Recognition of MHC class I molecules by allospecific CTL
Eur. J. Immunol. 1991.21: 2767-2774
Fumie AosaiO, a a e s Ohleno, Hans-Gwtaf LjunggrenO, Petter HoglundO, Lars Frankssonn, Hidde Ploeghv, Main TownsendA, Klas KiirreO and Hans J. StaussO Imperial Cancer Research Fund, Tumour Immunology Unito, University College London, London, Department of Tomoor Biologyo, Karolinska Institute, Stockholm, Institute of Molecular Medicine*, John RadclIffe Hospital, Headington, Oxford and The Netherlands Cancer Institutev, Amsterdam
2767
Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells Allospecific immune responses against the MHC of another individual are remarkably strong, due to a high number of respondingT cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class1 molecules, it has remained unclear whether MHC molecules can be recognized directly.We used the H-2b-derivedmurine lymphoma mutant RMA-S,which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Q p e A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse IWA-S. Up-regulation of the levels of H-2Kbdensity improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CIZ clones apparently can recognize class I molecules devoid of peptides,while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.
1 Introduction According to the “high ligand density” hypothesis proposed by Bevan [l], the ligands of alloreactiveTcellsare the MHC molecules alone, irrespective of the association with antigen (or in today’s terminology, irrespective of bound peptide). This means that all h4HC molecules on a target cell surface can act as ligands, and that this high determinant density can compensate for low-affinityTcRAigand interactions.Therecruitment of T cells that recognize MHC with low affinity, into the allorepertoire results in the high precursor frequency for any given foreign MHC. An alternative model, initially proposed by Matzinger and Bevan [2] postulated recognition of “multiple interaction antigens”. It has been realized more recently that interaction antigens are the equivalent of peptide/MHC complexes [3-51. The model assumes that allospecific CTL recognize specific peptide/MHC complexes just like nominal antigen-specific T cells. Since tolerance is self-MHC restricted [6,7], a vast number of endogenous peptides become immunogenicwhen presented by all0 MHC molecules. This results in a high frequency of alloresponsive Tcells, which can be said to recognize peptides in an allorestricted manner. Strong support for the “multiple interaction antigen” model comes from analysis of human alloreactive Tcell clones [S, 91 and from the observation of species and lineage specific allorecognition, i.e. T cell clones that recognize the
[I 95591 Correspoadence: Hans J. Stauss, I.C.R.F., H.T.I.G., Middlesex
School of Medicine, Courtauld Instituteof Biochemistry, 91 Riding House Street, London WlP 8BT, GB
Abbreviations: &m: f3z-Microglobulin SC: Spleen cells 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
relevant MHC molecules only when they are expressed in certain cell types [lo] or exposed to peptide extracts from cells of the correct species [ll]. If this model accounts alone for the strong allospecific responses, it must be assumed that all allospecific CTL follow the same rules as antigenspecific, self MHC-restricted clones, and require an intact processing pathway in the target cells. In contrast, the high-determinant-densityhypothesis predicts that some CTL clones should bc able to recognize processing defective targets, provided that the MHC density is high. The murine lymphoma mutant RMA-S has a defect that results in inefficient assembly of class I heavy chains with &-microglobulin (&m) [12, 131; only a small number of class I molecules reach the cell surface.This low cell surface expression of H-2 can be increased by incubating M A - S in the presence of H-2-binding peptides [12,14] or by culture at reduced temperature (23-31°C) [15,16]. The latter results in high level of cell surface expression of H-YB,m heterodimers that behave as if they were not occupied by peptides since they (a) are unstable at 37°C [16], (b) fail to present internally derived antigens [ E l , (c) present exogenously added peptides more efficiently than MHC molecules of the wild-type line [15] and (d) can be stabilized by the addition of H-2-binding peptides to the medium [12]. The low number of MHC molecules expressed by RMA-S at 37 “C had the same properties of peptide-deficient molecules [151. It has been shown that RMA-Swas resistant to H-Zrestricted CTL, although it was recognized by the same CTL if the appropriate peptides were added exogenously [12]. This suggests that RMA-S lacks a function required for presentation of inmcellular peptides in association with class I molecules. The mutant cells were rejected in H-2-mismatched recipient mice and lysed by allo-H-2-specificCTL in vbo [17],implying recognition of class I molecules lacking peptides by these CTL. RMA.S grew progressively and escaped H-2-restricted CTL when 0014-2980/91/1111-2767$3.50+ .25/0
2768
Eur. J. Immunol. 1991.21: 2767-2774
E Aosai, C. Owen, H.-G. Ljunggren et al.
with PlHTR-Kb (ll-clones) by LD of SC in 2Wpl microcultures containing 5 x 105 irradiated syngeneic feeder cells and 103 mitomycin C-treated Kb-transfected The purpose of the present study was to ask specificallyhow stimulator cells in DMEM tissue culture medium supM 2-ME, 200 m~ the density of MHC class1 molecules and their peptide plemented with 10% FCS, 5 X content will affect recognition by allospecific Cllclones. L-glutamine, nonessential amino acids, sodium pyruvate, RMA-S cells were used as peptide-deficient targets, and penicillin-streptomycin and 10 U/mlrIL2. Positive clones conditions were established which allowed to vary the were expanded by weekly stimulation with mitomycin density of class1 expression over a wide range. Some C-treated Kb-transfectants using the described medium anti-H-2KbCTL clones failed to lyse RMA-S cells indepen- except that rIL2 was replaced by 20% rat Con ASN. The dent of the level of Kbexpression. A portion of Cllclones Kb specificity of all clones was confirmed by their lysis of was able to lyse RMA-S target cells and analysis of these Kb-transfected cells but not of control transfectants. clones suggested that some may recognize peptidedeficient class I molecules. 2.4 CTL assay
assayed in an MHC-matched but multiple minor histocompatibility antigen-disparate combination [171.
2 Materials and methods 2.1 Mice and cell lines C3WHe and BALB/c mice were obtained from the ICRF animal colony and C57BL/6 mice were maintained at the Department of lhmor Biology, Karolinska Institute (Stockholm, Sweden). RBLS is a Rauscher virus-induced T cell lymphoma of C57BL/6 origin. RMA is a mutagenized but nonselected subline of RBL-5. RMA-S is an antiH-Zselected subline of RMA. The anti-H-2 selection and molecular analysis of the RMA-S line has been described in detail [13, 18-20]. RMA and RMA-S were maintained as suspension cultures in RPMI 1640 tissue culture medium containing 10% FCS and antibiotics. Transfection of the H-2Kb gene into human HeLa cells, murine DAP cells (subline of C3H-derived L cells) and murine PlHTR cells (subline of DBAD-derived P815 cells) has been described [21,22]. Transfected cells were grown in tissue culture medium containing 10% FCS and 500 pg/ml (HeLa) or 300 pg/ml (DAP, PlHTR) gentamycin (G418). The transfectantswere regularly tested for stable expression of Kbby FCM analysis.
2.2 mAb and staining Cells (2 x 105) were incubated for 30 min on ice in the presence of mAb 20-8-4 which recognize determinants in the al, a2 and domains of Kb [23]. Cells were then stained with second-layer fluorescein-labeled goat anti-mouse Ig and analyzed with a FACScan (Becton Diclcinson, Mountain View, CA). For control staining, cells were exposed to second-layer antibodies only.
Four-hour Wr-release assays were done at 37 "C or 31"C using 96-well microtiter plates containing 104 %r-labeled target cells and various numbers of effector CIZ (depending on the E/T ratio) in 200 pl medium per well. After 4 h 100 pl medium was removed and radioactivity was counted in a gamma counter. The percent specific lysis was determined using the equation: [(experimental release - spontaneous release)/(maxirnal release - spontaneous release)] X 100. CD8 blocking experiments were done by adding 0.3 pg anti-CD8mAb (YTS169.4, [24]) to each well at the beginning of the 4-h CTL assay. Control wells received 0.3 pg isotype-matched anti-CD4 mAb (GK1.5, [25]).The percent of anti-CD8mAb-mediated inhibition at each E/Tratio was calculated using the formula 11-(% lysis in the presence of anti-CD8 mAb/% lysis in the absence of mAb)] x 100.
2.5 Modulation of target cells For temperature induction of class I expression, RMA-S cells were cultured at 25 "Covernight and used the following day for FCM analyses and for CIZ assays. For induction of class I expression by human &m, RMA-S were cultured overnight at 37 "C or 25 "C in media containing 5 p~ human &m (Sigma, Poole, GB). Class I molecules induced by low temperature or human b m were loaded with Kb-binding peptides NP345-360 [12] or Db-binding peptides "366-379 [12] by adding 5 0 p ~peptide to overnight cultures. 51Cr labeling was done at the same peptide concentration and the CTL assay were done in the presence of 2 pM or 10 pM peptide. 2.6 LD analysis
2.3 Generation of CTL lines and clones
Bulk all0-H-2~-specificCI'L were generated from either naive BALB/c mice, or from mice 7-10days previously immunized with B6 spleen cells (SC). Responder SC were stimulated in v i m with irradiated B6 SC as described [17]. CIZ lines were established by weekly restimulation of the bulk cultures with the addition of 5 U/ml rIL2. Anti-B6 clones were isolated by LD and were stimulated weekly in the presence of 5 U/ml IL2. H-2Kb-specific CTL clones were established from C3H mice immunized with HeLa-Kb (3-, 9-, 24- and 37-clones) or from BALB/c mice immunized
SC from C3H mice immunized with HeLa-H-2Kbcells were restimulated in 96-well microtiter plates in 2 0 0 4 cultures containing 5 x 105irradiated syngeneicfeeder cells, 103 mitomycin C-treated Kb-transfected stimulator cells and responder SC ranging from 3 x 104 to 2.5 x 103 cells per well (at this cell density > 37% of the wells were negative when tested in a CTL assay 8 days later). At day 8 each well was split, and duplicates were analyzed in a Wr-release CTL assay using RMA and RMA-S as targets. Wells were recorded positive when the 51Cr release of RMA was greater than 3 SD above the spontaneous release of the labeled RMA cells.
Eur. J. Immunol. 1991.21: 2767-2774
Recognition of MHC class 1 molecules by allospecific CTL
2769
3 Results
lysed RMA targets well but did not show any significant lysis of RMA-S targets (Fig. 1E and F). CTL clone 9-25 and 9-26 showed reduced but significant lysis of RMA-S cells 3.1 CTL responses in a complete MHC mismatch relative to the efficient lysis of RMA cells (Fig. 1C and D). It has previously been shown that RMA-S can be lysed by We tested whether the inefficient RMA-S lysis observed allospecific CTL generated in primary and secondary bulk with four clones would improve after increasing the level of MLC against B6 SC [17]. This in vitro response correlated Kb expression. RMA-S cells were cultured at 25°C overwith rejection of RMA-S cells in allogeneic mice. The lysis night which resulted in a sigmficant upregulation of Kb of RMA-S by primary and secondary bulk CrrL cultures is expression (Fig. 2A); such temperature-induced cells are weaker than that of RMA as illustrated in Table 1. The referred to hereafter as RMA-S (25 "C). RMA-S (25 "C) inefficient lysis of RMA-S was maintained in CTL lines were used as targets in a 4-h CTL assay which was established from the bulk cultures. One possible explana- performed at 31°C [control staining of RMA-S (25°C) tion for the difference in RMA and RMA-S lysis was that cultured for 4 h at 31°C showed no decrease in Kb only a portion of CTL clones could lyse the mutant line and expression]. RMA-S (25 "C) were lysed more efficiently that different clonal distributions were maintained in the than RMA-S (37°C) by CTL clone 9-25 and 9-26 (Fig. 1C lines. Testing of a limited number of clones supported this and D). In contrast, temperature induction did not lead to (Table 1). Some CTL clones showed no lysis of RMA-S and enhanced CI'L lysis by clone 9-19 and 9-5 (Fig. 1E and some were able to lyse the mutant targets. These observa- F) tions prompted us to undertake a more extensive clonal analysis of allo-CTL responses with RMA and RMA-S as The clones shown in Fig. 1 represent typical examples of read out targets. In order to reduce the complexity in a CTL activities found among 56 anti-Kb clones. Based on response over a complete haplotype mismatch we chose to the ability to lyse RMA-S, we have divided the clones into work with a response to a single MHC class I allele, H-2Kb, three types (Table 2). Type A clones lysed RMA-S (37 "C) and RMA-S (25°C) very efficiently. Type B CTL showed expressed in transfected cells. clearly reduced lysis of RMA-S (37 "C).The lysis of RMA-S (25 "C) by these clones was more efficient but did not reach the level of lysis of RMA. Q p e C clones showed no Table 1. Ability of a11ti-H-2~ C T L to lyse R?viA-S target cells significant lysis of RMA-S (37 "C) or RMA-S (25 "C). Since RMA-S (25°C) expressed lower levels of Kb than RMA CrL % specific lysis of targes cells 1
population')
RMA 37°C
Bull.Mu:
m
Naive mice "N"
Immunized
5o:l 1O:l 77 65 83
82
5:l
1:l 72 75
RMA-S 37°C
2:l 5o:l 1O:l 63 22 13
2:l 10
76
66
48
5:l
1:l 15 41
67
Table2. Frequency of type A, B, C CI'L among long-term established clones and among short-term LD cultures from Kb-immunizedmice
mice "I"
CI'L lines " N
"r
Em
74 $0
28
62
T "I" "I" "1"
Shod--
56
16(28%)
30(54%)
282
29(10%)
174(62%)
lO(18%) 79(289k)
do&)
clones "W "W
Established clonesb)
31 52 31 64
17 27 13 31
7 2 6 4 58 37
4 2 4 11 16 22
1 0 2 6 8
11
a) Bulk ail0-H-2~-specificCTL were generated from either naive ("N") BAL,B/c mice, or from mice immunized 7-lodays previously ("I")with B6 spleen cells. c l z l i n e s and clones were established from the bulk cultures as described in Sect. 2.3.
3.2 H-2Kb-specificCTL dones show differences in their ability to lyse RMA-S We have established 56 CTL clones from C3H mice immunized in vivo with Kb-transfected HeLa cells (HeLa-Kb) [21] or from BAJ.,B/c mice immunized with Kb-tr&ected PlHTR cells [22]. The CTL activity of all clones against IwlA and RMA-S was determined. CTL clones 3-105 and 11-102lysed RMA and RMA-S target cells equally well (Fig. 1A and B). In contrast, clone 9-19 and 9-5
'Qpe A CTL activity was defined as specific lysis of M A - S (37 "C)2 75% of the specificlysis of =.Type B CTL activity was defined as specific lysis of RMA-S (37°C) ranging from 20%-75% of the lysis of Rh4A. 5 p e C CTL activity was defined as specific lysis of RMA-S(37 "C)
Eur. J. Immunol. 1991.21: 2767-2774
Fumie AosaiO, a a e s Ohleno, Hans-Gwtaf LjunggrenO, Petter HoglundO, Lars Frankssonn, Hidde Ploeghv, Main TownsendA, Klas KiirreO and Hans J. StaussO Imperial Cancer Research Fund, Tumour Immunology Unito, University College London, London, Department of Tomoor Biologyo, Karolinska Institute, Stockholm, Institute of Molecular Medicine*, John RadclIffe Hospital, Headington, Oxford and The Netherlands Cancer Institutev, Amsterdam
2767
Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells Allospecific immune responses against the MHC of another individual are remarkably strong, due to a high number of respondingT cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class1 molecules, it has remained unclear whether MHC molecules can be recognized directly.We used the H-2b-derivedmurine lymphoma mutant RMA-S,which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Q p e A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse IWA-S. Up-regulation of the levels of H-2Kbdensity improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CIZ clones apparently can recognize class I molecules devoid of peptides,while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.
1 Introduction According to the “high ligand density” hypothesis proposed by Bevan [l], the ligands of alloreactiveTcellsare the MHC molecules alone, irrespective of the association with antigen (or in today’s terminology, irrespective of bound peptide). This means that all h4HC molecules on a target cell surface can act as ligands, and that this high determinant density can compensate for low-affinityTcRAigand interactions.Therecruitment of T cells that recognize MHC with low affinity, into the allorepertoire results in the high precursor frequency for any given foreign MHC. An alternative model, initially proposed by Matzinger and Bevan [2] postulated recognition of “multiple interaction antigens”. It has been realized more recently that interaction antigens are the equivalent of peptide/MHC complexes [3-51. The model assumes that allospecific CTL recognize specific peptide/MHC complexes just like nominal antigen-specific T cells. Since tolerance is self-MHC restricted [6,7], a vast number of endogenous peptides become immunogenicwhen presented by all0 MHC molecules. This results in a high frequency of alloresponsive Tcells, which can be said to recognize peptides in an allorestricted manner. Strong support for the “multiple interaction antigen” model comes from analysis of human alloreactive Tcell clones [S, 91 and from the observation of species and lineage specific allorecognition, i.e. T cell clones that recognize the
[I 95591 Correspoadence: Hans J. Stauss, I.C.R.F., H.T.I.G., Middlesex
School of Medicine, Courtauld Instituteof Biochemistry, 91 Riding House Street, London WlP 8BT, GB
Abbreviations: &m: f3z-Microglobulin SC: Spleen cells 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
relevant MHC molecules only when they are expressed in certain cell types [lo] or exposed to peptide extracts from cells of the correct species [ll]. If this model accounts alone for the strong allospecific responses, it must be assumed that all allospecific CTL follow the same rules as antigenspecific, self MHC-restricted clones, and require an intact processing pathway in the target cells. In contrast, the high-determinant-densityhypothesis predicts that some CTL clones should bc able to recognize processing defective targets, provided that the MHC density is high. The murine lymphoma mutant RMA-S has a defect that results in inefficient assembly of class I heavy chains with &-microglobulin (&m) [12, 131; only a small number of class I molecules reach the cell surface.This low cell surface expression of H-2 can be increased by incubating M A - S in the presence of H-2-binding peptides [12,14] or by culture at reduced temperature (23-31°C) [15,16]. The latter results in high level of cell surface expression of H-YB,m heterodimers that behave as if they were not occupied by peptides since they (a) are unstable at 37°C [16], (b) fail to present internally derived antigens [ E l , (c) present exogenously added peptides more efficiently than MHC molecules of the wild-type line [15] and (d) can be stabilized by the addition of H-2-binding peptides to the medium [12]. The low number of MHC molecules expressed by RMA-S at 37 “C had the same properties of peptide-deficient molecules [151. It has been shown that RMA-Swas resistant to H-Zrestricted CTL, although it was recognized by the same CTL if the appropriate peptides were added exogenously [12]. This suggests that RMA-S lacks a function required for presentation of inmcellular peptides in association with class I molecules. The mutant cells were rejected in H-2-mismatched recipient mice and lysed by allo-H-2-specificCTL in vbo [17],implying recognition of class I molecules lacking peptides by these CTL. RMA.S grew progressively and escaped H-2-restricted CTL when 0014-2980/91/1111-2767$3.50+ .25/0
2768
Eur. J. Immunol. 1991.21: 2767-2774
E Aosai, C. Owen, H.-G. Ljunggren et al.
with PlHTR-Kb (ll-clones) by LD of SC in 2Wpl microcultures containing 5 x 105 irradiated syngeneic feeder cells and 103 mitomycin C-treated Kb-transfected The purpose of the present study was to ask specificallyhow stimulator cells in DMEM tissue culture medium supM 2-ME, 200 m~ the density of MHC class1 molecules and their peptide plemented with 10% FCS, 5 X content will affect recognition by allospecific Cllclones. L-glutamine, nonessential amino acids, sodium pyruvate, RMA-S cells were used as peptide-deficient targets, and penicillin-streptomycin and 10 U/mlrIL2. Positive clones conditions were established which allowed to vary the were expanded by weekly stimulation with mitomycin density of class1 expression over a wide range. Some C-treated Kb-transfectants using the described medium anti-H-2KbCTL clones failed to lyse RMA-S cells indepen- except that rIL2 was replaced by 20% rat Con ASN. The dent of the level of Kbexpression. A portion of Cllclones Kb specificity of all clones was confirmed by their lysis of was able to lyse RMA-S target cells and analysis of these Kb-transfected cells but not of control transfectants. clones suggested that some may recognize peptidedeficient class I molecules. 2.4 CTL assay
assayed in an MHC-matched but multiple minor histocompatibility antigen-disparate combination [171.
2 Materials and methods 2.1 Mice and cell lines C3WHe and BALB/c mice were obtained from the ICRF animal colony and C57BL/6 mice were maintained at the Department of lhmor Biology, Karolinska Institute (Stockholm, Sweden). RBLS is a Rauscher virus-induced T cell lymphoma of C57BL/6 origin. RMA is a mutagenized but nonselected subline of RBL-5. RMA-S is an antiH-Zselected subline of RMA. The anti-H-2 selection and molecular analysis of the RMA-S line has been described in detail [13, 18-20]. RMA and RMA-S were maintained as suspension cultures in RPMI 1640 tissue culture medium containing 10% FCS and antibiotics. Transfection of the H-2Kb gene into human HeLa cells, murine DAP cells (subline of C3H-derived L cells) and murine PlHTR cells (subline of DBAD-derived P815 cells) has been described [21,22]. Transfected cells were grown in tissue culture medium containing 10% FCS and 500 pg/ml (HeLa) or 300 pg/ml (DAP, PlHTR) gentamycin (G418). The transfectantswere regularly tested for stable expression of Kbby FCM analysis.
2.2 mAb and staining Cells (2 x 105) were incubated for 30 min on ice in the presence of mAb 20-8-4 which recognize determinants in the al, a2 and domains of Kb [23]. Cells were then stained with second-layer fluorescein-labeled goat anti-mouse Ig and analyzed with a FACScan (Becton Diclcinson, Mountain View, CA). For control staining, cells were exposed to second-layer antibodies only.
Four-hour Wr-release assays were done at 37 "C or 31"C using 96-well microtiter plates containing 104 %r-labeled target cells and various numbers of effector CIZ (depending on the E/T ratio) in 200 pl medium per well. After 4 h 100 pl medium was removed and radioactivity was counted in a gamma counter. The percent specific lysis was determined using the equation: [(experimental release - spontaneous release)/(maxirnal release - spontaneous release)] X 100. CD8 blocking experiments were done by adding 0.3 pg anti-CD8mAb (YTS169.4, [24]) to each well at the beginning of the 4-h CTL assay. Control wells received 0.3 pg isotype-matched anti-CD4 mAb (GK1.5, [25]).The percent of anti-CD8mAb-mediated inhibition at each E/Tratio was calculated using the formula 11-(% lysis in the presence of anti-CD8 mAb/% lysis in the absence of mAb)] x 100.
2.5 Modulation of target cells For temperature induction of class I expression, RMA-S cells were cultured at 25 "Covernight and used the following day for FCM analyses and for CIZ assays. For induction of class I expression by human &m, RMA-S were cultured overnight at 37 "C or 25 "C in media containing 5 p~ human &m (Sigma, Poole, GB). Class I molecules induced by low temperature or human b m were loaded with Kb-binding peptides NP345-360 [12] or Db-binding peptides "366-379 [12] by adding 5 0 p ~peptide to overnight cultures. 51Cr labeling was done at the same peptide concentration and the CTL assay were done in the presence of 2 pM or 10 pM peptide. 2.6 LD analysis
2.3 Generation of CTL lines and clones
Bulk all0-H-2~-specificCI'L were generated from either naive BALB/c mice, or from mice 7-10days previously immunized with B6 spleen cells (SC). Responder SC were stimulated in v i m with irradiated B6 SC as described [17]. CIZ lines were established by weekly restimulation of the bulk cultures with the addition of 5 U/ml rIL2. Anti-B6 clones were isolated by LD and were stimulated weekly in the presence of 5 U/ml IL2. H-2Kb-specific CTL clones were established from C3H mice immunized with HeLa-Kb (3-, 9-, 24- and 37-clones) or from BALB/c mice immunized
SC from C3H mice immunized with HeLa-H-2Kbcells were restimulated in 96-well microtiter plates in 2 0 0 4 cultures containing 5 x 105irradiated syngeneicfeeder cells, 103 mitomycin C-treated Kb-transfected stimulator cells and responder SC ranging from 3 x 104 to 2.5 x 103 cells per well (at this cell density > 37% of the wells were negative when tested in a CTL assay 8 days later). At day 8 each well was split, and duplicates were analyzed in a Wr-release CTL assay using RMA and RMA-S as targets. Wells were recorded positive when the 51Cr release of RMA was greater than 3 SD above the spontaneous release of the labeled RMA cells.
Eur. J. Immunol. 1991.21: 2767-2774
Recognition of MHC class 1 molecules by allospecific CTL
2769
3 Results
lysed RMA targets well but did not show any significant lysis of RMA-S targets (Fig. 1E and F). CTL clone 9-25 and 9-26 showed reduced but significant lysis of RMA-S cells 3.1 CTL responses in a complete MHC mismatch relative to the efficient lysis of RMA cells (Fig. 1C and D). It has previously been shown that RMA-S can be lysed by We tested whether the inefficient RMA-S lysis observed allospecific CTL generated in primary and secondary bulk with four clones would improve after increasing the level of MLC against B6 SC [17]. This in vitro response correlated Kb expression. RMA-S cells were cultured at 25°C overwith rejection of RMA-S cells in allogeneic mice. The lysis night which resulted in a sigmficant upregulation of Kb of RMA-S by primary and secondary bulk CrrL cultures is expression (Fig. 2A); such temperature-induced cells are weaker than that of RMA as illustrated in Table 1. The referred to hereafter as RMA-S (25 "C). RMA-S (25 "C) inefficient lysis of RMA-S was maintained in CTL lines were used as targets in a 4-h CTL assay which was established from the bulk cultures. One possible explana- performed at 31°C [control staining of RMA-S (25°C) tion for the difference in RMA and RMA-S lysis was that cultured for 4 h at 31°C showed no decrease in Kb only a portion of CTL clones could lyse the mutant line and expression]. RMA-S (25 "C) were lysed more efficiently that different clonal distributions were maintained in the than RMA-S (37°C) by CTL clone 9-25 and 9-26 (Fig. 1C lines. Testing of a limited number of clones supported this and D). In contrast, temperature induction did not lead to (Table 1). Some CTL clones showed no lysis of RMA-S and enhanced CI'L lysis by clone 9-19 and 9-5 (Fig. 1E and some were able to lyse the mutant targets. These observa- F) tions prompted us to undertake a more extensive clonal analysis of allo-CTL responses with RMA and RMA-S as The clones shown in Fig. 1 represent typical examples of read out targets. In order to reduce the complexity in a CTL activities found among 56 anti-Kb clones. Based on response over a complete haplotype mismatch we chose to the ability to lyse RMA-S, we have divided the clones into work with a response to a single MHC class I allele, H-2Kb, three types (Table 2). Type A clones lysed RMA-S (37 "C) and RMA-S (25°C) very efficiently. Type B CTL showed expressed in transfected cells. clearly reduced lysis of RMA-S (37 "C).The lysis of RMA-S (25 "C) by these clones was more efficient but did not reach the level of lysis of RMA. Q p e C clones showed no Table 1. Ability of a11ti-H-2~ C T L to lyse R?viA-S target cells significant lysis of RMA-S (37 "C) or RMA-S (25 "C). Since RMA-S (25°C) expressed lower levels of Kb than RMA CrL % specific lysis of targes cells 1
population')
RMA 37°C
Bull.Mu:
m
Naive mice "N"
Immunized
5o:l 1O:l 77 65 83
82
5:l
1:l 72 75
RMA-S 37°C
2:l 5o:l 1O:l 63 22 13
2:l 10
76
66
48
5:l
1:l 15 41
67
Table2. Frequency of type A, B, C CI'L among long-term established clones and among short-term LD cultures from Kb-immunizedmice
mice "I"
CI'L lines " N
"r
Em
74 $0
28
62
T "I" "I" "1"
Shod--
56
16(28%)
30(54%)
282
29(10%)
174(62%)
lO(18%) 79(289k)
do&)
clones "W "W
Established clonesb)
31 52 31 64
17 27 13 31
7 2 6 4 58 37
4 2 4 11 16 22
1 0 2 6 8
11
a) Bulk ail0-H-2~-specificCTL were generated from either naive ("N") BAL,B/c mice, or from mice immunized 7-lodays previously ("I")with B6 spleen cells. c l z l i n e s and clones were established from the bulk cultures as described in Sect. 2.3.
3.2 H-2Kb-specificCTL dones show differences in their ability to lyse RMA-S We have established 56 CTL clones from C3H mice immunized in vivo with Kb-transfected HeLa cells (HeLa-Kb) [21] or from BAJ.,B/c mice immunized with Kb-tr&ected PlHTR cells [22]. The CTL activity of all clones against IwlA and RMA-S was determined. CTL clones 3-105 and 11-102lysed RMA and RMA-S target cells equally well (Fig. 1A and B). In contrast, clone 9-19 and 9-5
'Qpe A CTL activity was defined as specific lysis of M A - S (37 "C)2 75% of the specificlysis of =.Type B CTL activity was defined as specific lysis of RMA-S (37°C) ranging from 20%-75% of the lysis of Rh4A. 5 p e C CTL activity was defined as specific lysis of RMA-S(37 "C)
