CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
54,
134-147 (1990)
Functional Human T Cell-B Cell Hybridomas Established Fusion of Normal T Cells and an EBV-Transformed B Cell Line’ HAJIME KIMAT.,*
from
JAMES BERENSON,~~ JONATHAN KAGAN,* AND ANDREW SAXON*$
*Division of Clinical Immunology and Aliergy, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024; fHematologylOncology Division, Wadsworth VA Medicai Center, Los Angeles, California 90073: and the SJonsson Cancer Center, UCLA School of Medicine, Los Angeles, California, 90024 Human T cell-B cell hybridomas containing 92 chromosomes were derived by fusing normal stimulated CD-depleted T cells with the EBV-transformed human B cell line 729-HF. These hybridomas coexpressed the T cell surface markers CD3. CD8, and CD2 and the B cell surface antigens CD19, CD20, CD23, DR, and DQ. They weakly and variably expressed surface IgM and TCR. Genomic analysis revealed the presence of rearranged Ig genes as well as B-T cell receptor genes that could be ascribed to the B and T cell parent, respectively. Analysis of TCR rearrangement suggests that the T-B hybridomas are, in fact, subclones of a single dominant clone. Although the hybrids expressed CDS and not CD4, following preincubation with PWM, some the of clones induced IgG synthesis from normal B cells while the parent B cell line failed to demonstrate this activity. Stimulation of the hybridomas with PMA down-regulated the T cell lineage-specific antigen display (CD3, CD8, and TCRl and increased IgM production from the hybrids without changing B cell surface antigen display. These hybridomas may be useful to dissect the steps involved in the ultimate commitment of a cell to the B or T cell lineages and will be made available to interested investigators. t: 1990 Academic Press. Inc.
INTRODUCTION
Established T and B cell lines have been very useful in the analysis of the phenotypic and functional changes induced upon lymphocyte activation. Agents such as phorbol 12-myristate 13-acetate (PMA)2 can induce differentiation of subclones of CEM, a leukemic T cell line, into CD3 + , CD8 + , and CD4 - cells with suppressor function (1). On the other hand, PMA down-regulates CD3 and CD4 and up-regulates CD25 and DR in alloreactive T cell clones (2), and downregulates CD20 in the Burkitt lymphoma B cell line, Raji (3). it has also been reported that after somatic hybridization, the T cell receptor is lost while B cell receptors (membrane Ig) remain on hybridomas derived from fusion of T and B cell lines (4). We have constructed T cell-B cell hybridomas between normal T cells and a B cell line (729-HF) and studied the phenotypic changes induced upon PMA stimulation. We have also studied functional aspects of the hybridoma ’ These studies were supported by USPHS Grants CA-12800, AI-15251, and AI-15332 ’ Abbreviations used: PMA, 12-myristate 12-acetate; CRPMI, complete RPM1 medium; PE, phycoerythin; APC, streptavidin allophycocyanin. 134 0090-1229/90 $1.50 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMAN
T-B
CELL
HYBRIDOMAS
135
clones and found that some hybridomas induce IgG synthesis by normal B cells whereas the parent 729-HF cells failed to do so. MATERIALS
AND METHODS
Cell separations and stimulation. Human mononuclear cells were obtained from heparinized blood of a normal donor by Ficoll-Hypaque separation. T cells were separated from the mononuclear cells by the 2-aminoethylisothiouronium bromide (Sigma, St. Louis, MO)-treated sheep red blood cell rosette technique (5, 6). Non-T cells were depleted of monocytes by plastic adherence and subsequently designated as B cells. T cells were resuspended in tissue culture flasks at a concentration of 1 X lO’?ml in RPM1 1640 (M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA), 2mM glutamine, 50 U/ml penicillin, and 50 kg/ml streptomycin (CRPMI). For CD4 cell separation, cells were first stimulated with phytohemagglutinin (PHA) (Burroughs-Wellcome, 1:200) and PMA (Sigma, 20 rig/ml) for 4 days, and then separated into CD4 + I - cells by immune adherence using Leu 3a (anti-CD4) antibody (Becton-Dickinson, Inc., Mountain View, CA) (7, 8). For the preparation of CD8 + cells, T cells were stimulated as above with Con A (10 &ml) and PMA (20 rig/ml), cultured for 4 days, and then separated by immune adherence using Leu 2a (anti-CDS) (Becton-Dickinson). Cell hybridization. After three washes with serum-free medium, purified CD4 + or CD8+ T cells were mixed with WI-L2/729-HF (729-HF, an HGPRT-negative subclone of the EBV-transformed B lymphoblastoid cell line WI-L2) (9) at a ratio of 2: 1 and fused in the presence of polyethylene glycol4000 (Sigma) as described (10). The fused cells were dispensed into microtiter plates (Linbro Chemical Co., New Haven, CT) at a concentration of 3 X lo5 cells/well. After overnight incubation, hypoxanthine, aminopterine, and thymidine (HAT) were added to each well. The HAT medium was changed twice weekly. Growing hybridomas were observed after 4 weeks and cloned by limiting dilution in the presence of 50 kg/ml of endothelial cell growth factor (Chemicon, Los Angeles, CA). Individual clones were then transferred to flasks and fed with CRPMI alone. Cloned hybridomas were shown to be negative for mycoplasma by DAPI and Hoecht DNA staining (11, 12) and had a mean number of 92 chromosomes. The morphology of the cells on Wright’s stain revealed the hybridomas to be large lymphoblastoid cells with very prominent nucleoli and vacuolization of the cytoplasm. Flow cytometric analysis of cell surface molecule display. The method used to quantitate subsets of lymphocytes by two-color flow cytometric analysis has been described previously (13). In the present experiments, phycoerythrin (PE) and fluorsecein isothiocyanate (FITC)-labeled antibodies to cell surface molecules were obtained from Becton-Dickinson. The CD23 antigen was detected using Mab 135 which was kindly provided by Dr. G. Delespesse, Notre Dame Hospital, University of Montreal, Montreal, Canada (14). Mab 135 was biotinylated by Dr. James Whitehead (Vector Laboratories, Burlingame, CA) and fluoresence visualized using either streptavidin PE (Becton-Dickinson) or streptavidin allophycocyanin (APC) (CALTAG, S. San Francisco, CA). Murine isotype control reagents used to evaluate nonspecific binding were IgG, FITC, IgG, PE (Becton-
136
KIMATA
ET AL.
Dickinson), and IgM FITC (Coulter Immunology, Hialeah, FL). Two-color flow cytometry was performed on a Coulter EPICS C (Coulter) equipped with an argon laser tuned to 488 nm and operating at 500 mW. Three-color analysis was performed on a FACS 440 (Becton-Dickinson) equipped with two lasers: and argon laser (488 nm, 500 mW) to excite FITC and PE and a rhodamine 6G dye laser (615 nm, 200 mW) pumped by the all lines output of a second argo laser to excite APC. Hybridoma stimulation. For studies of the hybridoma clones ability to provide helper cell function for lg production, cells (5 x 10S/ml) were stimulated with PMA (100 rig/ml) for 2 hr, washed thoroughly, and cultured overnight. To test the ability of the T-B hybridomas to induce IgG synthesis from normal B cells, hybrids (5 x 105/ml) were preincubated with 10 PI/ml of pokeweed mitogen (PWM, GIBCO) for 1 hr, washed thoroughly, irradiated (3000 rad), and then cultured with freshly obtained and enriched human blood B cells. The B cells ( I x 105) were cultured with the irradiated hybridoma cells (1 x 105) in microtiter plates for 10 days and lg in the culture supernatants was measured by ELISA (8). As controls, B cells were cultured with either 10 kl/rnl of PWM of T cell replacing factor (TRF, 10% vol/vol) which was obtained by stimulating irradiated (3000 rad) normal T cells with 10 pJ/ml of PWM for 1 day (1.5). This TRF contained no detectable lg. For studies of the hybridoma cells ability to produce IgM, cells (10,000/200 pJ/microtiter well) were cultured in CRPMI with 10% FCS for 7 days in the presence of medium alone, 50 ).&ml of cycloheximide. or 10 @ml of PMA. IgM produced was measured by ELISA (8). Southern blot analysis. Genomic DNA was prepared from the cell lines and normal granulocytes according to the method of Perry ef al. (16). The DNA was digested with restriction endonucleases (Bethesda Research Laboratories, MD). Ten micrograms of digested DNA was electrophoresed in 0.6% agarose gels at I to 2 V/cm for 42 hr and transferred to nylon filters by the method of Southern (17). Parallel lanes of similarly digested normal granulocyte (germ line) and cell line DNA were electrophoresed in parallel on the same gel. Probes for Ig and T cell receptor genes. The recombinant probe for the human immunoglobulin (lg) joining region (Jn) gene was kindly provided by Dr. Leroy Hood (California Institute of Technology, Pasadena, CA) and was subcloned in PBR322. The J, probe is a 3.2-kb pair, human genomic fragment extending from the EcoRl site in J,2 to the Hind111 site in the Jr.&~ intervening sequence (18). The B-TCR gene probe is a 0.4-kb Bglll-Bglll B-TCR cDNA fragment subcloned in Bluescribe (Vector Cloning Systems, San Diego, CA) and was derived from the JUR-P 2 clone which was donated by Dr. Tak Mak (University of Toronto, Canada) (19). Probe DNAs were labeled with [32P]dCTP (deoxycytidine triphosphate) by oligolabeling (20). Hybridization, washing, and autoradiography were then carried out as previously described (21). RESULTS Derivation of T-B cell human hybridomus. T cells were initially activated and then separated by positive and negative selection into CD4- or CDS-enriched subsets. These cells were separately fused with the EBV-transformed B cell parent 729-HF. By Day 34, all but 13 microtiter wells containing fused CD8 cells
HUMAN
137
T-B CELL HYBRIDOMAS
obtained by negative selection were dying. These wells were recombined into three wells, supplemented with 50 pg/ml of endothelial cell growth factor, and 21 days later, there was clear growth in one well. The cells from this well were separated into CD8 + and CD8 - fractions by panning with Leu 2a antibody. The resulting cells were cloned by limiting dilution with clones derived from the CDS+ fraction named KI clones (No. 1-9) and clones derived from the CD8called HK clones (No. 1-12). Results from the Southern blot analysis of the T cell receptor l3 gene suggest that at least the three clones tested (KI-9, HK-4, and HK-8) are all derived from the same progenitor cell. Surface phenotypic and functional characterization of T-B hybridomas. The hybrids expressed the B cell antigens CD19, CD20, and CD23; T cell antigens CD2, CD3, and CD8, variably depending upon the clone examined (Table 1); and MHC Class II molecules, DR and DQ. Three-color flow cytometric analysis of KI-9 revealed that the vast majority of these cells coexpressed the B cell antigen CD19 as well as the T cell markers, CD3 (Fig. IA) and CD8 (Fig. lB), when gated on all cells present. There were clearly both bright (65%) and dim (35%) CD8 + cells. Identical histograms were obtained for coexpression of CD19 and CD3 when gating on either bright or dim CDS+ cells (data not shown). Coexpression of both B (CD19, CD20, and DR) and T (CD2, CD3, and CD8) cell surface molecules has been consistent now for nearly 1 year in continuous culture. TABLE PHENOTYPICANALYSIS
OF
1 T-B HYBRIDOMAS % positive cells
Cells ParenV 729-HF CD8+ T cells Hybridoma clone KI-2 KI-3 KI-4 Kl-9 HK-2 HK-3 HK-4 HK-5 HK-6 HK-7 HK-8 HK-9 HK-IO HK-11 HK-12
CD19
CD23
CD2
CD3
CD4
CD8
92 0
98 0
0 99
0 99
0 0
0 99
94 82 71 89 65 81 85 64 74 68 89 89 92 71 72
95 88 86 85 83 90 88 86 93 95 94 99 94 93 94
98 98 89 95 92 93 99 94 95 94 93 92 94 95 96
98 86 83 94 74 57 84 77 82 85 97 91 93 90 91
1 1 0 1 0 1 1 1 1 1 0 1 1 0 0
5 4 19 88 14 11 23 18 6 9 4 8 9 16 24
” Cells consisted of the parental B cell line, 729HF, a control population of CD4 depleted and thereby CD8 enriched fresh T cells, or cells from the individual hybridoma clones derived as described under Materials and Methods. The results shown were obtained by single-color flow cytometric analysis.
138
KIMATA
Log
ET AL.
Green
Fluorescence CD3
64.
48.
I
I
16 Log
32
Red
48
64
Fluorescence CD8
FIG. 1. Three-color flow cytometric analysis of KI-9 cells. KI-9 cells were stained with anti-CD19 (Leu 12-PE), anti-CD8 (Leu 2a-biotin plus Strepavidin APC), or anti-Cd3 (Leu 4-FITC). (A) The coexpression of CD19 and CD3 with 85% of the cells staining positive for both markers. (B) The dual display of CD19, a B cell marker, and CD8. Eighty-nine percent of the cells coexpressed CD19 and CD8. The presence of both dim and bright CDS+ populations can clearly be seen. There was no difference in either the CD19 or CD3 display on the CD8 dim versus bright cells {data not shown).
HUMAN
T-B
CELL
139
HYBRIDOMAS
During the first 6 months of culture, up to 38% of cells (KI-9) stained positive for the T cell antigen receptor (TCR) b chain, although on the average, TCR was present on a lower percentage of cells from the other clones. TCR staining always appeared as a shift in the mean intensity of fluorescence rather than a discrete population of positive cells. Now, 1.5 years later, clones (including KI-9) continue to show variable TCR surface staining, as shown in Fig. 2, where 35% of HK-8 cells stained positive for TCR (Fig. 2B) in comparison to the isotype control (Fig. 2A). Notably, the intensity of TCR staining of the hybridoma cells was always diminished in comparison to fresh peripheral blood T cells (results not shown). We were able to reproducibly detect low levels (
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
54,
134-147 (1990)
Functional Human T Cell-B Cell Hybridomas Established Fusion of Normal T Cells and an EBV-Transformed B Cell Line’ HAJIME KIMAT.,*
from
JAMES BERENSON,~~ JONATHAN KAGAN,* AND ANDREW SAXON*$
*Division of Clinical Immunology and Aliergy, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024; fHematologylOncology Division, Wadsworth VA Medicai Center, Los Angeles, California 90073: and the SJonsson Cancer Center, UCLA School of Medicine, Los Angeles, California, 90024 Human T cell-B cell hybridomas containing 92 chromosomes were derived by fusing normal stimulated CD-depleted T cells with the EBV-transformed human B cell line 729-HF. These hybridomas coexpressed the T cell surface markers CD3. CD8, and CD2 and the B cell surface antigens CD19, CD20, CD23, DR, and DQ. They weakly and variably expressed surface IgM and TCR. Genomic analysis revealed the presence of rearranged Ig genes as well as B-T cell receptor genes that could be ascribed to the B and T cell parent, respectively. Analysis of TCR rearrangement suggests that the T-B hybridomas are, in fact, subclones of a single dominant clone. Although the hybrids expressed CDS and not CD4, following preincubation with PWM, some the of clones induced IgG synthesis from normal B cells while the parent B cell line failed to demonstrate this activity. Stimulation of the hybridomas with PMA down-regulated the T cell lineage-specific antigen display (CD3, CD8, and TCRl and increased IgM production from the hybrids without changing B cell surface antigen display. These hybridomas may be useful to dissect the steps involved in the ultimate commitment of a cell to the B or T cell lineages and will be made available to interested investigators. t: 1990 Academic Press. Inc.
INTRODUCTION
Established T and B cell lines have been very useful in the analysis of the phenotypic and functional changes induced upon lymphocyte activation. Agents such as phorbol 12-myristate 13-acetate (PMA)2 can induce differentiation of subclones of CEM, a leukemic T cell line, into CD3 + , CD8 + , and CD4 - cells with suppressor function (1). On the other hand, PMA down-regulates CD3 and CD4 and up-regulates CD25 and DR in alloreactive T cell clones (2), and downregulates CD20 in the Burkitt lymphoma B cell line, Raji (3). it has also been reported that after somatic hybridization, the T cell receptor is lost while B cell receptors (membrane Ig) remain on hybridomas derived from fusion of T and B cell lines (4). We have constructed T cell-B cell hybridomas between normal T cells and a B cell line (729-HF) and studied the phenotypic changes induced upon PMA stimulation. We have also studied functional aspects of the hybridoma ’ These studies were supported by USPHS Grants CA-12800, AI-15251, and AI-15332 ’ Abbreviations used: PMA, 12-myristate 12-acetate; CRPMI, complete RPM1 medium; PE, phycoerythin; APC, streptavidin allophycocyanin. 134 0090-1229/90 $1.50 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMAN
T-B
CELL
HYBRIDOMAS
135
clones and found that some hybridomas induce IgG synthesis by normal B cells whereas the parent 729-HF cells failed to do so. MATERIALS
AND METHODS
Cell separations and stimulation. Human mononuclear cells were obtained from heparinized blood of a normal donor by Ficoll-Hypaque separation. T cells were separated from the mononuclear cells by the 2-aminoethylisothiouronium bromide (Sigma, St. Louis, MO)-treated sheep red blood cell rosette technique (5, 6). Non-T cells were depleted of monocytes by plastic adherence and subsequently designated as B cells. T cells were resuspended in tissue culture flasks at a concentration of 1 X lO’?ml in RPM1 1640 (M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA), 2mM glutamine, 50 U/ml penicillin, and 50 kg/ml streptomycin (CRPMI). For CD4 cell separation, cells were first stimulated with phytohemagglutinin (PHA) (Burroughs-Wellcome, 1:200) and PMA (Sigma, 20 rig/ml) for 4 days, and then separated into CD4 + I - cells by immune adherence using Leu 3a (anti-CD4) antibody (Becton-Dickinson, Inc., Mountain View, CA) (7, 8). For the preparation of CD8 + cells, T cells were stimulated as above with Con A (10 &ml) and PMA (20 rig/ml), cultured for 4 days, and then separated by immune adherence using Leu 2a (anti-CDS) (Becton-Dickinson). Cell hybridization. After three washes with serum-free medium, purified CD4 + or CD8+ T cells were mixed with WI-L2/729-HF (729-HF, an HGPRT-negative subclone of the EBV-transformed B lymphoblastoid cell line WI-L2) (9) at a ratio of 2: 1 and fused in the presence of polyethylene glycol4000 (Sigma) as described (10). The fused cells were dispensed into microtiter plates (Linbro Chemical Co., New Haven, CT) at a concentration of 3 X lo5 cells/well. After overnight incubation, hypoxanthine, aminopterine, and thymidine (HAT) were added to each well. The HAT medium was changed twice weekly. Growing hybridomas were observed after 4 weeks and cloned by limiting dilution in the presence of 50 kg/ml of endothelial cell growth factor (Chemicon, Los Angeles, CA). Individual clones were then transferred to flasks and fed with CRPMI alone. Cloned hybridomas were shown to be negative for mycoplasma by DAPI and Hoecht DNA staining (11, 12) and had a mean number of 92 chromosomes. The morphology of the cells on Wright’s stain revealed the hybridomas to be large lymphoblastoid cells with very prominent nucleoli and vacuolization of the cytoplasm. Flow cytometric analysis of cell surface molecule display. The method used to quantitate subsets of lymphocytes by two-color flow cytometric analysis has been described previously (13). In the present experiments, phycoerythrin (PE) and fluorsecein isothiocyanate (FITC)-labeled antibodies to cell surface molecules were obtained from Becton-Dickinson. The CD23 antigen was detected using Mab 135 which was kindly provided by Dr. G. Delespesse, Notre Dame Hospital, University of Montreal, Montreal, Canada (14). Mab 135 was biotinylated by Dr. James Whitehead (Vector Laboratories, Burlingame, CA) and fluoresence visualized using either streptavidin PE (Becton-Dickinson) or streptavidin allophycocyanin (APC) (CALTAG, S. San Francisco, CA). Murine isotype control reagents used to evaluate nonspecific binding were IgG, FITC, IgG, PE (Becton-
136
KIMATA
ET AL.
Dickinson), and IgM FITC (Coulter Immunology, Hialeah, FL). Two-color flow cytometry was performed on a Coulter EPICS C (Coulter) equipped with an argon laser tuned to 488 nm and operating at 500 mW. Three-color analysis was performed on a FACS 440 (Becton-Dickinson) equipped with two lasers: and argon laser (488 nm, 500 mW) to excite FITC and PE and a rhodamine 6G dye laser (615 nm, 200 mW) pumped by the all lines output of a second argo laser to excite APC. Hybridoma stimulation. For studies of the hybridoma clones ability to provide helper cell function for lg production, cells (5 x 10S/ml) were stimulated with PMA (100 rig/ml) for 2 hr, washed thoroughly, and cultured overnight. To test the ability of the T-B hybridomas to induce IgG synthesis from normal B cells, hybrids (5 x 105/ml) were preincubated with 10 PI/ml of pokeweed mitogen (PWM, GIBCO) for 1 hr, washed thoroughly, irradiated (3000 rad), and then cultured with freshly obtained and enriched human blood B cells. The B cells ( I x 105) were cultured with the irradiated hybridoma cells (1 x 105) in microtiter plates for 10 days and lg in the culture supernatants was measured by ELISA (8). As controls, B cells were cultured with either 10 kl/rnl of PWM of T cell replacing factor (TRF, 10% vol/vol) which was obtained by stimulating irradiated (3000 rad) normal T cells with 10 pJ/ml of PWM for 1 day (1.5). This TRF contained no detectable lg. For studies of the hybridoma cells ability to produce IgM, cells (10,000/200 pJ/microtiter well) were cultured in CRPMI with 10% FCS for 7 days in the presence of medium alone, 50 ).&ml of cycloheximide. or 10 @ml of PMA. IgM produced was measured by ELISA (8). Southern blot analysis. Genomic DNA was prepared from the cell lines and normal granulocytes according to the method of Perry ef al. (16). The DNA was digested with restriction endonucleases (Bethesda Research Laboratories, MD). Ten micrograms of digested DNA was electrophoresed in 0.6% agarose gels at I to 2 V/cm for 42 hr and transferred to nylon filters by the method of Southern (17). Parallel lanes of similarly digested normal granulocyte (germ line) and cell line DNA were electrophoresed in parallel on the same gel. Probes for Ig and T cell receptor genes. The recombinant probe for the human immunoglobulin (lg) joining region (Jn) gene was kindly provided by Dr. Leroy Hood (California Institute of Technology, Pasadena, CA) and was subcloned in PBR322. The J, probe is a 3.2-kb pair, human genomic fragment extending from the EcoRl site in J,2 to the Hind111 site in the Jr.&~ intervening sequence (18). The B-TCR gene probe is a 0.4-kb Bglll-Bglll B-TCR cDNA fragment subcloned in Bluescribe (Vector Cloning Systems, San Diego, CA) and was derived from the JUR-P 2 clone which was donated by Dr. Tak Mak (University of Toronto, Canada) (19). Probe DNAs were labeled with [32P]dCTP (deoxycytidine triphosphate) by oligolabeling (20). Hybridization, washing, and autoradiography were then carried out as previously described (21). RESULTS Derivation of T-B cell human hybridomus. T cells were initially activated and then separated by positive and negative selection into CD4- or CDS-enriched subsets. These cells were separately fused with the EBV-transformed B cell parent 729-HF. By Day 34, all but 13 microtiter wells containing fused CD8 cells
HUMAN
137
T-B CELL HYBRIDOMAS
obtained by negative selection were dying. These wells were recombined into three wells, supplemented with 50 pg/ml of endothelial cell growth factor, and 21 days later, there was clear growth in one well. The cells from this well were separated into CD8 + and CD8 - fractions by panning with Leu 2a antibody. The resulting cells were cloned by limiting dilution with clones derived from the CDS+ fraction named KI clones (No. 1-9) and clones derived from the CD8called HK clones (No. 1-12). Results from the Southern blot analysis of the T cell receptor l3 gene suggest that at least the three clones tested (KI-9, HK-4, and HK-8) are all derived from the same progenitor cell. Surface phenotypic and functional characterization of T-B hybridomas. The hybrids expressed the B cell antigens CD19, CD20, and CD23; T cell antigens CD2, CD3, and CD8, variably depending upon the clone examined (Table 1); and MHC Class II molecules, DR and DQ. Three-color flow cytometric analysis of KI-9 revealed that the vast majority of these cells coexpressed the B cell antigen CD19 as well as the T cell markers, CD3 (Fig. IA) and CD8 (Fig. lB), when gated on all cells present. There were clearly both bright (65%) and dim (35%) CD8 + cells. Identical histograms were obtained for coexpression of CD19 and CD3 when gating on either bright or dim CDS+ cells (data not shown). Coexpression of both B (CD19, CD20, and DR) and T (CD2, CD3, and CD8) cell surface molecules has been consistent now for nearly 1 year in continuous culture. TABLE PHENOTYPICANALYSIS
OF
1 T-B HYBRIDOMAS % positive cells
Cells ParenV 729-HF CD8+ T cells Hybridoma clone KI-2 KI-3 KI-4 Kl-9 HK-2 HK-3 HK-4 HK-5 HK-6 HK-7 HK-8 HK-9 HK-IO HK-11 HK-12
CD19
CD23
CD2
CD3
CD4
CD8
92 0
98 0
0 99
0 99
0 0
0 99
94 82 71 89 65 81 85 64 74 68 89 89 92 71 72
95 88 86 85 83 90 88 86 93 95 94 99 94 93 94
98 98 89 95 92 93 99 94 95 94 93 92 94 95 96
98 86 83 94 74 57 84 77 82 85 97 91 93 90 91
1 1 0 1 0 1 1 1 1 1 0 1 1 0 0
5 4 19 88 14 11 23 18 6 9 4 8 9 16 24
” Cells consisted of the parental B cell line, 729HF, a control population of CD4 depleted and thereby CD8 enriched fresh T cells, or cells from the individual hybridoma clones derived as described under Materials and Methods. The results shown were obtained by single-color flow cytometric analysis.
138
KIMATA
Log
ET AL.
Green
Fluorescence CD3
64.
48.
I
I
16 Log
32
Red
48
64
Fluorescence CD8
FIG. 1. Three-color flow cytometric analysis of KI-9 cells. KI-9 cells were stained with anti-CD19 (Leu 12-PE), anti-CD8 (Leu 2a-biotin plus Strepavidin APC), or anti-Cd3 (Leu 4-FITC). (A) The coexpression of CD19 and CD3 with 85% of the cells staining positive for both markers. (B) The dual display of CD19, a B cell marker, and CD8. Eighty-nine percent of the cells coexpressed CD19 and CD8. The presence of both dim and bright CDS+ populations can clearly be seen. There was no difference in either the CD19 or CD3 display on the CD8 dim versus bright cells {data not shown).
HUMAN
T-B
CELL
139
HYBRIDOMAS
During the first 6 months of culture, up to 38% of cells (KI-9) stained positive for the T cell antigen receptor (TCR) b chain, although on the average, TCR was present on a lower percentage of cells from the other clones. TCR staining always appeared as a shift in the mean intensity of fluorescence rather than a discrete population of positive cells. Now, 1.5 years later, clones (including KI-9) continue to show variable TCR surface staining, as shown in Fig. 2, where 35% of HK-8 cells stained positive for TCR (Fig. 2B) in comparison to the isotype control (Fig. 2A). Notably, the intensity of TCR staining of the hybridoma cells was always diminished in comparison to fresh peripheral blood T cells (results not shown). We were able to reproducibly detect low levels (
