Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 432-435, January 1978
Immunology
Blocking of primary in vitro antibody responses to thymusindependent and thymus-dependent antigens with antiserum specific for IgM or IgD (receptor IgD/B cells)
J. C. CAMBIER, F. S. LIGLER, J. W. UHR, J. R. KETTMAN, AND E. S. VITETTA Department of Microbiology, University of Texas Southwestern Medical School, Dallas, Texas 75235
Communicated by H. Sherwood Lawrence, October 11, 1977
(GARIg) have been described (17). Rabbit anti-6 was judged to be monospecific by published criteria (18), including: (i) a single peak on sodium dodecyl sulfate/polyacrylamide gel electrophoresis after reaction with a lysate of iodinated splenocytes; (ii) immunofluorescent staining of the predicted numbers of cells from various lymphoid tissues; (iii) inability to stain splenocytes after their treatment with either anti-Ig or allotypic (19) anti-6; and (iv) independent capping of surface molecules on splenocytes with anti-6 and anti-A. Chromatographically purified fractions of normal rabbit serum (NRS), anti-,q, anti-Ig, or a 30% (NH4)2SO4 saturation precipitate of anti-6 was used for in vitro cell culture. The IgG fraction from GARIg was conjugated with fluorescein isothiocyanate (Sigma) (FITC-GARIg) and chromatographed on DEAE-cellulose (Whatman DE52) to give a fraction with molar fluorescein/ protein ratio of 2.6. Capping of Cell Surface Ig. Splenocytes were incubated for 30 min at 4° with NRS or the rabbit antisera described above. Cells were washed and exposed to GARIg (20) for 90 min at 370 in complete medium to achieve capping. Immunofluorescent Staining. Viable cells were prepared and treated with NRS or antisera specific for Ig, ,u, or 6 as described (20). After washing with balanced salt solution/azide, the cells were resuspended at 3 X 107 cells per ml in balanced salt solution/azide containing FITC-GARIg (0.1 mg/ml). After 10 min at 40, the cells were washed in balanced salt solution/ azide and fixed in 1% paraformaldehyde in phosphate-buffered saline. The cells were examined at X1000 magnification with a Leitz Ortholux no. 2 fluorescence microscope. For each sample, 100-200 cells were scored for fluorescence without knowledge of the identity of the sample. Culture Conditions. Splenocytes from adult BDF1 (C57BL/6 female X DBA/2 male F1) mice (obtained from Jackson Memorial Laboratory, Bar Harbor, ME) or neonatal mice (from our own breeding colony) were treated as described above to remove cell surface Ig. Cells were resuspended (107 viable cells per ml) in complete medium (IMEMZO, IBL, with 50,uM 2-mercaptoethanol) containing varying concentrations of either the same Ig antibody used to achieve capping or normal Ig. Preliminary experiments had indicated that capping alone with any of the anti-Igs used was ineffective in blocking subsequent immune responses, incubation with anti-Igs only (without prior capping) was marginally effective. Therefore, in the experiments described, control cells were capped with
ABSTRACT The effect of anti-M and anti-6 on the primary in vitro IgM response of murine splenocytes to thymus-dependent (trinitrophenylated erythrocytes) and thymus-independent (trinitrophenylated brucella) forms of trinitrophenyl was studied. The results indicate that either anti-M or anti-S can block the response of adult splenocytes to the thymus-dependent antigen. The thymus-dependent responses of neonatal splenocytes that bear a low concentration of IgD were also abrogated by treatment with anti-S. In contrast, anti-,u, but not anti-5, blocked the response of adult splenocytes to the thymus-independent antigen used. These results indicate that both IgM and IgD are receptors required for triggering cells by a thymus-dependent antigen but that only IgM receptors are required for triggering by the thymus-independent antigen used. Most splenic B cells bear both IgM and IgD (,M+b+) (1-3). On individual cells, both isotypes have identical specificity for antigen (4-7). jj+b+ cells appear to be the major precursors of the primary IgM antibody response to thymus-dependent (TD) antigens (8, 9). The roles played by each of the two isotypes on such cells are not known. One approach to elucidating the function of receptor IgM and IgD is to prevent their interaction with antigen during immunogenic stimulation by treating the cell with isotype-specific antibody. There are a number of reports in which anti-Igs have been used to block immune responsiveness. Both a primary IgM response and a secondary IgG response have been blocked by treatment of the cells with anti-,i (10-12), suggesting that IgM may be essential in "triggering" these responses. Mitogeninduced secretion of IgM and IgG have been blocked with anti-,u (13-15) or anti-S (15). However, anti-S did not block a secondary IgG response (12) and, when administered in vivo to rhesus monkeys, markedly increased IgM and IgG synthesis (16). It is difficult to make a unique interpretation of these observations. The purpose of the present study was to determine the effect of anti-,u and anti-S on the primary in vitro IgM response of murine splenocytes to TD [trinitrophenylated erythrocytes (TNP-SRBC)] and thymus-independent (TI) [trinitrophenylated brucella (TNP-brucella)] forms of trinitrophenylated antigens. The results indicate that IgD is required for responsiveness to TD antigen, whereas IgM is required for responsiveness to both forms of antigen.
MATERIALS AND METHODS Antisera. The preparation and specificity of rabbit antimouse Ig (anti-Ig), rabbit anti-,u, and goat anti-rabbit Ig
Abbreviations: TD, thymus-dependent; TNP-SRBC, trinitrophenylated erythrocytes; TI, thymus independent; TNP-brucella, trinitrophenylated Brucella abortus; GARIg, goat anti-rabbit Ig; NRS, normal rabbit serum; FITC, fluorescein isothiocyanate; PFC, plaque-forming cell.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemnent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 432
Immunology:
Antisera used for capping
NRS
Proc. Natl. Acad. Sci. USA 75 (1978)
Cambier et al.
Table 1. Comparison od cell surface Ig before and after capping Percentage of lymphocytes staining with antisera specific for: Ig IgD IgM After After Before Before Before After 43.0 + 11.3 41.5 ± 13.4 37.0 I 3.5 34.3 ± 1.1 27.3 + 2.3 26.7 + 3.0
433
Control (NRS) After Before 6.5 + 3.5
(3)
1.5 + 0.6
(2)
(2)
6.5 L 3.5
6.5 ± 0.7
(3)
(3)
(3)
(3)
(3)
Anti-,g
32.9 + 6.6
11.5 ± 7.6
41.4 + 6.7
39.2 + 8.3
43.2 I 8.5
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
Anti-6
32.9 + 7.4
35.6 i 7.7
41.4 + 7.7
12.0 ± 7.7
44.4 + 8.4
41.3 + 15.9
6.5 + 3.5
2.0 + 0.0
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
Anti-Ig
33.0 ± 8.5
7.8 ± 4.4
37.4 I 4.5
7.0 + 3.1
42.0 I 8.4
7.4 I 3.3
6.5 i 3.5
5.5 + 3.5
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
36.6 ±78.7
Splenocytes were stained with NRS or specific antisera before and after capping. Values are expressed as the mean ± SD; the number of experiments averaged is in parenthesis.
specific antiserum and then incubated with normal Ig. Cells cultured in 35-mm Falcon dishes, 0.8 ml per dish. To each culture was added 1 X 107 irradiated [1500 R (0.4 C/kg)], low-dose sheep red blood cell-primed (21) spleen cells in 0.1 ml of complete medium and 0.1 ml of 0.1% TNP-SRBC (22) (TD antigen) or 0.01% TNP-brucella (TI antigen) in medium (23). Cultures were incubated essentially as described by Mishell and Dutton (24) with use of the modification of Kettman and Dutton (22) to obtain a primary response. Assay. In all cases, direct plaque-forming cell (PFC) responses were assayed after a 4-day culture period, by using the microscope slide modification (25) of the hemolysis in gel technique of Jerne and Nordin (26). The anti-TNP PFC were determined by using trinitrophenylated horse erythrocytes (Colorado Serum CO) prepared by the method of Rittenberg and Pratt (27). Background PFC, determined using horse erythrocytes, were subtracted from trinitrophenylated horse erythrocyte PFC to give anti-TNP PFC. All data presented are averages of direct hemolytic PFC from duplicate assays obtained from pooled duplicate cultures. were
RESULTS Antibody-Induced Removal of Surface Ig. As shown in Table 1, the values for percentage of positive cells staining with isotype-specific sera were similar to those reported prevously (18, 28)-i.e., IgM = 33%, IgD = 39%, and Ig = 43%. As can be calculated from the data, treatment of splenocytes with anti-A, anti-6, or anti-Ig followed by GARIg decreased the number of cells bearing detectable quantities of the respective isotype(s) by 75-95%. Aliquots of these treated cells were used for studies of function. Effect of Incubation with Antibody on the Immune Responsiveness of Treated Splenocytes from Adult Mice. Treated cells were cultured with TNP-brucella or TNP-SRBC in the presence of varying concentrations (0-1000 ,ug/ml) of the Ig fraction from the same antiserum used for capping. The effect of such treatment on the primary antibody response is shown in Fig. 1. The TD responses to both TNP and SRBC were virtually abolished by treatment with anti-A, anti-6, or anti-Ig at 100-1000 ,ug of Ig per ml. As shown in Fig. 2, inhibition of the TD response did not occur when comparable concentrations of normal rabbit Ig were used, and inhibition was abrogated in the case of either anti-M or anti-Ig, but not anti-6, by addition of a mixture of IgM and IgG to the culture medium. These results suggest that both IgM and IgD receptors are required for activation of responsive cells by TD antigens. As also shown in Fig. 1, the primary TI response to TNP was blocked by anti-M and anti-Ig, but not anti-6, at >100 Ag of Ig
per ml. As shown in Fig. 2, normal rabbit Ig had no effect on the TI response and the blocking with anti-is and anti-Ig could be prevented by adding IgM and IgG to the medium. These results suggest that IgM but not IgD is required for a response to TNP on the TI carrier, brucella. Effect of Incubation with Anti-6 on the Immune Responsiveness of Treated Splenocytes from Neonatal Mice. The above studies suggest that IgD receptors on splenocytes of adult animals are mandatory for triggering a TD response. In contrast to adult mice, neonatal mice bear low levels of IgD on their splenocytes (28-31) and are only marginally responsive to TD antigens (32). The question thereby arises as to whether these small amounts of receptor IgD are required for TD responAnti-6 100 50
0
Anti-Mu
C
0
100
0
U-
50
-
C-
0
Anti-Ig 100
50-
0
1
10
100
1000
1g, jAg/mI FIG. 1. Effect of antibody concentration on the inhibition of in vitro IgM responses to TD (0, SRBC; 0, TNP) and TI (-, TNP) antigens. Cells were treated with the indicated antiserum and GARIg under capping conditions and cultured with an Ig fraction of the same antiserum used for capping. Responses of control cultures incubated without antibody, presented as PFC per 106 viable recovered cells, were as follows: anti-6 capped, SRBC 3408, TNP (TD) 434, TNP (TI) 342; anti-a capped, SRBC 3905, TNP (TD) 445, TNP (TI) 215; anti-Ig capped, SRBC 2672, TNP (TD) 418, TNP (TI) 506.
Proc. Nati. Acad. Sci. USA 75 (1978)
Immunology: Cambier et al.
434
Table 2. Effect of treatment with anti-6 on the immune responsiveness of splenocytes from neonatal mice
SRBC (TD) 44
PFC/106 viable recovered cells
L1 ML1.
z
Age of donor of B cells, days
Q
49
0
18 7
.0
C)
M 0
L._
rNr' I 1_
IME Anti-S Anti-Mu Anti-Ig Antibody used for capping
FIG. 2. Effect of antibody on the inhibition of in vitro IgM reControl cells were capped with specific antiserum and then were incubated with normal rabbit Ig at 400 ;g/ml (0). Capping alone was shown to have no effect on the subsequent antibody response presumably due to regeneration of receptors. The anti-g and anti-6 Ig preparations (a) were used at a concentration of 400 ;g/ml and the anti-Ig Ig fraction, at 250 jsg/ml (see Fig. 1). Where indicated (i), IgG (from normal serum) and IgM (from MOPC 104 secretions) were added at concentrations of approximately 800 ,g/ml and 200 ,g/ml, respectively, to cultures containing anti-g, anti-6, or anti-Ig.
sponses to TD and TI antigens.
siveness of neonatal splenocytes. An alternative possibility is that
the responses could be generated by cells bearing only IgM, which constitute the vast majority of cells in the spleens of animals less than 1 week of age. As shown in Table 2, treatment of splenocytes from 7- to 18-day-old mice with anti-b completely abolished the TD responses to both TNP and SRBC. These results suggest that the presence of receptor IgD is essential for triggering a neonatal cell with a TD antigen. We presume, therefore, that the responder cell bears both IgM and IgD, as in the adult. DISCUSSION The present studies demonstrate that either anti-At or anti-6 can block the primary in vitro immune response of adult splenocytes to the TD antigen, TNP-SRBC. In contrast, anti-M, but not anti-6, blocks the response to the TI antigen, TNP-brucella. Both responses can be blocked by anti-Ig serum. These observations have implications for the phenotype of the subset of lymphocytes responding to TD and TI antigens and for the role played by particular receptor isotypes in signaling virgin B cells during stimulation by antigen. These results represent convincing evidence that the major precursor for the primary IgM antibody response to a TD antigen is a 1i+b+ cell. Previous studies in mice indicated that (i) splenocytes stained with either anti-At or anti-b and positively sorted on the fluorescence-activated cell sorter gave virtually all of the primary IgM response in an adoptively transferred host (8), (ii) elimination of AL+ cells with anti-AL, complement, and azide abolished the primary IgM response (33), also in an adoptively transferred host, and (iii) either anti-AL or anti-b alone was ineffective in preventing antigen-induced suicide of the cell giving rise to the primary IgM antibody response (9).
SRBC Normal Anti-b rabbit Ig 5333 1476 302
238 0 0
TNP (TD) Normal rabbit Ig Anti-6
736 284 29
0 0 0
However, two critical experiments have not been performed: direct analysis of the contribution to the primary IgM response of 9+6+ cells and determination of the effect of deleting 6+ cells on the IgM response (the anti-b antisera in use are not cytotoxic). The present observations that blocking with either anti-,s or anti-b causes a reduction by over 90% of the primary response to TD antigens provide strong evidence that the precursor for the TD response bears both isotypes. The above results also indicate that the precursor for the TI response to TNP-brucella bears no functional IgD receptor. Earlier evidence that the precursor is a cell that bears predominantly IgM (;p+) is as follows: (i) neonatal splenocytes that contain only ,+p cells can give TI responses to certain antigens (34, 35) and (ii) large cells from adult spleens that are + (36) are enriched for TI responsiveness (37). The inability of anti-6 to block the primary IgM response argues either that no IgD is present on the precursor or that IgD is unnecessary for stimulation of the cell. The differences in time of acquisition of responsiveness to various TI antigens during ontogeny (34, 35) suggest that there could be certain TI antigens that require IgD receptors for triggering. The most striking implication of the present data is that both IgM and IgD are needed for triggering a B cell by a TD antigen. The role played by each isotype in immune activation is not known. One possibility is that each isotype gives an independent and necessary signal. Because T cells also provide an essential signal, triggering of TD-responsive B cells may require three signals. Another possibility is that the IgM and IgD receptors must interact with each other, possibly through additional membrane molecules to form a single triggering complex. Finally, the density of the receptors on TD responders may be relatively low, such that antigen interacting with either isotype is insufficient for delivering a signal; both must be involved in order to trigger. Immunofluorescence evidence obtained with the fluorescence-activated cell sorter (38) is consistent with the
latter possibility. There are several studies of embryonic or neonatal cells in culture that suggest that immune responsiveness to TD antigens develops before the acquisition of IgD receptors (39-43). Examination of the experimental protocols used in these studies indicates that, if ontogeny proceeds normally in culture, then immune responsiveness of fetal cells correlates with the expected time of appearance of surface IgD, 3-5 days after birth (30, 31). For example, 17-day fetal liver cells that secrete antibodies after 8 days in culture (41) would be analogous to B cells from 4-day-old neonates. Past studies are therefore consistent with the notion that receptor IgD is necessary for triggering virgin B cells with TD antigens. The present results provide further direct evidence for this idea because removal of receptor IgD completely abrogated the primary IgM response of neonatal splenocytes to SRBC and TNP. We cannot exclude the possibility that there is a trace contribution to the TD response by a p+ cell that is below the level of detection in our system. It should be emphasized that the TNP response of untreated
Immunology: Cambier et al. neonatal cells is markedly decreased compared to the Adtilt' response, suggesting that the TNP-responsive precursors in the neonate have recently appeared. A comparison of the effect of removing IgM or IgD on tolerance induction and immune responsiveness (44, 45) provides a provocative paradox. It appears as though acquisition of appreciable amounts of IgD on a A+ cell is responsible for markedly decreasing susceptibility to induction of tolerance of TD responders. Thus, removal of IgD by means of treatment with papain or anti-6 serum increases the tolerance susceptibility of the cell so that it behaves like neonatal TD responders which bear little IgD. Treatment with anti-M does not have a similar effect; indeed, preliminary experiments suggest that it may decrease the susceptibility of the IA+b+ cell to tolerance induction. In contrast, as mentioned above, both At and a appear to be essential for triggering a TD responder. It could be argued, therefore, that, after interaction with antigen, IgD can only give a triggering signal whereas IgM can give either a triggering signal or a tolerogenic one. Such an explanation is compatible with the results of tolerance induction and triggering of TI precursors which appear to be ,up+ cells. These cells are readily tolerized, yet they can be triggered for antibody formation by a TI antigen. IgM receptors are essential for each of these functions (45). The surface events that determine whether triggering or tolerance is induced require further study. We are grateful to Ms. G. Sloan, Mr. S. Lin, Ms. M. Bagby, Mr. Y. Chinn, Ms. S. Diase, and Ms. M. Neale for expert technical assistance and Ms. J. Hahn for secretarial assistance. J.C.C. is the recipient of Postdoctoral Grant AI-05021'from the National Institutes of Health. This work was supported by National Institutes of Health Grants Al10967, AI-11851, AI-12789, and AI-11893. 1. Pernis, B., Forni, L. & Knight, K. L. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, M., Preud'homme, J. L. -& Kourilsky, F. M. (North-Holland Publishing Co., Amsterdam), pp. 57-64. 2. Parkhouse, B. & Abney, E. R. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, J. L., Preud'homme, J. L. & Kourilsky, F. M. (North-Holland Publishing Co., Amsterdam), pp. 51-56. 3. Vitetta, E. S. & Uhr, J. W. (1976) Eur. J. Immunol. 6, 140143. 4. Salsano, F., Froland, S. S., Natvig, J. B. & Michaelsen, T. E. (1974) Scand, J. Immunol 3, 841-846. 5. Fu, S. M., Winchester, R. J. & Kunkel, H. G. (1975) J. Immunol. 114,250-252. 6. Stern, C. & McConnell, I. (1976) Eur. J. Immunol. 6, 225227. 7. Goding, J. W. & Layton, J. E. (1976) J. Exp. Med. 114, 852857. 8. Zan-Bar, I., Vitetta, E. S. & Strober, S. (1977) J. Exp. Med. 145, 1206-1215. 9. Coffman, R. L. & Cohn, M. (1977) J. Immunol. 118, 18061815. 10. Pierce, C. W., Solliday, S. M. & Asofsky, R. (1972) J. Exp. Med. 135,698-710. 11. Pierce, C. W., Solliday, S. M. & Asofsky, R. (1972) J. Exp. Med.
135,675-697.
Proc. Natl. Acad. Sci. USA 75 (1978)
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12. A Abney, E. R., Keeler, K. D., Parkhouse, R. M. E. & Willcox, H. N. A. (1976) Eur. J. Immunol. 6,443-450. 13. Melchers, F. & Andersson, J. (1973) Transplant. Rev. 14, 76130. 14. Andersson, J., Bullock, W. W. & Melchers, F. (1974) Eur. J. Immunol. 4, 715-722. 15. Finkelman, F. D. & Lipsky, P. E. (1977) J. Immunol., in press. 16. Pernis, B. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, M., Preud'homme, J. L. & Kourilsky, F. M. (NorthHolland Publishing Co., Amsterdam), pp. 25-26. 17. Vitetta, E. S., Forman, J. & Kettman, J. R. (1976) J. Exp. Med. 143, 1055-1066. 18. Zan-Bar, I., Strober, S. & Vitetta, E. S. (1977) J. Exp. Med. 145, 1188-1205. 19. Goding, J. W., Warr, G. W. & Warner, N. L. (1976) Proc. Natl. Acad. Sci. USA 73, 1305-1309. 20. Ligler, F. S., Vitetta, E. S. & Uhr, J. W. (1977) J. Immunol., 119, 1545-1546. 21. Falkoff, R. & Kettman, J. (1972) J. Immunol. 108,54-58. 22. Kettman, J. R. & Dutton, R. W. (1970) J. Immunol. 104, 1558-1561. 23. Cambier, J. C., Vitetta, E. S., Uhr, J. W. & Kettman, J. R. (1977) J. Exp. Med. 145, 778-783. 24. Mishell, R. I. & Dutton, R. W. (1967) J. Exp. Med. 126, 423454. 25. Mishell, R. & Dutton, R. W. (1966) Science 153, 1004-1005. 26. Jerne, N. K. & Nordin, A. A. (1963) Science 140,'405. 27. Rittenberg, M. R. & Pratt, K. L. (1969) Proc. Soc. Exp. Biol. Med. 132,575-581. 28. Goding, J. (1977) Contemporary Topics in Immunobiology (Plenum, New York), Vol. 8, in press. 29. Vitetta, E. S., Melcher, U., McWilliams, M., Phillips-Quagliata, J., Lamm, M. & Uhr, J. W. (1975) J. Exp. Med. 141, 206-216. 30. Abney, E. & Parkhouse, R. M. E. (1974) Nature 252, 600-602. 31. Kearney, J. F., Cooper, M. D., Klein, J. & Abney, E. R. (1977) J. Exp. Med. 146; 297-301. 32. Cambier, J. C., Kettman, J. R., Vitetta, E. S. & Uhr, J. W. (1976) J. Exp. Med. 144,293-297. 33. Yuan, D., Vitetta, E. S. & Kettman, J. (1977) J. Exp. Med. 145, 1421-1436. 34. Howard, J. G. & Hale, C. (1976) Eur. J. Immunol. 6, 486492. 35. Cambier, J. C., Kettman, J. R., Vitetta, E. S. & Uhr, J. W. (1977) Fed. Proc. Fed. Am. Soc. Biol. 36, 1302. 36. Qoodman, S. A., Vitetta, E. S., Melcher, U. & Uhr, J. W. (1975) J. Immunol. 114, 1646-1648. 37. Gorcyznski, R. M. & Feldman, M. (1975) Cell. Immunol. 18, 88-97. 38. Scher, I, Sharrow, S. O., Wastar, R., Jr., Asofsky, R. & Paul, W. E. (1976) J. Exp. Med. 144,494-506. 39. Press, J. & Klinman, N. (1973) J. Immunol. 111, 829-835. 40. Rosenberg, Y. J. & Cunningham, A. J. (1976) J. Immunol. 117, 1618-1621. 41. Phillips, R. A. & Melchers, F. (1976) J. Immunol. 117, 10991103. 42. Elson, C. J. (1977) Eur. J. Immunol. 7,6-10. 43. Stocker, J. W. (1977) Immunology 32,275-281. 44. Cambier, J. C., Vitetta, E. S., Kettman, J. R., Wetzel, G. M. & Uhr, J. W. (1977) J. Exp. Med. 146, 107-117. 45. Vitetta, E. S., Cambier, J. C., Ligler, F. S., Kettman, J. W. & Uhr, J. W. (1977) J. Exp. Med., in press.
Immunology
Blocking of primary in vitro antibody responses to thymusindependent and thymus-dependent antigens with antiserum specific for IgM or IgD (receptor IgD/B cells)
J. C. CAMBIER, F. S. LIGLER, J. W. UHR, J. R. KETTMAN, AND E. S. VITETTA Department of Microbiology, University of Texas Southwestern Medical School, Dallas, Texas 75235
Communicated by H. Sherwood Lawrence, October 11, 1977
(GARIg) have been described (17). Rabbit anti-6 was judged to be monospecific by published criteria (18), including: (i) a single peak on sodium dodecyl sulfate/polyacrylamide gel electrophoresis after reaction with a lysate of iodinated splenocytes; (ii) immunofluorescent staining of the predicted numbers of cells from various lymphoid tissues; (iii) inability to stain splenocytes after their treatment with either anti-Ig or allotypic (19) anti-6; and (iv) independent capping of surface molecules on splenocytes with anti-6 and anti-A. Chromatographically purified fractions of normal rabbit serum (NRS), anti-,q, anti-Ig, or a 30% (NH4)2SO4 saturation precipitate of anti-6 was used for in vitro cell culture. The IgG fraction from GARIg was conjugated with fluorescein isothiocyanate (Sigma) (FITC-GARIg) and chromatographed on DEAE-cellulose (Whatman DE52) to give a fraction with molar fluorescein/ protein ratio of 2.6. Capping of Cell Surface Ig. Splenocytes were incubated for 30 min at 4° with NRS or the rabbit antisera described above. Cells were washed and exposed to GARIg (20) for 90 min at 370 in complete medium to achieve capping. Immunofluorescent Staining. Viable cells were prepared and treated with NRS or antisera specific for Ig, ,u, or 6 as described (20). After washing with balanced salt solution/azide, the cells were resuspended at 3 X 107 cells per ml in balanced salt solution/azide containing FITC-GARIg (0.1 mg/ml). After 10 min at 40, the cells were washed in balanced salt solution/ azide and fixed in 1% paraformaldehyde in phosphate-buffered saline. The cells were examined at X1000 magnification with a Leitz Ortholux no. 2 fluorescence microscope. For each sample, 100-200 cells were scored for fluorescence without knowledge of the identity of the sample. Culture Conditions. Splenocytes from adult BDF1 (C57BL/6 female X DBA/2 male F1) mice (obtained from Jackson Memorial Laboratory, Bar Harbor, ME) or neonatal mice (from our own breeding colony) were treated as described above to remove cell surface Ig. Cells were resuspended (107 viable cells per ml) in complete medium (IMEMZO, IBL, with 50,uM 2-mercaptoethanol) containing varying concentrations of either the same Ig antibody used to achieve capping or normal Ig. Preliminary experiments had indicated that capping alone with any of the anti-Igs used was ineffective in blocking subsequent immune responses, incubation with anti-Igs only (without prior capping) was marginally effective. Therefore, in the experiments described, control cells were capped with
ABSTRACT The effect of anti-M and anti-6 on the primary in vitro IgM response of murine splenocytes to thymus-dependent (trinitrophenylated erythrocytes) and thymus-independent (trinitrophenylated brucella) forms of trinitrophenyl was studied. The results indicate that either anti-M or anti-S can block the response of adult splenocytes to the thymus-dependent antigen. The thymus-dependent responses of neonatal splenocytes that bear a low concentration of IgD were also abrogated by treatment with anti-S. In contrast, anti-,u, but not anti-5, blocked the response of adult splenocytes to the thymus-independent antigen used. These results indicate that both IgM and IgD are receptors required for triggering cells by a thymus-dependent antigen but that only IgM receptors are required for triggering by the thymus-independent antigen used. Most splenic B cells bear both IgM and IgD (,M+b+) (1-3). On individual cells, both isotypes have identical specificity for antigen (4-7). jj+b+ cells appear to be the major precursors of the primary IgM antibody response to thymus-dependent (TD) antigens (8, 9). The roles played by each of the two isotypes on such cells are not known. One approach to elucidating the function of receptor IgM and IgD is to prevent their interaction with antigen during immunogenic stimulation by treating the cell with isotype-specific antibody. There are a number of reports in which anti-Igs have been used to block immune responsiveness. Both a primary IgM response and a secondary IgG response have been blocked by treatment of the cells with anti-,i (10-12), suggesting that IgM may be essential in "triggering" these responses. Mitogeninduced secretion of IgM and IgG have been blocked with anti-,u (13-15) or anti-S (15). However, anti-S did not block a secondary IgG response (12) and, when administered in vivo to rhesus monkeys, markedly increased IgM and IgG synthesis (16). It is difficult to make a unique interpretation of these observations. The purpose of the present study was to determine the effect of anti-,u and anti-S on the primary in vitro IgM response of murine splenocytes to TD [trinitrophenylated erythrocytes (TNP-SRBC)] and thymus-independent (TI) [trinitrophenylated brucella (TNP-brucella)] forms of trinitrophenylated antigens. The results indicate that IgD is required for responsiveness to TD antigen, whereas IgM is required for responsiveness to both forms of antigen.
MATERIALS AND METHODS Antisera. The preparation and specificity of rabbit antimouse Ig (anti-Ig), rabbit anti-,u, and goat anti-rabbit Ig
Abbreviations: TD, thymus-dependent; TNP-SRBC, trinitrophenylated erythrocytes; TI, thymus independent; TNP-brucella, trinitrophenylated Brucella abortus; GARIg, goat anti-rabbit Ig; NRS, normal rabbit serum; FITC, fluorescein isothiocyanate; PFC, plaque-forming cell.
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Immunology:
Antisera used for capping
NRS
Proc. Natl. Acad. Sci. USA 75 (1978)
Cambier et al.
Table 1. Comparison od cell surface Ig before and after capping Percentage of lymphocytes staining with antisera specific for: Ig IgD IgM After After Before Before Before After 43.0 + 11.3 41.5 ± 13.4 37.0 I 3.5 34.3 ± 1.1 27.3 + 2.3 26.7 + 3.0
433
Control (NRS) After Before 6.5 + 3.5
(3)
1.5 + 0.6
(2)
(2)
6.5 L 3.5
6.5 ± 0.7
(3)
(3)
(3)
(3)
(3)
Anti-,g
32.9 + 6.6
11.5 ± 7.6
41.4 + 6.7
39.2 + 8.3
43.2 I 8.5
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
Anti-6
32.9 + 7.4
35.6 i 7.7
41.4 + 7.7
12.0 ± 7.7
44.4 + 8.4
41.3 + 15.9
6.5 + 3.5
2.0 + 0.0
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
Anti-Ig
33.0 ± 8.5
7.8 ± 4.4
37.4 I 4.5
7.0 + 3.1
42.0 I 8.4
7.4 I 3.3
6.5 i 3.5
5.5 + 3.5
(8)
(8)
(8)
(8)
(8)
(8)
(2)
(2)
36.6 ±78.7
Splenocytes were stained with NRS or specific antisera before and after capping. Values are expressed as the mean ± SD; the number of experiments averaged is in parenthesis.
specific antiserum and then incubated with normal Ig. Cells cultured in 35-mm Falcon dishes, 0.8 ml per dish. To each culture was added 1 X 107 irradiated [1500 R (0.4 C/kg)], low-dose sheep red blood cell-primed (21) spleen cells in 0.1 ml of complete medium and 0.1 ml of 0.1% TNP-SRBC (22) (TD antigen) or 0.01% TNP-brucella (TI antigen) in medium (23). Cultures were incubated essentially as described by Mishell and Dutton (24) with use of the modification of Kettman and Dutton (22) to obtain a primary response. Assay. In all cases, direct plaque-forming cell (PFC) responses were assayed after a 4-day culture period, by using the microscope slide modification (25) of the hemolysis in gel technique of Jerne and Nordin (26). The anti-TNP PFC were determined by using trinitrophenylated horse erythrocytes (Colorado Serum CO) prepared by the method of Rittenberg and Pratt (27). Background PFC, determined using horse erythrocytes, were subtracted from trinitrophenylated horse erythrocyte PFC to give anti-TNP PFC. All data presented are averages of direct hemolytic PFC from duplicate assays obtained from pooled duplicate cultures. were
RESULTS Antibody-Induced Removal of Surface Ig. As shown in Table 1, the values for percentage of positive cells staining with isotype-specific sera were similar to those reported prevously (18, 28)-i.e., IgM = 33%, IgD = 39%, and Ig = 43%. As can be calculated from the data, treatment of splenocytes with anti-A, anti-6, or anti-Ig followed by GARIg decreased the number of cells bearing detectable quantities of the respective isotype(s) by 75-95%. Aliquots of these treated cells were used for studies of function. Effect of Incubation with Antibody on the Immune Responsiveness of Treated Splenocytes from Adult Mice. Treated cells were cultured with TNP-brucella or TNP-SRBC in the presence of varying concentrations (0-1000 ,ug/ml) of the Ig fraction from the same antiserum used for capping. The effect of such treatment on the primary antibody response is shown in Fig. 1. The TD responses to both TNP and SRBC were virtually abolished by treatment with anti-A, anti-6, or anti-Ig at 100-1000 ,ug of Ig per ml. As shown in Fig. 2, inhibition of the TD response did not occur when comparable concentrations of normal rabbit Ig were used, and inhibition was abrogated in the case of either anti-M or anti-Ig, but not anti-6, by addition of a mixture of IgM and IgG to the culture medium. These results suggest that both IgM and IgD receptors are required for activation of responsive cells by TD antigens. As also shown in Fig. 1, the primary TI response to TNP was blocked by anti-M and anti-Ig, but not anti-6, at >100 Ag of Ig
per ml. As shown in Fig. 2, normal rabbit Ig had no effect on the TI response and the blocking with anti-is and anti-Ig could be prevented by adding IgM and IgG to the medium. These results suggest that IgM but not IgD is required for a response to TNP on the TI carrier, brucella. Effect of Incubation with Anti-6 on the Immune Responsiveness of Treated Splenocytes from Neonatal Mice. The above studies suggest that IgD receptors on splenocytes of adult animals are mandatory for triggering a TD response. In contrast to adult mice, neonatal mice bear low levels of IgD on their splenocytes (28-31) and are only marginally responsive to TD antigens (32). The question thereby arises as to whether these small amounts of receptor IgD are required for TD responAnti-6 100 50
0
Anti-Mu
C
0
100
0
U-
50
-
C-
0
Anti-Ig 100
50-
0
1
10
100
1000
1g, jAg/mI FIG. 1. Effect of antibody concentration on the inhibition of in vitro IgM responses to TD (0, SRBC; 0, TNP) and TI (-, TNP) antigens. Cells were treated with the indicated antiserum and GARIg under capping conditions and cultured with an Ig fraction of the same antiserum used for capping. Responses of control cultures incubated without antibody, presented as PFC per 106 viable recovered cells, were as follows: anti-6 capped, SRBC 3408, TNP (TD) 434, TNP (TI) 342; anti-a capped, SRBC 3905, TNP (TD) 445, TNP (TI) 215; anti-Ig capped, SRBC 2672, TNP (TD) 418, TNP (TI) 506.
Proc. Nati. Acad. Sci. USA 75 (1978)
Immunology: Cambier et al.
434
Table 2. Effect of treatment with anti-6 on the immune responsiveness of splenocytes from neonatal mice
SRBC (TD) 44
PFC/106 viable recovered cells
L1 ML1.
z
Age of donor of B cells, days
Q
49
0
18 7
.0
C)
M 0
L._
rNr' I 1_
IME Anti-S Anti-Mu Anti-Ig Antibody used for capping
FIG. 2. Effect of antibody on the inhibition of in vitro IgM reControl cells were capped with specific antiserum and then were incubated with normal rabbit Ig at 400 ;g/ml (0). Capping alone was shown to have no effect on the subsequent antibody response presumably due to regeneration of receptors. The anti-g and anti-6 Ig preparations (a) were used at a concentration of 400 ;g/ml and the anti-Ig Ig fraction, at 250 jsg/ml (see Fig. 1). Where indicated (i), IgG (from normal serum) and IgM (from MOPC 104 secretions) were added at concentrations of approximately 800 ,g/ml and 200 ,g/ml, respectively, to cultures containing anti-g, anti-6, or anti-Ig.
sponses to TD and TI antigens.
siveness of neonatal splenocytes. An alternative possibility is that
the responses could be generated by cells bearing only IgM, which constitute the vast majority of cells in the spleens of animals less than 1 week of age. As shown in Table 2, treatment of splenocytes from 7- to 18-day-old mice with anti-b completely abolished the TD responses to both TNP and SRBC. These results suggest that the presence of receptor IgD is essential for triggering a neonatal cell with a TD antigen. We presume, therefore, that the responder cell bears both IgM and IgD, as in the adult. DISCUSSION The present studies demonstrate that either anti-At or anti-6 can block the primary in vitro immune response of adult splenocytes to the TD antigen, TNP-SRBC. In contrast, anti-M, but not anti-6, blocks the response to the TI antigen, TNP-brucella. Both responses can be blocked by anti-Ig serum. These observations have implications for the phenotype of the subset of lymphocytes responding to TD and TI antigens and for the role played by particular receptor isotypes in signaling virgin B cells during stimulation by antigen. These results represent convincing evidence that the major precursor for the primary IgM antibody response to a TD antigen is a 1i+b+ cell. Previous studies in mice indicated that (i) splenocytes stained with either anti-At or anti-b and positively sorted on the fluorescence-activated cell sorter gave virtually all of the primary IgM response in an adoptively transferred host (8), (ii) elimination of AL+ cells with anti-AL, complement, and azide abolished the primary IgM response (33), also in an adoptively transferred host, and (iii) either anti-AL or anti-b alone was ineffective in preventing antigen-induced suicide of the cell giving rise to the primary IgM antibody response (9).
SRBC Normal Anti-b rabbit Ig 5333 1476 302
238 0 0
TNP (TD) Normal rabbit Ig Anti-6
736 284 29
0 0 0
However, two critical experiments have not been performed: direct analysis of the contribution to the primary IgM response of 9+6+ cells and determination of the effect of deleting 6+ cells on the IgM response (the anti-b antisera in use are not cytotoxic). The present observations that blocking with either anti-,s or anti-b causes a reduction by over 90% of the primary response to TD antigens provide strong evidence that the precursor for the TD response bears both isotypes. The above results also indicate that the precursor for the TI response to TNP-brucella bears no functional IgD receptor. Earlier evidence that the precursor is a cell that bears predominantly IgM (;p+) is as follows: (i) neonatal splenocytes that contain only ,+p cells can give TI responses to certain antigens (34, 35) and (ii) large cells from adult spleens that are + (36) are enriched for TI responsiveness (37). The inability of anti-6 to block the primary IgM response argues either that no IgD is present on the precursor or that IgD is unnecessary for stimulation of the cell. The differences in time of acquisition of responsiveness to various TI antigens during ontogeny (34, 35) suggest that there could be certain TI antigens that require IgD receptors for triggering. The most striking implication of the present data is that both IgM and IgD are needed for triggering a B cell by a TD antigen. The role played by each isotype in immune activation is not known. One possibility is that each isotype gives an independent and necessary signal. Because T cells also provide an essential signal, triggering of TD-responsive B cells may require three signals. Another possibility is that the IgM and IgD receptors must interact with each other, possibly through additional membrane molecules to form a single triggering complex. Finally, the density of the receptors on TD responders may be relatively low, such that antigen interacting with either isotype is insufficient for delivering a signal; both must be involved in order to trigger. Immunofluorescence evidence obtained with the fluorescence-activated cell sorter (38) is consistent with the
latter possibility. There are several studies of embryonic or neonatal cells in culture that suggest that immune responsiveness to TD antigens develops before the acquisition of IgD receptors (39-43). Examination of the experimental protocols used in these studies indicates that, if ontogeny proceeds normally in culture, then immune responsiveness of fetal cells correlates with the expected time of appearance of surface IgD, 3-5 days after birth (30, 31). For example, 17-day fetal liver cells that secrete antibodies after 8 days in culture (41) would be analogous to B cells from 4-day-old neonates. Past studies are therefore consistent with the notion that receptor IgD is necessary for triggering virgin B cells with TD antigens. The present results provide further direct evidence for this idea because removal of receptor IgD completely abrogated the primary IgM response of neonatal splenocytes to SRBC and TNP. We cannot exclude the possibility that there is a trace contribution to the TD response by a p+ cell that is below the level of detection in our system. It should be emphasized that the TNP response of untreated
Immunology: Cambier et al. neonatal cells is markedly decreased compared to the Adtilt' response, suggesting that the TNP-responsive precursors in the neonate have recently appeared. A comparison of the effect of removing IgM or IgD on tolerance induction and immune responsiveness (44, 45) provides a provocative paradox. It appears as though acquisition of appreciable amounts of IgD on a A+ cell is responsible for markedly decreasing susceptibility to induction of tolerance of TD responders. Thus, removal of IgD by means of treatment with papain or anti-6 serum increases the tolerance susceptibility of the cell so that it behaves like neonatal TD responders which bear little IgD. Treatment with anti-M does not have a similar effect; indeed, preliminary experiments suggest that it may decrease the susceptibility of the IA+b+ cell to tolerance induction. In contrast, as mentioned above, both At and a appear to be essential for triggering a TD responder. It could be argued, therefore, that, after interaction with antigen, IgD can only give a triggering signal whereas IgM can give either a triggering signal or a tolerogenic one. Such an explanation is compatible with the results of tolerance induction and triggering of TI precursors which appear to be ,up+ cells. These cells are readily tolerized, yet they can be triggered for antibody formation by a TI antigen. IgM receptors are essential for each of these functions (45). The surface events that determine whether triggering or tolerance is induced require further study. We are grateful to Ms. G. Sloan, Mr. S. Lin, Ms. M. Bagby, Mr. Y. Chinn, Ms. S. Diase, and Ms. M. Neale for expert technical assistance and Ms. J. Hahn for secretarial assistance. J.C.C. is the recipient of Postdoctoral Grant AI-05021'from the National Institutes of Health. This work was supported by National Institutes of Health Grants Al10967, AI-11851, AI-12789, and AI-11893. 1. Pernis, B., Forni, L. & Knight, K. L. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, M., Preud'homme, J. L. -& Kourilsky, F. M. (North-Holland Publishing Co., Amsterdam), pp. 57-64. 2. Parkhouse, B. & Abney, E. R. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, J. L., Preud'homme, J. L. & Kourilsky, F. M. (North-Holland Publishing Co., Amsterdam), pp. 51-56. 3. Vitetta, E. S. & Uhr, J. W. (1976) Eur. J. Immunol. 6, 140143. 4. Salsano, F., Froland, S. S., Natvig, J. B. & Michaelsen, T. E. (1974) Scand, J. Immunol 3, 841-846. 5. Fu, S. M., Winchester, R. J. & Kunkel, H. G. (1975) J. Immunol. 114,250-252. 6. Stern, C. & McConnell, I. (1976) Eur. J. Immunol. 6, 225227. 7. Goding, J. W. & Layton, J. E. (1976) J. Exp. Med. 114, 852857. 8. Zan-Bar, I., Vitetta, E. S. & Strober, S. (1977) J. Exp. Med. 145, 1206-1215. 9. Coffman, R. L. & Cohn, M. (1977) J. Immunol. 118, 18061815. 10. Pierce, C. W., Solliday, S. M. & Asofsky, R. (1972) J. Exp. Med. 135,698-710. 11. Pierce, C. W., Solliday, S. M. & Asofsky, R. (1972) J. Exp. Med.
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