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Use of monoclonal antibodies reactive with secretory epithelial cells for the immunocytochemical identification of plasma cells
b~tmunology Letters, 12 (1986) 295
300
Elsevier l m l e t 748
USE OF MONOCLONAL EPITHELIAL CELLS FOR
ANTIBODIES REACTIVE WITH SECRETORY THE IMMUNOCYTOCHEMICAL IDENTIFICATION OF PLASMA CELLS
G. D. J O H N S O N , N. R. LING, P. D. N A T H A N and D. L. H A R D I E Department of hnmunologv, The Medical School, Universio, of Birmingham, Vincent Drive, Birmingham B15 2TJ, U.K. (Received 6 December 1985) ( M o d i f i e d version received a n d accepted 5 February 1986)
1. Summary Six monoclonal antibodies (McAbs) were identified as plasma cell-reactive when screened on sections of h u m a n tonsil. They were all produced following immunisation of mice with cells of a h u m a n plasmacytoid line. Three of the antibodies also stained the cytoplasm (but not the surface) of blood B cells and were unreactive with other leucocytes; one McAb showed broad lymphocyte reactivity and two were completely unreactive with blood leucocytes; on testing with a panel of cell lines specificity for the plasmacytold line was demonstrated by three of the McAbs. In spite of the marked restriction shown by the reactivity of these antibodies in tests on cells of haemopoietic origin, tests on other human tissues - including thyroid and pancreas showed that a related antigen was present in the cytoplasm of secretory epithelial cells. The overall patterns of reactivity of the individual McAbs on various tissues and blood lymphocytes were different. Comparisons were made with the established McAb OKT10, which binds to plasma cells, early stern cells and activated lymphocytes; its binding to plasma cells was confirmed and it was shown that it did not stain secretory epithelia. The potent reactions obtained with the
new McAbs suggest that antibodies to antigens associated with epithelial cell secretory apparatus provide potentially useful reagents for studying plasma cells.
2. Introduction Cells of B-lineage which have reached the terminal stages of differentiation are sessile, nonproliferating antibody-secreting cells mainly of plasma cell morphology. Their functional role is different from that of the migrant antigenreactive B-cells from which they are derived. It would be expected that this dramatic change in function would be accompanied by differences in antigenic constitution of the two cell types and there are indications that this is so. Thus, M H C Class II antigens and antigens of the CD19-23 clusters are strongly expressed on B-cells but not on plasma cells. Conversely, the monoclonal t antibodies PC-1 [1], PCA-1 and PCA-2 [2], H A N PCI [3] and OKT10 [4] identify antigens present on plasma cells but not on B lymphocytes. However, the antigenic characterisation of plasma cells is at a very early stage of development. We describe here the identification of several new plasma cell-reactive McAbs and their use to demonstrate shared antigenicity between plasma ceils and secretory epithelia.
he), words." m o n o c l o n a l a n t i b o d i e s - p l a s m a cells - secretory epithelial cells -
immunocytochemistry
0 1 6 5 - 2 4 7 8 /' 86 .." $ 3.30 ~': 1986 Elsevier Science Publishers BN. (Biomedical Division)
295
3. Materials and Methods
3.1. Monoclonal antibodies Hybridomas were generated by fusion of cells of a BALB/c plasmacytoma cell line [P3-NS1/Ag4-1] with spleen cells of BALB/c mice which had been immunised i.p. with whole cells of the human myeloma cell line RPMI-8226 [5]. The fusion procedure was essentially that of Galfr6 et al. [6] modified as previously described [7]. Culture supernates were screened by immunofluorescence on preparations of the immunising cells and on cryostat sections of tonsil. Selected hybridomas were grown up in culture and in pristane-primed mice. Ascitic fluid was used in all subsequent studies. Antibodies binding to immunoglobulin determinants were excluded by retesting in the presence of human serum and by passive haemagglutination tests using red cells coated with a range of immunoglobulin preparations. The plasma cell-reactive McAb OKT10 was obtained from the American Type Culture Collection. A potent follicular dendritic cell-specific McAb produced in this Department, 2BFll [7], was used as a negative control for the analysis of plasma cell-reactive antibodies.
3.2.2. Cell suspensions Mononuclear (MN) cells were separated from peripheral blood of normal donors by density gradient centrifugation. Washed MN cells (1 × 106) were suspended in 100 txl of the antibody preparation (diluted 1:25) and rotated on ice for 30 rain. After staining with diluted anti-mouse immunoglobulin conjugate for 30 rain, the cells were resuspended in 50°7,0 glycerol containing DABCO. T-lymphocyteenriched and -depleted fractions were prepared by rosetting with sheep erythrocytes. Tests were also performed on acetone-fixed cytocentrifuge preparations obtained in a Shandon Cytospin 2. Similar techniques were employed for testing reactivity with cells from established cell lines as already described [7]. Complement-fixing activity was determined by IF as previously described [8]. 3.3. Mr of antigens Detergent extracts of RPMI-8226 cells were run in S D S - P A G E 10% cross-linked gels. Electroblotting onto nitrocellulose membrane was performed for 3 h at 20 V [10]. After exposure to the McAbs, washing and incubating with ~251sheep anti-mouse immunoglobulin, the labelled bands were revealed by autoradiography. M r standards were run simultaneously.
3.2. Immunofluorescence (IF) 3.2.1. Tissue sections The standard procedure used has been described [8]. Cryostat sections of human tonsil (two donors), thyroid and pancreas were cut at 6 ~m and fixed in acetone for 5 min at room temperature. Culture supernates were tested undiluted and ascitic fluids diluted 1:50 in phosphate-buffered saline containing 10°7,0 foetal calf serum. Double fluorescent staining was performed by applying paired mixtures of test antibodies having different immunoglobulin heavy chain isotypes, followed by staining with paired fluorescein and rhodamine conjugates of appropriate specificity for the mouse immunoglobulin isotypes. All IF tests were mounted in glycerol containing diazabicyclo-octane (DABCO) to retard fading during microscopy [91.
296
4. Results
4.1. Plasma cell reactivity The six new McAbs showed cytoplasmic staining of cells of the myeloma line RPMI-8226 in at least 75% of the cells (Table 1). Only two of the antibodies, however (BU9 and BUll), showed surface staining - of approximately 20°7o of the cells. When tested on tonsil sections all six showed cytoplasmic staining of plasma cells, notably in inter-follicular T-dependent areas, in and around germinal centres and in subepithelial areas (Fig. 1 and Table 2). Double staining established that the plasma cells identified by the different McAbs were coincident. Associated reactivity with other features of the tonsil sections was noted in five of the antibodies, as recorded in Table 2. Four of the McAbs stained
Table 1 Reactivity of the new monoclonal antibodies with peripheral blood cells and cell lines Clone
Test"
Peripheral blood lymphocytes T
BU5
C S
BU7
C S
BU9
C
S C
--
Non-T
Cell lines Pre-B (SM-SB)
Other cells
Burkitt (EB-4)
m
+
-4-
m
+
+
Wb
+
-
--
+
B-LCL (Ed-1)
Late B
T-cell (HUT)
Myeloid (HL60)
+
m
m
+
+
Plasmacytoid (RPMI-8226)
ARH-77
WM-I
m
m
m
-
-
-
q_
5%
W
+
+
+
+
+
10% W
10% W
6% +
-
22% +
S
6%
-
2OCHI
C S
_
_
21CC1
C S
20% + -
Bull
+
+
27% + _
_}_
5% W
-
+
3 %
-
-
+
,, c = fixed cytocentrifuge preparations; S = surface membrane staining of viable cells. ~' Weak staining of PMN and monocytes. + = Staining of >75% of the cells; W = weak staining; - = not tested. B - l y m p h o c y t e s in m a n t l e z o n e s a n d o n e o f t h e s e (BU9) also stained T-lymphocytes in interf o l l i c u l a r a r e a s . R e a c t i v i t y w i t h o t h e r cells a n d with tissue fibres was seen with three antibodies. T h e o n l y r e a c t i v i t y d e m o n s t r a b l e o n t o n s i l sec-
t i o n s w i t h O K T I 0 ( a p a r t f r o m p l a s m a cells) w a s w e a k s t a i n i n g o f l y m p h o i d cells in g e r m i n a l c e n t r e s . G e n e r a l f e a t u r e s o f t h e a n t i b o d i e s are a l s o s h o w n i n T a b l e 2. O n e M c A b ( 2 0 C H 1 ) was lgM class and this was the only antibody shown Table 2
G e n e r a l f e a t u r e s o f the new m o n o c l o n a l a n t i b o d i e s Clone
BU5 BU7
Antigen-'
M o u s e Ig
Complement
IF s t a i n i n g o f tonsil sections b
(kDa)
isotype
fixing activity
P l a s m a cells
B lymphocytes
-
+ + + +
+ +
+ +
+
+ -
62 (781 62 (78) (49) 135
GI
BUI 1 20CH1
72 62 (78)
GI M
+ +
+ + + +
+ -
21CC1
62 (78)
G1
-
+ +
-
BU9
G2b A
T lymphocytes
Other reactivity
S m o o t h m u s c l e fibres C o n n . tissue fibres and epithelium E n d o t h e l i u m a n d epithelial b a s a l cells
T h e first figure refers to the m o l e c u l a r mass o f the m a j o r b a n d a n d the b r a c k e t e d figure to a m i n o r b a n d . b I d e n t i c a l r e s u l t s were o b t a i n e d w i t h t o n s i l s f r o m t w o d o n o r s . +, moderate staining; + +, strong staining.
297
298
Fig. 1. l m m u n o f l u o r e s c e n t reactions o f m o n o c l o n a l a n t i b o d i e s on tissue sections. (a) P l a s m a cells in inter-follicular area o f tonsil; M c A b BUS ( × 4 0 0 ) . (b~ T h y r o i d a c i n a r epithelium; M c A b BUT ( × 160). (c) Pancreas a c i n a r a n d duct epithelium; M c A b BU9 ( × 160).
to be complement-fixing in its reaction with plasma cells. 4.2. Epithelial cell reactivity Very bright staining of the cytoplasm of acinar epithelial ceils in thyroid sections was obtained with five of the McAbs. All six stained pancreatic acinar epithelial cells but differences were seen in their reactivity with epithelial cells lining ducts and with islet cells (Fig. 1, Table 3). The most restricted reactivity was seen with 20CHI; OKT10 showed no staining of thyroid and pancreas. 4.3. Peripheral blood cells and haemopoietic cell lines 4.3.1. Blood M N cells Only one of the six McAbs (BU9) stained T cells (surface and cytoplasm); this antibody also stained monocytes and PMN weakly (Table 1). Non-T (E-rosette
negative) lymphocytes were stained by four antibodies in cytocentrifuge preparations and none of these gave surface staining. These results correlated with the staining of lymphocytes in tissue sections. Table 3 Reactivity o f the m o n o c l o n a l a n t i b o d i e s with sections o f epithelial tissue Clone
BU5 BU7 BU9 BUll 20C H 1 21CC1
Thyroid
Pancreas
Acinar epithelium
Acinar epithelium
Duct epithelium
Islet cells
+ + + +
+ + + + + + + +
+ + + + + + +
+ .' +a W
+ + + +
+ +
Selective. W, ; +,
W
; ++,
299
4.3.2. Cell lines One McAb (BU9) showed broad reactivity with the cell panel. The other five did not bind to cells of pre-B, T or myeloid lines. Three of the antibodies (BU5, BU7 and 20CHI) did not stain B-lymphoblastoid cells; of the others one showed broad reactivity with Bcell lines (BU9), 21CC1 stained some cells in the Ed-1 and ARH-77 lines and BUll stained 6% of the Ed-I cells.
Acknowledgements
5. Discussion
References
These findings demonstrate the occurrence of antigens shared by plasma cells and secretory epithelial cells. In addition, four of six new McAbs which showed this reactivity pattern also stained B and/or T lymphocytes in tissue sections and peripheral blood. The remaining two antibodies evidently recognise antigen(s) absent from haemopoietic cells (at least in peripheral tissue) and appearing only in the terminal stages of B-cell differentiation. The patterns of reactivity observed indicate heterogeneity of the antigens involved and this was confirmed by the M r analysis. The antigens revealed by these McAbs are evidently potent immunogens and in secretory cells may be associated with rough endoplasmic reticulum. A systematic study of the reactivity of such McAbs with secretory epithelial cells in a variety of tissues might show differences related to the secretory products of different cells. These antibodies appear to be potentially useful reagents for studying late differentiation in the Bcell lineage.
[l] Anderson, K. C., Bates, M. P., Slaughenhoupt, B., Schlossman, S. F. and Nadler, I_. M. (1984) J. lmmunol. 132, 3172-3179. [2] Anderson, K. C., Park, E. K., Bates, M. F., Leonard, R. C. E, Hardy, R., Schlossman, S. F. and Nadler, L. M. (1983) J. Immunol. 130, 1132-1138. [3] Mertens, N., Dehou, M. F., Vanderbruggen, H., Van Riet, I. and Van Camp, B. (1985) in: Protides Biological Fluids, pp. 891-895, Pergamon Press, Oxford. [4] Reinherz, E. L. and Schlossman, S. E (1981) Immunol. Today 2, 69-75. [5] Matsuoka, ~'\, Moore, G. E., Yagi, Y. and Pressman, D. (1967) Proc. Soc. Exp. Biol. Med. 125, 1246-1250. [6] Galfr6, G., Howe, S. C., Milstein, S., Butcher, G. W. and Howard, J. C. (1977) Nature 266, 550 552. [7] Johnson, G. D., Hardie, D. I,., Ling, N. R. and Maci,ennan, 1. C. N.I. (1986) J. Clin. Exp. hnmunol., in press. [8] Johnson, G. D. and Holborow, E. J. 11986) in: Handbook of Experimental Immunology (D. M. Weir and L. A. Herzenberg, Eds.) 4th Edn, Ch. 31, Blackwells, Edinburgh and Oxford, in press. [91 Johnson, G. D., Davidson, R. S., McNamee, K. C., Ru,sell, G., Gooduin, D. and Hoborow, E. J. 11982) J. Immunol. Methods 55, 231 242. [10] Towbin, H., Staehelin, T. and Gordon, J. (19791. Proc. Natl. Acad. Sci. U.S.A. 76(9), 4350-4354.
300
This work was supported by Programme Grant PG 840 2371 from the Medical Research Council and by a grant from Unilever. G.D.J. is a member of the M.R.C. external scientific staff. The secretarial assistance of Ms. E O'Reilly is gratefully acknowledged. P.D.N. is in receipt of an M.R.C. Studentship.
300
Elsevier l m l e t 748
USE OF MONOCLONAL EPITHELIAL CELLS FOR
ANTIBODIES REACTIVE WITH SECRETORY THE IMMUNOCYTOCHEMICAL IDENTIFICATION OF PLASMA CELLS
G. D. J O H N S O N , N. R. LING, P. D. N A T H A N and D. L. H A R D I E Department of hnmunologv, The Medical School, Universio, of Birmingham, Vincent Drive, Birmingham B15 2TJ, U.K. (Received 6 December 1985) ( M o d i f i e d version received a n d accepted 5 February 1986)
1. Summary Six monoclonal antibodies (McAbs) were identified as plasma cell-reactive when screened on sections of h u m a n tonsil. They were all produced following immunisation of mice with cells of a h u m a n plasmacytoid line. Three of the antibodies also stained the cytoplasm (but not the surface) of blood B cells and were unreactive with other leucocytes; one McAb showed broad lymphocyte reactivity and two were completely unreactive with blood leucocytes; on testing with a panel of cell lines specificity for the plasmacytold line was demonstrated by three of the McAbs. In spite of the marked restriction shown by the reactivity of these antibodies in tests on cells of haemopoietic origin, tests on other human tissues - including thyroid and pancreas showed that a related antigen was present in the cytoplasm of secretory epithelial cells. The overall patterns of reactivity of the individual McAbs on various tissues and blood lymphocytes were different. Comparisons were made with the established McAb OKT10, which binds to plasma cells, early stern cells and activated lymphocytes; its binding to plasma cells was confirmed and it was shown that it did not stain secretory epithelia. The potent reactions obtained with the
new McAbs suggest that antibodies to antigens associated with epithelial cell secretory apparatus provide potentially useful reagents for studying plasma cells.
2. Introduction Cells of B-lineage which have reached the terminal stages of differentiation are sessile, nonproliferating antibody-secreting cells mainly of plasma cell morphology. Their functional role is different from that of the migrant antigenreactive B-cells from which they are derived. It would be expected that this dramatic change in function would be accompanied by differences in antigenic constitution of the two cell types and there are indications that this is so. Thus, M H C Class II antigens and antigens of the CD19-23 clusters are strongly expressed on B-cells but not on plasma cells. Conversely, the monoclonal t antibodies PC-1 [1], PCA-1 and PCA-2 [2], H A N PCI [3] and OKT10 [4] identify antigens present on plasma cells but not on B lymphocytes. However, the antigenic characterisation of plasma cells is at a very early stage of development. We describe here the identification of several new plasma cell-reactive McAbs and their use to demonstrate shared antigenicity between plasma ceils and secretory epithelia.
he), words." m o n o c l o n a l a n t i b o d i e s - p l a s m a cells - secretory epithelial cells -
immunocytochemistry
0 1 6 5 - 2 4 7 8 /' 86 .." $ 3.30 ~': 1986 Elsevier Science Publishers BN. (Biomedical Division)
295
3. Materials and Methods
3.1. Monoclonal antibodies Hybridomas were generated by fusion of cells of a BALB/c plasmacytoma cell line [P3-NS1/Ag4-1] with spleen cells of BALB/c mice which had been immunised i.p. with whole cells of the human myeloma cell line RPMI-8226 [5]. The fusion procedure was essentially that of Galfr6 et al. [6] modified as previously described [7]. Culture supernates were screened by immunofluorescence on preparations of the immunising cells and on cryostat sections of tonsil. Selected hybridomas were grown up in culture and in pristane-primed mice. Ascitic fluid was used in all subsequent studies. Antibodies binding to immunoglobulin determinants were excluded by retesting in the presence of human serum and by passive haemagglutination tests using red cells coated with a range of immunoglobulin preparations. The plasma cell-reactive McAb OKT10 was obtained from the American Type Culture Collection. A potent follicular dendritic cell-specific McAb produced in this Department, 2BFll [7], was used as a negative control for the analysis of plasma cell-reactive antibodies.
3.2.2. Cell suspensions Mononuclear (MN) cells were separated from peripheral blood of normal donors by density gradient centrifugation. Washed MN cells (1 × 106) were suspended in 100 txl of the antibody preparation (diluted 1:25) and rotated on ice for 30 rain. After staining with diluted anti-mouse immunoglobulin conjugate for 30 rain, the cells were resuspended in 50°7,0 glycerol containing DABCO. T-lymphocyteenriched and -depleted fractions were prepared by rosetting with sheep erythrocytes. Tests were also performed on acetone-fixed cytocentrifuge preparations obtained in a Shandon Cytospin 2. Similar techniques were employed for testing reactivity with cells from established cell lines as already described [7]. Complement-fixing activity was determined by IF as previously described [8]. 3.3. Mr of antigens Detergent extracts of RPMI-8226 cells were run in S D S - P A G E 10% cross-linked gels. Electroblotting onto nitrocellulose membrane was performed for 3 h at 20 V [10]. After exposure to the McAbs, washing and incubating with ~251sheep anti-mouse immunoglobulin, the labelled bands were revealed by autoradiography. M r standards were run simultaneously.
3.2. Immunofluorescence (IF) 3.2.1. Tissue sections The standard procedure used has been described [8]. Cryostat sections of human tonsil (two donors), thyroid and pancreas were cut at 6 ~m and fixed in acetone for 5 min at room temperature. Culture supernates were tested undiluted and ascitic fluids diluted 1:50 in phosphate-buffered saline containing 10°7,0 foetal calf serum. Double fluorescent staining was performed by applying paired mixtures of test antibodies having different immunoglobulin heavy chain isotypes, followed by staining with paired fluorescein and rhodamine conjugates of appropriate specificity for the mouse immunoglobulin isotypes. All IF tests were mounted in glycerol containing diazabicyclo-octane (DABCO) to retard fading during microscopy [91.
296
4. Results
4.1. Plasma cell reactivity The six new McAbs showed cytoplasmic staining of cells of the myeloma line RPMI-8226 in at least 75% of the cells (Table 1). Only two of the antibodies, however (BU9 and BUll), showed surface staining - of approximately 20°7o of the cells. When tested on tonsil sections all six showed cytoplasmic staining of plasma cells, notably in inter-follicular T-dependent areas, in and around germinal centres and in subepithelial areas (Fig. 1 and Table 2). Double staining established that the plasma cells identified by the different McAbs were coincident. Associated reactivity with other features of the tonsil sections was noted in five of the antibodies, as recorded in Table 2. Four of the McAbs stained
Table 1 Reactivity of the new monoclonal antibodies with peripheral blood cells and cell lines Clone
Test"
Peripheral blood lymphocytes T
BU5
C S
BU7
C S
BU9
C
S C
--
Non-T
Cell lines Pre-B (SM-SB)
Other cells
Burkitt (EB-4)
m
+
-4-
m
+
+
Wb
+
-
--
+
B-LCL (Ed-1)
Late B
T-cell (HUT)
Myeloid (HL60)
+
m
m
+
+
Plasmacytoid (RPMI-8226)
ARH-77
WM-I
m
m
m
-
-
-
q_
5%
W
+
+
+
+
+
10% W
10% W
6% +
-
22% +
S
6%
-
2OCHI
C S
_
_
21CC1
C S
20% + -
Bull
+
+
27% + _
_}_
5% W
-
+
3 %
-
-
+
,, c = fixed cytocentrifuge preparations; S = surface membrane staining of viable cells. ~' Weak staining of PMN and monocytes. + = Staining of >75% of the cells; W = weak staining; - = not tested. B - l y m p h o c y t e s in m a n t l e z o n e s a n d o n e o f t h e s e (BU9) also stained T-lymphocytes in interf o l l i c u l a r a r e a s . R e a c t i v i t y w i t h o t h e r cells a n d with tissue fibres was seen with three antibodies. T h e o n l y r e a c t i v i t y d e m o n s t r a b l e o n t o n s i l sec-
t i o n s w i t h O K T I 0 ( a p a r t f r o m p l a s m a cells) w a s w e a k s t a i n i n g o f l y m p h o i d cells in g e r m i n a l c e n t r e s . G e n e r a l f e a t u r e s o f t h e a n t i b o d i e s are a l s o s h o w n i n T a b l e 2. O n e M c A b ( 2 0 C H 1 ) was lgM class and this was the only antibody shown Table 2
G e n e r a l f e a t u r e s o f the new m o n o c l o n a l a n t i b o d i e s Clone
BU5 BU7
Antigen-'
M o u s e Ig
Complement
IF s t a i n i n g o f tonsil sections b
(kDa)
isotype
fixing activity
P l a s m a cells
B lymphocytes
-
+ + + +
+ +
+ +
+
+ -
62 (781 62 (78) (49) 135
GI
BUI 1 20CH1
72 62 (78)
GI M
+ +
+ + + +
+ -
21CC1
62 (78)
G1
-
+ +
-
BU9
G2b A
T lymphocytes
Other reactivity
S m o o t h m u s c l e fibres C o n n . tissue fibres and epithelium E n d o t h e l i u m a n d epithelial b a s a l cells
T h e first figure refers to the m o l e c u l a r mass o f the m a j o r b a n d a n d the b r a c k e t e d figure to a m i n o r b a n d . b I d e n t i c a l r e s u l t s were o b t a i n e d w i t h t o n s i l s f r o m t w o d o n o r s . +, moderate staining; + +, strong staining.
297
298
Fig. 1. l m m u n o f l u o r e s c e n t reactions o f m o n o c l o n a l a n t i b o d i e s on tissue sections. (a) P l a s m a cells in inter-follicular area o f tonsil; M c A b BUS ( × 4 0 0 ) . (b~ T h y r o i d a c i n a r epithelium; M c A b BUT ( × 160). (c) Pancreas a c i n a r a n d duct epithelium; M c A b BU9 ( × 160).
to be complement-fixing in its reaction with plasma cells. 4.2. Epithelial cell reactivity Very bright staining of the cytoplasm of acinar epithelial ceils in thyroid sections was obtained with five of the McAbs. All six stained pancreatic acinar epithelial cells but differences were seen in their reactivity with epithelial cells lining ducts and with islet cells (Fig. 1, Table 3). The most restricted reactivity was seen with 20CHI; OKT10 showed no staining of thyroid and pancreas. 4.3. Peripheral blood cells and haemopoietic cell lines 4.3.1. Blood M N cells Only one of the six McAbs (BU9) stained T cells (surface and cytoplasm); this antibody also stained monocytes and PMN weakly (Table 1). Non-T (E-rosette
negative) lymphocytes were stained by four antibodies in cytocentrifuge preparations and none of these gave surface staining. These results correlated with the staining of lymphocytes in tissue sections. Table 3 Reactivity o f the m o n o c l o n a l a n t i b o d i e s with sections o f epithelial tissue Clone
BU5 BU7 BU9 BUll 20C H 1 21CC1
Thyroid
Pancreas
Acinar epithelium
Acinar epithelium
Duct epithelium
Islet cells
+ + + +
+ + + + + + + +
+ + + + + + +
+ .' +a W
+ + + +
+ +
Selective. W, ; +,
W
; ++,
299
4.3.2. Cell lines One McAb (BU9) showed broad reactivity with the cell panel. The other five did not bind to cells of pre-B, T or myeloid lines. Three of the antibodies (BU5, BU7 and 20CHI) did not stain B-lymphoblastoid cells; of the others one showed broad reactivity with Bcell lines (BU9), 21CC1 stained some cells in the Ed-1 and ARH-77 lines and BUll stained 6% of the Ed-I cells.
Acknowledgements
5. Discussion
References
These findings demonstrate the occurrence of antigens shared by plasma cells and secretory epithelial cells. In addition, four of six new McAbs which showed this reactivity pattern also stained B and/or T lymphocytes in tissue sections and peripheral blood. The remaining two antibodies evidently recognise antigen(s) absent from haemopoietic cells (at least in peripheral tissue) and appearing only in the terminal stages of B-cell differentiation. The patterns of reactivity observed indicate heterogeneity of the antigens involved and this was confirmed by the M r analysis. The antigens revealed by these McAbs are evidently potent immunogens and in secretory cells may be associated with rough endoplasmic reticulum. A systematic study of the reactivity of such McAbs with secretory epithelial cells in a variety of tissues might show differences related to the secretory products of different cells. These antibodies appear to be potentially useful reagents for studying late differentiation in the Bcell lineage.
[l] Anderson, K. C., Bates, M. P., Slaughenhoupt, B., Schlossman, S. F. and Nadler, I_. M. (1984) J. lmmunol. 132, 3172-3179. [2] Anderson, K. C., Park, E. K., Bates, M. F., Leonard, R. C. E, Hardy, R., Schlossman, S. F. and Nadler, L. M. (1983) J. Immunol. 130, 1132-1138. [3] Mertens, N., Dehou, M. F., Vanderbruggen, H., Van Riet, I. and Van Camp, B. (1985) in: Protides Biological Fluids, pp. 891-895, Pergamon Press, Oxford. [4] Reinherz, E. L. and Schlossman, S. E (1981) Immunol. Today 2, 69-75. [5] Matsuoka, ~'\, Moore, G. E., Yagi, Y. and Pressman, D. (1967) Proc. Soc. Exp. Biol. Med. 125, 1246-1250. [6] Galfr6, G., Howe, S. C., Milstein, S., Butcher, G. W. and Howard, J. C. (1977) Nature 266, 550 552. [7] Johnson, G. D., Hardie, D. I,., Ling, N. R. and Maci,ennan, 1. C. N.I. (1986) J. Clin. Exp. hnmunol., in press. [8] Johnson, G. D. and Holborow, E. J. 11986) in: Handbook of Experimental Immunology (D. M. Weir and L. A. Herzenberg, Eds.) 4th Edn, Ch. 31, Blackwells, Edinburgh and Oxford, in press. [91 Johnson, G. D., Davidson, R. S., McNamee, K. C., Ru,sell, G., Gooduin, D. and Hoborow, E. J. 11982) J. Immunol. Methods 55, 231 242. [10] Towbin, H., Staehelin, T. and Gordon, J. (19791. Proc. Natl. Acad. Sci. U.S.A. 76(9), 4350-4354.
300
This work was supported by Programme Grant PG 840 2371 from the Medical Research Council and by a grant from Unilever. G.D.J. is a member of the M.R.C. external scientific staff. The secretarial assistance of Ms. E O'Reilly is gratefully acknowledged. P.D.N. is in receipt of an M.R.C. Studentship.