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A modified plaque test for the detection of cells forming antibody to alloantigens
Journal of Immunological Methods 2 (1973) 213-219. © North-Holland Publishing Company
A MODIFIED PLAQUE TEST FOR THE DETECTION OF CELLS FORMING ANTIBODY TO ALLOANTIGENS G.M. T A Y L O R and Joan BENNETT * Charles Salt Research Centre, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, S Y I O 7A G, England
Received 1 November 1972
Accepted 8 November 1972
Cells secreting antibody to H-2 alloantigens can be detected in vitro by the alloantibody plaque assay. This test has been modified to allow the earlier and more objective determination of plaques by staining the lysed target cells with trypan blue. Details are given of optimum conditions necessary for the assay.
1. INTRODUCTION Cells secreting antibody can be detected in vitro by the haemolytic plaque technique (Jerne et al., 1963; lngraham and Bussard, 1964), in which lymphoid cell suspensions from recipients immunised with sheep red blood cells (SRBC) are mixed with target SRBC in a gel supporting medium and spread thinly in a petri dish or on a microscope slide. A n t i b o d y plaque forming cells (PFC) are denoted by areas of specific localised haemolysis in the gel following the addition of complement. This method has been widely used to study the kinetics of antibody formation. ( F o r review see Abdou and Richter, 1970.) The problems associated with the development of a plaque assay for alloantibody secreting cells centre around the selection o f a suitable target cell, and the method o f plaque detection. Hildeman and Pinkerton (1966) detected plaques in immunised mice using erythrocytes as target cells. Fuji et al. (1971a) had difficulty in reproducing these results, but detected plaques in mice immunised with 0 antigens using allogeneic (i.e. 0 incompatible) thymocytes. Recently, two laboratories have reported on the successful use of ascitic tumour cells as targets for alloantibody secreting cells (Fuji et al., 1971b; Nordin et al., 197t). Lymphoid cells from mice immunised with H-2 incompatible tissues were mixed with ascitic tumour target cells and incubated in a gel in the presence of complement. The clear areas of localised cytolysis which resulted were similar in appearance to haemolytic plaques. * Joan Bennett is a student from the Trent Polytechnic, Nottingham.
214
G.M. TAYLOR and J. BENNETT
In a modified alloantibody plaque assay we have made use of the permeability to trypan blue of nucleated cells killed by alloantibody and complement (Gofer and O'Gorman, 1955). We report here experiments which were undertaken to determine the optimum conditions for plaque development.
2. MATERIALS AND METHODS
2.1. Animals and immunisations BALB/c (H-2 d) mice bred in this laboratory were used throughout this study. The mice were immunised by intraperitoneal inoculation of 2 X 107 C57B1 spleen or EL4 lymphoma (H-2 b) cells 0.1 ml Eagle's medium (MEM).
2.2. Target cells Tumour target cells for use in the plaque test were EIA (H-2 b) ascites lymphoma cells maintained by intraperitoneal passage in C57B1 mice.
2.3. Complement Serum from New Zealand White rabbits was used as a source of complement diluted 1/10 with MEM. At this dilution rabbit serum was found to be non-toxic for EL4 cells.
2.4. Preparation of cells Spleens were removed after 8 - 1 1 days when peak numbers of plaque forming cells were found (Taylor and Bennett, in preparation). The spleen cells were brought into suspension in cooled Eagle's medium + 1% foetal calf serum (MEM + 1% FCS) by gentle pressure in a loose fitting glass homogeniser, filtered through a stainless steel 200 mesh gauze filter, and spun at 800 rpm for 3 - 4 min. The supernatant was removed, the cells resuspended in MEM + 1% FCS and spun again, after which the concentration was adjusted to 120 × 106/ml or as stated. The target cells were harvested in cold MEM + 1% FCS, filtered and washed as described above, and adjusted to 400 X 106/ml or as stated.
2.5. Plaque assay Equal volumes (0.15 ml) of spleen and target cell suspensions were mixed and heated to 3 9 - 4 0 ° C in a water bath. To this was added 1.2 ml of 0.5% agarose containing MEM, prepared as follows: 50 mg of agarose (Serva) was melted in 8.5 ml sterile distilled water on boiling water bath, the gel cooled to 40°C, and 1 ml
Modified alloantibody plaque assay
215
of prewarmed 10X concentrated Eagle's medium and 0.25 ml of 4.4% NaHCO 3 added. The plastic petri dishes (60 × 15 mm) receiving the agarose/cell mixtures were precoated with 1 ml of 1% agarose in phosphate buffered saline (PBS) and sterilised under ultraviolet light. The cell mixtures were drawn into prewarmed 2 ml plastic syringes, and 0.5 ml dispensed on to each dish. The mixture was spread evenly by swirling the dish, and allowed to set at 4°C in an atmosphere of 95% air/5% CO 2. Following the addition of 5 ml MEM + 1% FCS to each dish, incubation was carried out at 37°C for 1 hr in the above atmosphere. The dishes were then washed briefly in sterile phosphate buffered saline and 5 ml of MEM containing normal rabbit serum at a dilution of 1/10 added to each dish. The dishes were gassed and incubated for a further 1 hr, then washed twice with 5 ml volumes of PBS, and stained with 5 ml of 0.16% trypan blue in saline for 15 rain, followed by a further 2 or 3 washes with PBS. Plaques were counted immediately under a binocular microscope. Geometric means of plaque counts for 3 plates from each spleen were calculated according to the method of Dresser and Wortis (1967). 3. RESULTS We spread agarose mixtures of lymphoid cells from mice immunised with H-2 incompatible cells and target lymphoma cells in petri dishes and incubated them for
Fig. 1. Alloantibody plaque. Tumour cells killed by antibody and C' stained with trypan blue, surrounded by unstained live ceils. (Phase contrast X75)
216
G.M. TAYLOR and J. BENNETT Table 1 Effect of incubation and target cell numbers on the appearance of alloantibody plaques.
No. spleen cells/ml X 106
No. target cells/ml × 106
120 120 120
400 200 100
Appearance of plaques at: * (hr) 0
l
2
+++ ++
++
++
* Plates examined 0, 1, 2 hr after the complement incubation stage. 3 plates examined for each time and cell concentration, and scored thus: +++, plaques clear, well stained; ++ plaques diffuse and/or high background mortality; - plaques indistinct or absent. 1 hr, followed by a further hr treated with c o m p l e m e n t . On the addition of trypan blue, dense localised loci or plaques of blue stained cells in a background o f mainly unstained cells could be seen (fig. 1). The plaques, which measure 0 . 1 - 0 . 2 m m in diameter, are fairly uniform in size, and stain strongly at their centres and weakly at the periphery. There was no appreciable difference in the n u m b e r o f plaques developed using rabbit serum diluted 1/5 or 1/10 and we have elected to use the latter dilution. As the n u m b e r o f target cells was progressively reduced, or the time o f incubation after the addition of c o m p l e m e n t prolonged, the ease o f visualisation o f the plaques greatly diminished (table 1). The n u m b e r of dead t u m o u r cells after 24 hr was too great to allow adequate identification o f plaques. As e x p e c t e d , the n u m b e r o f plaques increased in p r o p o r t i o n to the increase in the n u m b e r o f spleen cells, although the n u m b e r o f plaques at the highest spleen cell c o n c e n t r a t i o n was lower than e x p e c t e d (table 2) and suggests an inhibitory effect. For routine tests a spleen cell c o n c e n t r a t i o n o f 120 × 106/ml is the m o s t useful, e x c e p t w h e n assaying l y m p h nodes w h e n the whole suspension is divided b e t w e e n 2 dishes. The effect o f c o m p l e m e n t and m e r c a p t o e t h a n o l on plaque f o r m a t i o n is shown in table 3. R a b b i t serum diluted 1/10 or normal MEM + 1% FCS was added to dishes Table 2 Effect of numbers of spleen cells on the numbers of plaques. No. spleen ceUs/ml ~ × 106
No. target cells/ml X 106
No. PFC/plate *
Nos. PFC/106 cells *
75 150 300
400 400 400
127.3 207.5 292.3
43.6 26.6 18.7
* Geometric mean of three plates. t BALB/c 9 received 2 X 107 C57B1 spleen cells 8 days previously.
Modified alloantibody plaque assay
217
Table 3 In vitro requirements for alloantibody plaque formation. Exp. No.
Treatment
No. PFC/plate
l *
+ C' -C' - 2-Me + 2-Me
281,243,334 0,0,0 233,287,269 0, 0, 0
2
• BALB/c (2 received 2 × 107 C57B1 spleen cells 11 (Exp. 1) or 10 (Exp. 2) days previously. with the same sample of spleen cells. Plaque formation was inhibited when complement was absent. In the second experiment 2-mercaptoethanol (0.1 M in MEM)or normal MEM was added to dishes for 1 hr at 37°C prior to the addition of complement. Plaque development was completely abolished by 2-mercaptoethanol without there being any toxic effect on the cells in the agarose gel, compared with tile plates treated with normal MEM, as ascertained by a lack of staining with trypan blue. It thus seems likely that plaque formation studied here is due to an IgM complement fixing alloantibody. Table 4 Specificity of plaque formation. Inoculum *
-
BALB/c spleen Sheep red cells C57B1 spleen
No. animals tested
Mean plaques/spleen +ve **
12 3 3 7
11.0 -+6.2 0 0 8677.9 -+ 2954.0
* Groups of BALB/c mice received 2 × 107 washed cells 10 11 days previously. ** Arithmetic means. Finally, we have examined the specificity of immunisation, the results of which are depicted in table 4. The spleens of 4 out of 12 normal untreated BALB/c mice had small numbers of plaque forming cells, ranging from 8 - 6 7 PFC/spleen. These naturally occurring plaques were generally smaller than plaques from stimulated mice. Taken with the 8 mice which were negative the arithmetic mean for the group is I 1.0 PFC/spleen. Neither the group receiving BALB/c spleen cells nor sheep red blood cells had PFC, whilst the mean for the group injected with C57B1 spleen was 8577, with a m a x i m u m - m i n i m u m range of 1 4 1 2 - 2 2 , 9 4 1 .
218
G.M. TAYLORand J. BENNETT
4. DISCUSSION The essential feature of the alloantibody plaque assay described here is that the tumour target cells lysed by IgM alloantibody and complement have been stained with trypan blue, and the foci of stained cells thus developed shown to contrast sharply with the unstained background target cells. Since cytolysis by alloantibody and complement results in the permeability of cells to trypan blue (Gorer and O'Gorman, 1955) the blue staining plaques should indicate the presence of alloantibody secreting cells. The fact that plaque formation was inhibited in the absence of complement, and abolished by treatment with 0.2 M-2-mercaptoethanot bears out this conclusion. Plaque formation was optimal at a target cell concentration of 400 X 106/ml (that is 20 X 106 cells/plate), and did not require overnight incubation as Nordin et al. (1971) describe in their assay. Moreover, the plates needed no further treatment after staining, other than brief washings with PBS to remove excess trypan blue. The concentration of spleen cells is of minimal importance in the assay, except at high levels, where fewer plaques were noted. This contrasts with the view of Fuji et al. (1971a) that a ratio of 5 tumour cells to 1 spleen cell was essential, since a ratio of 3.3 : 1 was consistently used here without any variation in plaquing efficiency. Although the exact specificity of the alloantibody secreted by spleen cells in this test has not been established, the evidence of Nordin et al. (1971) using different strains immunised with the same target cell, and Fuji et al. (1971b) using congenic strains indicates that H-2 alloantigens are involved. During assays of normal nonimmunised BALB/c mice, plaque forming cells were detected in 33% of the mice. The specificity of these natural allo- or possibly auto-antibody forming cells is as yet unknown except that they fit the requirements of plaque development as defined here. The range of PFC in mice stimulated with allogenic target cells was surprisingly large at the peak response, with considerable variation between individual mice which could not be assigned to low plaquing efficiency. Similar results were obtained by Fuji et al. (1971b), who administered allogeneic spleen cells intravenously. It seems possible that a graft-versus-host reaction on the part of the donor spleen cells could depress the host PFC response, though information on this is not available. We have examined harvests of EL4 cells from tumour bearers and plated them without allogeneic plaque forming cells. No plaques have ever been seen, which further indentifies the allogeneic spleen cells as the source of PFC. This does not rule out the possibility that PFC could be developed against turnout specific antigens of the syngeneic EL4 lymphoma. Indeed, in preliminary studies we have found small numbers of probable tumour-specific PFC in the spleens of routine C57B1 tumour bearers.
Modified alloantibody plaque assay
219
ACKNOWLEDGEMENTS We thank Mr. N.W. Nisbet, F.R.C.S.,for hisinterest, and the Medical Research Council for a grant.
REFERENCES Abdou, N.L. and M. Richter, 1970, Adv. lmmunol. 12,201. Dresser, D.W. and H.H. Wortis, 1967, in: Handbook of experimental immunology, ed. D.M. Weir (Blackweil Scientific Publications, Oxford) p. 1054. Fuji, H., M. Zaleski and F. Milgrom, 1971a, J. Immunol. 106, 56. Fuji, H., M. Zaleski and F. Milgrom, 1971b, Proc. Soc. Exptl. Biol. Med. 136,239. Gorer, P.A. and P. O'Gorman, 1955, Trnspl. Bull. 2, 124. Hildeman, W.H. and W. Pinkerton, 1966, J. Exptl. Med. 124, 885. Ingraham, J.S. and A. Bussard, 1964, J. Exptl. Med. 119,667. Jerne, N.K., A.A. Nordin, C. Henry, 1963, in: Cell bound antibodies, ed. B. Amos and H. Koprowski (Wistar Institute Press) p. 109. Nordin, A.A., J.-C. Cerottini and K.T. Brunner, 1971, Eur. J. lmmunol. 1,55.
A MODIFIED PLAQUE TEST FOR THE DETECTION OF CELLS FORMING ANTIBODY TO ALLOANTIGENS G.M. T A Y L O R and Joan BENNETT * Charles Salt Research Centre, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, S Y I O 7A G, England
Received 1 November 1972
Accepted 8 November 1972
Cells secreting antibody to H-2 alloantigens can be detected in vitro by the alloantibody plaque assay. This test has been modified to allow the earlier and more objective determination of plaques by staining the lysed target cells with trypan blue. Details are given of optimum conditions necessary for the assay.
1. INTRODUCTION Cells secreting antibody can be detected in vitro by the haemolytic plaque technique (Jerne et al., 1963; lngraham and Bussard, 1964), in which lymphoid cell suspensions from recipients immunised with sheep red blood cells (SRBC) are mixed with target SRBC in a gel supporting medium and spread thinly in a petri dish or on a microscope slide. A n t i b o d y plaque forming cells (PFC) are denoted by areas of specific localised haemolysis in the gel following the addition of complement. This method has been widely used to study the kinetics of antibody formation. ( F o r review see Abdou and Richter, 1970.) The problems associated with the development of a plaque assay for alloantibody secreting cells centre around the selection o f a suitable target cell, and the method o f plaque detection. Hildeman and Pinkerton (1966) detected plaques in immunised mice using erythrocytes as target cells. Fuji et al. (1971a) had difficulty in reproducing these results, but detected plaques in mice immunised with 0 antigens using allogeneic (i.e. 0 incompatible) thymocytes. Recently, two laboratories have reported on the successful use of ascitic tumour cells as targets for alloantibody secreting cells (Fuji et al., 1971b; Nordin et al., 197t). Lymphoid cells from mice immunised with H-2 incompatible tissues were mixed with ascitic tumour target cells and incubated in a gel in the presence of complement. The clear areas of localised cytolysis which resulted were similar in appearance to haemolytic plaques. * Joan Bennett is a student from the Trent Polytechnic, Nottingham.
214
G.M. TAYLOR and J. BENNETT
In a modified alloantibody plaque assay we have made use of the permeability to trypan blue of nucleated cells killed by alloantibody and complement (Gofer and O'Gorman, 1955). We report here experiments which were undertaken to determine the optimum conditions for plaque development.
2. MATERIALS AND METHODS
2.1. Animals and immunisations BALB/c (H-2 d) mice bred in this laboratory were used throughout this study. The mice were immunised by intraperitoneal inoculation of 2 X 107 C57B1 spleen or EL4 lymphoma (H-2 b) cells 0.1 ml Eagle's medium (MEM).
2.2. Target cells Tumour target cells for use in the plaque test were EIA (H-2 b) ascites lymphoma cells maintained by intraperitoneal passage in C57B1 mice.
2.3. Complement Serum from New Zealand White rabbits was used as a source of complement diluted 1/10 with MEM. At this dilution rabbit serum was found to be non-toxic for EL4 cells.
2.4. Preparation of cells Spleens were removed after 8 - 1 1 days when peak numbers of plaque forming cells were found (Taylor and Bennett, in preparation). The spleen cells were brought into suspension in cooled Eagle's medium + 1% foetal calf serum (MEM + 1% FCS) by gentle pressure in a loose fitting glass homogeniser, filtered through a stainless steel 200 mesh gauze filter, and spun at 800 rpm for 3 - 4 min. The supernatant was removed, the cells resuspended in MEM + 1% FCS and spun again, after which the concentration was adjusted to 120 × 106/ml or as stated. The target cells were harvested in cold MEM + 1% FCS, filtered and washed as described above, and adjusted to 400 X 106/ml or as stated.
2.5. Plaque assay Equal volumes (0.15 ml) of spleen and target cell suspensions were mixed and heated to 3 9 - 4 0 ° C in a water bath. To this was added 1.2 ml of 0.5% agarose containing MEM, prepared as follows: 50 mg of agarose (Serva) was melted in 8.5 ml sterile distilled water on boiling water bath, the gel cooled to 40°C, and 1 ml
Modified alloantibody plaque assay
215
of prewarmed 10X concentrated Eagle's medium and 0.25 ml of 4.4% NaHCO 3 added. The plastic petri dishes (60 × 15 mm) receiving the agarose/cell mixtures were precoated with 1 ml of 1% agarose in phosphate buffered saline (PBS) and sterilised under ultraviolet light. The cell mixtures were drawn into prewarmed 2 ml plastic syringes, and 0.5 ml dispensed on to each dish. The mixture was spread evenly by swirling the dish, and allowed to set at 4°C in an atmosphere of 95% air/5% CO 2. Following the addition of 5 ml MEM + 1% FCS to each dish, incubation was carried out at 37°C for 1 hr in the above atmosphere. The dishes were then washed briefly in sterile phosphate buffered saline and 5 ml of MEM containing normal rabbit serum at a dilution of 1/10 added to each dish. The dishes were gassed and incubated for a further 1 hr, then washed twice with 5 ml volumes of PBS, and stained with 5 ml of 0.16% trypan blue in saline for 15 rain, followed by a further 2 or 3 washes with PBS. Plaques were counted immediately under a binocular microscope. Geometric means of plaque counts for 3 plates from each spleen were calculated according to the method of Dresser and Wortis (1967). 3. RESULTS We spread agarose mixtures of lymphoid cells from mice immunised with H-2 incompatible cells and target lymphoma cells in petri dishes and incubated them for
Fig. 1. Alloantibody plaque. Tumour cells killed by antibody and C' stained with trypan blue, surrounded by unstained live ceils. (Phase contrast X75)
216
G.M. TAYLOR and J. BENNETT Table 1 Effect of incubation and target cell numbers on the appearance of alloantibody plaques.
No. spleen cells/ml X 106
No. target cells/ml × 106
120 120 120
400 200 100
Appearance of plaques at: * (hr) 0
l
2
+++ ++
++
++
* Plates examined 0, 1, 2 hr after the complement incubation stage. 3 plates examined for each time and cell concentration, and scored thus: +++, plaques clear, well stained; ++ plaques diffuse and/or high background mortality; - plaques indistinct or absent. 1 hr, followed by a further hr treated with c o m p l e m e n t . On the addition of trypan blue, dense localised loci or plaques of blue stained cells in a background o f mainly unstained cells could be seen (fig. 1). The plaques, which measure 0 . 1 - 0 . 2 m m in diameter, are fairly uniform in size, and stain strongly at their centres and weakly at the periphery. There was no appreciable difference in the n u m b e r o f plaques developed using rabbit serum diluted 1/5 or 1/10 and we have elected to use the latter dilution. As the n u m b e r o f target cells was progressively reduced, or the time o f incubation after the addition of c o m p l e m e n t prolonged, the ease o f visualisation o f the plaques greatly diminished (table 1). The n u m b e r of dead t u m o u r cells after 24 hr was too great to allow adequate identification o f plaques. As e x p e c t e d , the n u m b e r o f plaques increased in p r o p o r t i o n to the increase in the n u m b e r o f spleen cells, although the n u m b e r o f plaques at the highest spleen cell c o n c e n t r a t i o n was lower than e x p e c t e d (table 2) and suggests an inhibitory effect. For routine tests a spleen cell c o n c e n t r a t i o n o f 120 × 106/ml is the m o s t useful, e x c e p t w h e n assaying l y m p h nodes w h e n the whole suspension is divided b e t w e e n 2 dishes. The effect o f c o m p l e m e n t and m e r c a p t o e t h a n o l on plaque f o r m a t i o n is shown in table 3. R a b b i t serum diluted 1/10 or normal MEM + 1% FCS was added to dishes Table 2 Effect of numbers of spleen cells on the numbers of plaques. No. spleen ceUs/ml ~ × 106
No. target cells/ml X 106
No. PFC/plate *
Nos. PFC/106 cells *
75 150 300
400 400 400
127.3 207.5 292.3
43.6 26.6 18.7
* Geometric mean of three plates. t BALB/c 9 received 2 X 107 C57B1 spleen cells 8 days previously.
Modified alloantibody plaque assay
217
Table 3 In vitro requirements for alloantibody plaque formation. Exp. No.
Treatment
No. PFC/plate
l *
+ C' -C' - 2-Me + 2-Me
281,243,334 0,0,0 233,287,269 0, 0, 0
2
• BALB/c (2 received 2 × 107 C57B1 spleen cells 11 (Exp. 1) or 10 (Exp. 2) days previously. with the same sample of spleen cells. Plaque formation was inhibited when complement was absent. In the second experiment 2-mercaptoethanol (0.1 M in MEM)or normal MEM was added to dishes for 1 hr at 37°C prior to the addition of complement. Plaque development was completely abolished by 2-mercaptoethanol without there being any toxic effect on the cells in the agarose gel, compared with tile plates treated with normal MEM, as ascertained by a lack of staining with trypan blue. It thus seems likely that plaque formation studied here is due to an IgM complement fixing alloantibody. Table 4 Specificity of plaque formation. Inoculum *
-
BALB/c spleen Sheep red cells C57B1 spleen
No. animals tested
Mean plaques/spleen +ve **
12 3 3 7
11.0 -+6.2 0 0 8677.9 -+ 2954.0
* Groups of BALB/c mice received 2 × 107 washed cells 10 11 days previously. ** Arithmetic means. Finally, we have examined the specificity of immunisation, the results of which are depicted in table 4. The spleens of 4 out of 12 normal untreated BALB/c mice had small numbers of plaque forming cells, ranging from 8 - 6 7 PFC/spleen. These naturally occurring plaques were generally smaller than plaques from stimulated mice. Taken with the 8 mice which were negative the arithmetic mean for the group is I 1.0 PFC/spleen. Neither the group receiving BALB/c spleen cells nor sheep red blood cells had PFC, whilst the mean for the group injected with C57B1 spleen was 8577, with a m a x i m u m - m i n i m u m range of 1 4 1 2 - 2 2 , 9 4 1 .
218
G.M. TAYLORand J. BENNETT
4. DISCUSSION The essential feature of the alloantibody plaque assay described here is that the tumour target cells lysed by IgM alloantibody and complement have been stained with trypan blue, and the foci of stained cells thus developed shown to contrast sharply with the unstained background target cells. Since cytolysis by alloantibody and complement results in the permeability of cells to trypan blue (Gorer and O'Gorman, 1955) the blue staining plaques should indicate the presence of alloantibody secreting cells. The fact that plaque formation was inhibited in the absence of complement, and abolished by treatment with 0.2 M-2-mercaptoethanot bears out this conclusion. Plaque formation was optimal at a target cell concentration of 400 X 106/ml (that is 20 X 106 cells/plate), and did not require overnight incubation as Nordin et al. (1971) describe in their assay. Moreover, the plates needed no further treatment after staining, other than brief washings with PBS to remove excess trypan blue. The concentration of spleen cells is of minimal importance in the assay, except at high levels, where fewer plaques were noted. This contrasts with the view of Fuji et al. (1971a) that a ratio of 5 tumour cells to 1 spleen cell was essential, since a ratio of 3.3 : 1 was consistently used here without any variation in plaquing efficiency. Although the exact specificity of the alloantibody secreted by spleen cells in this test has not been established, the evidence of Nordin et al. (1971) using different strains immunised with the same target cell, and Fuji et al. (1971b) using congenic strains indicates that H-2 alloantigens are involved. During assays of normal nonimmunised BALB/c mice, plaque forming cells were detected in 33% of the mice. The specificity of these natural allo- or possibly auto-antibody forming cells is as yet unknown except that they fit the requirements of plaque development as defined here. The range of PFC in mice stimulated with allogenic target cells was surprisingly large at the peak response, with considerable variation between individual mice which could not be assigned to low plaquing efficiency. Similar results were obtained by Fuji et al. (1971b), who administered allogeneic spleen cells intravenously. It seems possible that a graft-versus-host reaction on the part of the donor spleen cells could depress the host PFC response, though information on this is not available. We have examined harvests of EL4 cells from tumour bearers and plated them without allogeneic plaque forming cells. No plaques have ever been seen, which further indentifies the allogeneic spleen cells as the source of PFC. This does not rule out the possibility that PFC could be developed against turnout specific antigens of the syngeneic EL4 lymphoma. Indeed, in preliminary studies we have found small numbers of probable tumour-specific PFC in the spleens of routine C57B1 tumour bearers.
Modified alloantibody plaque assay
219
ACKNOWLEDGEMENTS We thank Mr. N.W. Nisbet, F.R.C.S.,for hisinterest, and the Medical Research Council for a grant.
REFERENCES Abdou, N.L. and M. Richter, 1970, Adv. lmmunol. 12,201. Dresser, D.W. and H.H. Wortis, 1967, in: Handbook of experimental immunology, ed. D.M. Weir (Blackweil Scientific Publications, Oxford) p. 1054. Fuji, H., M. Zaleski and F. Milgrom, 1971a, J. Immunol. 106, 56. Fuji, H., M. Zaleski and F. Milgrom, 1971b, Proc. Soc. Exptl. Biol. Med. 136,239. Gorer, P.A. and P. O'Gorman, 1955, Trnspl. Bull. 2, 124. Hildeman, W.H. and W. Pinkerton, 1966, J. Exptl. Med. 124, 885. Ingraham, J.S. and A. Bussard, 1964, J. Exptl. Med. 119,667. Jerne, N.K., A.A. Nordin, C. Henry, 1963, in: Cell bound antibodies, ed. B. Amos and H. Koprowski (Wistar Institute Press) p. 109. Nordin, A.A., J.-C. Cerottini and K.T. Brunner, 1971, Eur. J. lmmunol. 1,55.