308
Immunology Today, vol. ,3, No. 11, 1982
Humoral immune response to liposomes Stephen C. Kinsky, Tatsuji Yasuda, Takushi Tadakuma Department of Pediatrics, National Jewish Hospital, Denver, Colorado, U.S.A.; Department of Cell Chemistry, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Department of Microbiology, Keio University School of Medicine, Tokyo, Japan Since the introduction of liposomes in 1965, these model membranes have influenced research in every biological discipline that can lay claim to membraneassociated phenomena. The first immunological application (reviewed in Ref. 1) was the elaboration of liposomes that could serve as targets for humoral mediated membrane damage via the antibody-dependent (classical) or antibody-independent (alternative) complement pathways. Unfortunately, attempts to develop analogous liposomes that can function as surrogate substrates for either antibody-dependent or -independent cell mediated lysis have been so far notably less successful. More recently, liposomes have been used to study the induction of afferent humoral and cellular responses. In this article, we shall focus attention on several features of antibody formation elicited by liposomes that have been made immunogenic (sensitized) by a novel class of synthetic lipid antigens: N-(hapten)-substituted derivatives of phosphatidylethanolamine (PE). Our intention is to review certain aspects of liposomal immunogenicity that have been reasonably well established and to point out a few areas that, in our opinion, are controversial and merit additional study.
General properties of liposomes containing hapten-substituted PE derivatives Hapten-substituted PE derivatives were originally devised as substitutes for the naturally occurring glycolipid antigens (e.g., ceramide antigens such as Forssman) that had been previously employed to sensitize liposomes to the action of antibody plus cm3qplement 2. One of the earliest compounds synthesized for this purpose was N-(2,4-dinitrophenyl-6-Naminocaproyl)-phosphatidylethanolamine3; this was also the first derivative (hereafter abbreviated DNPCap-PE) whose immunogenic properties were thoroughly investigated. The initial in-vivo studies in guinea pigs 4 showed free DNP-Cap-PE (i.e., the derivative not incorporated into liposomes) to be a weak immunogen even when administered in Freund's complete adjuvant (FCA). In this regard, DNP-Cap-PE resembles most natural lipid antigens which also do not induce appreciable antibody formation in pure form. The immunogenicity of DNP-Cap-PE was, however, enhanced 50-100 fold by insertion into liposomes containing beef sphingomyelin (in addition to cholesterol and a charged amphiphile), but not by incorporation into ~ Elsevier Biomedical Press 1')82 ()167~1919/82/00004000/$ 1.00
liposomes prepared with egg lecithin. This constituted the first indication that phospholipid composition plays a crucial role in the humoral response to these model membranes. Guinea pigs immunized with the sphingomyelin-containing liposomes elaborated antibody responses comparable in magnitude to those obtained with conventional dinitrophenylated protein immunogens. However, unlike the protein immunogens, DNP-Cap-PE sensitized liposomes in FCA elicited relatively restricted antibodies as demonstrated by hapten inhibition of plaque development and isoelectric focusing of serum antibodiesL This phenomenon is presumably a consequence of the similar, if not identical, conformation shared by all the repetitive DNP determinants present in the liposomal bilayers. Anti-DNP antibodies were also produced when DNP-Cap-lyso PE (lysophosphatidylethanolamine) was used in place of DNP-Cap-PE to sensitize sphingomyelin liposomes, but not when DNP-Cap-GPE (glycerophosphorylethanolamine) was employed 4. In other words, only the amphipathic DNP derivatives were effective, consistent with a requirement for a nonpolar region that noneovalently anchors the determinant to the lipid bilayers to render the liposomes immunogenic. It should be noted, in this connection, that a variety of N-substituted PE derivatives bearing different determinants (e.g., fluorescein) have since been shown to induce hapten-specific antibodies not only in guinea pigs 4 but also in rabbits 6 and mice (see below). Furthermore, liposomal immunogenicity is not limited to a humoral response exclusively: a cellular response, as revealed by delayed hypersensitivity reactions, can be induced in either guinea pigs 7-9 or mice l°,n by proper choice of a hapten substituent (e.g., mono(azobenzenearsonic acid)-tyrosine) attached to PE.
Characterization of liposomes as t h y m u s i n d e p e n d e n t type 1 or type 2 immunogens Additional features of the humoral response to liposomes have been subsequently elucidated by extensive studies in mice, both in vivo and in vilro. When administered in saline, DNP-Cap-PE sensitized Iiposomes (as well as liposomes containing other derivatives) were found to fulfil the classical criteria of thymus-independent (TI) immunogens. Thus, antiDNP antibodies were produced in T-cell-depleted (thymectomized, irradiated, bone-marrow-recon-
Immunology Today, voL 3, No. 11, !982
stituted) and T-cell deficient (nude) mice 12,13. This response in vivo was characterized by the preponderance of direct (IgM) over indirect (IgG) plaque-forming cells (PFC) in the spleen, with the concomitant appearance of mercaptoethanol-sensitive (IgM) antibodies in the serum 12. These features of TI immunogens have been confirmed by examining the in-vitro response to DNP-Cap-PE sensitized liposomes in cultures of spleen cells obtained from normal and
309
Liposomal immunogenicity in mice can be increased nearly 10 fold by incorporating the B-cell mitogen, lipid A, into the same liposomal bitayers that carry various N-substituted PE derivatives 16,17. Insertion of lipid A has several other profound effects on the immunogenic properties of liposomes. One is the transformation of these model membranes from a TItype 2 to a TI-type 1 immunogen (as defined in Ref. 18). For example, TNP-Cap-PE sensitized liposomes prepared without lipid A were unable to elicit significant responses in (CBA/N x BALB/c)F I male mice but did so in F~ female animals; in contrast the liposomes prepared with lipid A were immunogenic in F1 mice of either sex~% Also, a monoclonal anti-6 (murine IgD) antibody could inhibit the in-vitro response of BDFj cells to liposomes lacking lipid A, but had no effect on the immunogenicity of liposomes containing lipid A. Application of other criteria (e.g., ontogeny of the response) showed that liposomes minus lipid A resemble the prototype TI-2 immunogen, TNP-'Ficoll', whereas liposomes plus lipid A are comparable to the prototype TI-1 immunogen, TNPBrucella abortus. Inter alia, these findings imply that TI1 immunogens may differ from TI-2 irnmunogens by their capacity to act as B-cell mitogens. This, in turn, suggests that lipid bilayers per se (i.e., unsensitized liposomes) are non-mitogenic and cannot function as polyclonal B-cell activators, an aspect of liposomal immunogenicity that warrants further investigation.
liposomes prepared without and with lipid A, as suggested also by the studies described below. Although the participation of T cells has now become an unsettled question, in-vitro experiments have clearly shown that macrophages (adherent cells) are required for a response to liposomes lacking lipid A (Ret~. 14, 15). Macrophages are generally believed to play a key role in antigen 'processing' which we have interpreted to mean metabolic degradation of an immunogen to a simple product bearing the determinant and perhaps introduction of a lipophilic group(s) to facilitate incorporation of the product into the macrophage membrane for presentation to lymphocytes. Liposomes obviously possess determinants in a very simple amphipathic form (the PE derivative) that is noncovalently attached to the carrier (the lipid bilayers). It was therefore initially expected that macrophages would not be involved in liposomal immunogenicity on the assumption that the manner in which the determinants are situated in the lipid bilayers would correspond to the way that they are eventually presented on the macrophage surface. As such, this hypothesis does not take into consideration the possibility that the determinant may have to be recognized by lymphocytes together with some other intrinsic macrophage membrane component. This hypothesis thus needs to be again examined by determining if, for example, the macrophage requirement can be circumvented with sensitized liposomes which also contain an appropriate Ia antigen. Alternatively, macrophages may be required because these cells directly or indirectly provide a soluble factor(s) (interleukin?) necessary for liposomal immunogenicity. This possibility is supported by the observation that supernatant solutions-from cultures of mouse spleen cells, which have been stimulated with concanavalin A (a T-celt mitogen) can replace macrophages in the response to DNP-Cap-PE sensitized liposomes 22.
Involvement of T cells and macrophages It must be emphasized that transformation of liposomes from a TI-2 to a TI-1 immunogen by inclusion of lipid A was achieved under conditions in which the epitope density of TNP-Cap-PE was kept constant. Conversion was not obtained by merely increasing the amount of derivative incorporated into fiposomes lacking lipid A. However, increasing the density of TNP groups conjugated to polyacrylamide beads (PAB) transforms this immunogen from type 2 to type 12o. Moreover, if mouse spleen cells are extensively depleted of T cells, the response to TNPPAB may be totally or partially contingent on the presence of these cells, depending on epitope density 2°,21. Collectively, these observations imply that the function of T cells (or factors derived therefi'om) may be circumvented by the presence of a B-cell mitogen such as lipid A. They further indicate the necessity of reexamining the involvement o f t cells in the response to
Interactive effects of phospholipid composition, epitope density, and lipid A Limitations of space prevent detailed discussion of additional parameters that influence liposomal immunogenicity. It should, however, be noted that the pronounced difference between the response to beef sphingomyelin (SM) and egg phosphatidylcholine (PC) liposomes, which was initially detected in guinea pigs (see above), also occurs in mice 23. In extending these observations, it was found 2~ that liposomes prepared with the synthetic: phosphatidylcholines, dioleoyl-PC or dilauryl-PC, elicited essentially no response (like egg PC liposomes), whereas liposomes made with dimyristoyl-PC, dipatmitoyl-PC, or distearoyl-PC were significantly immunogenic (like beef SM liposomes). This phenomenon could possibly reflect an effect on the mobility (diffusion) of DNPCap-PE in the plane of the bilayer since the phospholipids which give rise to immunogenic liposomes possess transition temperatures (23-57°C) that are
n u d e mice 14,15.
310 higher t h a n those w h i c h do not ( - 2 2 - 0 ° C ) . I n h e r e n t in this hypothesis is the oft-made a s s u m p t i o n that activation of B lymphocytes requires cross-linking of a m i n i m a l n u m b e r of m e m b r a n e i m m u n o g l o b u l i n receptors. C i r c u m s t a n t i a l evidence that receptor cross-linking m a y be involved comes from the finding that liposomes are not i m m u n o g e n i c either in vivo or in vitro unless the epitope density of a P E derivative (taken as an indirect m e a s u r e of the distance b e t w e e n d e t e r m i n a n t s ) exceeds a critical threshold value 2~. It is also significant that i n c o r p o r a t i o n of lipid A can m o d u l a t e the d e p e n d e n c e of liposomal i m m u n o genicity on epitope density. T h u s , inclusion of the Bcell m i t o g e n causes a d r a m a t i c reduction in the a m o u n t of P E derivative that m u s t be i n c o r p o r a t e d to induce a response I7, a c c o m p a n i e d by a b o l i s h m e n t of the critical epitope density r e q u i r e m e n t ( u n p u b l i s h e d observations). T h e s e observations suggest that crosslinking of i m m u n o g l o b u l i n receptors m a y no longer be necessary to obtain a response to liposomes that c o n t a i n lipid A, a n d are c o m p a t i b l e with the proposal that, u n d e r these circumstances, the d e t e r m i n a n t merely serves to focus a mitogenic signal on to B cells. However, high epitope densities ha~)e been r e p o r t e d ~5 to inhibit the in-vitro responses to doses of liposomes considerably greater t h a n those used in other experim e n t s 24 so that extensive speculation on this point seems u n w a r r a n t e d at the p r e s e n t time.
Concluding comment A l t h o u g h outside the i m m e d i a t e scope of this review, reference should he m a d e to investigations showing that liposomes, w h i c h c o n t a i n either h u m a n H L A or mouse H-2 antigens, can elicit the p r o d u c t i o n of cytotoxic T cells in s e c o n d a r y in vitro cultures (see, for example, Ref. 25). Indeed, such liposomes have been elegantly exploited to confirm that a fbreign antigen (e.g., Sendal viral protein) must be recognized in the context of a histocompatibility antigen (i.e., a p p r o p r i a t e H-2) for the in-vitro induction of effector cells that can lyse virally infected syngeneic cells 26. However, with one exception 27, we know of no p a p e r to date d e m o n s t r a t i n g that liposomes, sensitized with a histocompatibility antigen, can evoke a p r i m a r y h u m o r a l or cellular response either in vivo or in vitro. Such systems need to be established to d e t e r m i n e if the p a r a m e t e r s (e.g., p h o s p h o l i p i d composition) that
ImmunolvgyToday, vol. 3, No. 11, 1982
attect the i m m u n o g e n i c i t y of synthetic lipid antigens (i.e., PE derivatives) in liposomes also influence the response to m e m b r a n e - l o c a l i z e d antigens w h i c h have more obvious medical significance.
References 1 Kinsky, S. C. (1972) Biochim. Biophys. Acta 265, 1-23 2 Uemura, K. and Kinsky, S. C. (1972) Biochemistry 11, 4085-4094 3 Six, H. R., Uemura, K. and Kinsky, S. C. (1973) Biochemistry 12, 4003-4011 4 Uemura, K., Nicolotti, R. A., Six, H. R. and Kinsky, S. C. (1974) Biochemistry 13, 1572-1578 5 Uemura, K., Claflin, J. L., Davie, J. M. and Kinsky, S. C. (1975).7. Imraunol. 114,958-961 6 Chan, S. W., Tan, C. T. and Hsai, J. C. (1977) Biochem. Biophys. Res. Commu~. 79,631 634 7 Nicolotti, R. A. and Kinsky, S. C. (1975) Biochemistry 14, 2331-2337 8 Kochibe, N., Nicolotti, R. A., Davie, J. M. and Kinsky, S. C. (1975) Proc. Natl Acad. Sci. U.S.A. 11, 4582-4586 9 Nieolotti, R. A., Kochibe, N. and Kinsky, S. C. (1976) J. Immunol. 117, 1898 1902 10 van Houte, A. J., Snippe, H., Peulen, G. T. M. and Willers, J. M. N. (1981) Immunology42, 165-173 11 van Houte, A. J., Snippe, H., Peulen, G. T. M. and Willers, J. M. N. (1981) Immunology 42, 233-239 12 Yasuda, T., Dancey, G. F. and Kinsky, S. C. (1977)J. Immunol. 119, 1863-1867 13 van Houte, A. J., Snippe, H. and Willers, J. M. N. (1979) Imraunology 37, 505-514 14 Yasuda, T., Tadakuma, T., Pierce, C. W. and Kinsky, S. C. (1979), 7. [mmunol. 123, i535-1539 15 Humphries, G. M. K. (1979). 7. Immunol. 123, 2126-2132 I6 Dancey, G. F., Yasuda, T. and Kinsky, S. C. (1977),7. lmraunol. 119, 1868-1873 17 Honegger, J. L., Isakson, P. C. and Kinsky, S. C. (1980) `7. Immunol. 124, 669-675 18 Mosier, D. E., Zitron, I. M., Mond, J. J., Ahmed, A., Scher, I. and Paul, W. E. (1977) Iramunol. Rev. 37, 89-104 19 Tadakuma, T., Yasuda, T., Tamauchi, H., Saito, K., Tsumita, T. and Kinsky, S. C. (1982).7. [mmunol. 128,206-210 20 Mond, J. J., Stein, K. g., Subbarao, B. and Paul, W. E. (1979) J. lmmunol. 123,239-245 21 Pure, E. and Vitetta, E. (1980) J. Iramunol. 125,420-427 22 Humphries, G. M. K. (1981) J. lmmunol. 126,688-692 23 Yasuda, T., Dancey, G. F. and Kinsky, S. C. (1977) Proc. Nail Acad. Sci. U.S.A. 74, 1234-1236 24 Tadakuma, T., Yasuda, T., Kinsky, S. C. and Picrce, C. W. (1980) J. Immunol. 124, 2175-2179 25 Hale, A. H., Lyles, D. S. and Fan, D. P. (1980) J. Immunol. 124, 724-731 26 Hale, A. H., Reubush, M. J. and Harris, D. T. (1980).7. Iramunol. 125,428-430 27 Hale, A. H. (1980) Cell. lmmunol. 55,328-341
Immunology Today, vol. ,3, No. 11, 1982
Humoral immune response to liposomes Stephen C. Kinsky, Tatsuji Yasuda, Takushi Tadakuma Department of Pediatrics, National Jewish Hospital, Denver, Colorado, U.S.A.; Department of Cell Chemistry, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Department of Microbiology, Keio University School of Medicine, Tokyo, Japan Since the introduction of liposomes in 1965, these model membranes have influenced research in every biological discipline that can lay claim to membraneassociated phenomena. The first immunological application (reviewed in Ref. 1) was the elaboration of liposomes that could serve as targets for humoral mediated membrane damage via the antibody-dependent (classical) or antibody-independent (alternative) complement pathways. Unfortunately, attempts to develop analogous liposomes that can function as surrogate substrates for either antibody-dependent or -independent cell mediated lysis have been so far notably less successful. More recently, liposomes have been used to study the induction of afferent humoral and cellular responses. In this article, we shall focus attention on several features of antibody formation elicited by liposomes that have been made immunogenic (sensitized) by a novel class of synthetic lipid antigens: N-(hapten)-substituted derivatives of phosphatidylethanolamine (PE). Our intention is to review certain aspects of liposomal immunogenicity that have been reasonably well established and to point out a few areas that, in our opinion, are controversial and merit additional study.
General properties of liposomes containing hapten-substituted PE derivatives Hapten-substituted PE derivatives were originally devised as substitutes for the naturally occurring glycolipid antigens (e.g., ceramide antigens such as Forssman) that had been previously employed to sensitize liposomes to the action of antibody plus cm3qplement 2. One of the earliest compounds synthesized for this purpose was N-(2,4-dinitrophenyl-6-Naminocaproyl)-phosphatidylethanolamine3; this was also the first derivative (hereafter abbreviated DNPCap-PE) whose immunogenic properties were thoroughly investigated. The initial in-vivo studies in guinea pigs 4 showed free DNP-Cap-PE (i.e., the derivative not incorporated into liposomes) to be a weak immunogen even when administered in Freund's complete adjuvant (FCA). In this regard, DNP-Cap-PE resembles most natural lipid antigens which also do not induce appreciable antibody formation in pure form. The immunogenicity of DNP-Cap-PE was, however, enhanced 50-100 fold by insertion into liposomes containing beef sphingomyelin (in addition to cholesterol and a charged amphiphile), but not by incorporation into ~ Elsevier Biomedical Press 1')82 ()167~1919/82/00004000/$ 1.00
liposomes prepared with egg lecithin. This constituted the first indication that phospholipid composition plays a crucial role in the humoral response to these model membranes. Guinea pigs immunized with the sphingomyelin-containing liposomes elaborated antibody responses comparable in magnitude to those obtained with conventional dinitrophenylated protein immunogens. However, unlike the protein immunogens, DNP-Cap-PE sensitized liposomes in FCA elicited relatively restricted antibodies as demonstrated by hapten inhibition of plaque development and isoelectric focusing of serum antibodiesL This phenomenon is presumably a consequence of the similar, if not identical, conformation shared by all the repetitive DNP determinants present in the liposomal bilayers. Anti-DNP antibodies were also produced when DNP-Cap-lyso PE (lysophosphatidylethanolamine) was used in place of DNP-Cap-PE to sensitize sphingomyelin liposomes, but not when DNP-Cap-GPE (glycerophosphorylethanolamine) was employed 4. In other words, only the amphipathic DNP derivatives were effective, consistent with a requirement for a nonpolar region that noneovalently anchors the determinant to the lipid bilayers to render the liposomes immunogenic. It should be noted, in this connection, that a variety of N-substituted PE derivatives bearing different determinants (e.g., fluorescein) have since been shown to induce hapten-specific antibodies not only in guinea pigs 4 but also in rabbits 6 and mice (see below). Furthermore, liposomal immunogenicity is not limited to a humoral response exclusively: a cellular response, as revealed by delayed hypersensitivity reactions, can be induced in either guinea pigs 7-9 or mice l°,n by proper choice of a hapten substituent (e.g., mono(azobenzenearsonic acid)-tyrosine) attached to PE.
Characterization of liposomes as t h y m u s i n d e p e n d e n t type 1 or type 2 immunogens Additional features of the humoral response to liposomes have been subsequently elucidated by extensive studies in mice, both in vivo and in vilro. When administered in saline, DNP-Cap-PE sensitized Iiposomes (as well as liposomes containing other derivatives) were found to fulfil the classical criteria of thymus-independent (TI) immunogens. Thus, antiDNP antibodies were produced in T-cell-depleted (thymectomized, irradiated, bone-marrow-recon-
Immunology Today, voL 3, No. 11, !982
stituted) and T-cell deficient (nude) mice 12,13. This response in vivo was characterized by the preponderance of direct (IgM) over indirect (IgG) plaque-forming cells (PFC) in the spleen, with the concomitant appearance of mercaptoethanol-sensitive (IgM) antibodies in the serum 12. These features of TI immunogens have been confirmed by examining the in-vitro response to DNP-Cap-PE sensitized liposomes in cultures of spleen cells obtained from normal and
309
Liposomal immunogenicity in mice can be increased nearly 10 fold by incorporating the B-cell mitogen, lipid A, into the same liposomal bitayers that carry various N-substituted PE derivatives 16,17. Insertion of lipid A has several other profound effects on the immunogenic properties of liposomes. One is the transformation of these model membranes from a TItype 2 to a TI-type 1 immunogen (as defined in Ref. 18). For example, TNP-Cap-PE sensitized liposomes prepared without lipid A were unable to elicit significant responses in (CBA/N x BALB/c)F I male mice but did so in F~ female animals; in contrast the liposomes prepared with lipid A were immunogenic in F1 mice of either sex~% Also, a monoclonal anti-6 (murine IgD) antibody could inhibit the in-vitro response of BDFj cells to liposomes lacking lipid A, but had no effect on the immunogenicity of liposomes containing lipid A. Application of other criteria (e.g., ontogeny of the response) showed that liposomes minus lipid A resemble the prototype TI-2 immunogen, TNP-'Ficoll', whereas liposomes plus lipid A are comparable to the prototype TI-1 immunogen, TNPBrucella abortus. Inter alia, these findings imply that TI1 immunogens may differ from TI-2 irnmunogens by their capacity to act as B-cell mitogens. This, in turn, suggests that lipid bilayers per se (i.e., unsensitized liposomes) are non-mitogenic and cannot function as polyclonal B-cell activators, an aspect of liposomal immunogenicity that warrants further investigation.
liposomes prepared without and with lipid A, as suggested also by the studies described below. Although the participation of T cells has now become an unsettled question, in-vitro experiments have clearly shown that macrophages (adherent cells) are required for a response to liposomes lacking lipid A (Ret~. 14, 15). Macrophages are generally believed to play a key role in antigen 'processing' which we have interpreted to mean metabolic degradation of an immunogen to a simple product bearing the determinant and perhaps introduction of a lipophilic group(s) to facilitate incorporation of the product into the macrophage membrane for presentation to lymphocytes. Liposomes obviously possess determinants in a very simple amphipathic form (the PE derivative) that is noncovalently attached to the carrier (the lipid bilayers). It was therefore initially expected that macrophages would not be involved in liposomal immunogenicity on the assumption that the manner in which the determinants are situated in the lipid bilayers would correspond to the way that they are eventually presented on the macrophage surface. As such, this hypothesis does not take into consideration the possibility that the determinant may have to be recognized by lymphocytes together with some other intrinsic macrophage membrane component. This hypothesis thus needs to be again examined by determining if, for example, the macrophage requirement can be circumvented with sensitized liposomes which also contain an appropriate Ia antigen. Alternatively, macrophages may be required because these cells directly or indirectly provide a soluble factor(s) (interleukin?) necessary for liposomal immunogenicity. This possibility is supported by the observation that supernatant solutions-from cultures of mouse spleen cells, which have been stimulated with concanavalin A (a T-celt mitogen) can replace macrophages in the response to DNP-Cap-PE sensitized liposomes 22.
Involvement of T cells and macrophages It must be emphasized that transformation of liposomes from a TI-2 to a TI-1 immunogen by inclusion of lipid A was achieved under conditions in which the epitope density of TNP-Cap-PE was kept constant. Conversion was not obtained by merely increasing the amount of derivative incorporated into fiposomes lacking lipid A. However, increasing the density of TNP groups conjugated to polyacrylamide beads (PAB) transforms this immunogen from type 2 to type 12o. Moreover, if mouse spleen cells are extensively depleted of T cells, the response to TNPPAB may be totally or partially contingent on the presence of these cells, depending on epitope density 2°,21. Collectively, these observations imply that the function of T cells (or factors derived therefi'om) may be circumvented by the presence of a B-cell mitogen such as lipid A. They further indicate the necessity of reexamining the involvement o f t cells in the response to
Interactive effects of phospholipid composition, epitope density, and lipid A Limitations of space prevent detailed discussion of additional parameters that influence liposomal immunogenicity. It should, however, be noted that the pronounced difference between the response to beef sphingomyelin (SM) and egg phosphatidylcholine (PC) liposomes, which was initially detected in guinea pigs (see above), also occurs in mice 23. In extending these observations, it was found 2~ that liposomes prepared with the synthetic: phosphatidylcholines, dioleoyl-PC or dilauryl-PC, elicited essentially no response (like egg PC liposomes), whereas liposomes made with dimyristoyl-PC, dipatmitoyl-PC, or distearoyl-PC were significantly immunogenic (like beef SM liposomes). This phenomenon could possibly reflect an effect on the mobility (diffusion) of DNPCap-PE in the plane of the bilayer since the phospholipids which give rise to immunogenic liposomes possess transition temperatures (23-57°C) that are
n u d e mice 14,15.
310 higher t h a n those w h i c h do not ( - 2 2 - 0 ° C ) . I n h e r e n t in this hypothesis is the oft-made a s s u m p t i o n that activation of B lymphocytes requires cross-linking of a m i n i m a l n u m b e r of m e m b r a n e i m m u n o g l o b u l i n receptors. C i r c u m s t a n t i a l evidence that receptor cross-linking m a y be involved comes from the finding that liposomes are not i m m u n o g e n i c either in vivo or in vitro unless the epitope density of a P E derivative (taken as an indirect m e a s u r e of the distance b e t w e e n d e t e r m i n a n t s ) exceeds a critical threshold value 2~. It is also significant that i n c o r p o r a t i o n of lipid A can m o d u l a t e the d e p e n d e n c e of liposomal i m m u n o genicity on epitope density. T h u s , inclusion of the Bcell m i t o g e n causes a d r a m a t i c reduction in the a m o u n t of P E derivative that m u s t be i n c o r p o r a t e d to induce a response I7, a c c o m p a n i e d by a b o l i s h m e n t of the critical epitope density r e q u i r e m e n t ( u n p u b l i s h e d observations). T h e s e observations suggest that crosslinking of i m m u n o g l o b u l i n receptors m a y no longer be necessary to obtain a response to liposomes that c o n t a i n lipid A, a n d are c o m p a t i b l e with the proposal that, u n d e r these circumstances, the d e t e r m i n a n t merely serves to focus a mitogenic signal on to B cells. However, high epitope densities ha~)e been r e p o r t e d ~5 to inhibit the in-vitro responses to doses of liposomes considerably greater t h a n those used in other experim e n t s 24 so that extensive speculation on this point seems u n w a r r a n t e d at the p r e s e n t time.
Concluding comment A l t h o u g h outside the i m m e d i a t e scope of this review, reference should he m a d e to investigations showing that liposomes, w h i c h c o n t a i n either h u m a n H L A or mouse H-2 antigens, can elicit the p r o d u c t i o n of cytotoxic T cells in s e c o n d a r y in vitro cultures (see, for example, Ref. 25). Indeed, such liposomes have been elegantly exploited to confirm that a fbreign antigen (e.g., Sendal viral protein) must be recognized in the context of a histocompatibility antigen (i.e., a p p r o p r i a t e H-2) for the in-vitro induction of effector cells that can lyse virally infected syngeneic cells 26. However, with one exception 27, we know of no p a p e r to date d e m o n s t r a t i n g that liposomes, sensitized with a histocompatibility antigen, can evoke a p r i m a r y h u m o r a l or cellular response either in vivo or in vitro. Such systems need to be established to d e t e r m i n e if the p a r a m e t e r s (e.g., p h o s p h o l i p i d composition) that
ImmunolvgyToday, vol. 3, No. 11, 1982
attect the i m m u n o g e n i c i t y of synthetic lipid antigens (i.e., PE derivatives) in liposomes also influence the response to m e m b r a n e - l o c a l i z e d antigens w h i c h have more obvious medical significance.
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