International Immunology. Vol. 4, No. 10, pp.
1113-1121
© 1992 Oxford University Press
High efficiency of endogenous antigen presentation by MHC class II molecules Veronique Calin-Laurens, Frederique Forquet, Suzanne Lombard-Platet, Patrick Bertolino, Isabelle Chretien, Marie-Claude Trescol-Blemont, Denis Gerlier, and Chantal Rabourdin-Combe Immunobiologie Moleculaire, UMR 49, CNRS-ENS Lyon, 69364 Lyon Cedex 07, France Key words: antigen presentation, endogenous antigen, hemagglutinin, hen egg lysozyme, MHC class II molecules
Abstract MHC class II molecules are Involved in the presentation of both exogenous and endogenous antigens to CD4 T cells. Using the trans-membrane hemagglutinin (HA) from measles virus and the secreted hen egg lysozyme (HEL) as antigen models, we have compared the efficiency of MHC class II presentation by naive antigen presenting cells (APCs) pulsed with exogenous antigen with that of their transfected counterparts synthesizing endogenous antigen. B cells expressing even a very low amount of trans-membrane HA were found to present endogenous HA to I-Ed restricted T cell hybrldomas with a high efficiency whereas their naive counterparts required to be pulsed with a comparatively high amount of exogenous HA. Similarly, MHC class II presentation of endogenous secreted HEL was found to be much more efficient when compared with that of exogenous HEL. Biochemical studies did not reveal any enhanced intracellular degradation of endogenous HEL. As expected, HEL was released in the surrounding medium within < 1 h. MHC class II presentation of endogenous HEL could not be explained by re-uptake by bystander APCs of HEL secreted In the surrounding medium. No sensitlzatlon of naive APCs could be observed either when co-cultured with HEL secreting cells or when cultured for 10 days with a sub-threshold amount of exogenous HEL. At the cell surface, I-Ed molecules immunopreclpitated from HEL secreting cells were found to be slightly enriched In SDS-reslstant forms. These data raised the question of how peptides derived from endogenous transmembrane and secreted antigens can so efficiently reach an MHC class II loading compartment.
Introduction T cells expressing an a0 TCR do not recognize an isolated nominal antigen but, rather, a complex associating MHC molecules with antigen derived peptides on an antigen presenting cell (APC) surface. Formation of peptide-MHC complexes in APCs thus requires antigen degradation into peptides and their association with MHC class I or class II molecules. Beside their distinct polypeptide structure, tissue expression, and respective CD8-TCR and CD4-TCR ligands, MHC class I and class II molecules differ by their intracellular trafficking. MHC class I molecules follow the 'default' secretory pathway (1,2) and MHC class II molecules, transiently associated with the invariant chain (li) (3), are routed to the endosomal compartment before expression at the cell surface (2,4-7). This difference if likely to be the key parameter which determines two functional sets of antigen derived peptides reaching the loading compartment of either MHC class I or MHC class II molecules. Indeed, accumulative data strongly support the endoplasmic reticulum
(ER) as the functional loading compartment for MHC class I molecules (reviewed in 8) and the endosomal compartment has been postulated from very early experiments to be the major if not the only functional loading compartment for MHC class II molecules (9 -12). Thus an important issue is to determine which antigen can give rise to peptides capable of reaching the functional MHC loading compartments and how they get there. We have undertaken a systematic approach using a given antigen to study the sensitization of APCs when the antigen is exogenousty supplied or endogenously synthesized and targeted to various APC compartments after transfection with the appropriate recombinant expression vector. We have previously reported that secreted and cytosolic endogenous hen egg lysozyme (HEL) but not exogenous HEL are presented by MHC class I molecules (13). In contrast, exogenous and secreted endogenous HEL but not cytosolic endogenous HEL are presented by MHC class II molecules (14). Accumulating reports
Correspondence to: D. Gerlier Transmitting editor: B. Malissen
Received 18 February 1992, accepted 22 June 1992
1114 Antigen presentation of MHC class II molecules have recently confirmed that MHC class II presentation occurs not only for exogenous antigens but also for endogenous antigens (14 - 21). This led us to ask what is the relative efficiency of MHC class II presentation of exogenous and endogenous antigens. We report here that, quite unexpectedly, both a trans-membrane and a secreted endogenous antigen appeared to be very efficiently presented by MHC class II molecules when compared with their exogenous counterparts.
Methods Antigen and APCs HEL was purchased from Sigma Chemical Co. (St Louis, MO) and hemagglutinin (HA) was purified from measles virus infected cells and incorporated into disearoyl-phosphatidylcholine liposomes according to Gerlier et al. (22). The following cell lines maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 6% FCS, 10 mM HEPES and 5 x 10~5 M /Smercaptoethanol were used as naive APCs: H-^ M 12.4.1 and H-2k CH27 B lymphoma cells. From these cell lines, stable HEL secreting and HA expressing clones were derived after transfection with pHMG-HELs eukaryotic expression vector containing full-length HEL cDNA (14) and with pHMG-HA eukaryotic expression vector containing HA cDNA (23) respectively. As markers of selection, pAG475/2 coding for hygromycine resistance and pRSV5 coding for mycophenolic acid resistance (24) were co-transfected. B cells were transfected by electroporation using the BioRad Gene Pulser® (BioRad, Richmond, CA). Briefly, 107M12.4.1 cells in 400 /il of DMEM supplemented with 10 mM HEPES containing 20 ^g of pHMG-HELs and 20 p.Q of pRSV5 were given an electric pulse of 250 V with the capacitance set at 960 /*F in a 4 mm width Gene Pulser® cuvette. Electroporation of CH27 cells was performed using a 270 V electric pulse and pAG475/2 as selection marker. One day after transfection, cells were washed once then cultured for 3 days before cloning in the presence of the relevant antibiotic. Stable transfectants secreting HEL or expression HA were selected either by detecting the protein [secreted HEL (HELs) or cell surface HA] or by deteoting the corresponding mRNA using either a RNA cytodot or a Northern blot assay previously described (13,14). The following clones were used: M12.HELs3, CH27.HELs10, M12.HA.5.1, and M12.HA.25. In one experiment, M 12.4.1 cells transfected with pHMG-HELc vector containing a signal sequence deleted HEL cDNA (13,14) and expressing a cytosol targeted HEL (clone M12.HELc4) was also used as a control. The cell lines were regularly checked for the absence of mycoplasma contamination by growth on microbial medium. Before their use in the T cell stimulation assay, fibroblastic L cells were grown at high density. Radiolabelling and immunoprecipitation Cells were metabolically radidabelled and solubilized according to Calin-Laurens et al. (14). Briefly, 106 cells were washed twice in serum-free medium without methionine then labelled at 37°C for 3 h with 125 /tCi of [^Jmethionine (Amersham, Les Ulis, France) in 0.5 ml of medium. The cells were then washed in PBS and lysed in buffer containing 2 % Triton X100. After ultracentrifugation, supernatants were pre-cleared with normal mouse serum for 1.5hat4°C, then with 0.1 mg of Protein A - Sepharose
(Pharmacia, Uppsala, Sweden) previously saturated with 50 ng of polyclonal rabbit anti-mouse antibodies (Biosys, Compiegne, France) for 1.5 h at 4°C. After centrifugation and elimination of Sepharose beads, immunoprecipitation was then performed with 50 ng of monoclonal mouse anti-HA antibody (cl55) (25), coupled with rabbit anti-mouse and Sepharose beads as above. After 2 h incubation at 4°C, Sepharose beads were recovered, washed twice in lysis buffer and processed for SDS - PAGE analysis. For pulse-chase studies, 15X10 6 cells were washed, incubated in methionine and cysteine free medium for 1 h and pelleted. The cell pellet was re-suspended in 50 /d (37 MBq) of pSJmethionine and pSJcysteine (Expre 3 ^ 3 ^, NEN-Dupont, Paris, France). After 15 min labelling, cells were quickly washed in medium, re-suspended in complete culture medium supplemented with 5 mM of cold methionine and cysteine, and incubated for up to 24 h. The cells were lysed as indicated above, and the extracts and cell-free supernatants were immunoprecipitated with a pool of antibodies directed against native and denatured HEL (14) in the presence of Protein A-Sepharose beads previously saturated with rabbit anti-mouse Ig antibodies. After extensive washes, the material was eluted from Protein A -Sepharose beads and loaded onto SDS polyacrylamide gel for electrophoresis. The material immunoprecipitated was revealed by autoradiography To study the SDS-resistant forms of the MHC class II expressed at the cell surface, 5 x 106 M12.4.1 and M12.HELs3 viable cells were lodinated at 4°C with 0.5 mCi125l using the lactoperoxydase procedure, washed, lysed in 2% NP-40, 6 mM CHAPS buffer (26), and then centrifuged. The supernatants were immunoprecipitated with I-Ed specific 14.4.4.S mAbs and Protein A - Sepharose according to the procedure described by Germain and Hendrix (26). After washes, the immunoprecipitates were eluted in Laemli sample buffer without /3-mercaptoethanol for 30 min at room temperature. Half of the immunoprecipitates were boiled at 100°C for 2 min before analysis onto 12.5% SDS - PAGE and autoradiography. The autoradiographies from unboiled samples were scanned using a densitometer to determine the percentage of I-Ed molecules in SDS-resistant forms, the total amount of I-Ed molecules immunoprecipitated being determined by adding the signal of SDS-resistant forms (C forms) to that of free MHC class II chains (U forms). T cell hybndomas The two HEL-specific I-Ak restricted 3A9 (27) and I-Ed restncted B10D24.42 (kindly provided by E. Sercarz) were used in this study. The HEL epitope has been determined, HEL 46-61 for 3A9 (27) and HEL 106-116 for B10D24.42 T cell hybridomas (E. Sercarz, personal communication). The HA-specific T cell clone TH5.124 was obtained as previously described (23). 7" cell functional assay Specific antigen stimulation of the T cell hybridomas was performed by co-cultivating 10s hybridoma cells and various amounts of APCs with or without various amounts of antigen in a final volume of 200 y\. After incubation for 20 h at 37 °C in 96-well microplates, IL-2 production in supernatants was measured in a biological assay using the growth of IL-2dependent CTL-L2 cell line revealed by using the MTT assay (14). Standard deviation of replicates was usually below 5%. In one experiment, M 12.4.1 cells were grown in tissue culture
Antigen presentation of MHC class II molecules medium containing 10 ^g HEL/ml with re-seeding every 3 - 4 days at 5 x 10" cells/ml before their use in the T cell functional assay. Co-cultures were performed by seeding 2 x 1 0 5 H-2d M12-HELs cells with 3 x 10* H-2* CH27 cells in 200 p\ of culture medium. After 24 h of incubation, 105 I-Ak restricted 3A9 T cells were added. As controls, 3A9 T cells were stimulated with 3 x 10" CH27 cells together with various amounts of HEL and various numbers of M12-HELs were used to stimulate I-Ed restricted B10D24.42 T cells. To quantify the relative efficiency of MHC class II presentation of exogenous and secreted endogenous antigen, the following calculations were done. The amount of HEL produced within 24 h by one HELs cell was determined by measuring the amount of HEL produced within 24 h by 105 cells in 200 pi of culture medium using an HEL-specific ELISA. In addition, HEL contents in HELs cells was estimated after running cell extracts onto SDS-PAGE and analysis by Western blot as previously described (14). Results APCs expressing a very low amount of cell-surface HA can efficiently stimulate MHC class ll-restricted T cells M 12.4.1 B cells were used to present exogenous HA or endogenous transmembrane HA after transfection with pHMG.HA recombinant expression vector to HA-specific T cell
1115
hybridomas. Both naive M12.4.1 B cells pulsed with an optimum concentration of HA and M12.HA.24 cells synthesizing a high amount of HA were able to stimulate the HA-specific TH5.124 T cell hybridoma (Fig. 1) as well as four other independent T cell hybridomas (data not shown). Moreover, MHC class II presentation of endogenous HA by B cells was found to be very efficient since only a few hundred M12.HA.24 cells stimulated the B10D24.42 T cells (Fig. 1B). In contrast, to induce a similar level of T cell activation, 3 x 10" naive M 12.4.1 cells had to be pulsed with 3 /xg/ml of exogenous HA (Fig. 1A). In addition, a similar efficiency of MHC class II presentation of endogenous HA was observed when M12.HA.5.1, another HA transfectant expressing very little HA, was used to stimulate TH5.124 T cells (Fig. 1B). This was not due to the isolation of an M12 sub-clone particularly potent as APCs, since when M12.HA.24 and M12.HA.5.1 were used to present exogenous HEL to an I-Ed restricted and HELspecific T cell hybridoma, they were as potent APCs as the parental M 12.4.1 cells (data not shown). The amount of HA synthesized by M12HA.24 and M12HA.5.1 cell clones, as evidenced after metabolic radiolabelling immunoprecipitation, and SDS - PAGE analysis (Fig. 2B), was found to correlate with the amount of HA mRNA as determined in a Northern blot analysis (Fig. 2A). This indicates that the very low amount of HA (see the faint band in lane 2 of Fig 2B) detected in M12.HA.5.1 cell extracts is not due to synthesis of an unstable HA overdegraded in this particular sub-clone. The amount of HA in M12.HA.24 cells could not be directly determined due to the high cross-reactivity on the parental M 12.4.1 cells of every anti-HA antibodies we have tested. These data indicated not only that endogenous trans-
0.4
o
d
0.2-
0.0
0
1
2
3
4
0.4-
12
0.2-
0.0
1 0z 1 03 cell number/well
10'
3 4
M
10"
Fig. 1. Efficient presentation of endogenous HA to the MHC class llrestricted T cells TH5.124 hybridoma T cells (10s) were stimulated with 3X10 4 M 12.4.1 B cells pulsed with exogenous HA (A) or with various numbers ol M12.HA (B) expressing low (M12HA5.1, open squares) or high(M12.HA.24, closed squares) levels of endogenous HA. After 24 h of stimulation, IL-2 was measured in the supernatants using the CTL-L2 bio-assay revealed by MTT assay
Fig. 2. Synthesis of endogenous HA in cells transfected with pHMG.HA expression vector. (A and B) The amount of HA synthesized by M12.HA.5.1 and M12.HA.24 cells clones correlates with the amount of HA mRNA. (A) Northern blot of 20 pQ of total RNA extracted from M12.4.1 (lane 1), M12.HA.5.1 (lane 2), and M12.HA.24 (lane 3) cells analysed using HA cDNA probe. (B) Immunoprecipitation after metabolic radiolabelling of M12.HA.5.1 (lanes 1 and 2) and M.12.HA.24 (lanes 3 and 4) cells using normal mouse serum (lanes 1 and 3) or HA specific monoclonal antibody (lanes 2 and 4). M: 69 kDa molecular weight marker.
1116 Antigen presentation of MHC class II molecules 12
3
4
5
4 5
10 l l O ' l i g HEL 3.3 > 10 4 ng HEL
1.1 i IO 4 ng HEL
12
3
4
5
6
7
8
9
10
Fig. 3. Synthesis and fate of endogenous HEL in cells transfected with pHMG.HEL expression vectors. (A) HEL mRNA expression in M12.4 1 (lane 1), M12.HELc4 (lane 2), M12.HELs3 (lane 3). CH27 (lanes 4 and 40, and CH27 HELsiO (lanes 5 and 50 as detected by RNA cytodot. Rapid RNA extraction was performed for 108 cells, and each sample (two dilutions) was dotted on nitrocellulose then hybridized with ^P-radiolabelled BamH\ HELc cDNA probe. HEL mRNA was detected by autoradiography after 16 h (lanes 1 - 5 ) or 3 week (lanes 4' and 50 exposure. (B) Pulse-chase study of HEL secreted by M12 HELs3. Cells were pulsed with ["^Jmethionine and [^Icysteine for 15 min followed by a chase with an excess of cold methionine and cysteine for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 3 (lanes 4 and 9), and 6 (lanes 5 and 10) h. Cell extracts (lanes 1 - 5) and cell free supernatants (lanes 6-10) were immunopreciprtated with HEL specific antibodies and analysed by SDS-PAGE. Molecular weight markers: 30, 21 5,14.3,6.5, and 3.4 kDa.
membrane HA was presented by MHC class II molecules but also that presentation of endogenous HA might be even more efficient than presentation of its exogenous counterpart. This prompted us to investigate the efficiency of MHC class II presentation of another endogenous antigen which would not be retained within the APCs but secreted. Endogenous HELs is very efficiently presented to MHC class IIresthcted T cells H-2* M 12.4.1 and H-2k CH27 B cells were transfected with pHMG.HELs recombinant expression vector coding for a secreted form of HEL and, as control, M12.4.1 cells were also transfected with pHMG.HELc recombinant expression vector coding for an HEL form lacking the signal peptide and thus targeted to the cytoso) (14). The M12.HELs3 clone secreting up to 300 ng HEL/106 cells/24 h and the CH27.HELs10 secreting < 1 ng HEL/106 cells/24 h were used in this study. The amount of HEL mRNA expressed in these transfectants correlates with the amount of HELs since in M12.HELs3 cells, HEL mRNA was detected after only few hours of autoradiographic exposure of the RNA cytodot (Fig. 3A, lane 2) whereas a 3 week exposure was necessary to detect HEL mRNA in CH27.HELs10 cells (Fig. 3A, lanes 5 and 50. As a control, M12.HELc4 expressing the cytosolic form of HEL contains as much HEL mRNA as M12.HELs3 cells (Fig. 3A, lane 3). We have previously reported
0.34 slO 4 Dg HEl
10* I 0.3
call* ag HCLi In c t l t i
30 ng ••crttad HEL / 2 4 boars
Fig. 4. Quantitative comparison between the I-Ed restricted presentation of exogenous HEL and that of endogenous HELs B10D24.42 T cells (105) were stimulated with vanous numbers of (A) naive H-2? M 12.4.1 B cells and various amount of exogenous HEL or (B) M12.HELs3 cells secreting endogenous HEL (closed squares) in a final volume of 200 pi. As a control, various numbers of M12.HELc4 cells synthesizing cytosoJ targeted HEL (open squares) were used as APCs. To allow a quantitative comparison between the efficiency of MHC class II presentation of exogenous versus endogenous HEL, the amounts of HEL and the amount of HEL secreted in the surrounding medium for 24 h (i.e. the length of the T cell stimulation assay) are indicated as two additional scales on the abscissa. These scales are valid only for M12 HELs3 cells (closed squares) and not for M12 HELc4 cells (open squares).
(14) that HEL targeted to the cytosol is very short-lived (ty, < 5 mm) and is not secreted. The fate of HEL synthesized by M12.HELs3 cells was studied by pulse - chase analysis (Fig. 3B). After 15 min of radiolabelling, neo-synthesized HEL was found in cell extract and not in the supernatant. After a 1 h chase with cold amino acids, most of the radiolabelled HEL had already been released into the supernatant, and the release was completed after 3 h. If the intracellular HEL contents (10 ng/106 M12.HELs3 cells) are released every hour, we would expect an accumulation of 240 ng of HEL in the surrounding medium after 24 h, which is in agreement with the amount of HEL found to be secreted per 24 h (300 ng/106 M12.HELs3 cells). Further incubation of the cells never resulted in detectable re-uptake of radiolabelled HEL even after 24 h (data not shown) and no detectable degradation of radiolabelled HEL was observed in the supernatant A pulse-chase study using CH27.HELs10 cells showed a similar result (data not shown). M12.HELs3 cells were used as APCs to stimulate the I-Edrestricted HEL-speciflc B10D24.42 T cell hybridoma. As an
Antigen presentation of MHC class II molecules 1117
SO£0Ong KEL
«7ngHB.
10J
10'
Fig. 5. Quantitative comparison between the I-Ak restricted presentation of exogenous HEL and that of endogenous HELs. 3A9 T 10s cells were stimulated with various numbers of (A) naive H-2k CH27 B cells and various amount of exogenous HEL or (B) CH27.HELs10 cells secreting endogenous HEL (closed squares) in a final volume of 200 y\
approach to compare the efficiency of MHC class II presentation of endogenous HELs and that of exogenous HEL, 105 B10D24.42 cells were either stimulated with various numbers of M 12.4.1 cells in the presence of various amounts of exogenous HEL or with various numbers of M12.HELs3 cells (Fig. 4). Endogenous HELs was very efficiently presented by MHC class II molecules since as few as 100 transfected cells were able to significantly stimulate B10D24.42 T cells (Fig. 4B). We then tried to compare the efficiency of MHC class II presentation of endogenous HELs with that of exogenous HEL. A similar T cell activation signal was observed when B10D24.42 T cells were stimulated (in a final volume of 200 #d) either with 2X10 3 M 12.4.1 cells fed with 105 ng of exogenous HEL for 24 h (i.e. the duration of the T cell stimulation) (Fig. 4A) or with 2 x 103 M12.HELS3 cells secreting only 0.6 ng of HEU24 h and containing 0.02 ng of HEL (on its way to being secreted) (Fig. 4B). In contrast, cells expressing HEL targeted to the cytosol (M12.HELc.4) failed to stimulate the B10D24.42 T cell hybridoma (Fig. 4B). This was not due to an inherent inability of M12.HELc4 cells to process and present HEL, since they were as efficient as their parental counterpart in presenting exogenous HEL (data not shown). This high efficiency of MHC class II presentation of endogenous HELs was confirmed using the CH27 transfectant secreting a barely detectable amount of HEL (Fig. 5). CH27.HELs10, 5 x 104, (secreting
1113-1121
© 1992 Oxford University Press
High efficiency of endogenous antigen presentation by MHC class II molecules Veronique Calin-Laurens, Frederique Forquet, Suzanne Lombard-Platet, Patrick Bertolino, Isabelle Chretien, Marie-Claude Trescol-Blemont, Denis Gerlier, and Chantal Rabourdin-Combe Immunobiologie Moleculaire, UMR 49, CNRS-ENS Lyon, 69364 Lyon Cedex 07, France Key words: antigen presentation, endogenous antigen, hemagglutinin, hen egg lysozyme, MHC class II molecules
Abstract MHC class II molecules are Involved in the presentation of both exogenous and endogenous antigens to CD4 T cells. Using the trans-membrane hemagglutinin (HA) from measles virus and the secreted hen egg lysozyme (HEL) as antigen models, we have compared the efficiency of MHC class II presentation by naive antigen presenting cells (APCs) pulsed with exogenous antigen with that of their transfected counterparts synthesizing endogenous antigen. B cells expressing even a very low amount of trans-membrane HA were found to present endogenous HA to I-Ed restricted T cell hybrldomas with a high efficiency whereas their naive counterparts required to be pulsed with a comparatively high amount of exogenous HA. Similarly, MHC class II presentation of endogenous secreted HEL was found to be much more efficient when compared with that of exogenous HEL. Biochemical studies did not reveal any enhanced intracellular degradation of endogenous HEL. As expected, HEL was released in the surrounding medium within < 1 h. MHC class II presentation of endogenous HEL could not be explained by re-uptake by bystander APCs of HEL secreted In the surrounding medium. No sensitlzatlon of naive APCs could be observed either when co-cultured with HEL secreting cells or when cultured for 10 days with a sub-threshold amount of exogenous HEL. At the cell surface, I-Ed molecules immunopreclpitated from HEL secreting cells were found to be slightly enriched In SDS-reslstant forms. These data raised the question of how peptides derived from endogenous transmembrane and secreted antigens can so efficiently reach an MHC class II loading compartment.
Introduction T cells expressing an a0 TCR do not recognize an isolated nominal antigen but, rather, a complex associating MHC molecules with antigen derived peptides on an antigen presenting cell (APC) surface. Formation of peptide-MHC complexes in APCs thus requires antigen degradation into peptides and their association with MHC class I or class II molecules. Beside their distinct polypeptide structure, tissue expression, and respective CD8-TCR and CD4-TCR ligands, MHC class I and class II molecules differ by their intracellular trafficking. MHC class I molecules follow the 'default' secretory pathway (1,2) and MHC class II molecules, transiently associated with the invariant chain (li) (3), are routed to the endosomal compartment before expression at the cell surface (2,4-7). This difference if likely to be the key parameter which determines two functional sets of antigen derived peptides reaching the loading compartment of either MHC class I or MHC class II molecules. Indeed, accumulative data strongly support the endoplasmic reticulum
(ER) as the functional loading compartment for MHC class I molecules (reviewed in 8) and the endosomal compartment has been postulated from very early experiments to be the major if not the only functional loading compartment for MHC class II molecules (9 -12). Thus an important issue is to determine which antigen can give rise to peptides capable of reaching the functional MHC loading compartments and how they get there. We have undertaken a systematic approach using a given antigen to study the sensitization of APCs when the antigen is exogenousty supplied or endogenously synthesized and targeted to various APC compartments after transfection with the appropriate recombinant expression vector. We have previously reported that secreted and cytosolic endogenous hen egg lysozyme (HEL) but not exogenous HEL are presented by MHC class I molecules (13). In contrast, exogenous and secreted endogenous HEL but not cytosolic endogenous HEL are presented by MHC class II molecules (14). Accumulating reports
Correspondence to: D. Gerlier Transmitting editor: B. Malissen
Received 18 February 1992, accepted 22 June 1992
1114 Antigen presentation of MHC class II molecules have recently confirmed that MHC class II presentation occurs not only for exogenous antigens but also for endogenous antigens (14 - 21). This led us to ask what is the relative efficiency of MHC class II presentation of exogenous and endogenous antigens. We report here that, quite unexpectedly, both a trans-membrane and a secreted endogenous antigen appeared to be very efficiently presented by MHC class II molecules when compared with their exogenous counterparts.
Methods Antigen and APCs HEL was purchased from Sigma Chemical Co. (St Louis, MO) and hemagglutinin (HA) was purified from measles virus infected cells and incorporated into disearoyl-phosphatidylcholine liposomes according to Gerlier et al. (22). The following cell lines maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 6% FCS, 10 mM HEPES and 5 x 10~5 M /Smercaptoethanol were used as naive APCs: H-^ M 12.4.1 and H-2k CH27 B lymphoma cells. From these cell lines, stable HEL secreting and HA expressing clones were derived after transfection with pHMG-HELs eukaryotic expression vector containing full-length HEL cDNA (14) and with pHMG-HA eukaryotic expression vector containing HA cDNA (23) respectively. As markers of selection, pAG475/2 coding for hygromycine resistance and pRSV5 coding for mycophenolic acid resistance (24) were co-transfected. B cells were transfected by electroporation using the BioRad Gene Pulser® (BioRad, Richmond, CA). Briefly, 107M12.4.1 cells in 400 /il of DMEM supplemented with 10 mM HEPES containing 20 ^g of pHMG-HELs and 20 p.Q of pRSV5 were given an electric pulse of 250 V with the capacitance set at 960 /*F in a 4 mm width Gene Pulser® cuvette. Electroporation of CH27 cells was performed using a 270 V electric pulse and pAG475/2 as selection marker. One day after transfection, cells were washed once then cultured for 3 days before cloning in the presence of the relevant antibiotic. Stable transfectants secreting HEL or expression HA were selected either by detecting the protein [secreted HEL (HELs) or cell surface HA] or by deteoting the corresponding mRNA using either a RNA cytodot or a Northern blot assay previously described (13,14). The following clones were used: M12.HELs3, CH27.HELs10, M12.HA.5.1, and M12.HA.25. In one experiment, M 12.4.1 cells transfected with pHMG-HELc vector containing a signal sequence deleted HEL cDNA (13,14) and expressing a cytosol targeted HEL (clone M12.HELc4) was also used as a control. The cell lines were regularly checked for the absence of mycoplasma contamination by growth on microbial medium. Before their use in the T cell stimulation assay, fibroblastic L cells were grown at high density. Radiolabelling and immunoprecipitation Cells were metabolically radidabelled and solubilized according to Calin-Laurens et al. (14). Briefly, 106 cells were washed twice in serum-free medium without methionine then labelled at 37°C for 3 h with 125 /tCi of [^Jmethionine (Amersham, Les Ulis, France) in 0.5 ml of medium. The cells were then washed in PBS and lysed in buffer containing 2 % Triton X100. After ultracentrifugation, supernatants were pre-cleared with normal mouse serum for 1.5hat4°C, then with 0.1 mg of Protein A - Sepharose
(Pharmacia, Uppsala, Sweden) previously saturated with 50 ng of polyclonal rabbit anti-mouse antibodies (Biosys, Compiegne, France) for 1.5 h at 4°C. After centrifugation and elimination of Sepharose beads, immunoprecipitation was then performed with 50 ng of monoclonal mouse anti-HA antibody (cl55) (25), coupled with rabbit anti-mouse and Sepharose beads as above. After 2 h incubation at 4°C, Sepharose beads were recovered, washed twice in lysis buffer and processed for SDS - PAGE analysis. For pulse-chase studies, 15X10 6 cells were washed, incubated in methionine and cysteine free medium for 1 h and pelleted. The cell pellet was re-suspended in 50 /d (37 MBq) of pSJmethionine and pSJcysteine (Expre 3 ^ 3 ^, NEN-Dupont, Paris, France). After 15 min labelling, cells were quickly washed in medium, re-suspended in complete culture medium supplemented with 5 mM of cold methionine and cysteine, and incubated for up to 24 h. The cells were lysed as indicated above, and the extracts and cell-free supernatants were immunoprecipitated with a pool of antibodies directed against native and denatured HEL (14) in the presence of Protein A-Sepharose beads previously saturated with rabbit anti-mouse Ig antibodies. After extensive washes, the material was eluted from Protein A -Sepharose beads and loaded onto SDS polyacrylamide gel for electrophoresis. The material immunoprecipitated was revealed by autoradiography To study the SDS-resistant forms of the MHC class II expressed at the cell surface, 5 x 106 M12.4.1 and M12.HELs3 viable cells were lodinated at 4°C with 0.5 mCi125l using the lactoperoxydase procedure, washed, lysed in 2% NP-40, 6 mM CHAPS buffer (26), and then centrifuged. The supernatants were immunoprecipitated with I-Ed specific 14.4.4.S mAbs and Protein A - Sepharose according to the procedure described by Germain and Hendrix (26). After washes, the immunoprecipitates were eluted in Laemli sample buffer without /3-mercaptoethanol for 30 min at room temperature. Half of the immunoprecipitates were boiled at 100°C for 2 min before analysis onto 12.5% SDS - PAGE and autoradiography. The autoradiographies from unboiled samples were scanned using a densitometer to determine the percentage of I-Ed molecules in SDS-resistant forms, the total amount of I-Ed molecules immunoprecipitated being determined by adding the signal of SDS-resistant forms (C forms) to that of free MHC class II chains (U forms). T cell hybndomas The two HEL-specific I-Ak restricted 3A9 (27) and I-Ed restncted B10D24.42 (kindly provided by E. Sercarz) were used in this study. The HEL epitope has been determined, HEL 46-61 for 3A9 (27) and HEL 106-116 for B10D24.42 T cell hybridomas (E. Sercarz, personal communication). The HA-specific T cell clone TH5.124 was obtained as previously described (23). 7" cell functional assay Specific antigen stimulation of the T cell hybridomas was performed by co-cultivating 10s hybridoma cells and various amounts of APCs with or without various amounts of antigen in a final volume of 200 y\. After incubation for 20 h at 37 °C in 96-well microplates, IL-2 production in supernatants was measured in a biological assay using the growth of IL-2dependent CTL-L2 cell line revealed by using the MTT assay (14). Standard deviation of replicates was usually below 5%. In one experiment, M 12.4.1 cells were grown in tissue culture
Antigen presentation of MHC class II molecules medium containing 10 ^g HEL/ml with re-seeding every 3 - 4 days at 5 x 10" cells/ml before their use in the T cell functional assay. Co-cultures were performed by seeding 2 x 1 0 5 H-2d M12-HELs cells with 3 x 10* H-2* CH27 cells in 200 p\ of culture medium. After 24 h of incubation, 105 I-Ak restricted 3A9 T cells were added. As controls, 3A9 T cells were stimulated with 3 x 10" CH27 cells together with various amounts of HEL and various numbers of M12-HELs were used to stimulate I-Ed restricted B10D24.42 T cells. To quantify the relative efficiency of MHC class II presentation of exogenous and secreted endogenous antigen, the following calculations were done. The amount of HEL produced within 24 h by one HELs cell was determined by measuring the amount of HEL produced within 24 h by 105 cells in 200 pi of culture medium using an HEL-specific ELISA. In addition, HEL contents in HELs cells was estimated after running cell extracts onto SDS-PAGE and analysis by Western blot as previously described (14). Results APCs expressing a very low amount of cell-surface HA can efficiently stimulate MHC class ll-restricted T cells M 12.4.1 B cells were used to present exogenous HA or endogenous transmembrane HA after transfection with pHMG.HA recombinant expression vector to HA-specific T cell
1115
hybridomas. Both naive M12.4.1 B cells pulsed with an optimum concentration of HA and M12.HA.24 cells synthesizing a high amount of HA were able to stimulate the HA-specific TH5.124 T cell hybridoma (Fig. 1) as well as four other independent T cell hybridomas (data not shown). Moreover, MHC class II presentation of endogenous HA by B cells was found to be very efficient since only a few hundred M12.HA.24 cells stimulated the B10D24.42 T cells (Fig. 1B). In contrast, to induce a similar level of T cell activation, 3 x 10" naive M 12.4.1 cells had to be pulsed with 3 /xg/ml of exogenous HA (Fig. 1A). In addition, a similar efficiency of MHC class II presentation of endogenous HA was observed when M12.HA.5.1, another HA transfectant expressing very little HA, was used to stimulate TH5.124 T cells (Fig. 1B). This was not due to the isolation of an M12 sub-clone particularly potent as APCs, since when M12.HA.24 and M12.HA.5.1 were used to present exogenous HEL to an I-Ed restricted and HELspecific T cell hybridoma, they were as potent APCs as the parental M 12.4.1 cells (data not shown). The amount of HA synthesized by M12HA.24 and M12HA.5.1 cell clones, as evidenced after metabolic radiolabelling immunoprecipitation, and SDS - PAGE analysis (Fig. 2B), was found to correlate with the amount of HA mRNA as determined in a Northern blot analysis (Fig. 2A). This indicates that the very low amount of HA (see the faint band in lane 2 of Fig 2B) detected in M12.HA.5.1 cell extracts is not due to synthesis of an unstable HA overdegraded in this particular sub-clone. The amount of HA in M12.HA.24 cells could not be directly determined due to the high cross-reactivity on the parental M 12.4.1 cells of every anti-HA antibodies we have tested. These data indicated not only that endogenous trans-
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Fig. 1. Efficient presentation of endogenous HA to the MHC class llrestricted T cells TH5.124 hybridoma T cells (10s) were stimulated with 3X10 4 M 12.4.1 B cells pulsed with exogenous HA (A) or with various numbers ol M12.HA (B) expressing low (M12HA5.1, open squares) or high(M12.HA.24, closed squares) levels of endogenous HA. After 24 h of stimulation, IL-2 was measured in the supernatants using the CTL-L2 bio-assay revealed by MTT assay
Fig. 2. Synthesis of endogenous HA in cells transfected with pHMG.HA expression vector. (A and B) The amount of HA synthesized by M12.HA.5.1 and M12.HA.24 cells clones correlates with the amount of HA mRNA. (A) Northern blot of 20 pQ of total RNA extracted from M12.4.1 (lane 1), M12.HA.5.1 (lane 2), and M12.HA.24 (lane 3) cells analysed using HA cDNA probe. (B) Immunoprecipitation after metabolic radiolabelling of M12.HA.5.1 (lanes 1 and 2) and M.12.HA.24 (lanes 3 and 4) cells using normal mouse serum (lanes 1 and 3) or HA specific monoclonal antibody (lanes 2 and 4). M: 69 kDa molecular weight marker.
1116 Antigen presentation of MHC class II molecules 12
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10 l l O ' l i g HEL 3.3 > 10 4 ng HEL
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Fig. 3. Synthesis and fate of endogenous HEL in cells transfected with pHMG.HEL expression vectors. (A) HEL mRNA expression in M12.4 1 (lane 1), M12.HELc4 (lane 2), M12.HELs3 (lane 3). CH27 (lanes 4 and 40, and CH27 HELsiO (lanes 5 and 50 as detected by RNA cytodot. Rapid RNA extraction was performed for 108 cells, and each sample (two dilutions) was dotted on nitrocellulose then hybridized with ^P-radiolabelled BamH\ HELc cDNA probe. HEL mRNA was detected by autoradiography after 16 h (lanes 1 - 5 ) or 3 week (lanes 4' and 50 exposure. (B) Pulse-chase study of HEL secreted by M12 HELs3. Cells were pulsed with ["^Jmethionine and [^Icysteine for 15 min followed by a chase with an excess of cold methionine and cysteine for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 3 (lanes 4 and 9), and 6 (lanes 5 and 10) h. Cell extracts (lanes 1 - 5) and cell free supernatants (lanes 6-10) were immunopreciprtated with HEL specific antibodies and analysed by SDS-PAGE. Molecular weight markers: 30, 21 5,14.3,6.5, and 3.4 kDa.
membrane HA was presented by MHC class II molecules but also that presentation of endogenous HA might be even more efficient than presentation of its exogenous counterpart. This prompted us to investigate the efficiency of MHC class II presentation of another endogenous antigen which would not be retained within the APCs but secreted. Endogenous HELs is very efficiently presented to MHC class IIresthcted T cells H-2* M 12.4.1 and H-2k CH27 B cells were transfected with pHMG.HELs recombinant expression vector coding for a secreted form of HEL and, as control, M12.4.1 cells were also transfected with pHMG.HELc recombinant expression vector coding for an HEL form lacking the signal peptide and thus targeted to the cytoso) (14). The M12.HELs3 clone secreting up to 300 ng HEL/106 cells/24 h and the CH27.HELs10 secreting < 1 ng HEL/106 cells/24 h were used in this study. The amount of HEL mRNA expressed in these transfectants correlates with the amount of HELs since in M12.HELs3 cells, HEL mRNA was detected after only few hours of autoradiographic exposure of the RNA cytodot (Fig. 3A, lane 2) whereas a 3 week exposure was necessary to detect HEL mRNA in CH27.HELs10 cells (Fig. 3A, lanes 5 and 50. As a control, M12.HELc4 expressing the cytosolic form of HEL contains as much HEL mRNA as M12.HELs3 cells (Fig. 3A, lane 3). We have previously reported
0.34 slO 4 Dg HEl
10* I 0.3
call* ag HCLi In c t l t i
30 ng ••crttad HEL / 2 4 boars
Fig. 4. Quantitative comparison between the I-Ed restricted presentation of exogenous HEL and that of endogenous HELs B10D24.42 T cells (105) were stimulated with vanous numbers of (A) naive H-2? M 12.4.1 B cells and various amount of exogenous HEL or (B) M12.HELs3 cells secreting endogenous HEL (closed squares) in a final volume of 200 pi. As a control, various numbers of M12.HELc4 cells synthesizing cytosoJ targeted HEL (open squares) were used as APCs. To allow a quantitative comparison between the efficiency of MHC class II presentation of exogenous versus endogenous HEL, the amounts of HEL and the amount of HEL secreted in the surrounding medium for 24 h (i.e. the length of the T cell stimulation assay) are indicated as two additional scales on the abscissa. These scales are valid only for M12 HELs3 cells (closed squares) and not for M12 HELc4 cells (open squares).
(14) that HEL targeted to the cytosol is very short-lived (ty, < 5 mm) and is not secreted. The fate of HEL synthesized by M12.HELs3 cells was studied by pulse - chase analysis (Fig. 3B). After 15 min of radiolabelling, neo-synthesized HEL was found in cell extract and not in the supernatant. After a 1 h chase with cold amino acids, most of the radiolabelled HEL had already been released into the supernatant, and the release was completed after 3 h. If the intracellular HEL contents (10 ng/106 M12.HELs3 cells) are released every hour, we would expect an accumulation of 240 ng of HEL in the surrounding medium after 24 h, which is in agreement with the amount of HEL found to be secreted per 24 h (300 ng/106 M12.HELs3 cells). Further incubation of the cells never resulted in detectable re-uptake of radiolabelled HEL even after 24 h (data not shown) and no detectable degradation of radiolabelled HEL was observed in the supernatant A pulse-chase study using CH27.HELs10 cells showed a similar result (data not shown). M12.HELs3 cells were used as APCs to stimulate the I-Edrestricted HEL-speciflc B10D24.42 T cell hybridoma. As an
Antigen presentation of MHC class II molecules 1117
SO£0Ong KEL
«7ngHB.
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Fig. 5. Quantitative comparison between the I-Ak restricted presentation of exogenous HEL and that of endogenous HELs. 3A9 T 10s cells were stimulated with various numbers of (A) naive H-2k CH27 B cells and various amount of exogenous HEL or (B) CH27.HELs10 cells secreting endogenous HEL (closed squares) in a final volume of 200 y\
approach to compare the efficiency of MHC class II presentation of endogenous HELs and that of exogenous HEL, 105 B10D24.42 cells were either stimulated with various numbers of M 12.4.1 cells in the presence of various amounts of exogenous HEL or with various numbers of M12.HELs3 cells (Fig. 4). Endogenous HELs was very efficiently presented by MHC class II molecules since as few as 100 transfected cells were able to significantly stimulate B10D24.42 T cells (Fig. 4B). We then tried to compare the efficiency of MHC class II presentation of endogenous HELs with that of exogenous HEL. A similar T cell activation signal was observed when B10D24.42 T cells were stimulated (in a final volume of 200 #d) either with 2X10 3 M 12.4.1 cells fed with 105 ng of exogenous HEL for 24 h (i.e. the duration of the T cell stimulation) (Fig. 4A) or with 2 x 103 M12.HELS3 cells secreting only 0.6 ng of HEU24 h and containing 0.02 ng of HEL (on its way to being secreted) (Fig. 4B). In contrast, cells expressing HEL targeted to the cytosol (M12.HELc.4) failed to stimulate the B10D24.42 T cell hybridoma (Fig. 4B). This was not due to an inherent inability of M12.HELc4 cells to process and present HEL, since they were as efficient as their parental counterpart in presenting exogenous HEL (data not shown). This high efficiency of MHC class II presentation of endogenous HELs was confirmed using the CH27 transfectant secreting a barely detectable amount of HEL (Fig. 5). CH27.HELs10, 5 x 104, (secreting
