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Separation of various B-cell subpopulations from mouse spleen
CELLULAR IMMUNOLOGY 46, 170-177 (1979)
Separation
of Various
II. Depletion
of Antigen-Specific B Cells by Rosetting with GlutaraldehydeFixed, Antigen-Coupled Red Blood Cells1 SHARYN
B-Cell Subpopulations
M. WALKERS AND WILLIAM
from Mouse Spleen
0. WEIGLE
Department of Immunopathology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037 Received
December
5, 1978
Mouse spleen cells were depleted of antigen-specific B cells (ASC) by rosetting with glutaraldehyde-fixed RBC coupled with specific antigen. Removal of the rosetted cells by density gradient centrifugation resulted in the depletion of more than 99% of all the ASC. Depletion was shown by the failure of rosette-depleted, primed spleen cells to generate antigen-specific antibody-producing cells. Functional assays showed that deither macrophages or helper T cells were removed. In comparison to other procedures used to deplete of ASC, rosetting with antigen-coupled RBC has advantages in terms of recovery, simplicity of the procedure, and efficiency of ASC depletion.
INTRODUCTION The relatively high density of surface Ig on B cells has permitted the development of methods to deplete antigen-specific B cells from mouse spleen. For some time it has been appreciated that surface Ig reflects the potential Ig product synthesized by these cells when triggered by antigen. This has been shown by the capacity of highly radiolabeled antigen to kill or “suicide” B cells with specificity for the antigen (l), the selective depletion of antigen-specific B cells (ASC)3 by passage over antigen columns (2-6), and the selective depletion of ASC treated with fluoresceinated antigen by passage through the fluorescence-activated cell sorter (FACS) (7). Use of this latter procedure has shown that surface Ig reflects not only the specificity, but also the avidity of the potential Ig product (8). These methods have been adopted and modified by others (9- 11) as a means to deplete and enrich for ASC for the study of cell-cell interactions in the immune response. This paper details a simple rosetting procedure to deplete ASC from spleen cells primed to a soluble protein antigen, turkey y-globulin (TGG). The outstanding 1 This is Publication No. 1669 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California. This work was supported in part by United States Public Health Service Grant AI-07007, American Cancer Society Grant IM42-H, and Biomedical Research Support Program Grant (RRO-5514). * Recipient of a Fellowship from the Arthritis Foundation. 3 Abbreviations used: ASC, antigen-specific B cells; FACS, fluorescence-activated cell sorter; TGG, turkey y-globulin; PFC, plaque-forming cell; GA, glutaraldehyde; ET, endotoxin. 170 0008-8749/79/090170-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
ROSETTE DEPLETION
OF ANTIGEN-SPECIFIC
B CELLS
171
feature of the procedure is the extreme effectiveness in the removal of ASC, while involving minimal manipulation and time. MATERIALS
AND METHODS
Zmmunizarion of mice. Male A/J mice (Jackson Laboratory, Bar Harbor, Me.) were primed at 6- 10 weeks of age with 0.1 ml of 100 to 200 pg alum-precipitated TGG in 0.1 ml complete Freund’s adjuvant given intraperitoneally (11). Generation and assay of antibody-producing cells. Spleen cells from mice primed with TGG 6 weeks or more prior were cultured and assayed as described in the preceding paper (12). TGG was added to the cultures indirectly by incubating the spleen cells at 4°C for 30-60 min with soluble TGG and then removing the unbound TGG with multiple washes. Plaque-forming cells (PFC) were measured after 6 days, with only indirect PFC measured in most experiments as the IgM response to TGG was insignificant. Results are expressed as the mean PFC of lo7 original spleen cells of duplicate cell pools with the standard error. Assay for endotoxin (ET)-induced proliferation. Spleen cells were cultured as for the generation of antibody-producing cells except that mercaptoethanol was omitted. Endotoxin, 055:B5 trichloroacetic acid extract, Difco Laboratories, Detroit, Michigan, was added at culture initiation at 50 I.Lg/ml.After 2 days culture, proliferation was measured by a [3H]thymidine pulse (13). Removal of TGG-specific B cells. Spleen cells were rosetted at 4°C with glutaraldehyde (GA)-fixed, TGG-coupled RBC and the rosetted cells removed by centrifugation on a density gradient similar to the rosetting procedure for the removal of B cells described in the preceding paper (12). RBC were fixed as described therein, except that a finer grade of GA was used to reduce nonspecific rosetting. Rosetting with TGG coupled to RBC fixed with a practical grade of GA averaged 4% rosettes, while TGG coupled to RBC fixed with a finer grade of GA averaged 1%. Briefly, 1.5 ml of 8% GA (8% Ultrapure-TEM Grade, Tousimis Research Corp., Rockville, Md., Catalog No. 1058) was neutralized with 0.050 ml 10% sodium carbonate and 5 ml 0.15 M sodium chloride added, followed by 1 ml 0.015 M sodium phosphate buffer, pH 8.0. Fixation was continued for 24 hr at 4°C with continuous stirring; the RBC were then washed, treated with tannic acid, and coupled with TGG as described for coupling Ig (12). Recovery of rosette-depleted spleen cells averaged 70%. RESULTS Depletion
of TGG-Specific
B Cells
Spleen cells primed to TGG were incubated at 4°C with GA-fixed, TGG-coupled RBC in an attempt to rosette the B cells specific for TGG. After removal of the rosetted cells, the remaining cells were assayed for their capacity to generate PFC after culture with TGG for 6 days. Table 1 shows that the control response to TGG was greater than 19,000 PFC per 10’ spleen cells put into culture, while the TGG rosette-depleted cultures failed to show a significant response above background. The depletion of TGG-specific rosetting cells did not remove appreciable numbers of TGG-specific T cells, as shown by the ability of rosettedepleted cells to reconstitute the response of T-cell-depleted, primed spleen cells from less than 52 to 13,071 PFC. Depletion of TGG-specific rosetting cells was
172
WALKER AND WEIGLE TABLE 1 Capacity of GA-Fixed, TGG-Coupled RBC to Remove B Cells Specific for TGG from Spleen Cells Primed to TGG
Exp. No. 1
2
Spleen cells (5 x 10s) Control Control TGG rosette depleted T cell depleted TGG rosette depleted (2.5 x 105) + T cell depleted (2.5 x 105) HSA rosette depleted Control Control TGG rosette depleted
TGG in culture
PFC
-
52 19,372 f 8,016 78 k 26 ~52
+ + +
+ +
13,971 + 933 18,203 f 7,313
TGG-treated adherent cells” TGG-treated adherent cells”
~61 5,368 2 488 <61
a TGG-treated adherent cells were prepared by adsorbing 6 x lo5 TGG-coupled spleen cells to individual wells in 0.06 ml medium with 10% FCS for 30 min at 37°C and washing six times vigorously to remove nonadherent cells.
dependent on rosetting with TGG-coupled RBC as shown by the failure of human serum albumin (HSA)-coupled RBC to remove TGG-responding cells. To determine if the reduced PFC response was due partially or entirely to removal of antigen-presenting cells, such as macrophages with cytophilic antibody for TGG, rather than TGG-specific B cells, attempts were made to reconstitute the response of rosette-depleted cells with adherent cells. TGG was presented to the cultures on non-TGG-primed spleen cells allowed to adhere to the culture wells before addition of the primed spleen cells. The last two lines of Table 1 show that TGCJpresented in this manner permitted control, non-rosette-depleted spleen cells to respond, but did not enable TGG rosette-depleted spleen cells to respond. In addition, nonspecific esterase staining of rosette-depleted sp1ee.ncells showed no decrease in macrophage number, making the loss of antigen-presenting cells unlikely. Thus, it can be concluded that the rosetting procedure eliminated the antibody response of TGG-primed spleen cells by depleting TGG-specific B cells and not an accessory cell. Eflect of the Temperature of Rosetting The rosetting procedure in the experiments described to this point was carried out at 4°C during the pelleting and gradient separation procedures. Table 2 shows that this temperature was optimal when compared to 37°C as TGG-primed spleen cells which had been depleted of cells rosetting at 37°C with HSA-coupled RBC failed to generate a response to TGG. This is in contrast to the results in Table 1 in which rosetting at 4°C with HSA-coupled RBC had no effect on the in vitro TGG response. Relatively large numbers of HSA rosettes were formed at 37°C about 20%, while experiments done at 4°C averaged only 0.2%. It can also be seen in Table 2 that the 37°C incubated HSA rosette-depleted cells did not respond to TGG even when TGG-adherent cells were added as a means of presenting antigen, indicating that large numbers of TGG-specific B cells were removed nonspecifically
ROSETTE DEPLETION
OF ANTIGEN-SPECIFIC
173
B CELLS
TABLE 2 Effect of Rosetting at 4°C versus 37°C on the Removal of Specific B Cells Spleen cells
TGG in culture
PFC
Control Control TGG rosette depleted at 4°C HSA rosette depleted 37°C HSA rosette depleted 37°C
+ + + TGG-treated adherent cells”
<61 5368 k 488 ~61 <61 <61
a TGG-treated adherent cells were prepared as described in Table 1.
by rosetting at 37°C. Thus, to obtain antigen-specific rosette depletion of AX, the procedure was carried out at low temperatures in all subsequent experiments. Determination of the B-Cell Content of TGG Rosette-Depleted Spleen Cells The B-cell content of TGG ‘rosette-depleted cultures was next quantified to see if the removal of TGG-specific B cells appreciably removed other B cells nonspecifically. A potent B-cell mitogen, ET, was added to TGG rosette-depleted spleen cells and the proliferative response measured 2 days later. Table 3 shows that rosetting removed all the potential PFC to TGG, while having no effect on ET-elicited proliferation. This is not surprising, since the number of TGG rosettes formed averaged about 1% of the spleen cells, while B cells constituted about 50% of the spleen. Even though the B-cell content of TGG rosette-depleted cultures was normal as measured by mitogenesis, the procedure could still have removed a number of non-TGG-specific B cells not detectable by ET-induced proliferation. Thus, to assess the specificity of TGG rosette depletion, the response of TGG rosettedepleted spleen cells to another antigen, SRBC, was measured as well. Table 4 shows that the TGG rosette-depleted spleen cells failed to generate PFC to TGG, but gave a normal response to SRBC. Thus, the deletion procedure was highly selective. It may be noted that since TGG was coupled to SRBC, one might have expected SRBC-specific cells to have been removed as well. However, the process of fixaTABLE 3 Determination of the B-Cell Content of Specific B-Cell-Depleted Spleen Cells as Measured by the Proliferative Response to Endotoxin (ET) [3H]Thymidine UptakeC (cpm) Spleen cells”
TGG in culture
PFCb
-
ET
Control TGG rosette depleted
+ +
40,541 ? 853 1103
5,063 ? 1,219 3,307 zt 1,833
90,239 k 1,060 93,122 + 5,924
a Primed to TGG. * Day 6. c Day 2.
174
WALKER AND WEIGLE TABLE 4 Specificity of Depletion of TGG-Specific B Cells from Mouse Spleen PFC TGG in culture
SRBC in culture
vs SRBC
Spleen cells” Control Control Control Control TGG rosette depleted TGG rosette depleted
+ + + +
+ + +
<40 9,260 ? 564 <40 5,676 f 740 <40 7,488 + 241
vs TGG (I&)
(W)
5,234 _’ 1,691 2,012 ? 1,208 42,193 f 2,254 43,482 2 8,052 <160 ~160
a Spleen cells were from TGG-primed mice given iv 0.02 ml of 0.01% SRBC suspension to prime T cells to SRBC (14).
tion of the SRBC and coupling with protein reduced the antigenicity of the SRBC by greater than 99% as assayed by hemagglutination with antiserum to SRBC. This should account for the failure of SRBC-specific cells to be removed. Titration of Helper T-Cell Activity in TGG Rosette-Depleted Cultures Even though in Table 1 it was shown that significant numbers of T cells specific for TGG were not removed by rosetting with TGG-coupled RBC, it was still possible that some specific T cells were removed. Thus, it was determined if the rosetting procedure removed any antigen-specific T cells as assayed by a titration of helper T-cell activity. Figure 1 compares the capacity of TGG rosette-depleted primed spleen cells and non-TGG rosette-depleted primed spleen cells to reconstitute the response of T-cell-depleted primed spleen cells. Whether the source of T-cell help was rosette- or non-rosette-depleted spleen cells, as the percentage of T cells was increased, PFC generated by the TGG-specific B cells increased to similar values, indicating equivalent helper T-cell contents in the two spleen cell populations. Thus, the depletion of TGG-specific rosetting cells reduced the response to TGG through removal of specific B cells, but not specific helper T cells. DISCUSSION A rosetting procedure was described for depletion of mouse B cells primed to a soluble protein antigen. Others have used rosetting procedures for removal of RBC-specific ASC (15,16) or hapten-specific ASC (17). However, rosette depletion of ASC specific for RBC coupled with relatively large, soluble protein antigens, such as TGG, has not been described before. The rosette depletion method removed specific B cells, but not helper T cells. Methods employing antigen columns (2, 3) or the FACS (7,8) also do not remove helper T cells, although both T and B cells have been found to rosette with SRBC antigen (15, 18). Excluding RBC antigens, it is uncertain why antigen-binding depletion procedures fail to remove specific T cells, because T cells do have the capacity to bind antigen, as shown by suicide experiments with highly radio-
ROSETTE DEPLETION OF ANTIGEN-SPECIFIC
B CELLS
175
160-
FIG. 1. Titration of helper T-cell activity in rosette-depleted versus non-rosette-depleted spleen cells. Increasing numbers of either TGG-primed spleen cells (0) or TGG rosette-depleted TGGprimed spleen cells (0) as a source of TGG-specific helper T cells were cultured with T-cell-depleted, TGG-primed spleen cells as a source of TGG-specific B cells. The total number of spleen cells per culture was held constant at 6 x 105.For purposes of calculation it was assumed that nonmanipulated spleen cells contained 50% T and 50% B cells, and that TGG rosette-depleted spleen cells contained a normal complement of T cells and B cells, except for the TGG-specific B cells which were removed, as shown by the failure of these cells to give a PFC response. The indirect antibody response measured after 6 days was normalized to PFC/lOr non-TGG rosette-depleted B cells.
labeled antigen (19) and radioautography (20). However, there are differences between T-cell and B-cell antigen binding; e.g., T cells bind about five times less antigen per cell than B cells (20), bind antigen more efficiently at 37°C rather than at 4°C (21), the temperature used in most B-cell depletion procedures, and appear to bind antigen more effectively in association with cell recognition products, such as those associated with macrophages (22). Thus, it is possible that as the binding characteristics of soluble protein antigens to T cells are better understood, helper T cells may be able to be removed by antigen binding. However, under the conditions described in this study, the rosetting procedure for AX depletion is restricted to B cells. Rosetting with antigen-coupled RBC has advantages in terms of recovery, simplicity of the procedure, and efficiency of ASC depletion when compared to other procedures. Antigen-coupled glass bead columns retain significant numbers of lymphoid cells nonspecifically, reducing the recovery of AX-depleted cells (2). In addition, other antigen-coupled matrices, such as Sephadex, may remove plasma cells and macrophages (23), thus reducing their selectivity for ASC. Other antigen matrices have been developed io increase cell recovery to greater than 80% (6), which is comparable to the 70% recovery observed by rosetting, but the process of coupling antigen to these matrices is complex (6). Preparation of the rosetting reagent involves only a few steps, with RBC being a convenient matrix for antigen coupling. In addition, the use of fixed RBC stabilizes the antigencoupled RBC reagent so that it is constantly available for routine depletion of ASC. When prepared sterilely, the reagent has the same long-term stability prop-
176
WALKER AND WEIGLE
erties as the anti-Ig rosetting reagent described in the preceding paper (12). Finally, the capacity of various AX depletion procedures to remove potential antibodyproducing cells varies from 50-90% (9, lo), while the rosette procedure averaged >99%. Rosette depletion may be more efficient at removing ASC since the antigencoupled RBC and ASC are forcibly brought together by centrifugation. This may overcome possible repulsive negative cell surface charges of the RBC and ASC, allowing ASC to bind to the antigen. The rosetting procedure was designed for the removal of ASC, not their enrichment, in contrast to several other procedures. The rosetted B cells are not recoverable by hypotonic lysis, because the RBC are stabilized by GA fixation. Attempts to dissociate the TGG-specific rosettes with soluble TGG have failed (unpublished observation), which may be because the antigen-coupled RBC bind with high avidity through multipoint attachments to the cell surface Ig (24). Despite this limitation, the main advantage of the procedure is the removal of specific B cells to undetectable levels as assayed functionally. Other procedures have concentrated on the recovery of ASC, rather than optimizing the conditions for depletion (4-6, 9, 10, 17). Thus, the rosetting procedure is highly applicable to experimental situations in- which efficient elimination of ASC is desired. Currently, methods to deplete B cells in general vary in effectiveness (12) which has led to debate as to the role of B cells in various immune phenomena. For example, there is discussion as to the necessity for specific B cells in the generation of helper T cells (25). Also, the role of antibody, and thus B cells, in various autoimmune diseases and in response to tumors is not certain. Because the rosetting procedure has the potential to deplete specific B cells highly effectively, it may be applicable to clarifying the role of specific B cells in various immune responses. ACKNOWLEDGMENTS The authors wish to express their appreciation to Joy Sturtevant for her outstanding technical assistance, and to Chris VanLeeuwen for her excellent secretarial work in the preparation of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Byrt, P., and Ada, G. L., Immunology 17, 503, 1969. Wigzell, H., and Andersson, B., J. Exp. Med. 129, 23, 1969. Wigzell, H., and Makehi, O., J. Exp. Med. 132, 110, 1970. Edelman, G. M., Rutishauser, U., and Millette, E. F., Proc. Nut. Acad. Sci. USA 6X3,2153, 1971. Choi, T. K., Slight, D., and Nisonoff, A., .Z. Exp. Med. 139, 761, 1974. Truffa-Bachi, P., and Wofsy, L., Proc. Nat. Acad. Sci. USA 66, 685, 1970. Julius, M. H., Masuda, T., and Herzenberg, L. A., Proc. Nat. Acad. Sci. USA 69, 1934, 1972. Julius, M. H., and Herzenberg, L. A., .Z. Exp. Med. 140, 904, 1974. Haas, W., Schrader, J. W., and Szenberg, A., Eur. J. Immunol. 4, 565, 1974. Scott, D. W., J. Exp. Med. 144, 69, 1976. Walker, S. M., Meinke, G. C., and Weigle, W. O., J. Exp. Med. 146, 445, 1977. Walker, S. M., Meinke, G. C., and Weigle, W. O., Cell. Immunol. 46, 158, 1979. Walker, S. M., and Weigle, W. O., Cell. Immunol. 36, 78, 1978. Falkoff, R., and Kettman, J. R., J. Zmmunol. 108, 54, 1972. Bach, J. F., and Dardenne, M., CeN. Zmmunol. 3, 1, 1972. Kenny, J. J., Merrill, J. E., and Ashman, R. F., J. Zmmunol. 120, 1233, 1978. Nossal, G. J. V., and Pike, B. L., J. Zmmunol. 120, 145, 1978. 18. Elliott, B. E., Haskill, J. S., and Axelrad, M. A., Z. Exp. Med. 138, 1133, 1973. 19. Basten, A., Miller, J. F. A. P., Warner, N. L., and Pye, J., Nature New Biol. 231, 104, 1971. 20. Hammerling, G. J., and McDevitt, H. O., J. Immunol. 112, 1726, 1974.
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B CELLS
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21. Hammerling, G., Lonai, P., and McDevitt, H. O., In “Membrane Receptors of Lymphocytes” (M. Seligmann, J. L. Preud’homme, and F. M. Kourilsky, Eds.), p. 117. American Elsevier, New York, 1975. 22. Paul, W. E., and Benaceraff, B., Science 195, 1293, 1977. 23. Ly, I. A., and Mishell, R. I., J. Immunol. Mefhods 5, 239, 1974. 24. Bystryn, J.-C., Siskind, G. W., and Uhr, J. W., J. Exp. Med. 141, 1227, 1975. 25. Playfair, J. H. L., Clin. Exp. Zmmunol. 17, 1, 1974.
Separation
of Various
II. Depletion
of Antigen-Specific B Cells by Rosetting with GlutaraldehydeFixed, Antigen-Coupled Red Blood Cells1 SHARYN
B-Cell Subpopulations
M. WALKERS AND WILLIAM
from Mouse Spleen
0. WEIGLE
Department of Immunopathology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037 Received
December
5, 1978
Mouse spleen cells were depleted of antigen-specific B cells (ASC) by rosetting with glutaraldehyde-fixed RBC coupled with specific antigen. Removal of the rosetted cells by density gradient centrifugation resulted in the depletion of more than 99% of all the ASC. Depletion was shown by the failure of rosette-depleted, primed spleen cells to generate antigen-specific antibody-producing cells. Functional assays showed that deither macrophages or helper T cells were removed. In comparison to other procedures used to deplete of ASC, rosetting with antigen-coupled RBC has advantages in terms of recovery, simplicity of the procedure, and efficiency of ASC depletion.
INTRODUCTION The relatively high density of surface Ig on B cells has permitted the development of methods to deplete antigen-specific B cells from mouse spleen. For some time it has been appreciated that surface Ig reflects the potential Ig product synthesized by these cells when triggered by antigen. This has been shown by the capacity of highly radiolabeled antigen to kill or “suicide” B cells with specificity for the antigen (l), the selective depletion of antigen-specific B cells (ASC)3 by passage over antigen columns (2-6), and the selective depletion of ASC treated with fluoresceinated antigen by passage through the fluorescence-activated cell sorter (FACS) (7). Use of this latter procedure has shown that surface Ig reflects not only the specificity, but also the avidity of the potential Ig product (8). These methods have been adopted and modified by others (9- 11) as a means to deplete and enrich for ASC for the study of cell-cell interactions in the immune response. This paper details a simple rosetting procedure to deplete ASC from spleen cells primed to a soluble protein antigen, turkey y-globulin (TGG). The outstanding 1 This is Publication No. 1669 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California. This work was supported in part by United States Public Health Service Grant AI-07007, American Cancer Society Grant IM42-H, and Biomedical Research Support Program Grant (RRO-5514). * Recipient of a Fellowship from the Arthritis Foundation. 3 Abbreviations used: ASC, antigen-specific B cells; FACS, fluorescence-activated cell sorter; TGG, turkey y-globulin; PFC, plaque-forming cell; GA, glutaraldehyde; ET, endotoxin. 170 0008-8749/79/090170-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
ROSETTE DEPLETION
OF ANTIGEN-SPECIFIC
B CELLS
171
feature of the procedure is the extreme effectiveness in the removal of ASC, while involving minimal manipulation and time. MATERIALS
AND METHODS
Zmmunizarion of mice. Male A/J mice (Jackson Laboratory, Bar Harbor, Me.) were primed at 6- 10 weeks of age with 0.1 ml of 100 to 200 pg alum-precipitated TGG in 0.1 ml complete Freund’s adjuvant given intraperitoneally (11). Generation and assay of antibody-producing cells. Spleen cells from mice primed with TGG 6 weeks or more prior were cultured and assayed as described in the preceding paper (12). TGG was added to the cultures indirectly by incubating the spleen cells at 4°C for 30-60 min with soluble TGG and then removing the unbound TGG with multiple washes. Plaque-forming cells (PFC) were measured after 6 days, with only indirect PFC measured in most experiments as the IgM response to TGG was insignificant. Results are expressed as the mean PFC of lo7 original spleen cells of duplicate cell pools with the standard error. Assay for endotoxin (ET)-induced proliferation. Spleen cells were cultured as for the generation of antibody-producing cells except that mercaptoethanol was omitted. Endotoxin, 055:B5 trichloroacetic acid extract, Difco Laboratories, Detroit, Michigan, was added at culture initiation at 50 I.Lg/ml.After 2 days culture, proliferation was measured by a [3H]thymidine pulse (13). Removal of TGG-specific B cells. Spleen cells were rosetted at 4°C with glutaraldehyde (GA)-fixed, TGG-coupled RBC and the rosetted cells removed by centrifugation on a density gradient similar to the rosetting procedure for the removal of B cells described in the preceding paper (12). RBC were fixed as described therein, except that a finer grade of GA was used to reduce nonspecific rosetting. Rosetting with TGG coupled to RBC fixed with a practical grade of GA averaged 4% rosettes, while TGG coupled to RBC fixed with a finer grade of GA averaged 1%. Briefly, 1.5 ml of 8% GA (8% Ultrapure-TEM Grade, Tousimis Research Corp., Rockville, Md., Catalog No. 1058) was neutralized with 0.050 ml 10% sodium carbonate and 5 ml 0.15 M sodium chloride added, followed by 1 ml 0.015 M sodium phosphate buffer, pH 8.0. Fixation was continued for 24 hr at 4°C with continuous stirring; the RBC were then washed, treated with tannic acid, and coupled with TGG as described for coupling Ig (12). Recovery of rosette-depleted spleen cells averaged 70%. RESULTS Depletion
of TGG-Specific
B Cells
Spleen cells primed to TGG were incubated at 4°C with GA-fixed, TGG-coupled RBC in an attempt to rosette the B cells specific for TGG. After removal of the rosetted cells, the remaining cells were assayed for their capacity to generate PFC after culture with TGG for 6 days. Table 1 shows that the control response to TGG was greater than 19,000 PFC per 10’ spleen cells put into culture, while the TGG rosette-depleted cultures failed to show a significant response above background. The depletion of TGG-specific rosetting cells did not remove appreciable numbers of TGG-specific T cells, as shown by the ability of rosettedepleted cells to reconstitute the response of T-cell-depleted, primed spleen cells from less than 52 to 13,071 PFC. Depletion of TGG-specific rosetting cells was
172
WALKER AND WEIGLE TABLE 1 Capacity of GA-Fixed, TGG-Coupled RBC to Remove B Cells Specific for TGG from Spleen Cells Primed to TGG
Exp. No. 1
2
Spleen cells (5 x 10s) Control Control TGG rosette depleted T cell depleted TGG rosette depleted (2.5 x 105) + T cell depleted (2.5 x 105) HSA rosette depleted Control Control TGG rosette depleted
TGG in culture
PFC
-
52 19,372 f 8,016 78 k 26 ~52
+ + +
+ +
13,971 + 933 18,203 f 7,313
TGG-treated adherent cells” TGG-treated adherent cells”
~61 5,368 2 488 <61
a TGG-treated adherent cells were prepared by adsorbing 6 x lo5 TGG-coupled spleen cells to individual wells in 0.06 ml medium with 10% FCS for 30 min at 37°C and washing six times vigorously to remove nonadherent cells.
dependent on rosetting with TGG-coupled RBC as shown by the failure of human serum albumin (HSA)-coupled RBC to remove TGG-responding cells. To determine if the reduced PFC response was due partially or entirely to removal of antigen-presenting cells, such as macrophages with cytophilic antibody for TGG, rather than TGG-specific B cells, attempts were made to reconstitute the response of rosette-depleted cells with adherent cells. TGG was presented to the cultures on non-TGG-primed spleen cells allowed to adhere to the culture wells before addition of the primed spleen cells. The last two lines of Table 1 show that TGCJpresented in this manner permitted control, non-rosette-depleted spleen cells to respond, but did not enable TGG rosette-depleted spleen cells to respond. In addition, nonspecific esterase staining of rosette-depleted sp1ee.ncells showed no decrease in macrophage number, making the loss of antigen-presenting cells unlikely. Thus, it can be concluded that the rosetting procedure eliminated the antibody response of TGG-primed spleen cells by depleting TGG-specific B cells and not an accessory cell. Eflect of the Temperature of Rosetting The rosetting procedure in the experiments described to this point was carried out at 4°C during the pelleting and gradient separation procedures. Table 2 shows that this temperature was optimal when compared to 37°C as TGG-primed spleen cells which had been depleted of cells rosetting at 37°C with HSA-coupled RBC failed to generate a response to TGG. This is in contrast to the results in Table 1 in which rosetting at 4°C with HSA-coupled RBC had no effect on the in vitro TGG response. Relatively large numbers of HSA rosettes were formed at 37°C about 20%, while experiments done at 4°C averaged only 0.2%. It can also be seen in Table 2 that the 37°C incubated HSA rosette-depleted cells did not respond to TGG even when TGG-adherent cells were added as a means of presenting antigen, indicating that large numbers of TGG-specific B cells were removed nonspecifically
ROSETTE DEPLETION
OF ANTIGEN-SPECIFIC
173
B CELLS
TABLE 2 Effect of Rosetting at 4°C versus 37°C on the Removal of Specific B Cells Spleen cells
TGG in culture
PFC
Control Control TGG rosette depleted at 4°C HSA rosette depleted 37°C HSA rosette depleted 37°C
+ + + TGG-treated adherent cells”
<61 5368 k 488 ~61 <61 <61
a TGG-treated adherent cells were prepared as described in Table 1.
by rosetting at 37°C. Thus, to obtain antigen-specific rosette depletion of AX, the procedure was carried out at low temperatures in all subsequent experiments. Determination of the B-Cell Content of TGG Rosette-Depleted Spleen Cells The B-cell content of TGG ‘rosette-depleted cultures was next quantified to see if the removal of TGG-specific B cells appreciably removed other B cells nonspecifically. A potent B-cell mitogen, ET, was added to TGG rosette-depleted spleen cells and the proliferative response measured 2 days later. Table 3 shows that rosetting removed all the potential PFC to TGG, while having no effect on ET-elicited proliferation. This is not surprising, since the number of TGG rosettes formed averaged about 1% of the spleen cells, while B cells constituted about 50% of the spleen. Even though the B-cell content of TGG rosette-depleted cultures was normal as measured by mitogenesis, the procedure could still have removed a number of non-TGG-specific B cells not detectable by ET-induced proliferation. Thus, to assess the specificity of TGG rosette depletion, the response of TGG rosettedepleted spleen cells to another antigen, SRBC, was measured as well. Table 4 shows that the TGG rosette-depleted spleen cells failed to generate PFC to TGG, but gave a normal response to SRBC. Thus, the deletion procedure was highly selective. It may be noted that since TGG was coupled to SRBC, one might have expected SRBC-specific cells to have been removed as well. However, the process of fixaTABLE 3 Determination of the B-Cell Content of Specific B-Cell-Depleted Spleen Cells as Measured by the Proliferative Response to Endotoxin (ET) [3H]Thymidine UptakeC (cpm) Spleen cells”
TGG in culture
PFCb
-
ET
Control TGG rosette depleted
+ +
40,541 ? 853 1103
5,063 ? 1,219 3,307 zt 1,833
90,239 k 1,060 93,122 + 5,924
a Primed to TGG. * Day 6. c Day 2.
174
WALKER AND WEIGLE TABLE 4 Specificity of Depletion of TGG-Specific B Cells from Mouse Spleen PFC TGG in culture
SRBC in culture
vs SRBC
Spleen cells” Control Control Control Control TGG rosette depleted TGG rosette depleted
+ + + +
+ + +
<40 9,260 ? 564 <40 5,676 f 740 <40 7,488 + 241
vs TGG (I&)
(W)
5,234 _’ 1,691 2,012 ? 1,208 42,193 f 2,254 43,482 2 8,052 <160 ~160
a Spleen cells were from TGG-primed mice given iv 0.02 ml of 0.01% SRBC suspension to prime T cells to SRBC (14).
tion of the SRBC and coupling with protein reduced the antigenicity of the SRBC by greater than 99% as assayed by hemagglutination with antiserum to SRBC. This should account for the failure of SRBC-specific cells to be removed. Titration of Helper T-Cell Activity in TGG Rosette-Depleted Cultures Even though in Table 1 it was shown that significant numbers of T cells specific for TGG were not removed by rosetting with TGG-coupled RBC, it was still possible that some specific T cells were removed. Thus, it was determined if the rosetting procedure removed any antigen-specific T cells as assayed by a titration of helper T-cell activity. Figure 1 compares the capacity of TGG rosette-depleted primed spleen cells and non-TGG rosette-depleted primed spleen cells to reconstitute the response of T-cell-depleted primed spleen cells. Whether the source of T-cell help was rosette- or non-rosette-depleted spleen cells, as the percentage of T cells was increased, PFC generated by the TGG-specific B cells increased to similar values, indicating equivalent helper T-cell contents in the two spleen cell populations. Thus, the depletion of TGG-specific rosetting cells reduced the response to TGG through removal of specific B cells, but not specific helper T cells. DISCUSSION A rosetting procedure was described for depletion of mouse B cells primed to a soluble protein antigen. Others have used rosetting procedures for removal of RBC-specific ASC (15,16) or hapten-specific ASC (17). However, rosette depletion of ASC specific for RBC coupled with relatively large, soluble protein antigens, such as TGG, has not been described before. The rosette depletion method removed specific B cells, but not helper T cells. Methods employing antigen columns (2, 3) or the FACS (7,8) also do not remove helper T cells, although both T and B cells have been found to rosette with SRBC antigen (15, 18). Excluding RBC antigens, it is uncertain why antigen-binding depletion procedures fail to remove specific T cells, because T cells do have the capacity to bind antigen, as shown by suicide experiments with highly radio-
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160-
FIG. 1. Titration of helper T-cell activity in rosette-depleted versus non-rosette-depleted spleen cells. Increasing numbers of either TGG-primed spleen cells (0) or TGG rosette-depleted TGGprimed spleen cells (0) as a source of TGG-specific helper T cells were cultured with T-cell-depleted, TGG-primed spleen cells as a source of TGG-specific B cells. The total number of spleen cells per culture was held constant at 6 x 105.For purposes of calculation it was assumed that nonmanipulated spleen cells contained 50% T and 50% B cells, and that TGG rosette-depleted spleen cells contained a normal complement of T cells and B cells, except for the TGG-specific B cells which were removed, as shown by the failure of these cells to give a PFC response. The indirect antibody response measured after 6 days was normalized to PFC/lOr non-TGG rosette-depleted B cells.
labeled antigen (19) and radioautography (20). However, there are differences between T-cell and B-cell antigen binding; e.g., T cells bind about five times less antigen per cell than B cells (20), bind antigen more efficiently at 37°C rather than at 4°C (21), the temperature used in most B-cell depletion procedures, and appear to bind antigen more effectively in association with cell recognition products, such as those associated with macrophages (22). Thus, it is possible that as the binding characteristics of soluble protein antigens to T cells are better understood, helper T cells may be able to be removed by antigen binding. However, under the conditions described in this study, the rosetting procedure for AX depletion is restricted to B cells. Rosetting with antigen-coupled RBC has advantages in terms of recovery, simplicity of the procedure, and efficiency of ASC depletion when compared to other procedures. Antigen-coupled glass bead columns retain significant numbers of lymphoid cells nonspecifically, reducing the recovery of AX-depleted cells (2). In addition, other antigen-coupled matrices, such as Sephadex, may remove plasma cells and macrophages (23), thus reducing their selectivity for ASC. Other antigen matrices have been developed io increase cell recovery to greater than 80% (6), which is comparable to the 70% recovery observed by rosetting, but the process of coupling antigen to these matrices is complex (6). Preparation of the rosetting reagent involves only a few steps, with RBC being a convenient matrix for antigen coupling. In addition, the use of fixed RBC stabilizes the antigencoupled RBC reagent so that it is constantly available for routine depletion of ASC. When prepared sterilely, the reagent has the same long-term stability prop-
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erties as the anti-Ig rosetting reagent described in the preceding paper (12). Finally, the capacity of various AX depletion procedures to remove potential antibodyproducing cells varies from 50-90% (9, lo), while the rosette procedure averaged >99%. Rosette depletion may be more efficient at removing ASC since the antigencoupled RBC and ASC are forcibly brought together by centrifugation. This may overcome possible repulsive negative cell surface charges of the RBC and ASC, allowing ASC to bind to the antigen. The rosetting procedure was designed for the removal of ASC, not their enrichment, in contrast to several other procedures. The rosetted B cells are not recoverable by hypotonic lysis, because the RBC are stabilized by GA fixation. Attempts to dissociate the TGG-specific rosettes with soluble TGG have failed (unpublished observation), which may be because the antigen-coupled RBC bind with high avidity through multipoint attachments to the cell surface Ig (24). Despite this limitation, the main advantage of the procedure is the removal of specific B cells to undetectable levels as assayed functionally. Other procedures have concentrated on the recovery of ASC, rather than optimizing the conditions for depletion (4-6, 9, 10, 17). Thus, the rosetting procedure is highly applicable to experimental situations in- which efficient elimination of ASC is desired. Currently, methods to deplete B cells in general vary in effectiveness (12) which has led to debate as to the role of B cells in various immune phenomena. For example, there is discussion as to the necessity for specific B cells in the generation of helper T cells (25). Also, the role of antibody, and thus B cells, in various autoimmune diseases and in response to tumors is not certain. Because the rosetting procedure has the potential to deplete specific B cells highly effectively, it may be applicable to clarifying the role of specific B cells in various immune responses. ACKNOWLEDGMENTS The authors wish to express their appreciation to Joy Sturtevant for her outstanding technical assistance, and to Chris VanLeeuwen for her excellent secretarial work in the preparation of the manuscript.
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