CELLULAR
IMMUNOLOGY
126,196-2 10 ( 1990)
T Cell Activation II. Activation of Human T Lymphoma Cells by Cross-Linking of Their MHC Class I Antigens’
STEEN DISSING,* CARSTEN GEISLER,? BENT RUBIN,t TORBEN PLESNER,$ AND M~GENS H. CLAESSON@’ *Institute qfGeneral Physiology, the §Department of Medical Anatomy A, tthe Institute of Experimental Immunology. University of Copenhagen, and the *Department of Internal Medicine and Hematology C, KAS Gentofte, Copenhagen, Denmark ReceivedAugust 25, 1989; accepted October 12, 1989 The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cd+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Caf’ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when C$’ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CDS-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules. 0 1990 Academic press, Inc.
INTRODUCTION MHC3 class I molecules have recently been shown to associate with several different cell surface molecules to form intermolecular complexes. Thus, MHC class I molecules appear to interact with various cell surface receptors for peptide hormones and growth factors ( l-6). In T lymphocytes, class I molecules have been shown to associate with adhesion molecules such as the CD4 and CDS receptors (7-9) and the IL-2 receptor ( 10). On the surface of leukemic T cells, class I antigens form an intermolecular complex with a CD3 associated, T-cell receptor-like structure ( 11). On the basis of these and other reports ( 12, 13) it thus appears that the class I molecules in addition ’ This work was supported by grants from The Danish Medical Research council, NOVO’s Foundation, and Ingeborg and Leo Dannin Foundation. 2 To whom correspondence should be addressed at Lab. Exp. Hematol. Immunol., The Department of Medical Anatomy A, the Panum Institute, Blegdamsvej 3,220O N, Copenhagen, Denmark. 3 Abbreviations used: MHC, major histocompatibility complex; TS-Ti, TCR-CD3, or TCR, the T cell receptor for MHC/antigen; Ca,2+, intracellular ionized calcium; RAM, rabbit anti-mouse Ig; SwAR, swine anti-rabbit Ig; P2m, fl,-microglobulin; RAP,m or Raf12m, rabbit ant$,m antibody; IL-2, interleukin 2; IL2R, interleukin 2 receptor; BBM.1, monoclonal antibody against P2m; W6/32, monoclonal antibody against monomorphic HLA class I antigens; FITC, fluorescein-isothiocyanate; PE, phycoerytrine. 196 0008-8749/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved
T-CELL
ACTIVATION
BY MHC CLASS I CROSS-LINKING
197
to their capability to associate with and present endogeneously synthesized peptide fragments to cytotoxic T lymphocytes ( 14, 15) also exert a nonimmunological function related to the expression and perhaps function of important cell surface receptors. We ( 16) and others ( 17) have recently shown that immobilized anti-class I antibody in concert with immobilized anti-CD3 antibody exerts a strong costimulatory effect on T helper and cytotoxic lymphocytes as judged from IL-2 receptor (IL-2R) expression, proliferative response to IL-2, IL-2 production, and in the case of cytotoxic T cells, the release of serine esterase. The results of these studies suggest that the T cell receptor complex for MHC/antigen (TCR, T3-Ti) is functionally linked to the class I molecules and perhaps, that the class I molecules by themselves can act as signal transducing elements. The aim of the present work was to further explore this possibility. We have studied the increase in cytoplasmic free calcium (Ca”), IL-2 production, and IG2R expression of normal (CD3’) and mutant (CD3-) Jurkat T lymphoma cells after exposure of cells to anti-MHC class I antibodies followed by crosslinking by a second-step antibody. Our data show that immunological cross-linking of class I antigens induces a specific prolonged increase in Ca”. In the presence of the ester, phorbol 12-myristate 13-acetate (PMA), cross-linking of class I antigens induces IL-2R expression and IL-2 secretion provided that the cells express a functional TCR complex. MATERIALS
AND METHODS
Jurkat cells and derivation of CD3- mutants. The IL-2 producing Jurkat cell line was obtained from ATCC (Rockville, MA, USA) and cultured in RPMI- 1640 culture medium including 10% fetal calf serum, fresh glutamine and antibiotics. The cells were tested free of mycoplasma contamination using the Mycotect system (Mycotect, GIBCO, USA). CD3- Jurkat variant cell clones (540 and 579) were obtained by ethan methylsulfonate mutagenesis followed by immunoselection with anti-CD3 antibody (see below) and passage through Ig anti-Ig columns. Passed cells were cloned at limiting dilution and growing clones were analyzed for TCR-CD3 expression using antiCD3 antibodies (see below) and FACS analysis ( 18). Northern blot analysis and immunoprecipitation followed by two-dimensional SDS-PAGE showed that both 540 and 579 Jurkat cell clones are CD3-Zeta-negative variants. In addition, we found no changes in surface markers CD2, CD4, CD5, CD7, CD8, CD9, CD1 la, CD54, or CD58 relative to the parental TCR-CD3+ Jurkat cells (( 19); Geisler et al., submitted for publication). Measurement of IL-2. IL-2 activity was assayed by the method of Gillis et al. (20) using the IL-2-dependent T cell line CTLL-2. In brief, the CTLL-2 cells were washed and adjusted to 5 X 104/ml culture medium. The cells were then added to flat-bottomed 96-well microculture plates in volumes of 100 ~1. Five to 25% of supematants from activated Jurkat cell cultures (see below) was added. After 20 hr culture, 0.5 &i tritiated thymidine (NEN, Boston, USA) was added per culture for 4 hr. The cultures were then harvested on filter paper and counted in a scintillation counter. A titration of an IL-2 standard was always included and the activity was expressed as units/ml. One unit is the activity which induces 50% of maximal tritiated thymidine incorporation of 5000 CTLL-2 cells. Measurements of the concentration of Ca”. Fura-2/AM was added to the cell suspension at a final concentration of 1@4. The Fura- loading period lasted for approx-
198
DISSING ET AL.
imately 20 min whereupon small portions (3 ml) of the cell suspension were removed and treated as described below. Generally, all experiments were performed within 60 min after the Fura- loading period. The cells were separated from the medium by centrifugation, the supernatant was removed and the cells resuspended in KrebsRinger bicarbonate buffer and washed. The measurements of Ca” in a suspension of cells in a cuvette was performed as described previously (2 I). Measurements were performed in a Perkin-Elmer LS-SB fluorescence spectrophotometer with a thermostatically controlled (37°C) l-cm path length cuvette. The cell suspension was continuously stirred by use of a teflon-coated magnet driven by a motor attached to the cuvette house. The measurements of Ca” were performed by excitation at two different wavelengths (340 and 380 nm) and measuring emission at 5 10 nm. A 450nm cutoff filter was placed in the emission path in order to decrease light scatter. The CaF+ concentrations were calculated from the measurements of the ratio of fluorescence intensities (“R values”) obtained at 340 and 380 nm, respectively, according to the equation (22): Ca” = Kd X (R - Rmin/Rmax - R) X (Sf,/Sb,) where Kd is the dissociation constant and R,,, and Rmin are ratios of fluorescence intensities of Fura- at 340 and 380 nm excitation at saturating calcium concentrations and in the absence of Ca*+, respectively. The Kd values were obtained by fitting measured R values to intracellular, free Ca*+ concentrations. The relation between R and Ca” was obtained by suspending the cells in a high K+ medium (135 mM KCl, 10 mMNaC1, 1 mMEGTA and 5 mMHepes at pH 7.25) and varying concentrations of Ca2+ (for details see 2 1). One PLM of ionomycin was added to the suspension which equilibrates Ca*’ across the membrane without rupture of the cell membrane. The values obtained from the cells were 20.0 for R,,, and 1.06 for Rmin . The proportionality coefficient (Sf,/Sb,) amounted to 7.0. A value for the apparent Kd of calciumbinding of Fura- of 170 + 20 nM (average -+ SE) was obtained. From the experiments with cell suspensions in the cuvette the calculations of Ca?’ were done by calculating the actual R values from lines drawn by eye through each set of fluorescence data points obtained by exciting at 340 and 380 nm and substracting the autofluorescence signal at a particular wavelength. This was determined by separating the cells from the medium and measuring the fluorescence signal from the supernatant. Finally the R values were related to the standard curve and the actual Ca” concentrations (in nM) were calculated. Images of Ca” in single cells were obtained by means of a digital image processing system (Quantex Corp., San Jose, CA, USA) and a fluorescence microscope (Zeiss IM35). Cells were placed on a thermostatically controlled microscope stand in a specially constructed chamber with the appropriate buffers. The cells were excited by a Xenon lamp at 340 and 380 nm by means of filter selector which changed the excitation filters with a frequency of 1 Hz. The objective used was a Zeiss Ultrafluor (100X, 1.25 NA) and the image was recorded with a SIT camera (Dage, MTI, USA) and transferred to image processing and an analysis system. The computers form the ratios of images at 340 and 380 nm excitation, which are then recorded on a video tape recorder. The gray value in each pixel was transformed into free Ca*’ concentration by sending the video recorded ratioed image into the computer and through a lookup table with a constructed standard curve as described above. An average of 10 frames of the ratioed image was obtained in order to construct an image. A three-
T-CELL
ACTIVATION
BY
MHC
CLASS
I CROSS-LINKING
199
dimensional plot was constructed from the image and the color codes were imposed on the plot to further illustrate the free calcium concentrations (see Fig. 4). Antibodies and chemicals. The monoclonal antibody against F1O1.O1 has been characterized recently and recognizes a conformational epitope on the CD3-TCR complex (23). Unconjugated and phycoerytrin (PE) conjugated anti-Leu-4 (antiCD3), fluorescein-thiocyanate (FITC)-conjugated monoclonal anti&m antibody, and fluorescein-conjugated anti-CD25 (IL-2R) antibody were purchased from Becton-Dickinson, Sonny Vale, CA. Mouse monoclonal antibodies against monomorphic HLA class I antigen, W6/32, and ,&2-microglobulin (&m), BBM. 1, were produced by the hybridomas, HB95 and HB28, respectively, purchased from ATCC. Antibodies were purified from hybridoma culture supernatants on Protein A-Sepharose (Pharmacia, Uppsala, Sweden). Polyclonal rabbit anti-mouse Ig (RAM), rabbit anti-human &rn (R&m), and swine anti-rabbit Ig (SwAR) antibodies were purchased from Dakopatts, Copenhagen, Denmark. All antibodies used for activation studies were dialyzed against phosphate-buffered saline and used at saturating levels (at least 10 pug/ml). Hepes, EGTA, digitonin, ionomycin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemicals, St. Louis, MO. Fura- was purchased from Molecular Probes Inc., Eugene, OR. Activation of Jurkat cells. Jurkat cells, 2 X lo5 per well, were cultured in volumes of 0.2 ml in the presence PMA (20 rig/ml) and ionomycin (500 rig/ml) or the various cross-linking antibodies for 24 hr whereupon the culture supernatants were assayed for IL-2 activity. The cells were harvested and stained with anti-CD25 antibody and examined in the fluorescence microscope for expression of IL-2R. In experiments with cross-linked CD3 or MHC class I antigens the cells were reacted with the appropriate first step antibodies for 1 hr at 4°C then washed, and cultured as above in the presence of the second step cross-linking antibody. In experiments with determination of Ca” the antibodies were added directly to unlabeled Jurkat cells or to cells prelabeled with first-step antibody as described above. FACS analysis. Cells were harvested during periods of optimal growth, washed in RPM1 1640 culture medium with 10% FCS, and labeled with saturating amounts of PE-conjugated anti-Leu4 and FITC conjugated anti&m antibody. Fluorescence was estimated by flow cytometry in a FACScan calibrated with CaliBrite fluorescent beads and the AutoComp computer program (Becton-Dickinson). Gatings were set in forward versus side scatter to exclude cell debris and agglutinated cells from the measurements. Irrelevant FITC and PE labeled monoclonal antibodies (Simultest, Becton-Dickinson) were used as negative controls. RESULTS Rise in Cal+ following anti-CD3 binding and MHC class I crosslinking. Fig. IA shows that Jurkat cells exposed to anti-CD3 antibody exhibited a sharp rise in Caf+ after a short lag period of approximately 25 set followed by a backregulation toward the prestimulatory level within the following minutes. When Jurkat cells were prereacted with anti-CD3 antibody and allowed to back-regulate Caf+ to near prestimulatory levels, the following exposure to a second step polyclonal rabbit anti-mouse Ig antibody (RAM) resulted in an immediate rise in Ca” also followed by back-regulation of Ca?+. It was generally observed that direct anti-CD3 binding resulted in a lag period of’ approximately 25 set whereas cross-linking generated an immediate response. Similar results were obtained with the two anti-CD3 antibodies, F. 10 1.O1 and Leu-4.
500
100
r
A
I
a- CD3 1
t I 0
01
I 1
I 2
I 3
I 4
I 5
Time (min) ICa.*‘lI
nM
300 -
200 ---I W6 132*RAM4./ -.-.a.
100 -
0'
Ra-O
’
m 1
I
I
I
I
I
I
I
I
I
0
1
2
3
4
5
6
7
8
Time (min) FIG. 1.Ca,‘+ in Jurkat cells. (A) Solid curve represents stimulation with mouse anti-human CD3 antibody (FIOI .Ol). Dashed curve represents stimulation with rabbit anti-mouse antibody (RAM) in cells prelabeled with FIO 1.O1. RAM and FIO 1.O1 were added at time 0 (arrows). (B) Solid curve represents stimulation with rabbit anti-human &rn antibody (Ra&m). Dashed and stippled-dashed curves represent stimulation with swine anti-rabbit (SwAR) and RAM, respectively, in cells prelabeled with R&m and mouse antihuman MHC class I heavy chain antibody (W6/32), respectively. Stimulatory antibodies were added at time 0 (arrows) and in addition, anti-CD3 antibody was added after 5 min (open arrow heads). (C) Solid and dashed curves represent stimulation with anti-CD3 and SwAR (prelabeled with R&m), respectively, at 1 mA4extracellular Ca’+. Stippled and stippled-dashed curves represent stimulation with anti-CD3 and SwAR (prelabeled as above), respectively, at 100 nMextracellular Ca”. Stimulatory antibodies were added at time 0 (arrows).
T-CELL
ACTIVATION
201
BY MHC CLASS I CROSS-LINKING
I Cai21 nM 300
200
-c amcD Ra-f$m+SwAR/
,
IMMUNOLOGY
126,196-2 10 ( 1990)
T Cell Activation II. Activation of Human T Lymphoma Cells by Cross-Linking of Their MHC Class I Antigens’
STEEN DISSING,* CARSTEN GEISLER,? BENT RUBIN,t TORBEN PLESNER,$ AND M~GENS H. CLAESSON@’ *Institute qfGeneral Physiology, the §Department of Medical Anatomy A, tthe Institute of Experimental Immunology. University of Copenhagen, and the *Department of Internal Medicine and Hematology C, KAS Gentofte, Copenhagen, Denmark ReceivedAugust 25, 1989; accepted October 12, 1989 The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cd+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Caf’ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when C$’ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CDS-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules. 0 1990 Academic press, Inc.
INTRODUCTION MHC3 class I molecules have recently been shown to associate with several different cell surface molecules to form intermolecular complexes. Thus, MHC class I molecules appear to interact with various cell surface receptors for peptide hormones and growth factors ( l-6). In T lymphocytes, class I molecules have been shown to associate with adhesion molecules such as the CD4 and CDS receptors (7-9) and the IL-2 receptor ( 10). On the surface of leukemic T cells, class I antigens form an intermolecular complex with a CD3 associated, T-cell receptor-like structure ( 11). On the basis of these and other reports ( 12, 13) it thus appears that the class I molecules in addition ’ This work was supported by grants from The Danish Medical Research council, NOVO’s Foundation, and Ingeborg and Leo Dannin Foundation. 2 To whom correspondence should be addressed at Lab. Exp. Hematol. Immunol., The Department of Medical Anatomy A, the Panum Institute, Blegdamsvej 3,220O N, Copenhagen, Denmark. 3 Abbreviations used: MHC, major histocompatibility complex; TS-Ti, TCR-CD3, or TCR, the T cell receptor for MHC/antigen; Ca,2+, intracellular ionized calcium; RAM, rabbit anti-mouse Ig; SwAR, swine anti-rabbit Ig; P2m, fl,-microglobulin; RAP,m or Raf12m, rabbit ant$,m antibody; IL-2, interleukin 2; IL2R, interleukin 2 receptor; BBM.1, monoclonal antibody against P2m; W6/32, monoclonal antibody against monomorphic HLA class I antigens; FITC, fluorescein-isothiocyanate; PE, phycoerytrine. 196 0008-8749/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved
T-CELL
ACTIVATION
BY MHC CLASS I CROSS-LINKING
197
to their capability to associate with and present endogeneously synthesized peptide fragments to cytotoxic T lymphocytes ( 14, 15) also exert a nonimmunological function related to the expression and perhaps function of important cell surface receptors. We ( 16) and others ( 17) have recently shown that immobilized anti-class I antibody in concert with immobilized anti-CD3 antibody exerts a strong costimulatory effect on T helper and cytotoxic lymphocytes as judged from IL-2 receptor (IL-2R) expression, proliferative response to IL-2, IL-2 production, and in the case of cytotoxic T cells, the release of serine esterase. The results of these studies suggest that the T cell receptor complex for MHC/antigen (TCR, T3-Ti) is functionally linked to the class I molecules and perhaps, that the class I molecules by themselves can act as signal transducing elements. The aim of the present work was to further explore this possibility. We have studied the increase in cytoplasmic free calcium (Ca”), IL-2 production, and IG2R expression of normal (CD3’) and mutant (CD3-) Jurkat T lymphoma cells after exposure of cells to anti-MHC class I antibodies followed by crosslinking by a second-step antibody. Our data show that immunological cross-linking of class I antigens induces a specific prolonged increase in Ca”. In the presence of the ester, phorbol 12-myristate 13-acetate (PMA), cross-linking of class I antigens induces IL-2R expression and IL-2 secretion provided that the cells express a functional TCR complex. MATERIALS
AND METHODS
Jurkat cells and derivation of CD3- mutants. The IL-2 producing Jurkat cell line was obtained from ATCC (Rockville, MA, USA) and cultured in RPMI- 1640 culture medium including 10% fetal calf serum, fresh glutamine and antibiotics. The cells were tested free of mycoplasma contamination using the Mycotect system (Mycotect, GIBCO, USA). CD3- Jurkat variant cell clones (540 and 579) were obtained by ethan methylsulfonate mutagenesis followed by immunoselection with anti-CD3 antibody (see below) and passage through Ig anti-Ig columns. Passed cells were cloned at limiting dilution and growing clones were analyzed for TCR-CD3 expression using antiCD3 antibodies (see below) and FACS analysis ( 18). Northern blot analysis and immunoprecipitation followed by two-dimensional SDS-PAGE showed that both 540 and 579 Jurkat cell clones are CD3-Zeta-negative variants. In addition, we found no changes in surface markers CD2, CD4, CD5, CD7, CD8, CD9, CD1 la, CD54, or CD58 relative to the parental TCR-CD3+ Jurkat cells (( 19); Geisler et al., submitted for publication). Measurement of IL-2. IL-2 activity was assayed by the method of Gillis et al. (20) using the IL-2-dependent T cell line CTLL-2. In brief, the CTLL-2 cells were washed and adjusted to 5 X 104/ml culture medium. The cells were then added to flat-bottomed 96-well microculture plates in volumes of 100 ~1. Five to 25% of supematants from activated Jurkat cell cultures (see below) was added. After 20 hr culture, 0.5 &i tritiated thymidine (NEN, Boston, USA) was added per culture for 4 hr. The cultures were then harvested on filter paper and counted in a scintillation counter. A titration of an IL-2 standard was always included and the activity was expressed as units/ml. One unit is the activity which induces 50% of maximal tritiated thymidine incorporation of 5000 CTLL-2 cells. Measurements of the concentration of Ca”. Fura-2/AM was added to the cell suspension at a final concentration of 1@4. The Fura- loading period lasted for approx-
198
DISSING ET AL.
imately 20 min whereupon small portions (3 ml) of the cell suspension were removed and treated as described below. Generally, all experiments were performed within 60 min after the Fura- loading period. The cells were separated from the medium by centrifugation, the supernatant was removed and the cells resuspended in KrebsRinger bicarbonate buffer and washed. The measurements of Ca” in a suspension of cells in a cuvette was performed as described previously (2 I). Measurements were performed in a Perkin-Elmer LS-SB fluorescence spectrophotometer with a thermostatically controlled (37°C) l-cm path length cuvette. The cell suspension was continuously stirred by use of a teflon-coated magnet driven by a motor attached to the cuvette house. The measurements of Ca” were performed by excitation at two different wavelengths (340 and 380 nm) and measuring emission at 5 10 nm. A 450nm cutoff filter was placed in the emission path in order to decrease light scatter. The CaF+ concentrations were calculated from the measurements of the ratio of fluorescence intensities (“R values”) obtained at 340 and 380 nm, respectively, according to the equation (22): Ca” = Kd X (R - Rmin/Rmax - R) X (Sf,/Sb,) where Kd is the dissociation constant and R,,, and Rmin are ratios of fluorescence intensities of Fura- at 340 and 380 nm excitation at saturating calcium concentrations and in the absence of Ca*+, respectively. The Kd values were obtained by fitting measured R values to intracellular, free Ca*+ concentrations. The relation between R and Ca” was obtained by suspending the cells in a high K+ medium (135 mM KCl, 10 mMNaC1, 1 mMEGTA and 5 mMHepes at pH 7.25) and varying concentrations of Ca2+ (for details see 2 1). One PLM of ionomycin was added to the suspension which equilibrates Ca*’ across the membrane without rupture of the cell membrane. The values obtained from the cells were 20.0 for R,,, and 1.06 for Rmin . The proportionality coefficient (Sf,/Sb,) amounted to 7.0. A value for the apparent Kd of calciumbinding of Fura- of 170 + 20 nM (average -+ SE) was obtained. From the experiments with cell suspensions in the cuvette the calculations of Ca?’ were done by calculating the actual R values from lines drawn by eye through each set of fluorescence data points obtained by exciting at 340 and 380 nm and substracting the autofluorescence signal at a particular wavelength. This was determined by separating the cells from the medium and measuring the fluorescence signal from the supernatant. Finally the R values were related to the standard curve and the actual Ca” concentrations (in nM) were calculated. Images of Ca” in single cells were obtained by means of a digital image processing system (Quantex Corp., San Jose, CA, USA) and a fluorescence microscope (Zeiss IM35). Cells were placed on a thermostatically controlled microscope stand in a specially constructed chamber with the appropriate buffers. The cells were excited by a Xenon lamp at 340 and 380 nm by means of filter selector which changed the excitation filters with a frequency of 1 Hz. The objective used was a Zeiss Ultrafluor (100X, 1.25 NA) and the image was recorded with a SIT camera (Dage, MTI, USA) and transferred to image processing and an analysis system. The computers form the ratios of images at 340 and 380 nm excitation, which are then recorded on a video tape recorder. The gray value in each pixel was transformed into free Ca*’ concentration by sending the video recorded ratioed image into the computer and through a lookup table with a constructed standard curve as described above. An average of 10 frames of the ratioed image was obtained in order to construct an image. A three-
T-CELL
ACTIVATION
BY
MHC
CLASS
I CROSS-LINKING
199
dimensional plot was constructed from the image and the color codes were imposed on the plot to further illustrate the free calcium concentrations (see Fig. 4). Antibodies and chemicals. The monoclonal antibody against F1O1.O1 has been characterized recently and recognizes a conformational epitope on the CD3-TCR complex (23). Unconjugated and phycoerytrin (PE) conjugated anti-Leu-4 (antiCD3), fluorescein-thiocyanate (FITC)-conjugated monoclonal anti&m antibody, and fluorescein-conjugated anti-CD25 (IL-2R) antibody were purchased from Becton-Dickinson, Sonny Vale, CA. Mouse monoclonal antibodies against monomorphic HLA class I antigen, W6/32, and ,&2-microglobulin (&m), BBM. 1, were produced by the hybridomas, HB95 and HB28, respectively, purchased from ATCC. Antibodies were purified from hybridoma culture supernatants on Protein A-Sepharose (Pharmacia, Uppsala, Sweden). Polyclonal rabbit anti-mouse Ig (RAM), rabbit anti-human &rn (R&m), and swine anti-rabbit Ig (SwAR) antibodies were purchased from Dakopatts, Copenhagen, Denmark. All antibodies used for activation studies were dialyzed against phosphate-buffered saline and used at saturating levels (at least 10 pug/ml). Hepes, EGTA, digitonin, ionomycin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemicals, St. Louis, MO. Fura- was purchased from Molecular Probes Inc., Eugene, OR. Activation of Jurkat cells. Jurkat cells, 2 X lo5 per well, were cultured in volumes of 0.2 ml in the presence PMA (20 rig/ml) and ionomycin (500 rig/ml) or the various cross-linking antibodies for 24 hr whereupon the culture supernatants were assayed for IL-2 activity. The cells were harvested and stained with anti-CD25 antibody and examined in the fluorescence microscope for expression of IL-2R. In experiments with cross-linked CD3 or MHC class I antigens the cells were reacted with the appropriate first step antibodies for 1 hr at 4°C then washed, and cultured as above in the presence of the second step cross-linking antibody. In experiments with determination of Ca” the antibodies were added directly to unlabeled Jurkat cells or to cells prelabeled with first-step antibody as described above. FACS analysis. Cells were harvested during periods of optimal growth, washed in RPM1 1640 culture medium with 10% FCS, and labeled with saturating amounts of PE-conjugated anti-Leu4 and FITC conjugated anti&m antibody. Fluorescence was estimated by flow cytometry in a FACScan calibrated with CaliBrite fluorescent beads and the AutoComp computer program (Becton-Dickinson). Gatings were set in forward versus side scatter to exclude cell debris and agglutinated cells from the measurements. Irrelevant FITC and PE labeled monoclonal antibodies (Simultest, Becton-Dickinson) were used as negative controls. RESULTS Rise in Cal+ following anti-CD3 binding and MHC class I crosslinking. Fig. IA shows that Jurkat cells exposed to anti-CD3 antibody exhibited a sharp rise in Caf+ after a short lag period of approximately 25 set followed by a backregulation toward the prestimulatory level within the following minutes. When Jurkat cells were prereacted with anti-CD3 antibody and allowed to back-regulate Caf+ to near prestimulatory levels, the following exposure to a second step polyclonal rabbit anti-mouse Ig antibody (RAM) resulted in an immediate rise in Ca” also followed by back-regulation of Ca?+. It was generally observed that direct anti-CD3 binding resulted in a lag period of’ approximately 25 set whereas cross-linking generated an immediate response. Similar results were obtained with the two anti-CD3 antibodies, F. 10 1.O1 and Leu-4.
500
100
r
A
I
a- CD3 1
t I 0
01
I 1
I 2
I 3
I 4
I 5
Time (min) ICa.*‘lI
nM
300 -
200 ---I W6 132*RAM4./ -.-.a.
100 -
0'
Ra-O
’
m 1
I
I
I
I
I
I
I
I
I
0
1
2
3
4
5
6
7
8
Time (min) FIG. 1.Ca,‘+ in Jurkat cells. (A) Solid curve represents stimulation with mouse anti-human CD3 antibody (FIOI .Ol). Dashed curve represents stimulation with rabbit anti-mouse antibody (RAM) in cells prelabeled with FIO 1.O1. RAM and FIO 1.O1 were added at time 0 (arrows). (B) Solid curve represents stimulation with rabbit anti-human &rn antibody (Ra&m). Dashed and stippled-dashed curves represent stimulation with swine anti-rabbit (SwAR) and RAM, respectively, in cells prelabeled with R&m and mouse antihuman MHC class I heavy chain antibody (W6/32), respectively. Stimulatory antibodies were added at time 0 (arrows) and in addition, anti-CD3 antibody was added after 5 min (open arrow heads). (C) Solid and dashed curves represent stimulation with anti-CD3 and SwAR (prelabeled with R&m), respectively, at 1 mA4extracellular Ca’+. Stippled and stippled-dashed curves represent stimulation with anti-CD3 and SwAR (prelabeled as above), respectively, at 100 nMextracellular Ca”. Stimulatory antibodies were added at time 0 (arrows).
T-CELL
ACTIVATION
201
BY MHC CLASS I CROSS-LINKING
I Cai21 nM 300
200
-c amcD Ra-f$m+SwAR/
,
