Eur. J. Immunol. 1990.20: 637-644
Fabienne Mazerolleso+, Frangois AmblardA, Catherine LumbrosoO, Olivia Lecomteo, Pierre-FrangoisVan de MoorteleO, Christiane BarbatO, Dominique Piatier-Tonneaua, Charles AuffrayA and Alain FischerO Immunologie et Rhumatologie Pkdiatriques, INSERM U 132, H6pital des Enfants-Maladeso, Paris and Institut d'Embryologie du CNRS et du College de Francea, Nogent s/Marne
CDCHLA class I1 interaction
637
Regulation of T helper-B lymphocyte adhesion through CD4-HLA class I1 interaction* Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus (EBV)-transformed B cells is mediated by CD2Aymphocyte function-associated antigen (LFA)-3 and LFA-lhntracellular adhesion molecule (1CAM)-1. Although some anti-CD4 antibodies block the antigen-independent adhesion of CD4+ T lymphocytes, the CD4-HLA class I1 interaction does not appear to significantly contribute to the forces of cell adhesion since CD4+ Tcells equally bind HLA class 11+and HLA class II- mutant B cells. In addition, conjugates formed between CD4+ Tcells and HLA class II- B cells remain stable for at least 1h while CD4+T/HLA class 11+B cell conjugate percentages promptly drop off. Down-regulation of CD4 or spontaneous low expression of CD4 also results in a persistance of conjugates formed with B cells.The role of the CD4-HLA class I1 interaction has been further studied by investigating the inhibitory effect of synthetic 12-mer peptides analogous to HLA class I1 and containing the Arg-Phe-Asp-Ser sequence conserved in the p1 domain. These peptides were previously found to inhibit HLA class II-restricted Tcell responses, this sequence being thought to be involved in CD4-HLA class 11 interaction. These peptides block conjugate formation of CD4+ resting Tcells or clones but not of CD8+ T cells, by interacting with the T cells as shown by preincubation experiments. Down-regulation of CD4 or spontaneous low expression results in the loss of the inhibitory activity. The peptide-mediated inhibition is neutralized by a soluble dimeric CD4 molecule. Alteration within the Arg-Phe-Asp-Ser sequence results in a significant loss of inhibition. It is thus proposed that the CD4-HLA class I1 interaction negatively regulates antigen-independent adhesion of T cells, this interaction involving the highly conserved Arg-Phe-Asp-Ser sequence in the HLA class I1 p1 sequence as a CD4-binding site.
1 Introduction Adhesion of T lymphocytes to target cells is mediated by at least two antigen-independent pathways, i. e. CD2 which binds lymphocyte function-associated antigen (LFA)-3 and LFA- 1 which binds the intracellular adhesion molecule (1CAM)-1 [l-31. A third adhesion pathway involving CD4 and HLA class I1 molecules might be used by CD4 T h lymphocytes as suggested by cell adhesion between CD4 and'HLA class I1 expressing cells [4] and inhibition by anti-CD4 antibodies of mouse CD4+ Th lymphocyte adhe-
sion to B lymphocytes [5]. In addition, it has been suggested that the CD4 molecule may play a role in antigen-specific, MHC class II-restricted T cell activation by associating with the CD3-Ti complex [6-81. This effect may be mediated by the p56lCktyrosine kinase that was found to be associated to the cytoplasmic tail of CD4 [9, 101. Conversely, it has been proposed that cross-linking of CD4, in the absence of TcR cross-linking generates a negative signal that may induce cell separation [ l l , 121.
Binding of CD4 t o MHC class I1 molecules has been demonstrated by Doyle and Strominger using CV-1 monkey fibroblasts strongly expressing CD4 [4], and confirmed by using transfected COS cells by Clayton et al. [13]. In the latter study, different parts of the CD4 molecule were suggested to be involved in CD4-HLA class I1 interaction [I 80981 as shown by site-directed mutagenesis [13]. We have proposed that CD4 and HLA class I1 molecules * This work was supported by Institut National de la SantC et de la previously may interact, at least in part, through preserved amino acid Recherche MCdicale and Centre National de la Recherche sequences (RADS and RFDS, respectively) analogous to Scientifique, and in part by grants from Programme National de Recherches sur le SIDA, Association pour la Recherche sur le the fibronectin-cell attachment site R G D S [14, 151, and Cancer, Direction des Etudes et Techniques, the Korberpreis have shown that synthetic dodecapeptides containing the Foundation and Rhone Poulenc Santt. RFDS or R ADS sequences inhibit antigen-specific HLA Supported by the Ligue Nationale de Recherche sur le Can- class II-restricted T cell proliferation and help for antibody cer. production [16]. We have herein investigated the role of CD4 in the antigen-independent adhesion of CD4+ T cells Correspondence: Alain Fischer, INSERh4 U 132, HBpital des to HLA class 11+B cells as both an adhesion pathway and a Enfants-Malades, 149, rue de Skvres, F-75743 Paris Cedex 15, regulatory element of adhesion. In addition, we have asked France whether synthetic peptides derived from HLA class I1 Abbreviations: ICAM-1: Intracellular adhesion molecule 1 molecules and containing the R FD S sequence could LFA: Lymphocyte function-associated antigen LAD: Leukocyte influence binding of CD4+ T cells to HLA class 11+ B cells. adhesion deficiency +
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0303-0637$02.50/0
638
Eur. J. Immunol. 1990.20: 637-644
F. Mazerolles, F. Amblard, C. Lumbroso et al.
2 Materials and methods
2.3 Synthetic peptides
2.1 Cells
DR-l2a, DR-l2b, DQ-12 peptides are analogous to residues 35 to 46 from the PI domain of HLA-DR1,2 and HLA-DQl sequences, respectively [14].They were synthesized by Neosystem (Strasbourg, France) according to the solid-phase synthesis method of Merrifield, and were further purified by HPLC, resulting in a purity 2 9 6 % of the peptide as shown by HPLC analysis. Peptide sequences were validated by amino acid analysis of the purified peptides. DR-12a variants were similarly synthesized as well as a 12-mer peptide analogous to residues 43-54 of the P1 domain of HLA-DR, and the 7-mer and 6-mer DR-7 and DR-6 peptides [14-16] .The Lys amino acid residue in DR-7 is not present in HLA-DR but was added for the sake of coupling to egg albumin. The tetrapeptide RFDS was prepared by A. Tartar, Lille, France. The peptides analogous to the fibrinogen molecule RGDF, LGRGD, G GAKQAGDV and GASPSTGTEEWTC were produced by G. Marguerie, INSERM U127, Grenoble, France.The G R D GDS peptide was purchased from Sigma (St. Louis, MO).
PBMC were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation from normal adults. CD4+ T lymphocytes were isolated from PBMC by a two-step procedure, i.e. neuraminidase-treated RBC rosetting followed by negative selection by panning using a CD8specificmAb (BC8, J.W.Widjdenes, BesanGon, France) and an anti-mouse Ig antibody (GaMIgG; Biomakor, Rehovot, Israel). CD4+ T cell preparations contained less than 2% CD8+ Tcells. CD4+ Tcells were also purified from PBMC from a patient with leukocyte adhesion deficiency (LAD), in whom expression of LFA-1 on leukocytes was absent [17]. CD8+ T lymphocytes were similarly isolated using a CD4-specific mAb (IOT4, Immunotech, Marseille, France). EBV-transformed B cells were used including four different sources of B cells: (a) EBV-transformed B cell lines; (b) B cells derived from a Burkitt lymphoma (BL2) which have been shown to poorly express the LFA-1, LFA-3 and ICAM-1 membrane molecules [18, 191; (c) EBV-transformed B cell lines from immunodeficient patients with an inherited deficiency in HLA class I1 molecule expression [20,21] as well as (d) mutant HLA class 11- B cells RJ225 produced by Accolla et al. [22]. Helper CD4+ T lymphocyte clone 4H (alloreactive, DR1 specific), M3 (influenza virus specific) and CD8+ T lymphocyte clone 4AS (alloreactive) were cultured in RPMI 1640 medium supplemented with 10% AB+ human serum and 20 U/ml of IL2 (Boehringer-Mannheim, Mannheim, FRG). 4H and 4AS clones were obtained from a rejected human kidney allograft [23]. The Jurkat CD4+ T cell line and a spontaneously obtained variant of the Jurkat Tcell line with low expression of CD4 were also used. In some experiments CD4+ T lymphocytes were incubated for 30 min to 4 h with either medium of PMA (Sigma; 20 ng/ml) in order to down-regulate CD4 molecule expression. CD4+ T lymphocytes treated with PMA for 4 h were further incubated after washings for 24 to 48 h in culture medium for the study of CD4 re-expression. 2.2 mAb The following mAb were used in adhesion assays: 25-3 (IgG1 anti-CDlla LFA-1, D. Olive) [24], IOT11 (IgG1 anti-CD2, Immunotech), L eu 3 a (IgG1 anti-CD4, Becton Dickinson, Mountain View, CA), 111-366 and F142-63C23 (IgG2 anti-CD4, €! Poncelet, Marseille, France), OKT4a (IgGh, Ortho, Raritan, NJ), WT31 (IgGl anti-CD3Ei complex, a kind gift from Dr. W. Thx (Nijmegen, The Netherlands), Leu-1 (IgG2, anti-CD5, Becton Dickinson), 84H10 (IgG1 anti-ICAM-1, D. Olive) [25], TS2/9 (IgG1 anti-LFA3,T. Springer) [2], IOT8 (IgG1 anti-CD8, Immunotech), IOT2 (IgGZa anti-HLA class I, Immunotech), ALB9 (IgG1 anti-CD24, Immunotech), 206 (IgG2, antiHLA-DR) [26],Tu22 (IgG2, anti-HLA-DQ) [27] and B7/21 (IgG1 anti-HLA-DP) [28].
2.4 Soluble CD4 CD4 IgG2, consisted of a construct of the two external domains of CD4 with the murine CH2 and cH3 domains of IgG2,. It was kindly supplied by A. Traunecker (Basel, Switzerland) [29].
2.5 Immunofluorescence Cell fluorescence intensity was evaluated by FCM analysis using a FACStar plus (Becton Dickinson) as revealed by specific antibodies and by a GaMIg (Nordic, Tilburg, The Netherlands).
2.6 Conjugate formation Antigen-independent T-B conjugate formation was performed according to the method described by Onishi et al. [30]. T and B cells were incubated at 2 x 106/ml with hydroxyethydine (40 pg/ml; Polysciences,Wamngton, PA) for 10 min at 25°C and fluorescein diacetate (100 pg/ml; Molecular Probes Inc., Eugene, OR) for 10 min at 37°C. After washings, 5 x 105 T cells were mixed with 5 X lo5 B cells in a 500 pl volume of RPMI 1640 medium (Gibco Biocult , Paisley, Scotland). Preliminary experiments have determined that optimal conjugate formation took place after a 20-min incubation at 37 "C. Incubation was followed by centrifugation at 250 x g for 5 min and gentle resuspension before counting. Conjugates were identified as redgreen pairs of cells under fluorescence microscopy. Two hundred to 350 fluorescent cells were counted in each experiment. Cells were counted blind by two independent observers. T-B conjugates were expressed as the percent of T-B conjugates vs. the total number of Tcells. Results are expressed as the percentage of T-B conjugates among all T cells or as the inhibition of T-B conjugate formation [l-(TB/T :TB control/T control)] x 100.
Eur. J. Immunol. 1990. 20: 637-644
CD4-HLA class I1 interaction
3 Results 3.1 Role of membrane adhesion molecules in conjugate formation between CD4+ T lymphocytes and B lymphocytes We have investigated which membrane molecules expressed by CD4+ T lymphocytes are involved in antigenindependent cell conjugate formation with EBV-transformed B lymphocytes expressing HLA class I1antigens. A significant percentage of CD4+ T lymphocytes was shown to bind EBV-B cells. In contrast, poor binding of CD4+ T lymphocytes to HLA class 11+B lymphocytes expressing very low levels of LFA-3 and ICAM-1 was observed while the percentages of CD4+ T lymphocytes able to bind HLA class 11+and HLA class 11- B cells were similar (Fig. la). HLA class 11- B cells were found to express ICAM-1 and LFA-3 similarly to control EBV-B cells (data not shown). BL2 B cells and control EBV-B cells similarly express HLA class I1 molecules (data not shown). LFA-1- Tcells equally bound HLA class 11+and class 11- B cells [24] (6.1 f 1.7 vs. 6.5 k 1.5%, mean of three experiments) although the percentage of conjugates was lower compared to LFA-l+ T cells (Fig. 1).Preincubation of CD4+ T cells with CD2 or LFA-1-specific antibodies and of B cells with LFA-3- and ICAh4-1-specificantibodies resulted in partial inhibition of conjugate formation. In contrast, mAb specific for CD5, CD3, CD24, CD21 and HLA class I had no effect (Fig. 1). The L e u 3 a anti-CD4 antibody could inhibit the formation of most conjugates while LFA-1 and CD2-specific antibod-
639
ies (or ICAM-1- and LFA-3-specific antibodies) exerted an inhibitory activity that was less potent but additive to each other. The same pattern of inhibition was observed on conjugates formed between CD4+ T lymphocytes and HLA class 11- B lymphocytes derived from immunodeficient patients (Fig. lb) or obtained by mutation (data not shown).
3.2 Influence of CD4 and HLA class II expression on the kinetics of conjugate formation between CD4+ T and B lymphocytes A peak of conjugates was repeatedly found after a 20-min incubation. As shown in Fig. 2a, the percentage of conjugates formed between CD4+ and HLA class 11+ cells dropped off rapidly after a 20-min incubation whereas the percentage of conjugates formed between CD4+ and HLA class 11- cells remained at a plateau for at least 40 min after reaching maximal level (Fig. 2a). The absence or diminished CD4 expression onTcells using the CD41°WJurkat line (Fig. 2d), or following down-regulation of CD4 by PMA (Fig. 2b) gave rise to a similar plateau of conjugates formed with HLA class II+ cells. Re-expression of CD4 after a 48-h incubation of PMAtreated cells led to a curve of conjugate formations comparable t o that of untreated CD4+ T cells (Fig. 2b). Similar findings were made using LFA-1- T cells isolated from a patient with an inherited effective expression of LAD [20,21]. This indicates that PMA treatment did not modify kinetics of conjugate formation through LFA-1 expression alteration (Fig. 2c) [31].
3.3 Inhibition of CD4+ T lymphocyte-HLA class II+ B lymphocytes conjugate formation by synthetic peptides derived from HLA class I1 molecules We have previously shown that synthetic dodecapeptides derived from the sequences of the HLA-DR domain (DR-12 amino acids 35-46, hereafter referred to as DR12a) containing the R F D S sequence, have immunosuppressive properties towards antigen-specific, HLA class IIrestricted Tcell proliferation and help for antibody production in in v i m assays [16]. We have investigated whether this peptide can also inhibit antigen-independent conjugate formation of CD4+ T lymphocytes. inhibitlon of con)ugato formation
(%I
Figure 1. Conjugate formation between CD4+ T lymphocytes and B lymphocytes. Role of membrane adhesion molecules. In panels a and b, CD4+ T lymphocytes were incubated with normal EBV-B cell lines (hached bars), HLA class 11- B cell lines (black bars) or LFA-3Iow,ICAM-1Iow HLA class II+ B cell lines (open bars). In (b) T and B lymphocytes were preincubated with each antibody (anti-LFA-1: 0.5 pg/ml; anti-CD2: 2 pg/ml; anti-CD4: 2.5 pglml; anti-CD3 and anti-CDS: 20 pg/ml, anti-ICAM-1 and anti-LFA-3 1/1@ dilution of ascites, anti-HLA class I 10 pglml; anti-CD24: 20 pg/ml) as indicated for 30 min at 4"C, then washed and incubated for conjugate formation.The OKT4A antibody gave similar inhibitory effects as Leu-3a (data not shown). In (b) the percentage of control T-B conjugates was 18 k 4%. Results are given as mean of 5 independent experiments k 1 SD.
The DR-12a peptide was shown to be able to strongly inhibit conjugate formation between CD4+ T lymphocytes and class 11+ B lymphocytes. The effect of the DR-12a peptide is dose dependent with a maximal activity at concentrations L 10 pg/ml (Fig. 3a). The inhibitory activities of DR-l2a, DR-12b and DQ-12 peptides were similarly observed on resting CD4+ T lymphocytes isolated by negative selection (CD8-), positive selection (CD4+; data not shown) and on cloned helper CD4+ T cells as well (Fig. 3a and b). In contrast, the same peptides did not exert any inhibitory effect on conjugate formation between CD8+ T cells isolated either by negative or positive selection or cloned CD8+ Tcells and B cells (Fig. 3c and
4.
Eur. J. Immunol. 1990. 20: 637-644
F. Mazerolles, F. Amblard, C. Lumbroso et al.
640
I
(b)
Figure 2. Influence of CD4 and HLA class I1 expression by T and B cells, respectively, on the kinetics of conjugate formation. (a) Kinetics of conjugate formation between CD4+ T cell and HLA class II+ (A) or HLA class 11- (A) B cells derived from a patient with an inherited deficiency in HLA class I1 molecule expression. B CI I(* PMA 4h+48h The percentage of conjugates formed significantly differ at 30 min and there40 60 after (p
Fabienne Mazerolleso+, Frangois AmblardA, Catherine LumbrosoO, Olivia Lecomteo, Pierre-FrangoisVan de MoorteleO, Christiane BarbatO, Dominique Piatier-Tonneaua, Charles AuffrayA and Alain FischerO Immunologie et Rhumatologie Pkdiatriques, INSERM U 132, H6pital des Enfants-Maladeso, Paris and Institut d'Embryologie du CNRS et du College de Francea, Nogent s/Marne
CDCHLA class I1 interaction
637
Regulation of T helper-B lymphocyte adhesion through CD4-HLA class I1 interaction* Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus (EBV)-transformed B cells is mediated by CD2Aymphocyte function-associated antigen (LFA)-3 and LFA-lhntracellular adhesion molecule (1CAM)-1. Although some anti-CD4 antibodies block the antigen-independent adhesion of CD4+ T lymphocytes, the CD4-HLA class I1 interaction does not appear to significantly contribute to the forces of cell adhesion since CD4+ Tcells equally bind HLA class 11+and HLA class II- mutant B cells. In addition, conjugates formed between CD4+ Tcells and HLA class II- B cells remain stable for at least 1h while CD4+T/HLA class 11+B cell conjugate percentages promptly drop off. Down-regulation of CD4 or spontaneous low expression of CD4 also results in a persistance of conjugates formed with B cells.The role of the CD4-HLA class I1 interaction has been further studied by investigating the inhibitory effect of synthetic 12-mer peptides analogous to HLA class I1 and containing the Arg-Phe-Asp-Ser sequence conserved in the p1 domain. These peptides were previously found to inhibit HLA class II-restricted Tcell responses, this sequence being thought to be involved in CD4-HLA class 11 interaction. These peptides block conjugate formation of CD4+ resting Tcells or clones but not of CD8+ T cells, by interacting with the T cells as shown by preincubation experiments. Down-regulation of CD4 or spontaneous low expression results in the loss of the inhibitory activity. The peptide-mediated inhibition is neutralized by a soluble dimeric CD4 molecule. Alteration within the Arg-Phe-Asp-Ser sequence results in a significant loss of inhibition. It is thus proposed that the CD4-HLA class I1 interaction negatively regulates antigen-independent adhesion of T cells, this interaction involving the highly conserved Arg-Phe-Asp-Ser sequence in the HLA class I1 p1 sequence as a CD4-binding site.
1 Introduction Adhesion of T lymphocytes to target cells is mediated by at least two antigen-independent pathways, i. e. CD2 which binds lymphocyte function-associated antigen (LFA)-3 and LFA- 1 which binds the intracellular adhesion molecule (1CAM)-1 [l-31. A third adhesion pathway involving CD4 and HLA class I1 molecules might be used by CD4 T h lymphocytes as suggested by cell adhesion between CD4 and'HLA class I1 expressing cells [4] and inhibition by anti-CD4 antibodies of mouse CD4+ Th lymphocyte adhe-
sion to B lymphocytes [5]. In addition, it has been suggested that the CD4 molecule may play a role in antigen-specific, MHC class II-restricted T cell activation by associating with the CD3-Ti complex [6-81. This effect may be mediated by the p56lCktyrosine kinase that was found to be associated to the cytoplasmic tail of CD4 [9, 101. Conversely, it has been proposed that cross-linking of CD4, in the absence of TcR cross-linking generates a negative signal that may induce cell separation [ l l , 121.
Binding of CD4 t o MHC class I1 molecules has been demonstrated by Doyle and Strominger using CV-1 monkey fibroblasts strongly expressing CD4 [4], and confirmed by using transfected COS cells by Clayton et al. [13]. In the latter study, different parts of the CD4 molecule were suggested to be involved in CD4-HLA class I1 interaction [I 80981 as shown by site-directed mutagenesis [13]. We have proposed that CD4 and HLA class I1 molecules * This work was supported by Institut National de la SantC et de la previously may interact, at least in part, through preserved amino acid Recherche MCdicale and Centre National de la Recherche sequences (RADS and RFDS, respectively) analogous to Scientifique, and in part by grants from Programme National de Recherches sur le SIDA, Association pour la Recherche sur le the fibronectin-cell attachment site R G D S [14, 151, and Cancer, Direction des Etudes et Techniques, the Korberpreis have shown that synthetic dodecapeptides containing the Foundation and Rhone Poulenc Santt. RFDS or R ADS sequences inhibit antigen-specific HLA Supported by the Ligue Nationale de Recherche sur le Can- class II-restricted T cell proliferation and help for antibody cer. production [16]. We have herein investigated the role of CD4 in the antigen-independent adhesion of CD4+ T cells Correspondence: Alain Fischer, INSERh4 U 132, HBpital des to HLA class 11+B cells as both an adhesion pathway and a Enfants-Malades, 149, rue de Skvres, F-75743 Paris Cedex 15, regulatory element of adhesion. In addition, we have asked France whether synthetic peptides derived from HLA class I1 Abbreviations: ICAM-1: Intracellular adhesion molecule 1 molecules and containing the R FD S sequence could LFA: Lymphocyte function-associated antigen LAD: Leukocyte influence binding of CD4+ T cells to HLA class 11+ B cells. adhesion deficiency +
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0303-0637$02.50/0
638
Eur. J. Immunol. 1990.20: 637-644
F. Mazerolles, F. Amblard, C. Lumbroso et al.
2 Materials and methods
2.3 Synthetic peptides
2.1 Cells
DR-l2a, DR-l2b, DQ-12 peptides are analogous to residues 35 to 46 from the PI domain of HLA-DR1,2 and HLA-DQl sequences, respectively [14].They were synthesized by Neosystem (Strasbourg, France) according to the solid-phase synthesis method of Merrifield, and were further purified by HPLC, resulting in a purity 2 9 6 % of the peptide as shown by HPLC analysis. Peptide sequences were validated by amino acid analysis of the purified peptides. DR-12a variants were similarly synthesized as well as a 12-mer peptide analogous to residues 43-54 of the P1 domain of HLA-DR, and the 7-mer and 6-mer DR-7 and DR-6 peptides [14-16] .The Lys amino acid residue in DR-7 is not present in HLA-DR but was added for the sake of coupling to egg albumin. The tetrapeptide RFDS was prepared by A. Tartar, Lille, France. The peptides analogous to the fibrinogen molecule RGDF, LGRGD, G GAKQAGDV and GASPSTGTEEWTC were produced by G. Marguerie, INSERM U127, Grenoble, France.The G R D GDS peptide was purchased from Sigma (St. Louis, MO).
PBMC were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation from normal adults. CD4+ T lymphocytes were isolated from PBMC by a two-step procedure, i.e. neuraminidase-treated RBC rosetting followed by negative selection by panning using a CD8specificmAb (BC8, J.W.Widjdenes, BesanGon, France) and an anti-mouse Ig antibody (GaMIgG; Biomakor, Rehovot, Israel). CD4+ T cell preparations contained less than 2% CD8+ Tcells. CD4+ Tcells were also purified from PBMC from a patient with leukocyte adhesion deficiency (LAD), in whom expression of LFA-1 on leukocytes was absent [17]. CD8+ T lymphocytes were similarly isolated using a CD4-specific mAb (IOT4, Immunotech, Marseille, France). EBV-transformed B cells were used including four different sources of B cells: (a) EBV-transformed B cell lines; (b) B cells derived from a Burkitt lymphoma (BL2) which have been shown to poorly express the LFA-1, LFA-3 and ICAM-1 membrane molecules [18, 191; (c) EBV-transformed B cell lines from immunodeficient patients with an inherited deficiency in HLA class I1 molecule expression [20,21] as well as (d) mutant HLA class 11- B cells RJ225 produced by Accolla et al. [22]. Helper CD4+ T lymphocyte clone 4H (alloreactive, DR1 specific), M3 (influenza virus specific) and CD8+ T lymphocyte clone 4AS (alloreactive) were cultured in RPMI 1640 medium supplemented with 10% AB+ human serum and 20 U/ml of IL2 (Boehringer-Mannheim, Mannheim, FRG). 4H and 4AS clones were obtained from a rejected human kidney allograft [23]. The Jurkat CD4+ T cell line and a spontaneously obtained variant of the Jurkat Tcell line with low expression of CD4 were also used. In some experiments CD4+ T lymphocytes were incubated for 30 min to 4 h with either medium of PMA (Sigma; 20 ng/ml) in order to down-regulate CD4 molecule expression. CD4+ T lymphocytes treated with PMA for 4 h were further incubated after washings for 24 to 48 h in culture medium for the study of CD4 re-expression. 2.2 mAb The following mAb were used in adhesion assays: 25-3 (IgG1 anti-CDlla LFA-1, D. Olive) [24], IOT11 (IgG1 anti-CD2, Immunotech), L eu 3 a (IgG1 anti-CD4, Becton Dickinson, Mountain View, CA), 111-366 and F142-63C23 (IgG2 anti-CD4, €! Poncelet, Marseille, France), OKT4a (IgGh, Ortho, Raritan, NJ), WT31 (IgGl anti-CD3Ei complex, a kind gift from Dr. W. Thx (Nijmegen, The Netherlands), Leu-1 (IgG2, anti-CD5, Becton Dickinson), 84H10 (IgG1 anti-ICAM-1, D. Olive) [25], TS2/9 (IgG1 anti-LFA3,T. Springer) [2], IOT8 (IgG1 anti-CD8, Immunotech), IOT2 (IgGZa anti-HLA class I, Immunotech), ALB9 (IgG1 anti-CD24, Immunotech), 206 (IgG2, antiHLA-DR) [26],Tu22 (IgG2, anti-HLA-DQ) [27] and B7/21 (IgG1 anti-HLA-DP) [28].
2.4 Soluble CD4 CD4 IgG2, consisted of a construct of the two external domains of CD4 with the murine CH2 and cH3 domains of IgG2,. It was kindly supplied by A. Traunecker (Basel, Switzerland) [29].
2.5 Immunofluorescence Cell fluorescence intensity was evaluated by FCM analysis using a FACStar plus (Becton Dickinson) as revealed by specific antibodies and by a GaMIg (Nordic, Tilburg, The Netherlands).
2.6 Conjugate formation Antigen-independent T-B conjugate formation was performed according to the method described by Onishi et al. [30]. T and B cells were incubated at 2 x 106/ml with hydroxyethydine (40 pg/ml; Polysciences,Wamngton, PA) for 10 min at 25°C and fluorescein diacetate (100 pg/ml; Molecular Probes Inc., Eugene, OR) for 10 min at 37°C. After washings, 5 x 105 T cells were mixed with 5 X lo5 B cells in a 500 pl volume of RPMI 1640 medium (Gibco Biocult , Paisley, Scotland). Preliminary experiments have determined that optimal conjugate formation took place after a 20-min incubation at 37 "C. Incubation was followed by centrifugation at 250 x g for 5 min and gentle resuspension before counting. Conjugates were identified as redgreen pairs of cells under fluorescence microscopy. Two hundred to 350 fluorescent cells were counted in each experiment. Cells were counted blind by two independent observers. T-B conjugates were expressed as the percent of T-B conjugates vs. the total number of Tcells. Results are expressed as the percentage of T-B conjugates among all T cells or as the inhibition of T-B conjugate formation [l-(TB/T :TB control/T control)] x 100.
Eur. J. Immunol. 1990. 20: 637-644
CD4-HLA class I1 interaction
3 Results 3.1 Role of membrane adhesion molecules in conjugate formation between CD4+ T lymphocytes and B lymphocytes We have investigated which membrane molecules expressed by CD4+ T lymphocytes are involved in antigenindependent cell conjugate formation with EBV-transformed B lymphocytes expressing HLA class I1antigens. A significant percentage of CD4+ T lymphocytes was shown to bind EBV-B cells. In contrast, poor binding of CD4+ T lymphocytes to HLA class 11+B lymphocytes expressing very low levels of LFA-3 and ICAM-1 was observed while the percentages of CD4+ T lymphocytes able to bind HLA class 11+and HLA class 11- B cells were similar (Fig. la). HLA class 11- B cells were found to express ICAM-1 and LFA-3 similarly to control EBV-B cells (data not shown). BL2 B cells and control EBV-B cells similarly express HLA class I1 molecules (data not shown). LFA-1- Tcells equally bound HLA class 11+and class 11- B cells [24] (6.1 f 1.7 vs. 6.5 k 1.5%, mean of three experiments) although the percentage of conjugates was lower compared to LFA-l+ T cells (Fig. 1).Preincubation of CD4+ T cells with CD2 or LFA-1-specific antibodies and of B cells with LFA-3- and ICAh4-1-specificantibodies resulted in partial inhibition of conjugate formation. In contrast, mAb specific for CD5, CD3, CD24, CD21 and HLA class I had no effect (Fig. 1). The L e u 3 a anti-CD4 antibody could inhibit the formation of most conjugates while LFA-1 and CD2-specific antibod-
639
ies (or ICAM-1- and LFA-3-specific antibodies) exerted an inhibitory activity that was less potent but additive to each other. The same pattern of inhibition was observed on conjugates formed between CD4+ T lymphocytes and HLA class 11- B lymphocytes derived from immunodeficient patients (Fig. lb) or obtained by mutation (data not shown).
3.2 Influence of CD4 and HLA class II expression on the kinetics of conjugate formation between CD4+ T and B lymphocytes A peak of conjugates was repeatedly found after a 20-min incubation. As shown in Fig. 2a, the percentage of conjugates formed between CD4+ and HLA class 11+ cells dropped off rapidly after a 20-min incubation whereas the percentage of conjugates formed between CD4+ and HLA class 11- cells remained at a plateau for at least 40 min after reaching maximal level (Fig. 2a). The absence or diminished CD4 expression onTcells using the CD41°WJurkat line (Fig. 2d), or following down-regulation of CD4 by PMA (Fig. 2b) gave rise to a similar plateau of conjugates formed with HLA class II+ cells. Re-expression of CD4 after a 48-h incubation of PMAtreated cells led to a curve of conjugate formations comparable t o that of untreated CD4+ T cells (Fig. 2b). Similar findings were made using LFA-1- T cells isolated from a patient with an inherited effective expression of LAD [20,21]. This indicates that PMA treatment did not modify kinetics of conjugate formation through LFA-1 expression alteration (Fig. 2c) [31].
3.3 Inhibition of CD4+ T lymphocyte-HLA class II+ B lymphocytes conjugate formation by synthetic peptides derived from HLA class I1 molecules We have previously shown that synthetic dodecapeptides derived from the sequences of the HLA-DR domain (DR-12 amino acids 35-46, hereafter referred to as DR12a) containing the R F D S sequence, have immunosuppressive properties towards antigen-specific, HLA class IIrestricted Tcell proliferation and help for antibody production in in v i m assays [16]. We have investigated whether this peptide can also inhibit antigen-independent conjugate formation of CD4+ T lymphocytes. inhibitlon of con)ugato formation
(%I
Figure 1. Conjugate formation between CD4+ T lymphocytes and B lymphocytes. Role of membrane adhesion molecules. In panels a and b, CD4+ T lymphocytes were incubated with normal EBV-B cell lines (hached bars), HLA class 11- B cell lines (black bars) or LFA-3Iow,ICAM-1Iow HLA class II+ B cell lines (open bars). In (b) T and B lymphocytes were preincubated with each antibody (anti-LFA-1: 0.5 pg/ml; anti-CD2: 2 pg/ml; anti-CD4: 2.5 pglml; anti-CD3 and anti-CDS: 20 pg/ml, anti-ICAM-1 and anti-LFA-3 1/1@ dilution of ascites, anti-HLA class I 10 pglml; anti-CD24: 20 pg/ml) as indicated for 30 min at 4"C, then washed and incubated for conjugate formation.The OKT4A antibody gave similar inhibitory effects as Leu-3a (data not shown). In (b) the percentage of control T-B conjugates was 18 k 4%. Results are given as mean of 5 independent experiments k 1 SD.
The DR-12a peptide was shown to be able to strongly inhibit conjugate formation between CD4+ T lymphocytes and class 11+ B lymphocytes. The effect of the DR-12a peptide is dose dependent with a maximal activity at concentrations L 10 pg/ml (Fig. 3a). The inhibitory activities of DR-l2a, DR-12b and DQ-12 peptides were similarly observed on resting CD4+ T lymphocytes isolated by negative selection (CD8-), positive selection (CD4+; data not shown) and on cloned helper CD4+ T cells as well (Fig. 3a and b). In contrast, the same peptides did not exert any inhibitory effect on conjugate formation between CD8+ T cells isolated either by negative or positive selection or cloned CD8+ Tcells and B cells (Fig. 3c and
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Eur. J. Immunol. 1990. 20: 637-644
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(b)
Figure 2. Influence of CD4 and HLA class I1 expression by T and B cells, respectively, on the kinetics of conjugate formation. (a) Kinetics of conjugate formation between CD4+ T cell and HLA class II+ (A) or HLA class 11- (A) B cells derived from a patient with an inherited deficiency in HLA class I1 molecule expression. B CI I(* PMA 4h+48h The percentage of conjugates formed significantly differ at 30 min and there40 60 after (p
