Xid Mice Lack Regulatory Immunoglobulin, Which Contributes to the Enhancement of SRBC Responses" S. D. SIMPSON, S. S. PECQUET, AND P. B. ERNST' Intestinal Disease Research Unit Department of Pathology McMaster University Hamilton, Ontario, Canada L8N 325
INTRODUCTION CBA/N xid mice have a number of immunodeficiencies that manifest as an increased susceptibility to different infections.' The basis for this immunodeficiency lies in large part with the absence of normally differentiated CD5 B cells that can constitute a large proportion of B cells in the intestine*and directly contribute to mucosal immune responses (Pecquet et al., submitted). Moreover, CD5 B cells are necessary to confer immunity to certain bacteria.' The increase in mucosal infections, however, may also reflect a deficiency in regulation. Mice with lack of X-linked immunodeficiencyare nonresponsive to pneumococcal polysaccharide type 111, but this is reversed by the adoptive transfer of activated contrasuppressor T cells.4This suggests they lack some element in the contrasuppressor pathway. As contrasuppression is activated by B cells and/or regulatory immunoglobulin (Ig)4.sthe absence of CD5 B cells in xid mice may be associated with the lack of contrasuppression. Thus, the purpose of these experiments was to determine if the regulatory Ig needed to activate contrasuppression was accountable for the absence of this activity in CBA/N xid mice.
MATERIAL AND METHODS Previously we have shown that antigen-pulsed macrophages (M4)cocultured with Th cells produce a supernatant containing contrasuppressive factors (CSF) that are necessary for the activation of contrasuppression. This is mediated in part by isotypespecific binding factors that interact with regulatory Ig.5 Thus, by substituting M4, T cells, or the regulatory Ig from CBA/N xid mice, we could test for the presence of each component.
=Thiswork was supported by the Medical Research Council of Canada and the Ligue Nationale Contre le Cancer. bAddresscorrespondence to Dr. P. B. Emst, Department of Pathology, Health Sciences Centre, Rm 4H17, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325. 179
ANNALS NEW YORK ACADEMY OF SCIENCES
180
CONTROL
Ts JJJ
IgM
JNJ
IgG
JJN IgA
NJJ NNJ N”
I 0
1000
I
,
2000
3000
I
4000
5000
SRBC-SPECIFIC PFC PER CULTURE
FIGURE 1. Characterization of the contrasuppression deficiency in xid mice. Using CBA/J mice, cells with suppressive activity were generated in vivo and added to cultures of SRBC-stimulated naive splenic responder cells.5 Forty microliters of supernatant from SRBC-pulsed M+ T-cell cocultures combined with 20 pL of regulatory Ig (denoted JJJ) were added to the suppressed cultures. Five days later, cultured cells were harvested, and SRBC-specific IgM, IgG, and IgA plaque forming cell responses were determined on control, suppressed, and contrasuppressed cultures. In order to characterize the deficiency of contrasuppression in xid mice, xid M+, T cells, and regulatory Ig were substituted to compare them to their CBA/J counterparts. After substitution with xid M+ (NJJ) and T cells (JNJ), contrasuppression was activated. The serum from the xid mice (JJN), however, did not contain any regulatory Ig. Xid M+,T cells, and regulatory Ig were unable to generate supernatants (NNN) with any activity, but when CBA/J regulatory Ig was substituted for xid serum, the supernatants (NNJ) had contrasuppressive activity. This suggests that xid MI$ and T cells can interact to generate functional supernatants, but the B cells cannot produce the regulatory Ig.
RESULTS AND DISCUSSION Both xid Mr$ and T cells could participate in the generation of CSF (FIG.1). When xid serum, however, or concentrated affinity-purified xid Ig was combined with the M4 T-cell coculture supernatants, neither provided the regulatory Ig required to activate contrasuppression. Furthermore, the K villosa-adherent contrasuppressor T cell is not deficient in these mice. Therefore the absence of contrasuppression in xid mice is primarily due to a defect in the production of regulatory Ig by B cells. This is consistent with other reports that contrasuppression is activated by B cells: but it may not be through “antigen presentation” but novel interactions with soluble Ig. As the interaction between the regulatory Ig and the T cells that generate the CSF is IgH-restricted,6 we also examined the effects of the Ig deficiency on IgH restriction. Using regulatory Ig from a variety of strains of mice, CBA/J T cells were restricted but CBA/N xid T cells were not. Thus, the deficiency in the restricting element, the regulatory Ig, also affects the IgH restriction of the T cells. Using this deficient strain of mice, one can reconstitute with different pools of B-cell precursors to assess the role of CD5 B cells in regulatory circuits.’
SIMPSON et al.: SRBC RESPONSES
181
ACKNOWLEDGMENTS We would like to thank Barb Bagnarol and Lenore Zettel for their technical assistance. REFERENCES 1. O'BRIEN, A. D., I SCHER,G. H. CAMPBELL, R. P. MACDERMOTT & S. B. FORMAL. 1979.
Susceptibility of CBA/N mice to infection with Salmonella typhimurium: influence of the X-linked gene controlling B lymphocyte function. J. Immunol. 123: 720-724. 2. KROESE,F. G. M., E. C. BUTCHER,A. M. STALL,P. A. LALOR,S. ADAMS& L. A. HERZENBERG. 1989. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int. Immunol. 1: 75-84. 0. GRAY& D. BRILES. 1982. Protection of mice from infection 3. YOTHER,J., C. FORMAN, with S f r e p f ~ ~ c u s p n e u m o n iby a eanti-phosphocholine antibody. Infect. Immun. 36 184188.
BRALEY-MULLEN, H. 1990. Requirement for B cells for activation of contrasuppressor T cells by type 111 pneumococcal polysaccharide. J. Immunol. 144: 2465-2472. 5. ERNST,P. B., S.-T. LEE, J. MAEBA,A. M. STANISZ& F. PARASKEVAS. 1989. A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit. J. Immunol. 143: 1426- 1432. 1988. Macrophage T cell interaction. Induction in 6. MAEBA,J., S.-T. LEE & F.PARASKEVAS. vitro of a mediator with potent antisuppressor activity. J. Immunol. Methods 106 7-17. 7. PECQUET,S. S.,J. ZAZULAK,S. D. SIMPSON& P. B. ERNST.1992. Ann. N. Y.Acad. Sci. This volume.
4.
INTRODUCTION CBA/N xid mice have a number of immunodeficiencies that manifest as an increased susceptibility to different infections.' The basis for this immunodeficiency lies in large part with the absence of normally differentiated CD5 B cells that can constitute a large proportion of B cells in the intestine*and directly contribute to mucosal immune responses (Pecquet et al., submitted). Moreover, CD5 B cells are necessary to confer immunity to certain bacteria.' The increase in mucosal infections, however, may also reflect a deficiency in regulation. Mice with lack of X-linked immunodeficiencyare nonresponsive to pneumococcal polysaccharide type 111, but this is reversed by the adoptive transfer of activated contrasuppressor T cells.4This suggests they lack some element in the contrasuppressor pathway. As contrasuppression is activated by B cells and/or regulatory immunoglobulin (Ig)4.sthe absence of CD5 B cells in xid mice may be associated with the lack of contrasuppression. Thus, the purpose of these experiments was to determine if the regulatory Ig needed to activate contrasuppression was accountable for the absence of this activity in CBA/N xid mice.
MATERIAL AND METHODS Previously we have shown that antigen-pulsed macrophages (M4)cocultured with Th cells produce a supernatant containing contrasuppressive factors (CSF) that are necessary for the activation of contrasuppression. This is mediated in part by isotypespecific binding factors that interact with regulatory Ig.5 Thus, by substituting M4, T cells, or the regulatory Ig from CBA/N xid mice, we could test for the presence of each component.
=Thiswork was supported by the Medical Research Council of Canada and the Ligue Nationale Contre le Cancer. bAddresscorrespondence to Dr. P. B. Emst, Department of Pathology, Health Sciences Centre, Rm 4H17, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325. 179
ANNALS NEW YORK ACADEMY OF SCIENCES
180
CONTROL
Ts JJJ
IgM
JNJ
IgG
JJN IgA
NJJ NNJ N”
I 0
1000
I
,
2000
3000
I
4000
5000
SRBC-SPECIFIC PFC PER CULTURE
FIGURE 1. Characterization of the contrasuppression deficiency in xid mice. Using CBA/J mice, cells with suppressive activity were generated in vivo and added to cultures of SRBC-stimulated naive splenic responder cells.5 Forty microliters of supernatant from SRBC-pulsed M+ T-cell cocultures combined with 20 pL of regulatory Ig (denoted JJJ) were added to the suppressed cultures. Five days later, cultured cells were harvested, and SRBC-specific IgM, IgG, and IgA plaque forming cell responses were determined on control, suppressed, and contrasuppressed cultures. In order to characterize the deficiency of contrasuppression in xid mice, xid M+, T cells, and regulatory Ig were substituted to compare them to their CBA/J counterparts. After substitution with xid M+ (NJJ) and T cells (JNJ), contrasuppression was activated. The serum from the xid mice (JJN), however, did not contain any regulatory Ig. Xid M+,T cells, and regulatory Ig were unable to generate supernatants (NNN) with any activity, but when CBA/J regulatory Ig was substituted for xid serum, the supernatants (NNJ) had contrasuppressive activity. This suggests that xid MI$ and T cells can interact to generate functional supernatants, but the B cells cannot produce the regulatory Ig.
RESULTS AND DISCUSSION Both xid Mr$ and T cells could participate in the generation of CSF (FIG.1). When xid serum, however, or concentrated affinity-purified xid Ig was combined with the M4 T-cell coculture supernatants, neither provided the regulatory Ig required to activate contrasuppression. Furthermore, the K villosa-adherent contrasuppressor T cell is not deficient in these mice. Therefore the absence of contrasuppression in xid mice is primarily due to a defect in the production of regulatory Ig by B cells. This is consistent with other reports that contrasuppression is activated by B cells: but it may not be through “antigen presentation” but novel interactions with soluble Ig. As the interaction between the regulatory Ig and the T cells that generate the CSF is IgH-restricted,6 we also examined the effects of the Ig deficiency on IgH restriction. Using regulatory Ig from a variety of strains of mice, CBA/J T cells were restricted but CBA/N xid T cells were not. Thus, the deficiency in the restricting element, the regulatory Ig, also affects the IgH restriction of the T cells. Using this deficient strain of mice, one can reconstitute with different pools of B-cell precursors to assess the role of CD5 B cells in regulatory circuits.’
SIMPSON et al.: SRBC RESPONSES
181
ACKNOWLEDGMENTS We would like to thank Barb Bagnarol and Lenore Zettel for their technical assistance. REFERENCES 1. O'BRIEN, A. D., I SCHER,G. H. CAMPBELL, R. P. MACDERMOTT & S. B. FORMAL. 1979.
Susceptibility of CBA/N mice to infection with Salmonella typhimurium: influence of the X-linked gene controlling B lymphocyte function. J. Immunol. 123: 720-724. 2. KROESE,F. G. M., E. C. BUTCHER,A. M. STALL,P. A. LALOR,S. ADAMS& L. A. HERZENBERG. 1989. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int. Immunol. 1: 75-84. 0. GRAY& D. BRILES. 1982. Protection of mice from infection 3. YOTHER,J., C. FORMAN, with S f r e p f ~ ~ c u s p n e u m o n iby a eanti-phosphocholine antibody. Infect. Immun. 36 184188.
BRALEY-MULLEN, H. 1990. Requirement for B cells for activation of contrasuppressor T cells by type 111 pneumococcal polysaccharide. J. Immunol. 144: 2465-2472. 5. ERNST,P. B., S.-T. LEE, J. MAEBA,A. M. STANISZ& F. PARASKEVAS. 1989. A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit. J. Immunol. 143: 1426- 1432. 1988. Macrophage T cell interaction. Induction in 6. MAEBA,J., S.-T. LEE & F.PARASKEVAS. vitro of a mediator with potent antisuppressor activity. J. Immunol. Methods 106 7-17. 7. PECQUET,S. S.,J. ZAZULAK,S. D. SIMPSON& P. B. ERNST.1992. Ann. N. Y.Acad. Sci. This volume.
4.
