Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5688-5691, November 1978
Immunology
Induction of secondary cytotoxic T lymphocytes by purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles (immune response/alloreactivity/xenoreactivity/liposomes/reconstitution of membrane function) VICTOR H. ENGELHARD*, JACK L. STROMINGER*, MATTHEW MESCHERt, AND STEVEN BURAKOFFt * The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138; and t Department of Pathology, Harvard Medical School, loston,Massachusetts 02115
Contributed by Jack L. Strominger, September 1, 1978
ABS1RACT Mouse cytotoxic T lymphocytes were induced by culturing primed spleen cells with cells, membranes, or detergent-sohubilized and dialyzed membranes from the human lymphoblastoid cell line JY. Cytotoxic T cells could also be induced by coculturing primed spleen cells with phospholipid vesicles containing purified HLA-A and -B antigens derived from JY cells. The induction of killer cells by al subcellular fractions demonstrated an optimal response as a function of the amount of material added and, in the case of ILA-containing liposomes, this optimum was dependent upon the density of the molecules in the liposomes. In addition, the mam*mal level of cytotoxicity elicited was also dependent upon this density. Finally, the cytotoxic cells demonstrated specificity as judged by a lower level of lysis on an HILA-unrelated lymphoblastoid cell line than on the appropriate target, JY. These results suggest that major histocompatibility antigens alone are sufficient for the induction of a secondary cytotoxic T lymphocyte response. It is proposed that this system may be useful in defining possible sites on the HLA molecule recognized by cytolytic T lymphocytes. There is now a large body of evidence that implicates the major histocompatibility antigens (H-2 in mice and HLA in humans) as both stimulators of and targets for the action of cytotoxic T lymphocytes (CTLs). Allogenic CTLs can be induced between two inbred mouse strains that differ only at the H-2K or D locus (1). Mutant strains of mice have been isolated whose lymphoid cells give rise to a cytotoxic T cell response when cultured together with the parental cells. These mutations have been mapped to the H-2K or D locus (2-4) and in two cases have been shown to reflect changes in the amino acid sequence of the H-2K antigens (5). Other strains, selected for the loss of serologically detectable H-2 antigens, neither stimulate CTL production nor act as targets for CTLs generated against the parental cell (6). Finally, induction of CTLs (7) and lysis of target cells by CTLs (8) can be inhibited by alloantisera specific for the H-2 antigens of the stimulator and target cells, respec-
tively. Although the above evidence is compelling, it does not eliminate the possibility that other antigens on the cell surface may be presented together with the histocompatibility antigens and thus form a part of the stimulating target antigen structure (9). It has therefore become necessary to approach the question from a biochemical point of view, by attempting to purify the molecules involved in the response. It has been demonstrated that isolated murine tumor plasma membranes retain the ability to stimulate a secondary CTL response (10), and this activity can be solubilized with deoxycholate (11, 12). The CTL-stimulating antigens copurify with H-2 antigens through lentil lectin affinity chromatography and gel filtrations but have not yet been obtained in highly purified form or in sufficient amounts
to allow preparation of well-characterized liposomes. Highly purified HLA antigens can be obtained in relatively large amounts, however, and their incorporation into well-characterized phospholipid vesicles has been studied (13). It has already been demonstrated that mouse CTLs could be generated to the human lymphoblastoid cell line JY (14). Utilizing the fact ,that xenogeneic CTLs appear to be similar to allogeneic CTLs in their specificity for major histocompatibility antigens (15-17), it was decided to investigate whether purified HLA-A and -B antigens isolated from JY cells and incorporated into vesicles retained the capacity to induce a CTL response. The results reported here demonstrate that this is indeed the case and that the CTLs stimulated by the purified material are specific for alloantigenic determinants expressed on the HLA molecules. MATERIALS AND METHODS C57BL/6 mice were obtained from the Jackson Laboratory. The human B lymphoblastoid cell lines JY (HLA-A2,2, -B7,7) and Raji (HLA-A3,10, -B18,uw35) were maintained in RPMI 1640 medium containing 10% fetal calf serum. Membranes were prepared from JY cells by repeated resuspension in 10 mM Tris/0.2 mM dithioerythritol, pH 8.0. Debris was removed by centrifugation at 5000 X g for 10 min, and the membranes were collected by centrifugation at 100,000 X g for 1 hr. Reconstituted membranes were prepared by solubilization with 0.5% deoxycholate/10 mM Tris/0.14 M NaCl, pH 8.0, at a detergent-to-protein ratio of 4:1, centrifugation for 100,000 X g for 1 hr, and dialysis of the supernatant against 10 mM Tris/0.14 NaCl/5 mM CaCld, pH 8.0, to remove the detergent. Purified HLA-A and -B antigens were prepared from JY membranes by detergent solubilization and affinity chromatography on anti-,32-microglobulin-Sepharose (18). In order to facilitate comparisons among these various fractions for their efficiency in inducing CTLs, amounts of HLA are expressed in terms of cell equivalents, based on yields of 90, 72, and 54% for membranes, reconstituted membranes, and purified HLA-A and -B antigens, respectively, from intact cells. This corresponds to about 150 ng of HLA per 106 cells. Reconstitution of purified HLA-A and -B antigens into phospholipid vesicles was as described (13). Briefly, the purified antigens in deoxycholate were added to a solution of JY cell membrane lipid in detergent, and the mixture was dialyzed against 10 mM Tris/0.14 M NaCl/5 mM CaCl2, pH 8.0. For the induction of cytotoxic T cells in C57BL/6 mice, animals were immunized by intraperitoneal injection of 2 X 107 human cells and reimmunized 1-2 months later. One week later, the spleens were removed, and spleen cells were co-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CTLs, cytotoxic T lymphocytes; E/T, effector-to-target cell ratio; HLA-liposomes, purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles. * L. Sherman, S. Burakoff, and M. Mescher, unpublished data.
5688
Immunology: Engelhard et al. cultured in vitro with varying amounts of cells or subcellular material as described (10). After 5 days, the cells were reisolated and tested for cytotoxicity on 5lCr-labeled targets. Results are expressed as percentage specific release = [(E - C)/(1 - C)] X 100, in which E = fraction of total 51Cr released in the presence of CTLs and C = fraction of total 51Cr released in the presence of nonimmune spleen cells. Total release was determined by freezing and thawing four times (10). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was carried out according to Laemmli (19). Protein was determined by the method of Lowry et al. (20) with bovine serum albumin as a standard or by using an extinction coefficient, A (units/mg per ml), for purified HLA antigens of 1.5 at 280 nm.
Proc. Natl. Acad. Sci. USA 75 (1978)
5689
44,000-
RESULTS It has been previously shown (13) that HLA-A and -B antigens, purified from the human lymphoblastoid cell line JY by detergent solubilization and affinity chromatography, could be incorporated into phospholipid vesicles (HLA-liposomes). These experiments also showed that, when the protein-to-phospholipid ratio was
Immunology
Induction of secondary cytotoxic T lymphocytes by purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles (immune response/alloreactivity/xenoreactivity/liposomes/reconstitution of membrane function) VICTOR H. ENGELHARD*, JACK L. STROMINGER*, MATTHEW MESCHERt, AND STEVEN BURAKOFFt * The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138; and t Department of Pathology, Harvard Medical School, loston,Massachusetts 02115
Contributed by Jack L. Strominger, September 1, 1978
ABS1RACT Mouse cytotoxic T lymphocytes were induced by culturing primed spleen cells with cells, membranes, or detergent-sohubilized and dialyzed membranes from the human lymphoblastoid cell line JY. Cytotoxic T cells could also be induced by coculturing primed spleen cells with phospholipid vesicles containing purified HLA-A and -B antigens derived from JY cells. The induction of killer cells by al subcellular fractions demonstrated an optimal response as a function of the amount of material added and, in the case of ILA-containing liposomes, this optimum was dependent upon the density of the molecules in the liposomes. In addition, the mam*mal level of cytotoxicity elicited was also dependent upon this density. Finally, the cytotoxic cells demonstrated specificity as judged by a lower level of lysis on an HILA-unrelated lymphoblastoid cell line than on the appropriate target, JY. These results suggest that major histocompatibility antigens alone are sufficient for the induction of a secondary cytotoxic T lymphocyte response. It is proposed that this system may be useful in defining possible sites on the HLA molecule recognized by cytolytic T lymphocytes. There is now a large body of evidence that implicates the major histocompatibility antigens (H-2 in mice and HLA in humans) as both stimulators of and targets for the action of cytotoxic T lymphocytes (CTLs). Allogenic CTLs can be induced between two inbred mouse strains that differ only at the H-2K or D locus (1). Mutant strains of mice have been isolated whose lymphoid cells give rise to a cytotoxic T cell response when cultured together with the parental cells. These mutations have been mapped to the H-2K or D locus (2-4) and in two cases have been shown to reflect changes in the amino acid sequence of the H-2K antigens (5). Other strains, selected for the loss of serologically detectable H-2 antigens, neither stimulate CTL production nor act as targets for CTLs generated against the parental cell (6). Finally, induction of CTLs (7) and lysis of target cells by CTLs (8) can be inhibited by alloantisera specific for the H-2 antigens of the stimulator and target cells, respec-
tively. Although the above evidence is compelling, it does not eliminate the possibility that other antigens on the cell surface may be presented together with the histocompatibility antigens and thus form a part of the stimulating target antigen structure (9). It has therefore become necessary to approach the question from a biochemical point of view, by attempting to purify the molecules involved in the response. It has been demonstrated that isolated murine tumor plasma membranes retain the ability to stimulate a secondary CTL response (10), and this activity can be solubilized with deoxycholate (11, 12). The CTL-stimulating antigens copurify with H-2 antigens through lentil lectin affinity chromatography and gel filtrations but have not yet been obtained in highly purified form or in sufficient amounts
to allow preparation of well-characterized liposomes. Highly purified HLA antigens can be obtained in relatively large amounts, however, and their incorporation into well-characterized phospholipid vesicles has been studied (13). It has already been demonstrated that mouse CTLs could be generated to the human lymphoblastoid cell line JY (14). Utilizing the fact ,that xenogeneic CTLs appear to be similar to allogeneic CTLs in their specificity for major histocompatibility antigens (15-17), it was decided to investigate whether purified HLA-A and -B antigens isolated from JY cells and incorporated into vesicles retained the capacity to induce a CTL response. The results reported here demonstrate that this is indeed the case and that the CTLs stimulated by the purified material are specific for alloantigenic determinants expressed on the HLA molecules. MATERIALS AND METHODS C57BL/6 mice were obtained from the Jackson Laboratory. The human B lymphoblastoid cell lines JY (HLA-A2,2, -B7,7) and Raji (HLA-A3,10, -B18,uw35) were maintained in RPMI 1640 medium containing 10% fetal calf serum. Membranes were prepared from JY cells by repeated resuspension in 10 mM Tris/0.2 mM dithioerythritol, pH 8.0. Debris was removed by centrifugation at 5000 X g for 10 min, and the membranes were collected by centrifugation at 100,000 X g for 1 hr. Reconstituted membranes were prepared by solubilization with 0.5% deoxycholate/10 mM Tris/0.14 M NaCl, pH 8.0, at a detergent-to-protein ratio of 4:1, centrifugation for 100,000 X g for 1 hr, and dialysis of the supernatant against 10 mM Tris/0.14 NaCl/5 mM CaCld, pH 8.0, to remove the detergent. Purified HLA-A and -B antigens were prepared from JY membranes by detergent solubilization and affinity chromatography on anti-,32-microglobulin-Sepharose (18). In order to facilitate comparisons among these various fractions for their efficiency in inducing CTLs, amounts of HLA are expressed in terms of cell equivalents, based on yields of 90, 72, and 54% for membranes, reconstituted membranes, and purified HLA-A and -B antigens, respectively, from intact cells. This corresponds to about 150 ng of HLA per 106 cells. Reconstitution of purified HLA-A and -B antigens into phospholipid vesicles was as described (13). Briefly, the purified antigens in deoxycholate were added to a solution of JY cell membrane lipid in detergent, and the mixture was dialyzed against 10 mM Tris/0.14 M NaCl/5 mM CaCl2, pH 8.0. For the induction of cytotoxic T cells in C57BL/6 mice, animals were immunized by intraperitoneal injection of 2 X 107 human cells and reimmunized 1-2 months later. One week later, the spleens were removed, and spleen cells were co-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CTLs, cytotoxic T lymphocytes; E/T, effector-to-target cell ratio; HLA-liposomes, purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles. * L. Sherman, S. Burakoff, and M. Mescher, unpublished data.
5688
Immunology: Engelhard et al. cultured in vitro with varying amounts of cells or subcellular material as described (10). After 5 days, the cells were reisolated and tested for cytotoxicity on 5lCr-labeled targets. Results are expressed as percentage specific release = [(E - C)/(1 - C)] X 100, in which E = fraction of total 51Cr released in the presence of CTLs and C = fraction of total 51Cr released in the presence of nonimmune spleen cells. Total release was determined by freezing and thawing four times (10). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was carried out according to Laemmli (19). Protein was determined by the method of Lowry et al. (20) with bovine serum albumin as a standard or by using an extinction coefficient, A (units/mg per ml), for purified HLA antigens of 1.5 at 280 nm.
Proc. Natl. Acad. Sci. USA 75 (1978)
5689
44,000-
RESULTS It has been previously shown (13) that HLA-A and -B antigens, purified from the human lymphoblastoid cell line JY by detergent solubilization and affinity chromatography, could be incorporated into phospholipid vesicles (HLA-liposomes). These experiments also showed that, when the protein-to-phospholipid ratio was
