Brief Communication
Activated human T cells express p2microglobulin-associated HLA-A,B,C molecules not recognized by W6/32 mAb T. Crepaldi, S. D’Alfonso, P. Richiardi. Activated human T cells express B2-microglobulin-associated HLA-A,B,C molecules not recognized by W6/32 mAb. Tissue Antigens 1991: 37: 138-140.
T. CNy8ld1, 8. D’AhMtr and P. Rlchiardi Bpartimento di Genetrca, Biologia e Chimica M e dm Serlone di Genetica. Universita’ di Torino
and Centro CNR per I’lmmunogenetica e Istocornpatibilita’. Torino, Italy
Key words PPmicrogbbulin - mAb
- HLA
Recerved 1 August, accepted loc puMition 7 December 1990
In humans, several serological reagents define nonHLA-A,B,C class I antigens that are expressed on activated cells but not on resting lymphocytes (l), thus showing a tissue distribution different from that of A, B, C molecules. It has been suggested that these alloantisera recognize beta 2-microglobulin (~2m)-associatedmolecules controlled by a series of alleles linked to HLA-A, named “HT” (2) or “HA” (3). By using sequential immunoprecipitation experiments, it was shown that these proteins do not react with the monoclonal antibody (mAb) W6/32, that recognizes a conformational monomorphic epitope of A,B,C molecules (3). The aim of the present work was to test for the presence on the cell surface of activated cells of W6/32negative, P2m-associated novel class I molecules. To this purpose sequential immunoprecipitations with W6/32 mAb and a locally produced anti-02rn mAb (R1.30) were performed on cell lysates prepared from ‘*’I surface-labelled resting lymphocytes (PBL) or PHA-activated cells (PHA-L). The immunoprecipitates were further characterized by one-dimensional isoelectric focusing (1 D IEF). As shown in Fig. 1, it was found that 10 sequential immunoprecipitations with W6/32, analyzed by SDS-PAGE, still left a great amount of R1.30reactive material in PHA-L extracts, thus confirming the existence on activated cells of P2m-associated molecules not recognized by W6/32. The same result was obtained using two different PHA-L samples. Contrarily, mAb W6/32 completely elim138
hated the R1.30-reactive material from a PBL extract. To assess if these W6/32-negative, R1.30reactive molecules could represent novel class I antigens expressed on the cell surface of activated lymphocytes, they were analyzed by one-dimensional isoelectric focusing (ID IEF) on the assumption that products of different loci are bound to have a different PI. Unexpectedly, the p2m-associated molecules left afterextemke preclearing with W6/32 were resolved as IEF bands indistinguishable from those of HLA-A,B antigens detected by W6/32 (Fig. 2). The same result was obtained in three experiments performed using PHA-L preparations from 3 different individuals. Contrarily, a locally produced anti-HLA class I mAb (01.65) was able to completely remove the R I .30-reactive material present in the cell lysate from one of the PHA-L samples, thus indicating that these results were not due to technical artifacts. The above data clearly showed that PHA-L carry on the cell membrane two populations of mature, p2m-associated class I antigens, one of which is W6/32-negative, although it is positive with a different anti-HLA class I mAb, 01.65. The identity of PI of the molecules precipitated by W6/32 and by R1/30 demonstrates that they have the same primary structure. Different conformations of the same gene products are, therefore, likely to be the cause of differential reactivity of antibodies. The W6132-defined antigenic determinant is particularly sensitive to conformational changes of the P2m-heavy chain com-
p2-m on activated T cells
Figure I . SDS-PAGE (12.5%) analysis of NP40-solubilized molecules obtained from ‘251-labelledPHA-L sequenticllly immunoprecipitated 10 times with mAb W6/32 and then once with the anti-P2m mAb R1.30 (lane a); immunoprecipitations with W6/ 3 2 1st (lane b). 10th (lane c), 9th (lane d). ( 5 days exposure).
plex. In fact, it can be acquired by class I heavy chain from different, non-human species following association with bovine P2m; on the other hand in some mouse strains, where the murine heavy chain is covalently bound to bovine P2m, the formation or the exposure of the W6/32 antigenic determinant is prevented (4). It can be hypothesized that a subpopulation of class I molecules undergoes conformational changes upon lymphocyte activation, thus preventing the expression or exposure of the W6/32 epitope. A murine class I1 epitope showing a differential expression in different cell types has been described, demonstrating that the same MHC molecules can acquire different conformations on the cell surface (5). A physical association between the HLA class I antigens and other molecules on the surface of activated lymphocytes has been reported (6, 7) and could result in a conformational modification of complexed class I molecules. Another explanation could be that different peptides binding to the same MHC class I molecules affect their final conformation, as suggested by the experiments of Townsend et al. (8).
Figure 2. NP40 (1%)-solubilized 1251-surfacelabelled PHA-L, obtained from an HLA-A2,30, B17,45donor, were sequentially immunoprecipitated 10 times with mAb W6/32 and then once with the anti-fl2m mAb R1.30. Resulting immunoprecipitates were treated with 0.1U Neuraminidase type VIII and analyzed by I D IEF on a pH 4-9 gradient following the method described by Neefjes et al. (9). a: mAb W6/32,1st immunoprecipitation; b: anti-fl2m mAb R1.30, 1Ith immunoprecipitation; c: mAb W6/32, 10th immunoprecipitation. (3 days exposure).
In conclusion, by ID IEF analysis, P2m-associated molecules solubilized from PHA-activated cells after complete depletion of W6/32-positive molecules are indistinguishable from A,B,C. Thus, in some cellular systems, mAb W6/32 does not recognize all HLA-A,B,C molecules, as previously thought. This observation should be kept in mind when attempting to identify new class I gene products after sequential precipitation with W6/32.
Acknowledgments Work supported by MPI 60% and by CNR, progetto finalhato Biotecnologie e Biostrumentazione. T. Crepaldi and S. D’Alfonso are recipients of a fellowship from “Comitato Gigi Ghirotti”. Refereices 1. Gazit E, Fauchet R, Jones M, et al. Antigen Society n. 31
139
Crepaldi et al. Report. In: Dupont B, ed. Immunobiology of HLA, vol I. New York: Springer Verlag, 1989: 281. 2. Gazit E, Terhorst C, Yunis E. The human 'T' genetic region of the HLA linkage group is a polymorphism detected on lectin-activated lymphocytes. Nature 1980: 2&1: 275. 3. Fauchet R, Boscher M, Bouhallier 0, et al. New class I in man: serological and molecular characterization. Human Immunology 1986: 17 3. 4.Kahn-Perles B, Boyer C, Arnold B, Sanderson AR, Femer P, Lemonnier FA. Acquisition of HLA class I W6/32 defined antigenic determinant by heavy chain from different species following association with bovin b2-microglobulin. J Immunoll987: 138:2190. 5. Murphy DB, Lo D, Rath S, et al. A novel MHC class I1 epitope expressed in thymic medulla but not cortex. Nature 1989: 338:765. 6. Blue ML, Craig KA, Anderson P, Branton KR, Schlossman SF. Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxiccells. Cell 1988: 54: 413.
140
7. Bushkin Y, Demaria S, Le J, Schwab R. Physical association between the CD8 and HLA Class I molecules on the surface of activated human T lymphocytes. Proc Natl Acad Sci USA 1988: 85: 3985. 8. Townsend A, Olden C, Bastin J, Ljunggren HG, Foster L, Karre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 1989: 340:443. 9. Neeijes JJ, Brew-Vriesendorp BS, Van Seventer GA, Ivanyi P, Ploegh HL. An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLA-class I subtypes. Human Immunology 1986 16 169. Address: Patricia Richiardi, PhD Dipartimento di Genetica Via Santena 19 10126 Torino Italy
Activated human T cells express p2microglobulin-associated HLA-A,B,C molecules not recognized by W6/32 mAb T. Crepaldi, S. D’Alfonso, P. Richiardi. Activated human T cells express B2-microglobulin-associated HLA-A,B,C molecules not recognized by W6/32 mAb. Tissue Antigens 1991: 37: 138-140.
T. CNy8ld1, 8. D’AhMtr and P. Rlchiardi Bpartimento di Genetrca, Biologia e Chimica M e dm Serlone di Genetica. Universita’ di Torino
and Centro CNR per I’lmmunogenetica e Istocornpatibilita’. Torino, Italy
Key words PPmicrogbbulin - mAb
- HLA
Recerved 1 August, accepted loc puMition 7 December 1990
In humans, several serological reagents define nonHLA-A,B,C class I antigens that are expressed on activated cells but not on resting lymphocytes (l), thus showing a tissue distribution different from that of A, B, C molecules. It has been suggested that these alloantisera recognize beta 2-microglobulin (~2m)-associatedmolecules controlled by a series of alleles linked to HLA-A, named “HT” (2) or “HA” (3). By using sequential immunoprecipitation experiments, it was shown that these proteins do not react with the monoclonal antibody (mAb) W6/32, that recognizes a conformational monomorphic epitope of A,B,C molecules (3). The aim of the present work was to test for the presence on the cell surface of activated cells of W6/32negative, P2m-associated novel class I molecules. To this purpose sequential immunoprecipitations with W6/32 mAb and a locally produced anti-02rn mAb (R1.30) were performed on cell lysates prepared from ‘*’I surface-labelled resting lymphocytes (PBL) or PHA-activated cells (PHA-L). The immunoprecipitates were further characterized by one-dimensional isoelectric focusing (1 D IEF). As shown in Fig. 1, it was found that 10 sequential immunoprecipitations with W6/32, analyzed by SDS-PAGE, still left a great amount of R1.30reactive material in PHA-L extracts, thus confirming the existence on activated cells of P2m-associated molecules not recognized by W6/32. The same result was obtained using two different PHA-L samples. Contrarily, mAb W6/32 completely elim138
hated the R1.30-reactive material from a PBL extract. To assess if these W6/32-negative, R1.30reactive molecules could represent novel class I antigens expressed on the cell surface of activated lymphocytes, they were analyzed by one-dimensional isoelectric focusing (ID IEF) on the assumption that products of different loci are bound to have a different PI. Unexpectedly, the p2m-associated molecules left afterextemke preclearing with W6/32 were resolved as IEF bands indistinguishable from those of HLA-A,B antigens detected by W6/32 (Fig. 2). The same result was obtained in three experiments performed using PHA-L preparations from 3 different individuals. Contrarily, a locally produced anti-HLA class I mAb (01.65) was able to completely remove the R I .30-reactive material present in the cell lysate from one of the PHA-L samples, thus indicating that these results were not due to technical artifacts. The above data clearly showed that PHA-L carry on the cell membrane two populations of mature, p2m-associated class I antigens, one of which is W6/32-negative, although it is positive with a different anti-HLA class I mAb, 01.65. The identity of PI of the molecules precipitated by W6/32 and by R1/30 demonstrates that they have the same primary structure. Different conformations of the same gene products are, therefore, likely to be the cause of differential reactivity of antibodies. The W6132-defined antigenic determinant is particularly sensitive to conformational changes of the P2m-heavy chain com-
p2-m on activated T cells
Figure I . SDS-PAGE (12.5%) analysis of NP40-solubilized molecules obtained from ‘251-labelledPHA-L sequenticllly immunoprecipitated 10 times with mAb W6/32 and then once with the anti-P2m mAb R1.30 (lane a); immunoprecipitations with W6/ 3 2 1st (lane b). 10th (lane c), 9th (lane d). ( 5 days exposure).
plex. In fact, it can be acquired by class I heavy chain from different, non-human species following association with bovine P2m; on the other hand in some mouse strains, where the murine heavy chain is covalently bound to bovine P2m, the formation or the exposure of the W6/32 antigenic determinant is prevented (4). It can be hypothesized that a subpopulation of class I molecules undergoes conformational changes upon lymphocyte activation, thus preventing the expression or exposure of the W6/32 epitope. A murine class I1 epitope showing a differential expression in different cell types has been described, demonstrating that the same MHC molecules can acquire different conformations on the cell surface (5). A physical association between the HLA class I antigens and other molecules on the surface of activated lymphocytes has been reported (6, 7) and could result in a conformational modification of complexed class I molecules. Another explanation could be that different peptides binding to the same MHC class I molecules affect their final conformation, as suggested by the experiments of Townsend et al. (8).
Figure 2. NP40 (1%)-solubilized 1251-surfacelabelled PHA-L, obtained from an HLA-A2,30, B17,45donor, were sequentially immunoprecipitated 10 times with mAb W6/32 and then once with the anti-fl2m mAb R1.30. Resulting immunoprecipitates were treated with 0.1U Neuraminidase type VIII and analyzed by I D IEF on a pH 4-9 gradient following the method described by Neefjes et al. (9). a: mAb W6/32,1st immunoprecipitation; b: anti-fl2m mAb R1.30, 1Ith immunoprecipitation; c: mAb W6/32, 10th immunoprecipitation. (3 days exposure).
In conclusion, by ID IEF analysis, P2m-associated molecules solubilized from PHA-activated cells after complete depletion of W6/32-positive molecules are indistinguishable from A,B,C. Thus, in some cellular systems, mAb W6/32 does not recognize all HLA-A,B,C molecules, as previously thought. This observation should be kept in mind when attempting to identify new class I gene products after sequential precipitation with W6/32.
Acknowledgments Work supported by MPI 60% and by CNR, progetto finalhato Biotecnologie e Biostrumentazione. T. Crepaldi and S. D’Alfonso are recipients of a fellowship from “Comitato Gigi Ghirotti”. Refereices 1. Gazit E, Fauchet R, Jones M, et al. Antigen Society n. 31
139
Crepaldi et al. Report. In: Dupont B, ed. Immunobiology of HLA, vol I. New York: Springer Verlag, 1989: 281. 2. Gazit E, Terhorst C, Yunis E. The human 'T' genetic region of the HLA linkage group is a polymorphism detected on lectin-activated lymphocytes. Nature 1980: 2&1: 275. 3. Fauchet R, Boscher M, Bouhallier 0, et al. New class I in man: serological and molecular characterization. Human Immunology 1986: 17 3. 4.Kahn-Perles B, Boyer C, Arnold B, Sanderson AR, Femer P, Lemonnier FA. Acquisition of HLA class I W6/32 defined antigenic determinant by heavy chain from different species following association with bovin b2-microglobulin. J Immunoll987: 138:2190. 5. Murphy DB, Lo D, Rath S, et al. A novel MHC class I1 epitope expressed in thymic medulla but not cortex. Nature 1989: 338:765. 6. Blue ML, Craig KA, Anderson P, Branton KR, Schlossman SF. Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxiccells. Cell 1988: 54: 413.
140
7. Bushkin Y, Demaria S, Le J, Schwab R. Physical association between the CD8 and HLA Class I molecules on the surface of activated human T lymphocytes. Proc Natl Acad Sci USA 1988: 85: 3985. 8. Townsend A, Olden C, Bastin J, Ljunggren HG, Foster L, Karre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 1989: 340:443. 9. Neeijes JJ, Brew-Vriesendorp BS, Van Seventer GA, Ivanyi P, Ploegh HL. An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLA-class I subtypes. Human Immunology 1986 16 169. Address: Patricia Richiardi, PhD Dipartimento di Genetica Via Santena 19 10126 Torino Italy
