thymus. Eur. J. immunoi., 19: 2297-2301. Howard, C.J., Sopp, P., Parsons, K.R. and Finch, J., 1989. In vivo depletion of BoT4 (CD4)

76

W.I. MORRISON AND W.C. DAVIS

and non-T4/T8 lymphocyte subsets in cattle with monoclonal antibodies. Eur. J. Immunol., 19: 757-764. MacKay, C.R., Maddox, J.F. and Brandon, M.R., 1986. Three distinct subpopulat~ons of sheep T iymphocytes. Eur. J. Immunol., 16:19-25. MacKay, C.R., Beya, M.F. and Matzinger, P., 1989. Gamma/delta T cells express a unique surface molecule appearing late during thymic development. Eur. J. Immunol., 19: 14771483. Morrison, W.i., MacHugh, N.D., Bensaid, A., Goddeeris, B.M., Teale, A.J. and McKeever, D.J., 1988. A monoclonal antibody which reacts specifically with a population of bovine lymphocytes lacking B cell and T cell markers. In: S. Fossum and B. Rolstad (Editors), Histophysiology of the Immune System. Plenum Press, New York, NY pp. 591-596.