- Email: [email protected]
Heterogeneity of antibodies produced by single hemolytic foci
CELLULAR
2, 250-258
IMMUNOLOGY
(1971)
Heterogeneity
of
Antibodies
Single
Hemolytic
P. A. Department
of Biology,
University
Produced Foci ’
CAMPBELL
of California, Recrived
by
’ San
Diego,
La Jolla,
California
92037
January 4,197l
Lethally irradiated male BDF, mice were injected with 10’ bone marrow cells, 8 X 10s spleen cells, and sheep or goat red blood cells as antigens. The cross-reactivity between these red blood cells is approximately 40%, as determined by injecting normal mice with either sheep or goat red blood cells, removing their spleens 4 days later, and assaying all spleens for both antisheep and antigoat plaque-forming cells. Eight days after injection of bone marrow, spleen, and antigen, the spleens of the irradiated recipients were removed and assayed for the presence of hemolytic foci by the Playfair technique, and were found to contain an average of 0.7 foci/spleen. Earlier studies had demonstrated that such foci could contain plaque-forming cells derived from more than one precursor cell. The positive pieces comprising a single hemolytic focus were removed, pooled, and aliquots were assayed for direct and indirect plaque-forming cells to the immunizing antigen. Several foci were found to contain 17-827 0 as many plaque-forming cells lysing the cross-reacting antigen as lysed the immunizing antigen. These data indicate that these foci contained plaque-forming cells of two specificities: (1) plaque-forming cells which responded to determinants on the immunizing antigen only, and (2) plaque-forming cells which responded to determinants on bo’th the immunizing and cross-reacting antigens. In addition, single foci were found which contained both direct and indirect plaqueforming cells lysing the immunizing antigen. Sixteen of 39 foci assayed contained more than twice as many indirect as direct plaque-forming cells. We concluded that a single hemolytic focus, probably derived from more than one precursor cell, could contain plaque-forming cells producing antibodies of more than one set of specificities and immunoglobulins of more than one type.
INTRODUCTION Mouse spleens contain all the cellular components required for initiation of an immune response to sheep red blood cell antigens. Among these cellular components are the thymus-derived cells (T cells), which initiate the immune response, and bone marrow-derived precursor cells (B cells), which are direct progenitors of 1 This research was AI-08795 to Dr. Richard 2 Supported s Kind,
by United
supported W. Dutton. States
P., and P. A. Campbell,
by
United
Public
Health
unpublished
States Service results. 250
Public
Health
Postdoctoral
Service Fellowship
Research -41-40639.
Grant
HETEROGENEOUS
ANTIBODIES
IN
HEMOLYTIC
FOCI
251
antibody-forming cells ( l-10). experiments examining these immunoD uring competent cells, it was discovered that injection of small numbers of normal spleen cells or thoracic duct lymphocytes and red blood cell antigens into lethally irradiated mice led to the appearance of distinct regions of hemolytic activity in the spleens of these mice after 8 days (11,12). The number of these hemolytic foci is directly proportional to the number of spleen cells or thoracic duct lymphocytes injected (1, lo-13), and is not increased by the addition of bone marrow cells to the inoculum (10, 14). However, addition of bone marrow does result in an increased splenic plaque-forming cell response (10, 14, 15). We have recently shown that this increase in plaque-forming cells upon addition of bone marrow is due, at least in part, to the presence of more than one precursor cell in single hemolytic foci (10). Spleen cells bearing H 2 bd histocompatibility antigens and bone marrow cells carrying H 2 d antigens were injected into lethally irradiated recipient H 2 bd mice. Using antisera directed against H 2 b or H 2 d antigens, it was shown that single hemolytic foci could contain some plaque-forming cells bearing antigens found on the injected spleen cells, and some plaque-forming cells bearing antigens on the injected bone marrow cells. These data are compatible with a model in which a single immunocompetent unit contains both a thymusderived cell, which is required to initiate the immune response, and one or more bone marrow-derived cells, which determine the magnitude of the response (5, 10, 16, 17). Therefore, the number of hemolytic foci would be directly proportional to the number of T cells injected, while the number of plaque-forming cells per focus would be dependent on the number of B cells injected. Since a single focus can contain progeny of more than one precursor cell, we asked the questions: Can the plaque-forming cells in a single hemolytic focus produce antibodies of more than one specificity ? Can the plaque-forming cells in a single hemolytic focus produce antibodies of more than one type; that is, both direct and indirect plaque-forming cells ? The experiments described below were designed to answer these questions. The data suggest that a single hemolytic focus can contain precursor cel!s whose plaqueforming cell progeny can produce antibodies of more than one specificity and more than one immunoglobulin type. MATERIALS
AND
METHODS
Uice. Male and female BDF, (C57B1/6J X DBA/2J) mice, aged 6-16 weeks, were used throughout these experiments. They were fed and watered ad Zibitum. The drinking water contained 16 ppm chlorine to decrease Pseudomonas infections. Antigens. Sheep red blood cells (SRBC) and goat red blood cells (GRBC) were purchased from Colorado Serum Company preserved in Alsever’s solution. A single lot of each antigen was used throughout the experiments. The cells were washed two to three times and resuspended in balanced salt solution (BSS) (18) prior to use. Irradiation. Mice were irradiated with 990-1024 R from a 6oCo source through the courtesy of Mr. Irving Goldman and the Salk Institute for Biological Studies. All mice were injected within 2-20 hr after irradiation. Plaque-forming cell assay. Plaque-forming cells were detected according to the
252
CAMPBELT.
method of Jerne and Nordin (19) using the microscope slide modification ( 18). All slides were incubated in a humidified environment at 37°C for 1 hr without complement, and 2 hr with complement with or without 1: 200 dilution of developing antiserum. The complement source used was a 1 : 10 dilution of guinea pig serum. Developing antiserum. The developing antiserum had been prepared and tested according to the following procedure.” Rabbit gamma globulin was precipitated from normal rabbit serum using 50% saturated ammonium sulfate. Following removal of the ammonium sulfate by dialysis, the rabbit gamma globulin was injected into LAF, mice over a period of several weeks. The immune mice were bled, and antibodies in their serum were precipitated with the rabbit gamma globulin antigen. Normal rabbits were injected with the washed immune precipitate containing rabbit gamma globulin and mouseantirabbit gamma globulin in water-inoil emulsion. The rabbits were bled after several injections, their sera were collected, and the developing antiserum was titrated. A dilution of 1 : 200 rabbit antiserum was found to give the maximum number of indirect plaque-forming cells and little or no inhibition of direct plaque-forming cells. Hemolytic foci. The presence of hemolytic foci in mouse spleens was detected by a modification (13) of the method of Playfair et al (11). In the experiments reported here, lethally irradiated mice were injected intravenously with 8 X lo5 spleen cells and 6.613.5 X lo6 bone marrow cells. The mice received 0.1 ml 20% SRBC or GRBC intravenously on Day 1 after irradiation and intraperitoneally 3 days later. On Day 8, the mice were killed by cervical dislocation and the spleen removed. Each spleen was cut transversely into 10-15 slices, then each slice was cut into three to 8 pieces, approximately 1-2 mm3 in size. The pieces were carefully placed in a petri dish on a base layer of lo/O agarose in BSS so that the orientation of the pieces in the original organ was maintained. The spleen pieces were overlaid with 4 ml of 0.5% agarose containing 0.2 ml 40% RBC with which they were immunized and incubated at 37°C for 1.5 hr. Ten percent guinea pig complement which had been absorbed one to three times with mouse spleen and thymus cells was added, and the plates incubated an additional l/2 hr at 37°C. The complement was then removed, the plates flooded with cold BSS, and the hemolytic foci counted. Zones of hemolysis around adjacent spleen pieces indicated the presence of a single hemolytic focus. RESULTS Experimental
Design
Lethally irradiated mice were injected with 8 X lo5 spleencells and with approximately lo7 bone marrow cells. In this manner, a single focus could be expected to contain more than one precursor cell, as reported earlier (10). The hemolytic foci were developed, as described above, and the pieces from a single hemolytic focus were removed from the agarose plate, pooled, and teased into a single-cell suspension.The cells were sedimented by centrifugation, and resuspendedin 0.3 ml BSS. One hundred ,pl of the cell suspensionwere then assayed for the number of direct plaque-forming cells responding to the immunizing antigen, 100 ~1 were assayed for plaque-forming cells responding to the cross-reacting antigen, and
HETEROGENEOUS
ANTIBODIES
IN
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253
25-100 ~1 of the remaining suspension were assayed for indirect plaque-forming cells responding to the immunizing antigen. In most experiments, the immunizing antigen was GRUC, while in some experiments SRBC were used as the immunizing antigen. In either case, recipient spleens contained less than one hemolytic focus, to avoid chance overlap of two foci. Each hemolytic focus contained an average of four to six positive pieces. Most mouse spleens were cut into a total of 40-50 pieces. Following the above procedure, we could determine whether plaque-forming cells in a single focus could make antibodies of more than one specificity or more than one immunoglobulin type. Percentage of cross-reacfivity of SRBC and GRBC. Two experiments were conducted to determine the percentage of cross-reactivity of the two lots of SRBC and GRBC used in these experiments. For each experiment, two mice were injected intravenously with 0.2 ml 10% SRBC, and two mice were injected intravenously with the same dose of GRBC. On Day 4 after injection in one experiment and Day 5 in the second experiment, the splenic plaque-forming cells responding to each antigen were assayed. There were 345% as many cells lysing GRBC as there were lysing SRBC in the spleens of the mice immunized with SRBC. The spleens of mice immunized with GRBC contained 475% as many plaque-forming cel!s aaginst SRIJC as against GRBC. That is, the cross-reactivity of GRBC with SRBC was 34%, and of SRBC with GRBC was 47%, for an average crossreactivity of 40%. Specificity of plaque-forwing cells &z single foci. A total of 191 mice injected with spleen and bone marrow were analyzed for the presence of hemolytic foci. The average number of foci per spleen in these mice was 0.72. Of the 92 foci whose pieces were removed for analysis, 53 contained less than 10 detectable direct plaque-forming cells to the immunizing antigen in the loo-p1 aliquot assayed. These foci presumably had less than 30 recoverable direct plaque-forming cells lysing the immunizing antigen, since one-third of each focus was assayed for these plaque-forming cells. The remaining foci are discussed below. The 39 foci presented in Table 1 represent data from eight experiments. All foci in which 10 or more plaque-forming cells responding to the immunizing antigen could be detected in the loo-p1 aliquot are reported; less than 10 plaque-forming cells was arbitrarily considered not significant. Foci containing less than eight plaque-forming cells lysing the cross-reacting RBC are listed together at the bottom of the table. The reason for the selection of eight as a minimum significant number of cross-reacting plaque-forming cells is illustrated in Fig. 1 which is a tlistribution graph of the number of plaque-forming cells making antibodies to the cross-reacting antigen in one-third of each focus listed in Table 1. A distinct demarcation is seen between foci containing three or less cross-reacting plaqueforming cells in each 100 ~1, and those containing eight or more cross-reacting plaque-forming cells. Figure 2 depicts the percentage of cross-reacting plaqueforming cells in each focus listed in Table 1. As illustrated in Table 1 and Fig. 2, 26/39 foci contained plaque-forming cells making antibodies which lysed the immunizing antigen only. These foci contained between 10 and 4x8 plaque-forming cells responding to the immunizing antigen in each 100 ~1~ and in all but one case, less than eight plaque-forming cells lysing the
254
CAMPBELL
TABLE SPECIFICITIES
1
OF PLAQUE-FORMING
CELLS
IN SINGLE
HEMOLYTIC
FOCI
Percent Immunizing
antigen
SRBC
SRBC
a
cross-reacting PFC
GRBC 22 8
72 150
31 5 11.5 108 100 82 76 68 62 50 32 30 30 17 <6 <6
GRBC (ii) b (10) (400 1
SRBC GRBC
c d
(ii) (10) (488)
(16)
(21)
(ii, 8 8 39
(& 16 25 128
(128) 11-188 o-3
($) o-3 lo-488
a Number of plaque-forming cells lysing SRBC per 100-J aliquot. b Foci in parentheses came from spleens with two foci, but each focus was quite distinct. percentage of positive pieces in these spleens never exceeded 29%. c Eleven foci in spleens of mice immunized with SRBC contained <67e plaque-forming lysing GRBC. d Fourteen foci in spleens of mice immunized with GRBC contained <6% plaque-forming lysing SRBC.
The cells cells
cross-reacting antigen in other lOO-,pl aliquots. In these foci, there were less than 6% as many cross-reacting plaque-forming cells as those directed to the immunizing antigen. The five foci with 3@-50% cross-reacting plaque-forming cells may be considered to be particularly significant. They contained an adequate number of plaqueforming cells in each aliquot analyzed, and were from spleens containing one focus only. In these foci, while some of the plaque-forming cells release an antibody which lysed the cross-reacting red blood cells as well as the immunizing red blood cells, the rest (SO-70%) of the plaque-forming cells in these single foci lysed the immunizing antigen only. For instance, the second focus in Table 1 contains 72
CROSS-REACTING , 1
: :
: :
2
3 4
f I IT0 5678910
x
: 1
20
30
50 70
100
r
200 300
PFC x
I
500
1
700 loo0
FIG. 1. The number of plaque-forming cells in 100 pl of each of the 39 foci listed in Table 2 which lysed the cross-reacting antigen. Based on the distribution of these plaque-forming cells lysing the cross-reacting antigen in each focus, as illustrated here, foci containing less than eight cross-reacting plaque-forming cells were not considered to have a significant number of crossreacting plaque-forming cells.
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255
plaque-forming cells lysing the immunizing antigen in 100 ~1, and 22 plaqueforming cells lysing the cross-reacting antigen in 100 ~1. This implies that in the IOO-~1 aliquot assayed against the immunizing antigen, 22 plaque-forming cells were the progeny of the precursor cell (or cells) which responded to determinants shared by the immunizing and cross-reacting antigens, and that the remaining 50 plaque-forming cells were the progeny of a precursor cell (or cells) responding only to determinants on the immunizing antigen. Such foci appear to contain plaque-forming cells making antibodies of more than one set of specificities; i.e., ( 1) plaque-forming cells directed against determinants on both SRBC and GRBC, and (2) plaque-forming cells directed against determinants on the immunizing antigen only. The foci in parentheses in Fig. 2 and Table 1 were from spleenscontaining two foci. All other foci came from spleenswith only one focus. IVhen foci were used from spleens containing two foci, they were quite separate and distinct. Spleens containing two foci which were not clearly separate were not analyzed. Throughout these experiments, negative pieces, that is, those not part of detectable hemolytic foci, were analyzed for plaque-forming cells. As many as 10 such adjacent pieces never contained more than five detectable plaque-forming cells to the immunizing and cross-reacting antigens in loo-p1 aliquots. Direct and indirect plaque-forming cells ilt siltgle foci. Most foci in the experiments discussedabove were also analyzed for the presence of indirect as well as direct plaque-forming cells lysing the immunizing red blood cell (20, 21). Table 2 lists all foci containing more than 10 plaque-forming cells to the immunizing antigen by either the direct or indirect assay. A twofold increase in plaque-forming cells upon addition of developing antiserum was arbitrarily considered to represent a significant number of indirect plaque-forming cells. All foci containing more than twice as many indirect as direct plaque-forming cells are listed separately and are considered to contain both types of plaque-forming cells. Foci considered to be making direct plaque-forming cells only, that is, those containing less than twice as many indirect plaque-forming cells, are listed together. Table 2 shows that 16/39 foci contained direct plaque-forming cells and, in addition, significant numbers of indirect plaque-forming cells. Twenty-three of 39 foci had less than twice as many indirect plaque-forming cells as direct plaqueforming cells, and were considered to be making direct plaque-forming cells only. These data demonstrate that a single focus can contain both direct and indirect plaque-forming cells.
PERCENT
CROSS-REACTING
PFC
2. The percentage of plaque-forming cells lysing the cross-reacting antigen in each focus listed in Table 2. Calculated from (percentage of cells lysing cross-reacting antigen X lOO)/ percentage of cells lysing immunizing antigen. Those foci in parentheses were taken from spleens containing two foci. All numbers are rounded off to the nearest 10%. FIG.
256
CAMPBELL
TABLE DIRECT
Immunizing SRBC
antigen
AND
INDIRECT
Direct
2
PLAQUE-FORMING
PFC (46)
*
CELLS
Indirect
c
PFC
(224) (440 1 16 320 97 90 928 280 (15) 288 (15)
(188) GRBC
IN
8 3 4 4 51 16
(2) 65 (4) 9 (4) 4 68 6
(ii) 10 136 12
INDIVIDUAL
FOCI
0
Indirect Direct
PFC/ PFC
4.9 2.3 2.0 106.7 24.3 22.5 18.2 17.5 7.5 4.4 3.8 2.7 2.5 2.5 2.0 2.0
n All foci containing two times as many indirect plaque-forming cells as direct plaque-forming cells are considered to have a significant number of indirect plaque-forming cells and are listed above. Four foci in mice immunized with SRBC and 19 foci in mice immunized with GRBC contained less than two times as many indirect plaque-forming cells as direct plaque-forming cells. These foci are considered to be homogeneous for direct plaque-forming cells and are not listed above. * Number of plaque-forming cells per loo-p1 aliquot. c The foci in parentheses came from spleens with two clearly distinct foci each.
DISCUSSION Earlier experiments demonstrated that a single hemolytic focus can contain the progeny of more than one precursor cell (10). The experiments described here demonstrate that such a hemolytic focus can contain plaque-forming cells producing antibodies of more than one set of specificities, and that a single focus can contain both direct and indirect plaque-forming cells. It is very unlikely that the presence of two kinds of precursor cells in a single focus resulted from the existence of two overlapping foci in the same spleen, since the number of foci seen per spleen averaged only 0.7. In addition, participation of background cells was very unlikely, since controls (published earlier (10) and also run in these experiments) injected with bone marrow and antigen only contained no hemolytic foci and very few plaque-forming cells. Many laboratories have previously reported that a single hemolytic focus makes antibodies of one specificity only (11, 22-25). N o one has yet reported the presence of plaque-forming cells making antibodies of different specificities in single hemolytic foci. However, Luzzati et al. (26) have demonstrated that a single piece of rabbit lymph node can synthesize antibodies which migrate as more than one discrete electrophoretic band, which they suggest may be interpreted as evidence for more than one precursor cell per focus. The experiments indicating one anti-
HETEROGENEOUS
ANTIRODIES
IN
ITEMOLYTIC
257
FOCI
body per focus were not conducted under conditions which would favor the presence of more than one precursor cell per focus, . i.e., conditions under which there were an excess number of precursor cells. In the experiments reported here, the thymus-derived cells were present only in the small inoculum of S X 10’ spleen cells. Bone marrow-derived precursor cells were provided in large excess, being present both in the spleen inoculum and also in the 10’ bone marrow cells injected. Under conditions such as these, many foci would be expected to contain more than one precursor cell, and could produce antibodies of more than one specificity. A further prediction of these data and our earlier work (10) is that the number of plaque-forming cells per hemolytic focus need not be constant, but is rather a function of the number of precursor cells in that focus. It has been reported that the number of plaque-forming cells per focus is constant (7, 27). Xlthough this may be true in a focus involving only one precursor cell, the number of plaqueforming cells per focus need not be constant when excess precursor cells are available (10, 14, 16). The second set of data reported in this paper demonstrates that a single focus can contain both direct and indirect plaque-forming cells. Although Celada and Wigzell suggested that a single focus may make antibodies of one class only (24), other authors have shown that a single focus can contain plaque-forming cells making more than one class of antibody (6, 22, 28). Our data confirm that a single focus can contain both direct and indirect plaque-forming cells. The direct and indirect plaque-forming cells could be the progeny of single different precursor cells committed to producing either direct or indirect plaque-forming cells, or they could represent a switch within single plaque-forming cells from 19s to 7s antibody formation. The data presented here do not allow differentiation between these possibilities. The data reported here, demonstrating that a single focus can contain plaqueforming cells making antibodies of more than one specificity, strongly suggest that it is the bone marrow-derived precursor cells and not the thymus-derived cells which determine the specificity of the antibodies produced. The data argue against transfer of information controlling antibody specificity from the thymus-derived cell to the bone marrow-derived precursor cells, but do suggest close contact between T cells and B cells. Our data are compatible with the following kind of model for the functional unit involved in the initiation of murine immune responses to RBC antigens: a thymus-derived cell, required for initiation of the hemolytic response, signals surrounding bone marrow-derived precursor cells to differentiate and divide into plaque-forming cells which make antibodies of the specificities dictated by the precursor cells from which they are derived. ACKNOWLEDGMENT The author wishes to thank were conducted, for his support
Dr. Richard W. Dutton, and for helpful discussion.
in whose
laboratory
these
experiments
REFERENCES 1. Claman, 1966.
H.
K.,
Chaperon,
E. S., and Triplett,
R. F., Proc.
Sot.
Exp.
Biol.
Med.
122. 1167,
258
CAMPBELL
2. Miller, J. F. A. P., and Mitchell, G. F., Nature, Londorz 216, 659, 1967. 3. Miiller, G., Ed., “Antigen-Sensitive Cells : Their Source and Differentiation,” Transplant. Rev. 5, 1969. 4. Cunningham, A. J., Immunology 17, 933, 1969. 5. Groves, D. S., Lever, W. E., and Makinodan, T., Nature, London 222, 95, 1969. 6. Shearer, G. M., Cudkowicz, G., and Priore, R. L., J. Exp. Med. 130, 467, 1969. 7. Shearer, G. M., and Cudkowicz, G., J. Exp. Med. 130, 1243, 1969. 8. Talmage, D. W., Radovich, J., and Hemingsen, H., J Allergy 43, 323, 1969. 9. Dutton, R. W., Campbell, P., Chan, E., Hirst, J., Hoffman, M., Kettman, J., Lesley, J., McCarthy, M., Mishell, R. I., Raidt, D. J., and Vann, D., Int. Comsocation Zmnzunol., 2nd, in press 10. Vann, D. C., and Campbell, P. A., J. Inanzunol. 105, 1584, 1970. 11. Playfair, J. H. L., Papermaster, B. W., and Cole, L. J., Science 149, 998, 1965. 12. Kennedy, J. C., Siminovitch, L., Till, J. E., and McCulloch, E. A., Proc. Sot. En-p. Biol. Med. 120, 868, 1965. 13. Kind, P., and Campbell, P. A., J. Immunol. 100, 55, 1968. 14. Mitchell, G. F., and Miller, J. F. A. P., J. Exp. Med. 126, 821, 1968. 15. Radovich, J., Hemingsen, H., and Talmage, D W., J Znamunol. 100, 756, 1968. 16. Gregory, C. J., and Lajtha, L. G., Nature, Londolz 216, 1079, 1968. 17. Makinodan, T., Sado, T., Groves, D. L., and Price, G., Curr. Top. Microbial. Immunol. 49, 81, 1969. 18. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. 19. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. 20. Sterzl, J., and Riha, I., Nature, London 206, 858, 1965. 21. Dresser, D. W., and Wortis, H. H., Nature, London 206, 859, 1965. 22. Papermaster, B. W., Cold Spring Harbor Symp. Quant. Biol. 32, 447, 1967. 23. Nakano, M., and Braun, W., Science 151, 338, 1966. 24. Celada, F., and Wigzell, H., Immunology 11,453, 1966. 25. Klinman, N. R., Inwnzlnochemistry 6, 757, 1969. 26. Luzzati, A. L., Tosi, R. M., and Carbonara, A. O., J. Exp. Med. 132, 199, 1970. 27. Shearer, G. M., and Cudkowicz, G., J. Exp. Med. 129, 935, 1969. 28. Cunningham, A. J., Awt. .I. Exp. Biol. Med. Sci. 47, 493, 1969.
2, 250-258
IMMUNOLOGY
(1971)
Heterogeneity
of
Antibodies
Single
Hemolytic
P. A. Department
of Biology,
University
Produced Foci ’
CAMPBELL
of California, Recrived
by
’ San
Diego,
La Jolla,
California
92037
January 4,197l
Lethally irradiated male BDF, mice were injected with 10’ bone marrow cells, 8 X 10s spleen cells, and sheep or goat red blood cells as antigens. The cross-reactivity between these red blood cells is approximately 40%, as determined by injecting normal mice with either sheep or goat red blood cells, removing their spleens 4 days later, and assaying all spleens for both antisheep and antigoat plaque-forming cells. Eight days after injection of bone marrow, spleen, and antigen, the spleens of the irradiated recipients were removed and assayed for the presence of hemolytic foci by the Playfair technique, and were found to contain an average of 0.7 foci/spleen. Earlier studies had demonstrated that such foci could contain plaque-forming cells derived from more than one precursor cell. The positive pieces comprising a single hemolytic focus were removed, pooled, and aliquots were assayed for direct and indirect plaque-forming cells to the immunizing antigen. Several foci were found to contain 17-827 0 as many plaque-forming cells lysing the cross-reacting antigen as lysed the immunizing antigen. These data indicate that these foci contained plaque-forming cells of two specificities: (1) plaque-forming cells which responded to determinants on the immunizing antigen only, and (2) plaque-forming cells which responded to determinants on bo’th the immunizing and cross-reacting antigens. In addition, single foci were found which contained both direct and indirect plaqueforming cells lysing the immunizing antigen. Sixteen of 39 foci assayed contained more than twice as many indirect as direct plaque-forming cells. We concluded that a single hemolytic focus, probably derived from more than one precursor cell, could contain plaque-forming cells producing antibodies of more than one set of specificities and immunoglobulins of more than one type.
INTRODUCTION Mouse spleens contain all the cellular components required for initiation of an immune response to sheep red blood cell antigens. Among these cellular components are the thymus-derived cells (T cells), which initiate the immune response, and bone marrow-derived precursor cells (B cells), which are direct progenitors of 1 This research was AI-08795 to Dr. Richard 2 Supported s Kind,
by United
supported W. Dutton. States
P., and P. A. Campbell,
by
United
Public
Health
unpublished
States Service results. 250
Public
Health
Postdoctoral
Service Fellowship
Research -41-40639.
Grant
HETEROGENEOUS
ANTIBODIES
IN
HEMOLYTIC
FOCI
251
antibody-forming cells ( l-10). experiments examining these immunoD uring competent cells, it was discovered that injection of small numbers of normal spleen cells or thoracic duct lymphocytes and red blood cell antigens into lethally irradiated mice led to the appearance of distinct regions of hemolytic activity in the spleens of these mice after 8 days (11,12). The number of these hemolytic foci is directly proportional to the number of spleen cells or thoracic duct lymphocytes injected (1, lo-13), and is not increased by the addition of bone marrow cells to the inoculum (10, 14). However, addition of bone marrow does result in an increased splenic plaque-forming cell response (10, 14, 15). We have recently shown that this increase in plaque-forming cells upon addition of bone marrow is due, at least in part, to the presence of more than one precursor cell in single hemolytic foci (10). Spleen cells bearing H 2 bd histocompatibility antigens and bone marrow cells carrying H 2 d antigens were injected into lethally irradiated recipient H 2 bd mice. Using antisera directed against H 2 b or H 2 d antigens, it was shown that single hemolytic foci could contain some plaque-forming cells bearing antigens found on the injected spleen cells, and some plaque-forming cells bearing antigens on the injected bone marrow cells. These data are compatible with a model in which a single immunocompetent unit contains both a thymusderived cell, which is required to initiate the immune response, and one or more bone marrow-derived cells, which determine the magnitude of the response (5, 10, 16, 17). Therefore, the number of hemolytic foci would be directly proportional to the number of T cells injected, while the number of plaque-forming cells per focus would be dependent on the number of B cells injected. Since a single focus can contain progeny of more than one precursor cell, we asked the questions: Can the plaque-forming cells in a single hemolytic focus produce antibodies of more than one specificity ? Can the plaque-forming cells in a single hemolytic focus produce antibodies of more than one type; that is, both direct and indirect plaque-forming cells ? The experiments described below were designed to answer these questions. The data suggest that a single hemolytic focus can contain precursor cel!s whose plaqueforming cell progeny can produce antibodies of more than one specificity and more than one immunoglobulin type. MATERIALS
AND
METHODS
Uice. Male and female BDF, (C57B1/6J X DBA/2J) mice, aged 6-16 weeks, were used throughout these experiments. They were fed and watered ad Zibitum. The drinking water contained 16 ppm chlorine to decrease Pseudomonas infections. Antigens. Sheep red blood cells (SRBC) and goat red blood cells (GRBC) were purchased from Colorado Serum Company preserved in Alsever’s solution. A single lot of each antigen was used throughout the experiments. The cells were washed two to three times and resuspended in balanced salt solution (BSS) (18) prior to use. Irradiation. Mice were irradiated with 990-1024 R from a 6oCo source through the courtesy of Mr. Irving Goldman and the Salk Institute for Biological Studies. All mice were injected within 2-20 hr after irradiation. Plaque-forming cell assay. Plaque-forming cells were detected according to the
252
CAMPBELT.
method of Jerne and Nordin (19) using the microscope slide modification ( 18). All slides were incubated in a humidified environment at 37°C for 1 hr without complement, and 2 hr with complement with or without 1: 200 dilution of developing antiserum. The complement source used was a 1 : 10 dilution of guinea pig serum. Developing antiserum. The developing antiserum had been prepared and tested according to the following procedure.” Rabbit gamma globulin was precipitated from normal rabbit serum using 50% saturated ammonium sulfate. Following removal of the ammonium sulfate by dialysis, the rabbit gamma globulin was injected into LAF, mice over a period of several weeks. The immune mice were bled, and antibodies in their serum were precipitated with the rabbit gamma globulin antigen. Normal rabbits were injected with the washed immune precipitate containing rabbit gamma globulin and mouseantirabbit gamma globulin in water-inoil emulsion. The rabbits were bled after several injections, their sera were collected, and the developing antiserum was titrated. A dilution of 1 : 200 rabbit antiserum was found to give the maximum number of indirect plaque-forming cells and little or no inhibition of direct plaque-forming cells. Hemolytic foci. The presence of hemolytic foci in mouse spleens was detected by a modification (13) of the method of Playfair et al (11). In the experiments reported here, lethally irradiated mice were injected intravenously with 8 X lo5 spleen cells and 6.613.5 X lo6 bone marrow cells. The mice received 0.1 ml 20% SRBC or GRBC intravenously on Day 1 after irradiation and intraperitoneally 3 days later. On Day 8, the mice were killed by cervical dislocation and the spleen removed. Each spleen was cut transversely into 10-15 slices, then each slice was cut into three to 8 pieces, approximately 1-2 mm3 in size. The pieces were carefully placed in a petri dish on a base layer of lo/O agarose in BSS so that the orientation of the pieces in the original organ was maintained. The spleen pieces were overlaid with 4 ml of 0.5% agarose containing 0.2 ml 40% RBC with which they were immunized and incubated at 37°C for 1.5 hr. Ten percent guinea pig complement which had been absorbed one to three times with mouse spleen and thymus cells was added, and the plates incubated an additional l/2 hr at 37°C. The complement was then removed, the plates flooded with cold BSS, and the hemolytic foci counted. Zones of hemolysis around adjacent spleen pieces indicated the presence of a single hemolytic focus. RESULTS Experimental
Design
Lethally irradiated mice were injected with 8 X lo5 spleencells and with approximately lo7 bone marrow cells. In this manner, a single focus could be expected to contain more than one precursor cell, as reported earlier (10). The hemolytic foci were developed, as described above, and the pieces from a single hemolytic focus were removed from the agarose plate, pooled, and teased into a single-cell suspension.The cells were sedimented by centrifugation, and resuspendedin 0.3 ml BSS. One hundred ,pl of the cell suspensionwere then assayed for the number of direct plaque-forming cells responding to the immunizing antigen, 100 ~1 were assayed for plaque-forming cells responding to the cross-reacting antigen, and
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253
25-100 ~1 of the remaining suspension were assayed for indirect plaque-forming cells responding to the immunizing antigen. In most experiments, the immunizing antigen was GRUC, while in some experiments SRBC were used as the immunizing antigen. In either case, recipient spleens contained less than one hemolytic focus, to avoid chance overlap of two foci. Each hemolytic focus contained an average of four to six positive pieces. Most mouse spleens were cut into a total of 40-50 pieces. Following the above procedure, we could determine whether plaque-forming cells in a single focus could make antibodies of more than one specificity or more than one immunoglobulin type. Percentage of cross-reacfivity of SRBC and GRBC. Two experiments were conducted to determine the percentage of cross-reactivity of the two lots of SRBC and GRBC used in these experiments. For each experiment, two mice were injected intravenously with 0.2 ml 10% SRBC, and two mice were injected intravenously with the same dose of GRBC. On Day 4 after injection in one experiment and Day 5 in the second experiment, the splenic plaque-forming cells responding to each antigen were assayed. There were 345% as many cells lysing GRBC as there were lysing SRBC in the spleens of the mice immunized with SRBC. The spleens of mice immunized with GRBC contained 475% as many plaque-forming cel!s aaginst SRIJC as against GRBC. That is, the cross-reactivity of GRBC with SRBC was 34%, and of SRBC with GRBC was 47%, for an average crossreactivity of 40%. Specificity of plaque-forwing cells &z single foci. A total of 191 mice injected with spleen and bone marrow were analyzed for the presence of hemolytic foci. The average number of foci per spleen in these mice was 0.72. Of the 92 foci whose pieces were removed for analysis, 53 contained less than 10 detectable direct plaque-forming cells to the immunizing antigen in the loo-p1 aliquot assayed. These foci presumably had less than 30 recoverable direct plaque-forming cells lysing the immunizing antigen, since one-third of each focus was assayed for these plaque-forming cells. The remaining foci are discussed below. The 39 foci presented in Table 1 represent data from eight experiments. All foci in which 10 or more plaque-forming cells responding to the immunizing antigen could be detected in the loo-p1 aliquot are reported; less than 10 plaque-forming cells was arbitrarily considered not significant. Foci containing less than eight plaque-forming cells lysing the cross-reacting RBC are listed together at the bottom of the table. The reason for the selection of eight as a minimum significant number of cross-reacting plaque-forming cells is illustrated in Fig. 1 which is a tlistribution graph of the number of plaque-forming cells making antibodies to the cross-reacting antigen in one-third of each focus listed in Table 1. A distinct demarcation is seen between foci containing three or less cross-reacting plaqueforming cells in each 100 ~1, and those containing eight or more cross-reacting plaque-forming cells. Figure 2 depicts the percentage of cross-reacting plaqueforming cells in each focus listed in Table 1. As illustrated in Table 1 and Fig. 2, 26/39 foci contained plaque-forming cells making antibodies which lysed the immunizing antigen only. These foci contained between 10 and 4x8 plaque-forming cells responding to the immunizing antigen in each 100 ~1~ and in all but one case, less than eight plaque-forming cells lysing the
254
CAMPBELL
TABLE SPECIFICITIES
1
OF PLAQUE-FORMING
CELLS
IN SINGLE
HEMOLYTIC
FOCI
Percent Immunizing
antigen
SRBC
SRBC
a
cross-reacting PFC
GRBC 22 8
72 150
31 5 11.5 108 100 82 76 68 62 50 32 30 30 17 <6 <6
GRBC (ii) b (10) (400 1
SRBC GRBC
c d
(ii) (10) (488)
(16)
(21)
(ii, 8 8 39
(& 16 25 128
(128) 11-188 o-3
($) o-3 lo-488
a Number of plaque-forming cells lysing SRBC per 100-J aliquot. b Foci in parentheses came from spleens with two foci, but each focus was quite distinct. percentage of positive pieces in these spleens never exceeded 29%. c Eleven foci in spleens of mice immunized with SRBC contained <67e plaque-forming lysing GRBC. d Fourteen foci in spleens of mice immunized with GRBC contained <6% plaque-forming lysing SRBC.
The cells cells
cross-reacting antigen in other lOO-,pl aliquots. In these foci, there were less than 6% as many cross-reacting plaque-forming cells as those directed to the immunizing antigen. The five foci with 3@-50% cross-reacting plaque-forming cells may be considered to be particularly significant. They contained an adequate number of plaqueforming cells in each aliquot analyzed, and were from spleens containing one focus only. In these foci, while some of the plaque-forming cells release an antibody which lysed the cross-reacting red blood cells as well as the immunizing red blood cells, the rest (SO-70%) of the plaque-forming cells in these single foci lysed the immunizing antigen only. For instance, the second focus in Table 1 contains 72
CROSS-REACTING , 1
: :
: :
2
3 4
f I IT0 5678910
x
: 1
20
30
50 70
100
r
200 300
PFC x
I
500
1
700 loo0
FIG. 1. The number of plaque-forming cells in 100 pl of each of the 39 foci listed in Table 2 which lysed the cross-reacting antigen. Based on the distribution of these plaque-forming cells lysing the cross-reacting antigen in each focus, as illustrated here, foci containing less than eight cross-reacting plaque-forming cells were not considered to have a significant number of crossreacting plaque-forming cells.
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255
plaque-forming cells lysing the immunizing antigen in 100 ~1, and 22 plaqueforming cells lysing the cross-reacting antigen in 100 ~1. This implies that in the IOO-~1 aliquot assayed against the immunizing antigen, 22 plaque-forming cells were the progeny of the precursor cell (or cells) which responded to determinants shared by the immunizing and cross-reacting antigens, and that the remaining 50 plaque-forming cells were the progeny of a precursor cell (or cells) responding only to determinants on the immunizing antigen. Such foci appear to contain plaque-forming cells making antibodies of more than one set of specificities; i.e., ( 1) plaque-forming cells directed against determinants on both SRBC and GRBC, and (2) plaque-forming cells directed against determinants on the immunizing antigen only. The foci in parentheses in Fig. 2 and Table 1 were from spleenscontaining two foci. All other foci came from spleenswith only one focus. IVhen foci were used from spleens containing two foci, they were quite separate and distinct. Spleens containing two foci which were not clearly separate were not analyzed. Throughout these experiments, negative pieces, that is, those not part of detectable hemolytic foci, were analyzed for plaque-forming cells. As many as 10 such adjacent pieces never contained more than five detectable plaque-forming cells to the immunizing and cross-reacting antigens in loo-p1 aliquots. Direct and indirect plaque-forming cells ilt siltgle foci. Most foci in the experiments discussedabove were also analyzed for the presence of indirect as well as direct plaque-forming cells lysing the immunizing red blood cell (20, 21). Table 2 lists all foci containing more than 10 plaque-forming cells to the immunizing antigen by either the direct or indirect assay. A twofold increase in plaque-forming cells upon addition of developing antiserum was arbitrarily considered to represent a significant number of indirect plaque-forming cells. All foci containing more than twice as many indirect as direct plaque-forming cells are listed separately and are considered to contain both types of plaque-forming cells. Foci considered to be making direct plaque-forming cells only, that is, those containing less than twice as many indirect plaque-forming cells, are listed together. Table 2 shows that 16/39 foci contained direct plaque-forming cells and, in addition, significant numbers of indirect plaque-forming cells. Twenty-three of 39 foci had less than twice as many indirect plaque-forming cells as direct plaqueforming cells, and were considered to be making direct plaque-forming cells only. These data demonstrate that a single focus can contain both direct and indirect plaque-forming cells.
PERCENT
CROSS-REACTING
PFC
2. The percentage of plaque-forming cells lysing the cross-reacting antigen in each focus listed in Table 2. Calculated from (percentage of cells lysing cross-reacting antigen X lOO)/ percentage of cells lysing immunizing antigen. Those foci in parentheses were taken from spleens containing two foci. All numbers are rounded off to the nearest 10%. FIG.
256
CAMPBELL
TABLE DIRECT
Immunizing SRBC
antigen
AND
INDIRECT
Direct
2
PLAQUE-FORMING
PFC (46)
*
CELLS
Indirect
c
PFC
(224) (440 1 16 320 97 90 928 280 (15) 288 (15)
(188) GRBC
IN
8 3 4 4 51 16
(2) 65 (4) 9 (4) 4 68 6
(ii) 10 136 12
INDIVIDUAL
FOCI
0
Indirect Direct
PFC/ PFC
4.9 2.3 2.0 106.7 24.3 22.5 18.2 17.5 7.5 4.4 3.8 2.7 2.5 2.5 2.0 2.0
n All foci containing two times as many indirect plaque-forming cells as direct plaque-forming cells are considered to have a significant number of indirect plaque-forming cells and are listed above. Four foci in mice immunized with SRBC and 19 foci in mice immunized with GRBC contained less than two times as many indirect plaque-forming cells as direct plaque-forming cells. These foci are considered to be homogeneous for direct plaque-forming cells and are not listed above. * Number of plaque-forming cells per loo-p1 aliquot. c The foci in parentheses came from spleens with two clearly distinct foci each.
DISCUSSION Earlier experiments demonstrated that a single hemolytic focus can contain the progeny of more than one precursor cell (10). The experiments described here demonstrate that such a hemolytic focus can contain plaque-forming cells producing antibodies of more than one set of specificities, and that a single focus can contain both direct and indirect plaque-forming cells. It is very unlikely that the presence of two kinds of precursor cells in a single focus resulted from the existence of two overlapping foci in the same spleen, since the number of foci seen per spleen averaged only 0.7. In addition, participation of background cells was very unlikely, since controls (published earlier (10) and also run in these experiments) injected with bone marrow and antigen only contained no hemolytic foci and very few plaque-forming cells. Many laboratories have previously reported that a single hemolytic focus makes antibodies of one specificity only (11, 22-25). N o one has yet reported the presence of plaque-forming cells making antibodies of different specificities in single hemolytic foci. However, Luzzati et al. (26) have demonstrated that a single piece of rabbit lymph node can synthesize antibodies which migrate as more than one discrete electrophoretic band, which they suggest may be interpreted as evidence for more than one precursor cell per focus. The experiments indicating one anti-
HETEROGENEOUS
ANTIRODIES
IN
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257
FOCI
body per focus were not conducted under conditions which would favor the presence of more than one precursor cell per focus, . i.e., conditions under which there were an excess number of precursor cells. In the experiments reported here, the thymus-derived cells were present only in the small inoculum of S X 10’ spleen cells. Bone marrow-derived precursor cells were provided in large excess, being present both in the spleen inoculum and also in the 10’ bone marrow cells injected. Under conditions such as these, many foci would be expected to contain more than one precursor cell, and could produce antibodies of more than one specificity. A further prediction of these data and our earlier work (10) is that the number of plaque-forming cells per hemolytic focus need not be constant, but is rather a function of the number of precursor cells in that focus. It has been reported that the number of plaque-forming cells per focus is constant (7, 27). Xlthough this may be true in a focus involving only one precursor cell, the number of plaqueforming cells per focus need not be constant when excess precursor cells are available (10, 14, 16). The second set of data reported in this paper demonstrates that a single focus can contain both direct and indirect plaque-forming cells. Although Celada and Wigzell suggested that a single focus may make antibodies of one class only (24), other authors have shown that a single focus can contain plaque-forming cells making more than one class of antibody (6, 22, 28). Our data confirm that a single focus can contain both direct and indirect plaque-forming cells. The direct and indirect plaque-forming cells could be the progeny of single different precursor cells committed to producing either direct or indirect plaque-forming cells, or they could represent a switch within single plaque-forming cells from 19s to 7s antibody formation. The data presented here do not allow differentiation between these possibilities. The data reported here, demonstrating that a single focus can contain plaqueforming cells making antibodies of more than one specificity, strongly suggest that it is the bone marrow-derived precursor cells and not the thymus-derived cells which determine the specificity of the antibodies produced. The data argue against transfer of information controlling antibody specificity from the thymus-derived cell to the bone marrow-derived precursor cells, but do suggest close contact between T cells and B cells. Our data are compatible with the following kind of model for the functional unit involved in the initiation of murine immune responses to RBC antigens: a thymus-derived cell, required for initiation of the hemolytic response, signals surrounding bone marrow-derived precursor cells to differentiate and divide into plaque-forming cells which make antibodies of the specificities dictated by the precursor cells from which they are derived. ACKNOWLEDGMENT The author wishes to thank were conducted, for his support
Dr. Richard W. Dutton, and for helpful discussion.
in whose
laboratory
these
experiments
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