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T hybridoma αβ gene transfected in a murine T cell hybridoma: Role of CD4 molecule in vitro and in vivo—Engraftment in SCID mice induces T cell maturation
CELLULAR
IMMUNOLOGY
144, 105-l 16 (1992)
T Hybridoma cr/p Gene Transfected in a Murine T Cell Hybridoma: Role of CD4 Molecule in Vitro and in Viva-Engraftment in SCID Mice Induces T Cell Maturation CHRISTINEBRUNAUD,* EDGARDODELFINOCAROSELLA,~ ANDBERNARDCHARPENTIER~ *Laboratoire
d’lmmunologie cellulaire, Pasteur Mhieux Shwns et Vaccins, Marcy L’Etoile, France; and TLaboratoire d’lmmnnologie cellulaire et de transplantation IRK, Villeju(~ France Received February 19. 1992: accepted June 22, 1992
In the present work, we tested in SCID and Balb/c mice the activity of T cell hybridoma transfected with T cell receptor (TCR) cr/P chain genes. A T cell hybridoma denoted DO1I107 was used as recipient for transfection of cytotoxic KB5C20 TCR a/p heterodimer genes by protoplast fusion or electroporation. After transfection, the parental DOI I 107 T cell line reexpressed CD5 and CD4 surface molecules. In vitro, we noted strong proliferation and unusual cytotoxic reactivities against H-2’ target cells although the transfected cell line does not express the CD8 molecule. The fate of parental and transfected cells was examined in severe combined immunodeficient (SCID) and Balb/c mice at Day 16after intravenous injection. Cells from bone marrow, thymus, and spleentissueswere analyzed by immunofluorescence.The transfectedT cell hybridoma was CD3+ Desire I+ CD4+ Thyl.2+. The SCID mice grafted with the transfected T cell hybridoma presenteda high percentageof CD3+ (15%),CD4+ (27%),Thyl.2+ (27.52%) and Desire I+ (8.74%) cells in the spleen. The percentagesof CD3+ (6.2%) and Thyl.2+ (5.06%) cells in the spleen from SCID mice grafted with parental T cell DO11107 and from untreated SCID were similar and lower (CD3+, 3.52%; Thyl.2+, 4.34%). It seemsthat transfected T cells hybridoma grafted in the SCID mice induce significant expression of CD4+ Thyl.2’ Desire l- cells (17%) in the spleen.. These results indicate that transfected T cells graft may allow T cell differentiation. In Balb/c mice, the percentage of different T cell subsetsin bone marrow, thymus, or spleen cells in mice injected with transfected T cells was similar to that in untreated mice. We did not observe any cytotoxic or significant allogeneic proliferation in vitro. 0 1992 Academic press, 1~.
INTRODUCTION The mechanisms regulating expression of T cell receptors (TCR) have now been studied in vitro after transfection of the a/p heterodimer by a number of investigators (l-3). Transfection has been a useful technique as it allows cells to acquire new functions (4, 5). For instance, Saito et af. (6) have shown that transfection of the a//3 chain genesof murine Ia-restricted TCR into the human cell line JURKAT induced expression of a functional human/murine CD3/TCR. This complex presents the antigen Ia specificity of the parental murine clone. SCID (severe combined immunodeficient) mice (7) represent a good experimental model system for studying human hematolymphoid differentiation in vivo (8, 9), as well as a number of autoimmune diseases(10, 11). The autosomal recessivescid/scid mutation leadsto a severedeficiency of T and B lymphocyte functions due to a defective 105 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
106
BRUNAUD,
CAROSELLA.
AND
CHARPENTIER
somatic rearrangement of antigen receptor genes( 12, 13). These mice display only a low level of circulating Igs (< 10 pg/ml) and a reduced percentage of cells with surface markers CD2, CD3/TCR, CD4, CD8, and Thy 1.2.Thus, SCID mice may be considered a good model for functional studies of.transfected T and B cells. In the present work, we have studied the in vivo activity in SCID and Balb/c mice of a T cell hybridoma (DO11107) (14) in which the o(and /I genesof a cytotoxic clone KB5C20 (15) were transfected. Transfection leads to the reexpression of the CD4 molecule on the transfected hybridoma. Thus, this transfected hybridoma has an unusual cytotoxic activity against H-2k cells and induces allogeneic spleen cell proliferation. The singular phenotype of this transfected clone offers a direct way of investigating through surface antigen expression the roles and effects of these proteins in vivo. SCID mice, but not Balb/c mice, grafted with the transfected T cell hybridoma displayed mature CD4+ CD3+ Thyl.2+ T lymphocytes. Grafting of transfected tumor T cells may have induced T cell differentiation. Nevertheless, we did not observe a reconstitution of murine T and B cells. In Balb/c mice, no difference in maturation could be detected. MATERIALS AND METHODS Hybridoma. The murine DO11107 hybridoma ( 14, 16- 18), kindly provided by Dr. B. Malissen (Luminy, France), was used as the target cell in gene transfer. This cell is a helper hybridoma derived by fusion of T cell blasts specific for chicken ovalbumin (OVA) (from Balb/c mice) and BW5 147 thymoma cells (from AKR mice) (17) of H2d’kphenotype. These cells were cultured in DMEM (GIBCO) medium supplemented with 2 mM glutamine, 2 mM pyruvate, 8% FCS, 5 X 1Op5M 2$ME, and 10’ U/liter penicillin-streptomycin solution at 37°C in a 10% COJair atmosphere. Gene transfection. TCR a/p genes from the cytotoxic clone (KBSCZO) carried by the plasmid pSV2neo were obtained from Dr. B. Malissen ( 15). Two techniques were used: (a) protoplast fusion following the methods of Mulligan and Berg (19) and (b) electroporation using the Progenetor system (Hoefer Scientific Instruments). Five million DO11107 parental cells/ml in 1X Hepes buffer (20 mM Hepes, pH 7.3, 137 mM NaCl, 5 mA4 KCl, 0.7 mM Na2HP04, 6 mA4 dextrose) were introduced in 24 flatbottom microtiter culture wells, 40 pg circular plasmid DNA was added to the cells, and cells were submitted to 1 to 8 impulsions at 750 V/cm/900 psec at room temperature. Following electroporation, cells were incubated under the same culture conditions described above. Twenty-four hours after transfection, selective medium containing 2 mg/ml of geneticin (G4 18, Sigma Chemical Co.) was applied. Two weeks later, KBSC20 TCR a//3’ cells were cloned by limiting dilution (at I cell/well). Transfected stable clones were screened at Day 80. Monoclonal antibody reagents and cytofluorimetric analysis. The percentages of CD4+, CDS+, CD8+, and Thy 1.2’ T cells were determined by direct immunofluorescence.Briefly, cells were incubated at 4°C for 30 min with the following fluoresceinated antibodies: L3T4 ( 10 pg/ 1O6cells) (Caltag, Tebu), Ly 1 ( 10 fig/ 1O6cells) (Becton-Dickinson), Ly2 (10 pg/ lo6 cells) (Caltag, Tebu), and Thyl.2 (10 pg/ lo6 cells) (Caltag, Tebu). Cells were then washed with phosphate-buffered saline (0.15 M NaCl, 0.0 1 A4 phosphate buffer, pH 7.4) + 2% FCS and analyzed on a FACS system. The percentageof TCR a//3’ T cells wasdetermined by indirect immunofluorescence assay.Briefly, cells were incubated at 4°C for 30 min with unconjugated hamster anti-
T HYBRIDOMA
a//3 GENE
TRANSFECTION
107
mouse CD3 t 145 2C 11 (kindly provided by Dr. Bluestone) (20) ( 10 pg/ 1O6cells) and with unconjugated anticlonotypic antibody Desire 1 (15) (from B. Malissen) (10 pg/ lo6 cells) specific for KB5C20 TCR. Cells were washed with phosphate-buffered saline and then incubated with FITCconjugated goat anti-mouse (Ortho Diagnostic System) or FITC-conjugated rabbit anti-hamster (for anti-CD3 c antibody) antibody. Cells were washed three more times before being analyzed. Double labeling of cells was achieved by incubating either with both FITC-conjugated mAb and phycoerythrin-conjugated mAb for two-color flow cytometry or with unconjugated Desire 1 or anti-CD3 t antibodies followed by the appropriate fluorescent conjugate. Flow cytofluorimetric analysis of surface markers was performed on a FACScan Becton-Dickinson cell sorter with a single Argon laser. Dead cells were excluded from analysis using a combination of low-angle and sideway light scatter. Data were obtained as percentage of positive viable cells after subtracting the number of stained cells from the fluorescence-conjugated cells. Mixed-lymphoq@ reaction (MLR). Spleen cells from various haplotypes (H-2d, H2b, H-2k, H-2dk) were used as responder cells and stimulator cells in MLR. Parental and transfected T cell hybridoma were used as stimulator cells. Spleen cells were freed from erythrocytes following Geys treatment and were washed twice in RPM1 1640 with 5% FCS. The responder cells (2 X lo5 cells/50 ~1) and stimulator cells (2 X 10’ cells/50 ~1)irradiated at 2000 rad were cocultured in 96 flat-bottom microtiter culture wells in a humidified atmosphere containing 5% CO1 at 32°C for 5 days and were pulsed on the last day with [3H]thymidine (1 &i/well) followed by harvesting 24 hr later. Incorporated radioactivity was measured by beta liquid scintillation counting. In vitro CTL assays. A group of parental and transfected T cell hybridomas was stimulated for 5 days in the presence of irradiated splenocytes from different strains of mice, and another group was not stimulated. The hybridomas were collected and tested for lytic activity against splenocyte cells used as targets. These splenocytes were incubated for 3 days with PHA. Hybridomas used as effector cells were incubated for an additional 4 hr with 5’Cr-labeled target splenocytes under different effector to target cell ratios. The percentageof specific 5’Cr releasewas defined as = 100 X (experimental release - spontaneous release)/(total release - spontaneous release). Mice. Homozygous CB-17/scid/scid mice designated as SCID mice (6) and kindly provided by Pr. Wagner (University of Ulm) were bred and maintained in our laboratory. Mice were housed in microisolator cagescontaining sterilized food and water. Animals were not treated with antibiotics and were healthy throughout these experiments. All manipulations were performed aseptically under a laminar flow hood. The serum lg concentration was tested in all mice before they were used. Mice whose Ig levels were greater than 3 pg/ml were eliminated. Homozygous strains of inbred Balb/ c (H-2d), C57Bl/lO (H-2’), CBA (H-2d), and Fl (Balb/c X AKR) (H-2dk) mice were purchased from IFFA CREDO Laboratories (France). Cell tram&. Nonirradiated SCID and Balb/c mice (6-to gweek-old male or female) were injected intravenously with 20 X 1O6DO11107parental cells or 20 X 1O6DO11107 transfected cells expressing Desire 1 antigen. Sixteen days after cell transfer, thymus, spleen, and bone marrow from treated and untreated SCID and Balb/c mice were removed aseptically. They were disrupted in the medium by pressing tissue fragments between two glass slides. Cell suspensions were filtered through cotton gauze and washed twice in the medium. Cell suspensions
108
BRUNAUD,
CAROSELLA, AND CHARPENTIER
were freed from erythrocytes by using Geys treatment, and then were washed twice in RPM1 1640 with 5% FCS. Cell phenotypes were examined by immunofluorescence analysis following the methods previously described. Cell culture. Cells from thymus, spleen, and bone marrow were cultured in six flatbottom wells at a final concentration of lo6 cells/ml in DMEM medium under the conditions described above. Cells were harvested after 24 hr or 9 days of culture, and the expression of lymphocyte surface antigens was evaluated by immunofluorescence. Cells from nonreconstituted KID and Balb/c mice were used as negative histogram control. RESULTS Phenotype analysis of transfected T cell clones. The stability of the different clones obtained by transfection was analyzed regularly over a 12-weekperiod by flow cytometry. No significant variation in cell surface markers (CD3, CD4, CD5, CD8, Thy I .2, and Desire 1) was observed (Table 1). We selected four clones by protoplast fusion, A8B 1, G7A4, H 1IA 1, and H 11H 11; and two clones by electroporation, C6A8 and C6F8. The latter were obtained after 6 impulsions of 900 psec at 750 V/cm. In tests using 1 to 5 impulsions (900 fisec at 750 V/cm), no stable clones could be recovered, whereas 7 to 8 impulsions resulted in complete cell lysis. Transfection frequencies with electroporation were notably lower than those observed with protoplast fusion (8 X lo-’ vs 5 X 10e5).The doubling time for growth of parental and transfected hybridomas was identical irrespective of the means of transfection. TABLE 1 Cell Surface Antigens on T Cell Clones before and after Transfection (3 Months of Culture) T cell clone (% of positive cells) Protoplast fusion
Parental Expression of Ag
DOll107”
GAM +,d CD3+ CD4+ CD5+ CD8+ CD2+ LFAl+ Thyl.2+ Desire 1+xe CD3+CD4+ CD3+CD5+ CD3+CD8+ CD3+ Thyl.2+ CD4+ desire l+ Thyl.2+ desire l+ Note. NT, not tested.
0.9f 99 8.4 0.9 1.1 32 93.7 97.4 3.8 2.5 0.2 0.5 96.2 2.3 5
A8Blb
G7A4
G7Hll
2.6 93.1 94.3 95.2 3.4 NT NT 87.5 98.6 94 94.2 2.3 99.1 99.5 99.3
0.4 91.6 97.6 94.9 5.9 40 99.76 89 98.5 98.4 99.6 2.5 97.1 99.2 99.5
1.9 99.2 99.2 99.4 2.3 98 NT NT 99.3 NT NT NT NT NT NT
HllAll 3 99 98.9 99.5 1.5 97.7 NT NT 98.9 NT NT NT NT NT NT
Electroporation Hl lH1
C6A8’
C6F8
1.7 99.4 99.2 99.2 3.2 89 NT NT 98.4 NT NT NT NT NT NT
2.5 84.9 54 45.5 3.1 86 NT NT 58.9 57.1 93.3 5.4 97.9 46.8 67.4
0.5 77.6 58.5 68.7 0.3 99.7 NT NT 78.4 NT NT NT 99.5 86.9 99.5
T HYBRIDOMA
109
(Y/P GENE TRANSFECTION
The parental hybridoma DO1I 107 expressed a low level of CD4 (8.42%) and did not express the CD8 molecule surface marker. However, it bears the Thy 1.2 antigen and the mature pan-T CD3 antigen (98 and 97%, respectively; Table 1). The percentage of CD3+, CD4+, CD5+, Thy 1.2+,and Desire 1+ cells was higher among clones obtained by protoplast fusion than among those obtained by electroporation (Table 1). High levels of Desire 1 are observed in all clones of transfected cells. These clones are also CD3+, Thyl.2+, and CDS-. On the other hand, transfected tumor clones now express the CD4 and CD5 antigens. Phenotypic analysis by double labeling confirms that >90% of transfected T cells are CD4+CD3+ and CD4+Desire I’. Functional analysis qf parental and transfected T cell clones. The ability of the parental and transfected T cell hybridomas to activate or not activate murine spleen cells from different strains of mice (H-2d, H-2b, H-2k, and H-2dk) is shown in Table 2. There was no significant difference in the proliferation of various H-2 haplotypes expressing spleen cells when the parental T cell hybridoma DO11107 was used as stimulator. The decreasevaried from 64% for H-2b-bearing spleen cells to as much as 80% for H-2k-bearing spleen cells compared with corresponding MLR with H-2dk stimulator spleen cells (2367 to 6508 cpm and 2672 to 14,673 cpm, respectively). In contrast, regardlessof MHC haplotype, significant proliferation was observed in mixed cultures of spleen cells with the transfected stimulator T cells of hybridoma G7A4. In particular, we observed a surprisingly high reactivity, from 2656 to 10,698 cpm with H-2dk responder spleen cells. Generally, the number of cpm does not significantly differ among the different H-2 haplotypes used as responder cells. It seems that this unusual activity is acquired after gene transfection. As shown in Table 1, the low percentage of CD8+ cells indicates that neither hybridoma expressesthis accessorymolecule. We analyzed the ability of DO11107 and G7A4 T cell hybridomas to direct lysis murine target spleencells (Table 3). As expected, in the absence of any stimulation by effector cells, none of the hybridoma CTLs significantly lysed the murine target cells whatever the haplotype. Nevertheless, as shown in Tables 3b and 3c, the transfected G7A4 hybridoma, cultured for 5 days with H-2k or H-2b cells, has a strong cytotoxic activity toward the identical target cells in CTL assays(from 20% against H-2b target spleen cells to 75% against H-2k target
TABLE 2 Allogeneic Responsewith Parental and Transfected T Cell Hybridoma Stimulator:
Spleen cells
T hybridoma
Responder: Spleen cells
Balb/c (H-2d)
CBA/J (H-2*)
C57B1/6 (H-2b)
Fl(Balb/c X AKR) (H-2d*)
WI II07 (H-2d’)
Balb/c (H-2d) CBA/J (H-2k) C57Bl/6 (H-2b) FI(Balb/c X AKR) (H-2dk)
2,243 + 151* 20,442 + 2520 5,598 + 148
19,189 + 4263 2,015 + 240 6,978 + 423
22,711 + 321 26,835 + 426 2,680 + 915
11,550 + 300 14,673 + 214 6,508 + 652
2418 + 36 2672 + 40 2367 + 52
3,409 + 476
3,093 + 787
3,469 f 644
2,656 + 745
112 + 45
G-IA4 (H-2d’) 14,533 t 67 13,916 + 107 14,978 + 123 0,698 + 80
Note. Responder cells (2 X 10’) were cultured for 5 days with irradiated spleen cells (2 X 105)and T cell hybridoma cell stimulators. These results are expressedas [ 3H]thymidine incorporated cpm. cpm, mean of three mice.
110
BRUNAUD, CAROSELLA, AND CHARPENTIER TABLE 3 Cytotoxicity Analysisof Parentaland TransfectedT Cell Hybridoma
a. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2dstrain mice % of specificlysis T:E* 150 I:25 I:12
I:01
DO11107 NT
DO11107 (stimulatedby H-2dcells)
G7A4 (NT)
13.3
G7A4 (stimulatedby H-2dcells)
b. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2b strain mice % of specificlysis DO1I107 (NT)
DO11107 (stimulatedby H-2bcells)
G7A4 (NT)
G7A4 (stimulatedby H-2bcells)
1:50 I:25 I:12
10 10 10
1:Ol
CO
T:E
c. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2’ strain mice % of specificlysis T:E I:50 I:25 1:12 I:01
DO11107 (NT) ‘co -co
DO11107 (stimulatedby H-2k cells)
G7A4 (NV
G7A4 (stimulatedby H-2Ircells)
to LO
Note.T:E. target/effectercell ratio; NT, not tested.Meanof three mice
spleen cells), compared to an absence of cytotoxicity with parental DO1 1107 hybridoma cells. Altogether, these results suggest that this particular unusual reactivity was acquired after transfection. Grqfls qfpurental and transficted cells in SCID mice. For cell graft experiments in SCID mice, we used the T cell clone hybridoma G7A4 obtained by protoplast fusion (see Table 1). Four SCID mice were grafted in each of the treated groups. The phenotypes of splenocytes were analyzed 16 days after engraftment. The CD3+ and Thy 1.2’ cells constituted a high percentage of the T cell population of SCID mouse splenocytes having received the clone G7A4 (Table 4). Mice engrafted with the G7A4 hybridoma
T HYBRIDOMA
a/P GENE
111
TRANSFECTION
TABLE 4 Analysis of Surface Antigens on Splenic Cells from Grafted and Ungrafted SCID Mice (at Day 16) SCID mice (% of positive cells) Expression of Ag CD3’ CD4’ CD5+ Thyl.2+ Desire I+
Untreated 3.52 0.42 0.62 4.34 0.8
f k k f f
0.5’ 0.1 0.2 0.1 0.2
Grafted with DO I 1107” 6.26 0.52 I .36 5.06 0.7
+ k i f f
l.ld 0. I 0.4 3.1 0.3
Grafted with G7A4h 15 + 21 + 10.8 + 27.52 + 8.74 +
2.3’ 2.1’ l.lR 2.1 0.6
Nom. The results are shown as means + SE by the Student Fisher test. a Day 0, engraftment of SCID mice with 25 million parental T cells DO1 1107. ’ Day 0, engraftment of SCID mice with 20 million transfected T cells G7A4. c vs d P < 0.005, c vs e P < 0.001. “VsgPi 0.01. fvs f P < 0.001.
present a high percentage of Desire l+ and CD4+ cells, while no difference could be observed between mice engrafted with the parental hybridoma and nontreated mice. Splenocytes from the treated and nontreated mice were then placed in culture and analyzed 24 hr later (Table 5). The nontreated group expressedlow levels of CD3 and Thy I .2 and presented a high cell mortality. Spleen cells from the two treated groups proliferated and had high levels of CD3+ and Thy 1.2’. Lymphocytes bearing Desire 1 are found only in cultures of spleen from mice treated with the transfected clone G7A4. We tested CD4 and CD5 markers only after 130 hr of culture (data not shown). Thus, we observed that CD5 and CD4 antigen expression appeared on splenocytes cultured from SCID grafted with G7A4 T cells and not on splenocytescultured from SCID grafted with the parental T cell hybridoma. It is likely that the cells proliferating in vitro were those that were grafted initially.
TABLE 5 Analysis of Surface Antigens on Culture Spleen Cells from Grafted and Ungrafted SCID Mice (after 24 hr of Culture) SCID mice (% of positive cells) Expression of Ag
Untreated
Grafted with DOI 1107”
Grafted with G7A4b
CD3+ CD5+ Thyl.2+ Desire l+
2.9‘ 0.8 5.6 3
68 NT 70. I 0.6
51 NT 41.7 42.5
Nofe. NT, not tested. ’ 16 days after engraftment of parental T cell DO I I 107. ’ 16 days after engraftment of transfected T cell G7A4. ‘ Mean of three mice.
112
BRUNAUD,
CAROSELLA,
AND
CHARPENTIER
The low number of thymocytes recovered did not allow us to carry out a similar analysis on this cell population. Engraftment ofparental and transfected cells in Balb/c mice. Five Balb/c mice were grafted in each of the treated groups. The phenotypes of bone marrow, thymus, and spleen cells from inoculated Balb/c mice were analyzed 16 days postgrafting (Table 6). No difference in the percentage of cells displaying the various markers could be detected between treated and nontreated mice, including the cells displaying Desire It and CD4+ (also in Balb/c mice treated with G7A4 cells). The Desire 1 antibody stains 9-10% of bone marrow cells from Balb/c mice treated or not treated with the transfected or the parental T cell hybridoma, revealing a background level. In the spleen we noted a high percentage (25%) of cells expressing KB5C20 TCR in control or mice treated with DO11107 cells, due to nonspecific staining of immunoglobulins carried by B lymphocyte membrane. After 9 days in culture, mice grafted with the DO11107 clone have a large population of CD3+CD4- and CD4-CDS- T cells in bone marrow, thymus, and spleen (Table 7). However, the cells in culture from mice grafted with transfected G7A4 clone have a majority of T cells bearing the antigens CD3+CD4+, CDS+Thy 1.2+, and Desire 1 in all tissues tested. This population also appears in culture from thymocytes. DISCUSSION The mouse hybridoma DO1110 was originally obtained by the fusion of AKR thymoma BW5147 to chicken ovalbumin-specific T cell blasts from Balb/c mice. This hybridoma secretesIL-2 when challenged with ovalbumin in the context of IAd- or IAb-presenting cells. Clone DO11107 is a derivative of DO1110 characterized by the nonexpression of the parental LY/@ heterodimer. Consequently, this hybridoma expresses only the BW5 147 TCR and does not respond to ovalbumin in the context of restricted IAd or IAb. KB5C20 is a cytotoxic clone from the BlO.BR mouse which recognizes Class I H2Kb molecules. Transfection of both cvand /I genes from a cytotoxic clone does not necessarily lead to CD8 expression, nor does it suffice to retarget the recipient toward a MHC Class I specificity as described by Ansell and Bancroft (12). Transfection techniques which utilize chemical agents (such as DEAE dextran and calcium phosphate) do not give high transfection efficiencies with T cell hybridoma (21). In the present study, two other techniques were employed: electroporation (22, 23) and protoplast fusion (19). Electroporation was not very efficient (one Desire l+ clone for 8 X 1O7transfected cells) compared to protoplast fusion (one Desire 1+ clone for 5 X lo5 transfected cells). However, both techniques gave rise to clones displaying similar levels of surface antigens. Transfection of TCR (Yand p chain genes from a cytotoxic clone into T DO1I 107 hybridoma induces CD4 and CD5 antigen reexpression. Moreover, transfected T cell hybridoma has particular cytotoxic reactions and induces spleen cell proliferation, whatever the haplotype. It seemsthat KB5C20 TCR a//3 was not implicated in this phenomenon but the role of the CD4 molecule has been questioned. Incubation of the stimulator G7A4 hybridoma with 1 pg mAb Desire I/ 1O6cells for 1 hr did not reveal any effect on the murine spleen cell response (24). Transfected T hybridoma treatment with 1 pg of L3T4 mAb/106 cells resulted in the inhibition
4 9.3 5 24.9 0.04 69.9 0.4
0.9
3.6 95.1
Thyl.Z+ Desire 1+ CD3+CD4+ CD3+CD4CD3-CD4+ CD3-CD4CD4+CD8+
CD4+CD8-
CD4-CD8+ CD4-CD8-
I.1 94.7
2.2
8.8 II 9.1 16.8 0.08 74 1.9
4.5
Grafted with DOl11107”
0.7 96.1
2
2.8 12.6 4.5 9.4 0.02 86.2 1.2
5.8
Grafted with G7A4b
12.3 21.1
19.6
94.6 5.2 54.5 15.7 10.9 18.9 46.3
26.7
Untreated
Note. NT, not tested. a Day = 0, engraftment of Balb/c mice with 20 million DO11107 cells. ’ Day = 0, engraftment of Balb/c mice with 20 million transfected G7A4 cells. ’ Mean of five mice.
4’
Untreated
CD5+
Balb/c surface Ag
Bone marrow cells
10.2 I2
14.4
97.5 1.9 66.4 11.3 IO 12.3 63.5
25.1
Grafted with D0111107”
Thymus cells
7.2 12.9
9.2
90. I 1.2 40.8 5.7 34 19.6 70.7
19.1
Grafted with G7A4’
Tissue cells (% of positive cells)
NT
NT
45.1 25 25.6 53.9 1.5 19.1 NT
30
Untreated
23.1 58.8
15.4
25.5 24.3 33.6 51.5 0.04 14.9 2.8
16.4
Grafted with D0111107”
Spleen cells
Cell Surface Antigens on Bone Marrow Cells, Thymocytes, and Splenocytes from Grafted and Ungrafted Balb/c Mice (at Day 16)
TABLE 6
25.2 59
12.1
25.3 23.3 22 50.5 0.08 21.4 3.7
14.4
Grafted with G7A4b
w
z
3
3 -i F z 4
F R s 0
2 % 5
-l
114
BRUNAUD,
CAROSELLA, AND CHARPENTIER TABLE 7
Surface Antigens on Bone Marrow, Thyrnocytes, and Spleen on Cultured Cells from Balb/c Mice Grafted with DO11107 Parental Cells or with G7A4-Transfected Cells (Tested 9 Davs after Culture) Tissue cells (W of positive cells) Bone marrow cells
Thymus cells
Spleen cells
Balb/c surface Ag
Grafted with DO11107”
Grafted with G7A4 b
Grafted with D011107”
Grafted with G7A4b
Grafted with D011107”
Grafted with G7A4 b
CD5+ Thyl.2+ Desire l+ CD3+CD4+ CD3+CD4CD3-CD4+ CD3-CD4CD4+CD8+ CD4+CD8CD4-CD8+ CD4-CD8-
0.6 99.1’ 1.5 0.5 90.4 0.02 9.1 0.02 0.02 0.02 99.9
63.4 99.4 89.7 92.4 4.5 0.02 3.1 0.02 99.7 0.08 0.2
1.1 99.5 1.1 0.6 92.5 0.02 6.9 0.02 0.02 0.03 99.9
80.9 97 92.6 99.5 0.02 0.42 0.02 0.02 98.5 0.02 1.4
1.4 97.4 0.3 9.3 76.6 0.02 14.1 0.02 0.8 1.8 97.3
32.6 98.7 84.6 74.1 21.4 0.3 4.3 6.5 65.2 1.2 27.1
u 16 days after engraftment of parental T cell DOI 1107. b 16 days after engraftment of transfected T cell G7A4. ’ Mean of five mice.
of >97% of spleen cell proliferation. In vitro CD4 molecule expression on transfected T cells leads to allogeneic proliferation, and we observed a CD4+ T cell hybridoma with an unusual cytotoxic reactivity. The activities of the parental or transfected hybridoma cells were tested in vivo in SCID and Balb/c mice. SCID mice that received transfected G7A4 cells present in the spleen, a large T cell population expressing CD3, CD4, CD5, Thy1.2, and Desire 1 antigens. We observed that 15% of the spleen cells from SCID mice express CD3 antigen, and 27% express Thy 1.2 and CD4 molecules. There is no corresponding CD3+ and Thy1.2’ population in mice administered with the parental DO11107 hybridoma becausethe percentage of spleen cells carrying these corresponding antigens was similar to that of the control. In vivo, we observed CD4’ T cell proliferation. In vitro, both DO11107 and G7A4 display similar percentagesof CD3 and Thy 1.2 cells (97 and 98%, respectively), as well as similar doubling times (data not shown). In vivo, we studied the phenotype of splenic cells from SCID mice 9 weeks after inoculation with parental and transfected hybridoma T cells. We observed similar Thy 1.2,CD4, and Desire 1 antigen expressionfound on cells from SCID mice engrafted with either the G7A4 or the DO11107 clone. This fact confirms that transfected cells do not grow more rapidly in vivo than parental tumor cell lines. We noted that spleen cells rescued from SCID mice grafted with G7A4 cells present a lower percentageof Desire 1 antigens (8.48%) compared to Thyl.2 or CD4 expression
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(27%) while these three markers are similarly expressedon G7A4 cells. This difference in percentagesof splenocytes could be explained by the following: 1. The exogenous tumor cells have undergone a change in vivo such as a relative instability in the expression of transfected genes. After 24 or 48 hr of culture without the presenceof G4 18 (geniticin), cultured cells from bone marrow, thymus, and spleen from SCID mice inoculated with G7A4 cells expressed a stable phenotype virtually identical to that of cell populations initially administered. Surface antigen expression was not modified during the in vivo state (Table 5 vs Table 1). The CD4+, Thy1.2+, and Desire l+ cell percentages show that it is not only due to a particular growth of this tumor T cell line. 2. T cells of SCID origin are elicited by infusion of the transfected T cell hybridoma. Expression of CD4 on the surface of the transfected clone could be accompanied by the appearanceof a T helper population in vivo and possibly by the secretion of various cytokines capable of inducing T cell maturation in these mice. SCID mice which received either the parental or the transfected clones did not display T and B cell reconstitution as could be observed after PBL inoculation of Balb/c mice (data not shown). The number of cells injected in SCID mice (20 X lo6 cells/SCID mice) induced splenomegaly and hepatomegaly. In Balb/c mice, whether engrafted or not, we could not detect any difference among the cell populations expressing surface markers CD3, CD4, CD5, Thy 1.2, and Desire 1. No T cell differentiation could be observed but we did observe a tolerance state which seemsto appear particularly in the thymus from grafted Balb/c mice (Table 7 vs Table 6). We observed CD4 T cell proliferation but no cytotoxic reactions in vivo after T cell hybridoma transfection in SCID or Balb/c mice, which means that. in viva, another factor could modify the activities revealed in vitro. Madsen et ul. (25) have recently reported heart allograft survival in Balb/c (H-2d) mice inoculated with L cells (H-2d) transfected with ClassI chain genesbearing different haplotypes. We were interested in evaluating the role of TCR cu/fl in skin allograft survival using transfected T cell hybridoma inoculation before skin graft. We were not able (data not shown) to decreaseskin allograft rejection. These data indicate that, in rive, the role of various components must be shown to allow the development of an animal model for transplantation. In conclusion, we have demonstrated that transfection of T cell hybridoma with TCR cu/pchain genesfrom a cytotoxic clone induces the helper effect (in vitro and in viva) and unusual cytotoxic activity seen (in vitro) due to CD4 molecule expression. In these studies, SCID mice may prove to be a good model for use in the testing of transfected T cell clones. ACKNOWLEDGMENTS This work was supported by grants from INSERM-CNAMTS, D&legation B la recherche clinique AP/HP and ARC. C. Brunaud received grants from Institut Pasteur MCrieux Strums et Vaccins.
REFERENCES I. Ohashi. P. S.. Tak. W. Mak. Van den Elsen, P. Yanagi, Y., Yoshikai. Y.. Calman. A. F.. Terhorst. C.. Stobo, .I. D., and Weiss. A.. Nufzw316, 606, 1985.
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2. Berkhout, B., Alarcon, B., Terhorst, C., J. Biol. Chem. 263, 8528, 1988. 3. Dembic, Z., Haas, W., Weiss,S., McCubrey, J., Kiefer, H., von Boehmer, H., and Steinmetz, M., Nature 320,232, 1986. 4. Seed,B., and Aruffo, A., Proc. Natl. Acad. Sci. USA 84, 3365, 1987. 5. Aruffo, A., and Seed, B., Embo J. 6, 3313, 1987. 6. Saito, T., Weiss, A., Miller, J., Norcross, M. A., and Germain, R. N., Nature 325, 125, 1987. 7. Bosma, G. C., Custer, R. P., and Bosma, M. J., Nature 301, 527, 1983. 8. Mosier, D. E., Gulizia, R. J., Baird, S. M., and Wilson, D. B., Nature 335, 256, 1988. 9. McCune, J. M., Kaneshima, H., Shultz, L. D., Lieberman, M., and Weissman, 1. L., Science241, 1632, 1988. IO. Duchosal, M. A., McConahey, P. J., Robinson, C. A., and Dixon, F. J., J. Exp. Med. 172, 985, 1990. 11. Namikawa, R., Kaneshima, H., Lieberman, M., Weissman, 1. L., and McCune, J. M., Science 242, 1684, 1988. 12. Ansell, J. D., and Bancroft, G. J., Immunol. Today 10, 322, 1989. 13. Carroll, A. M., Hardy, R. R., and Bosma, M. J., J. Immunol. 143, 1087, 1989. 14. Yague, J., White, J., Coleclough, C., Kappler, J., Palmer, E., and Marrack, P., CeN42, 81, 1985. 15. Gabert, J., Langlet, C., Zamoyska, R., Parnes,J. R., Schmitt Verhulst, A-M., and Malissen, B., Cell 50, 545, 1987. 16. Kappler, J. W., Skidmore, B., White, J., and Marrack, P., J. Exp. Med. 153, 1198, 1981. 17. Letourneur, F., and Malissen, B., Eur. J. Immunol. 19, 2269, 1989. 18. Malissen, M., Trucy, J., Letourneur, F., Rebai, N., Dunn, D. E., Fitch, F. W., Hood, L., and Malissen, B., Cell 55, 49, 1988. 19. Mulligan, R. C., and Berg, P., Proc. Natl. Acad. Sci. USA 78,2072, 1981. 20. Leo, O., Foo, M., Sachs,D. H., Salmeson, L. E., and Bluestone, J. A., Proc. Nat/. Acad. Sci. USA 84, 1374, 1987. 2 I. Ohtani, K., Nakamura, M., Saito, S., Nagata, K., Sugamura, K., and Hinuma, Y., Nucleic Acids Res. 17, 1589, 1989. 22. Knutson, J. C., and Yee, D., Anal. Biochem. 164, 44, 1987. 23. Hama-Inaba, H., Nishimoto, T., Ohtsubo, M., Sato, K., and Kasai, M., Nucleic Acids Res. 19, 1988. 24. Carosella, E. D., Brunaud, C., and Charpentier, B., International Immunol., 1992, in press. 25. Madsen, J. C., Superina, R. A., Wood, K. J., and Morris, P. J., Nature 332, 161, 1988.
IMMUNOLOGY
144, 105-l 16 (1992)
T Hybridoma cr/p Gene Transfected in a Murine T Cell Hybridoma: Role of CD4 Molecule in Vitro and in Viva-Engraftment in SCID Mice Induces T Cell Maturation CHRISTINEBRUNAUD,* EDGARDODELFINOCAROSELLA,~ ANDBERNARDCHARPENTIER~ *Laboratoire
d’lmmunologie cellulaire, Pasteur Mhieux Shwns et Vaccins, Marcy L’Etoile, France; and TLaboratoire d’lmmnnologie cellulaire et de transplantation IRK, Villeju(~ France Received February 19. 1992: accepted June 22, 1992
In the present work, we tested in SCID and Balb/c mice the activity of T cell hybridoma transfected with T cell receptor (TCR) cr/P chain genes. A T cell hybridoma denoted DO1I107 was used as recipient for transfection of cytotoxic KB5C20 TCR a/p heterodimer genes by protoplast fusion or electroporation. After transfection, the parental DOI I 107 T cell line reexpressed CD5 and CD4 surface molecules. In vitro, we noted strong proliferation and unusual cytotoxic reactivities against H-2’ target cells although the transfected cell line does not express the CD8 molecule. The fate of parental and transfected cells was examined in severe combined immunodeficient (SCID) and Balb/c mice at Day 16after intravenous injection. Cells from bone marrow, thymus, and spleentissueswere analyzed by immunofluorescence.The transfectedT cell hybridoma was CD3+ Desire I+ CD4+ Thyl.2+. The SCID mice grafted with the transfected T cell hybridoma presenteda high percentageof CD3+ (15%),CD4+ (27%),Thyl.2+ (27.52%) and Desire I+ (8.74%) cells in the spleen. The percentagesof CD3+ (6.2%) and Thyl.2+ (5.06%) cells in the spleen from SCID mice grafted with parental T cell DO11107 and from untreated SCID were similar and lower (CD3+, 3.52%; Thyl.2+, 4.34%). It seemsthat transfected T cells hybridoma grafted in the SCID mice induce significant expression of CD4+ Thyl.2’ Desire l- cells (17%) in the spleen.. These results indicate that transfected T cells graft may allow T cell differentiation. In Balb/c mice, the percentage of different T cell subsetsin bone marrow, thymus, or spleen cells in mice injected with transfected T cells was similar to that in untreated mice. We did not observe any cytotoxic or significant allogeneic proliferation in vitro. 0 1992 Academic press, 1~.
INTRODUCTION The mechanisms regulating expression of T cell receptors (TCR) have now been studied in vitro after transfection of the a/p heterodimer by a number of investigators (l-3). Transfection has been a useful technique as it allows cells to acquire new functions (4, 5). For instance, Saito et af. (6) have shown that transfection of the a//3 chain genesof murine Ia-restricted TCR into the human cell line JURKAT induced expression of a functional human/murine CD3/TCR. This complex presents the antigen Ia specificity of the parental murine clone. SCID (severe combined immunodeficient) mice (7) represent a good experimental model system for studying human hematolymphoid differentiation in vivo (8, 9), as well as a number of autoimmune diseases(10, 11). The autosomal recessivescid/scid mutation leadsto a severedeficiency of T and B lymphocyte functions due to a defective 105 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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somatic rearrangement of antigen receptor genes( 12, 13). These mice display only a low level of circulating Igs (< 10 pg/ml) and a reduced percentage of cells with surface markers CD2, CD3/TCR, CD4, CD8, and Thy 1.2.Thus, SCID mice may be considered a good model for functional studies of.transfected T and B cells. In the present work, we have studied the in vivo activity in SCID and Balb/c mice of a T cell hybridoma (DO11107) (14) in which the o(and /I genesof a cytotoxic clone KB5C20 (15) were transfected. Transfection leads to the reexpression of the CD4 molecule on the transfected hybridoma. Thus, this transfected hybridoma has an unusual cytotoxic activity against H-2k cells and induces allogeneic spleen cell proliferation. The singular phenotype of this transfected clone offers a direct way of investigating through surface antigen expression the roles and effects of these proteins in vivo. SCID mice, but not Balb/c mice, grafted with the transfected T cell hybridoma displayed mature CD4+ CD3+ Thyl.2+ T lymphocytes. Grafting of transfected tumor T cells may have induced T cell differentiation. Nevertheless, we did not observe a reconstitution of murine T and B cells. In Balb/c mice, no difference in maturation could be detected. MATERIALS AND METHODS Hybridoma. The murine DO11107 hybridoma ( 14, 16- 18), kindly provided by Dr. B. Malissen (Luminy, France), was used as the target cell in gene transfer. This cell is a helper hybridoma derived by fusion of T cell blasts specific for chicken ovalbumin (OVA) (from Balb/c mice) and BW5 147 thymoma cells (from AKR mice) (17) of H2d’kphenotype. These cells were cultured in DMEM (GIBCO) medium supplemented with 2 mM glutamine, 2 mM pyruvate, 8% FCS, 5 X 1Op5M 2$ME, and 10’ U/liter penicillin-streptomycin solution at 37°C in a 10% COJair atmosphere. Gene transfection. TCR a/p genes from the cytotoxic clone (KBSCZO) carried by the plasmid pSV2neo were obtained from Dr. B. Malissen ( 15). Two techniques were used: (a) protoplast fusion following the methods of Mulligan and Berg (19) and (b) electroporation using the Progenetor system (Hoefer Scientific Instruments). Five million DO11107 parental cells/ml in 1X Hepes buffer (20 mM Hepes, pH 7.3, 137 mM NaCl, 5 mA4 KCl, 0.7 mM Na2HP04, 6 mA4 dextrose) were introduced in 24 flatbottom microtiter culture wells, 40 pg circular plasmid DNA was added to the cells, and cells were submitted to 1 to 8 impulsions at 750 V/cm/900 psec at room temperature. Following electroporation, cells were incubated under the same culture conditions described above. Twenty-four hours after transfection, selective medium containing 2 mg/ml of geneticin (G4 18, Sigma Chemical Co.) was applied. Two weeks later, KBSC20 TCR a//3’ cells were cloned by limiting dilution (at I cell/well). Transfected stable clones were screened at Day 80. Monoclonal antibody reagents and cytofluorimetric analysis. The percentages of CD4+, CDS+, CD8+, and Thy 1.2’ T cells were determined by direct immunofluorescence.Briefly, cells were incubated at 4°C for 30 min with the following fluoresceinated antibodies: L3T4 ( 10 pg/ 1O6cells) (Caltag, Tebu), Ly 1 ( 10 fig/ 1O6cells) (Becton-Dickinson), Ly2 (10 pg/ lo6 cells) (Caltag, Tebu), and Thyl.2 (10 pg/ lo6 cells) (Caltag, Tebu). Cells were then washed with phosphate-buffered saline (0.15 M NaCl, 0.0 1 A4 phosphate buffer, pH 7.4) + 2% FCS and analyzed on a FACS system. The percentageof TCR a//3’ T cells wasdetermined by indirect immunofluorescence assay.Briefly, cells were incubated at 4°C for 30 min with unconjugated hamster anti-
T HYBRIDOMA
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mouse CD3 t 145 2C 11 (kindly provided by Dr. Bluestone) (20) ( 10 pg/ 1O6cells) and with unconjugated anticlonotypic antibody Desire 1 (15) (from B. Malissen) (10 pg/ lo6 cells) specific for KB5C20 TCR. Cells were washed with phosphate-buffered saline and then incubated with FITCconjugated goat anti-mouse (Ortho Diagnostic System) or FITC-conjugated rabbit anti-hamster (for anti-CD3 c antibody) antibody. Cells were washed three more times before being analyzed. Double labeling of cells was achieved by incubating either with both FITC-conjugated mAb and phycoerythrin-conjugated mAb for two-color flow cytometry or with unconjugated Desire 1 or anti-CD3 t antibodies followed by the appropriate fluorescent conjugate. Flow cytofluorimetric analysis of surface markers was performed on a FACScan Becton-Dickinson cell sorter with a single Argon laser. Dead cells were excluded from analysis using a combination of low-angle and sideway light scatter. Data were obtained as percentage of positive viable cells after subtracting the number of stained cells from the fluorescence-conjugated cells. Mixed-lymphoq@ reaction (MLR). Spleen cells from various haplotypes (H-2d, H2b, H-2k, H-2dk) were used as responder cells and stimulator cells in MLR. Parental and transfected T cell hybridoma were used as stimulator cells. Spleen cells were freed from erythrocytes following Geys treatment and were washed twice in RPM1 1640 with 5% FCS. The responder cells (2 X lo5 cells/50 ~1) and stimulator cells (2 X 10’ cells/50 ~1)irradiated at 2000 rad were cocultured in 96 flat-bottom microtiter culture wells in a humidified atmosphere containing 5% CO1 at 32°C for 5 days and were pulsed on the last day with [3H]thymidine (1 &i/well) followed by harvesting 24 hr later. Incorporated radioactivity was measured by beta liquid scintillation counting. In vitro CTL assays. A group of parental and transfected T cell hybridomas was stimulated for 5 days in the presence of irradiated splenocytes from different strains of mice, and another group was not stimulated. The hybridomas were collected and tested for lytic activity against splenocyte cells used as targets. These splenocytes were incubated for 3 days with PHA. Hybridomas used as effector cells were incubated for an additional 4 hr with 5’Cr-labeled target splenocytes under different effector to target cell ratios. The percentageof specific 5’Cr releasewas defined as = 100 X (experimental release - spontaneous release)/(total release - spontaneous release). Mice. Homozygous CB-17/scid/scid mice designated as SCID mice (6) and kindly provided by Pr. Wagner (University of Ulm) were bred and maintained in our laboratory. Mice were housed in microisolator cagescontaining sterilized food and water. Animals were not treated with antibiotics and were healthy throughout these experiments. All manipulations were performed aseptically under a laminar flow hood. The serum lg concentration was tested in all mice before they were used. Mice whose Ig levels were greater than 3 pg/ml were eliminated. Homozygous strains of inbred Balb/ c (H-2d), C57Bl/lO (H-2’), CBA (H-2d), and Fl (Balb/c X AKR) (H-2dk) mice were purchased from IFFA CREDO Laboratories (France). Cell tram&. Nonirradiated SCID and Balb/c mice (6-to gweek-old male or female) were injected intravenously with 20 X 1O6DO11107parental cells or 20 X 1O6DO11107 transfected cells expressing Desire 1 antigen. Sixteen days after cell transfer, thymus, spleen, and bone marrow from treated and untreated SCID and Balb/c mice were removed aseptically. They were disrupted in the medium by pressing tissue fragments between two glass slides. Cell suspensions were filtered through cotton gauze and washed twice in the medium. Cell suspensions
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were freed from erythrocytes by using Geys treatment, and then were washed twice in RPM1 1640 with 5% FCS. Cell phenotypes were examined by immunofluorescence analysis following the methods previously described. Cell culture. Cells from thymus, spleen, and bone marrow were cultured in six flatbottom wells at a final concentration of lo6 cells/ml in DMEM medium under the conditions described above. Cells were harvested after 24 hr or 9 days of culture, and the expression of lymphocyte surface antigens was evaluated by immunofluorescence. Cells from nonreconstituted KID and Balb/c mice were used as negative histogram control. RESULTS Phenotype analysis of transfected T cell clones. The stability of the different clones obtained by transfection was analyzed regularly over a 12-weekperiod by flow cytometry. No significant variation in cell surface markers (CD3, CD4, CD5, CD8, Thy I .2, and Desire 1) was observed (Table 1). We selected four clones by protoplast fusion, A8B 1, G7A4, H 1IA 1, and H 11H 11; and two clones by electroporation, C6A8 and C6F8. The latter were obtained after 6 impulsions of 900 psec at 750 V/cm. In tests using 1 to 5 impulsions (900 fisec at 750 V/cm), no stable clones could be recovered, whereas 7 to 8 impulsions resulted in complete cell lysis. Transfection frequencies with electroporation were notably lower than those observed with protoplast fusion (8 X lo-’ vs 5 X 10e5).The doubling time for growth of parental and transfected hybridomas was identical irrespective of the means of transfection. TABLE 1 Cell Surface Antigens on T Cell Clones before and after Transfection (3 Months of Culture) T cell clone (% of positive cells) Protoplast fusion
Parental Expression of Ag
DOll107”
GAM +,d CD3+ CD4+ CD5+ CD8+ CD2+ LFAl+ Thyl.2+ Desire 1+xe CD3+CD4+ CD3+CD5+ CD3+CD8+ CD3+ Thyl.2+ CD4+ desire l+ Thyl.2+ desire l+ Note. NT, not tested.
0.9f 99 8.4 0.9 1.1 32 93.7 97.4 3.8 2.5 0.2 0.5 96.2 2.3 5
A8Blb
G7A4
G7Hll
2.6 93.1 94.3 95.2 3.4 NT NT 87.5 98.6 94 94.2 2.3 99.1 99.5 99.3
0.4 91.6 97.6 94.9 5.9 40 99.76 89 98.5 98.4 99.6 2.5 97.1 99.2 99.5
1.9 99.2 99.2 99.4 2.3 98 NT NT 99.3 NT NT NT NT NT NT
HllAll 3 99 98.9 99.5 1.5 97.7 NT NT 98.9 NT NT NT NT NT NT
Electroporation Hl lH1
C6A8’
C6F8
1.7 99.4 99.2 99.2 3.2 89 NT NT 98.4 NT NT NT NT NT NT
2.5 84.9 54 45.5 3.1 86 NT NT 58.9 57.1 93.3 5.4 97.9 46.8 67.4
0.5 77.6 58.5 68.7 0.3 99.7 NT NT 78.4 NT NT NT 99.5 86.9 99.5
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The parental hybridoma DO1I 107 expressed a low level of CD4 (8.42%) and did not express the CD8 molecule surface marker. However, it bears the Thy 1.2 antigen and the mature pan-T CD3 antigen (98 and 97%, respectively; Table 1). The percentage of CD3+, CD4+, CD5+, Thy 1.2+,and Desire 1+ cells was higher among clones obtained by protoplast fusion than among those obtained by electroporation (Table 1). High levels of Desire 1 are observed in all clones of transfected cells. These clones are also CD3+, Thyl.2+, and CDS-. On the other hand, transfected tumor clones now express the CD4 and CD5 antigens. Phenotypic analysis by double labeling confirms that >90% of transfected T cells are CD4+CD3+ and CD4+Desire I’. Functional analysis qf parental and transfected T cell clones. The ability of the parental and transfected T cell hybridomas to activate or not activate murine spleen cells from different strains of mice (H-2d, H-2b, H-2k, and H-2dk) is shown in Table 2. There was no significant difference in the proliferation of various H-2 haplotypes expressing spleen cells when the parental T cell hybridoma DO11107 was used as stimulator. The decreasevaried from 64% for H-2b-bearing spleen cells to as much as 80% for H-2k-bearing spleen cells compared with corresponding MLR with H-2dk stimulator spleen cells (2367 to 6508 cpm and 2672 to 14,673 cpm, respectively). In contrast, regardlessof MHC haplotype, significant proliferation was observed in mixed cultures of spleen cells with the transfected stimulator T cells of hybridoma G7A4. In particular, we observed a surprisingly high reactivity, from 2656 to 10,698 cpm with H-2dk responder spleen cells. Generally, the number of cpm does not significantly differ among the different H-2 haplotypes used as responder cells. It seems that this unusual activity is acquired after gene transfection. As shown in Table 1, the low percentage of CD8+ cells indicates that neither hybridoma expressesthis accessorymolecule. We analyzed the ability of DO11107 and G7A4 T cell hybridomas to direct lysis murine target spleencells (Table 3). As expected, in the absence of any stimulation by effector cells, none of the hybridoma CTLs significantly lysed the murine target cells whatever the haplotype. Nevertheless, as shown in Tables 3b and 3c, the transfected G7A4 hybridoma, cultured for 5 days with H-2k or H-2b cells, has a strong cytotoxic activity toward the identical target cells in CTL assays(from 20% against H-2b target spleen cells to 75% against H-2k target
TABLE 2 Allogeneic Responsewith Parental and Transfected T Cell Hybridoma Stimulator:
Spleen cells
T hybridoma
Responder: Spleen cells
Balb/c (H-2d)
CBA/J (H-2*)
C57B1/6 (H-2b)
Fl(Balb/c X AKR) (H-2d*)
WI II07 (H-2d’)
Balb/c (H-2d) CBA/J (H-2k) C57Bl/6 (H-2b) FI(Balb/c X AKR) (H-2dk)
2,243 + 151* 20,442 + 2520 5,598 + 148
19,189 + 4263 2,015 + 240 6,978 + 423
22,711 + 321 26,835 + 426 2,680 + 915
11,550 + 300 14,673 + 214 6,508 + 652
2418 + 36 2672 + 40 2367 + 52
3,409 + 476
3,093 + 787
3,469 f 644
2,656 + 745
112 + 45
G-IA4 (H-2d’) 14,533 t 67 13,916 + 107 14,978 + 123 0,698 + 80
Note. Responder cells (2 X 10’) were cultured for 5 days with irradiated spleen cells (2 X 105)and T cell hybridoma cell stimulators. These results are expressedas [ 3H]thymidine incorporated cpm. cpm, mean of three mice.
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BRUNAUD, CAROSELLA, AND CHARPENTIER TABLE 3 Cytotoxicity Analysisof Parentaland TransfectedT Cell Hybridoma
a. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2dstrain mice % of specificlysis T:E* 150 I:25 I:12
I:01
DO11107 NT
DO11107 (stimulatedby H-2dcells)
G7A4 (NT)
13.3
G7A4 (stimulatedby H-2dcells)
b. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2b strain mice % of specificlysis DO1I107 (NT)
DO11107 (stimulatedby H-2bcells)
G7A4 (NT)
G7A4 (stimulatedby H-2bcells)
1:50 I:25 I:12
10 10 10
1:Ol
CO
T:E
c. Cytotoxic activity of parentaland transfectedT cell hybridomaon spleencellstargetsfrom H-2’ strain mice % of specificlysis T:E I:50 I:25 1:12 I:01
DO11107 (NT) ‘co -co
DO11107 (stimulatedby H-2k cells)
G7A4 (NV
G7A4 (stimulatedby H-2Ircells)
to LO
Note.T:E. target/effectercell ratio; NT, not tested.Meanof three mice
spleen cells), compared to an absence of cytotoxicity with parental DO1 1107 hybridoma cells. Altogether, these results suggest that this particular unusual reactivity was acquired after transfection. Grqfls qfpurental and transficted cells in SCID mice. For cell graft experiments in SCID mice, we used the T cell clone hybridoma G7A4 obtained by protoplast fusion (see Table 1). Four SCID mice were grafted in each of the treated groups. The phenotypes of splenocytes were analyzed 16 days after engraftment. The CD3+ and Thy 1.2’ cells constituted a high percentage of the T cell population of SCID mouse splenocytes having received the clone G7A4 (Table 4). Mice engrafted with the G7A4 hybridoma
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TABLE 4 Analysis of Surface Antigens on Splenic Cells from Grafted and Ungrafted SCID Mice (at Day 16) SCID mice (% of positive cells) Expression of Ag CD3’ CD4’ CD5+ Thyl.2+ Desire I+
Untreated 3.52 0.42 0.62 4.34 0.8
f k k f f
0.5’ 0.1 0.2 0.1 0.2
Grafted with DO I 1107” 6.26 0.52 I .36 5.06 0.7
+ k i f f
l.ld 0. I 0.4 3.1 0.3
Grafted with G7A4h 15 + 21 + 10.8 + 27.52 + 8.74 +
2.3’ 2.1’ l.lR 2.1 0.6
Nom. The results are shown as means + SE by the Student Fisher test. a Day 0, engraftment of SCID mice with 25 million parental T cells DO1 1107. ’ Day 0, engraftment of SCID mice with 20 million transfected T cells G7A4. c vs d P < 0.005, c vs e P < 0.001. “VsgPi 0.01. fvs f P < 0.001.
present a high percentage of Desire l+ and CD4+ cells, while no difference could be observed between mice engrafted with the parental hybridoma and nontreated mice. Splenocytes from the treated and nontreated mice were then placed in culture and analyzed 24 hr later (Table 5). The nontreated group expressedlow levels of CD3 and Thy I .2 and presented a high cell mortality. Spleen cells from the two treated groups proliferated and had high levels of CD3+ and Thy 1.2’. Lymphocytes bearing Desire 1 are found only in cultures of spleen from mice treated with the transfected clone G7A4. We tested CD4 and CD5 markers only after 130 hr of culture (data not shown). Thus, we observed that CD5 and CD4 antigen expression appeared on splenocytes cultured from SCID grafted with G7A4 T cells and not on splenocytescultured from SCID grafted with the parental T cell hybridoma. It is likely that the cells proliferating in vitro were those that were grafted initially.
TABLE 5 Analysis of Surface Antigens on Culture Spleen Cells from Grafted and Ungrafted SCID Mice (after 24 hr of Culture) SCID mice (% of positive cells) Expression of Ag
Untreated
Grafted with DOI 1107”
Grafted with G7A4b
CD3+ CD5+ Thyl.2+ Desire l+
2.9‘ 0.8 5.6 3
68 NT 70. I 0.6
51 NT 41.7 42.5
Nofe. NT, not tested. ’ 16 days after engraftment of parental T cell DO I I 107. ’ 16 days after engraftment of transfected T cell G7A4. ‘ Mean of three mice.
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CHARPENTIER
The low number of thymocytes recovered did not allow us to carry out a similar analysis on this cell population. Engraftment ofparental and transfected cells in Balb/c mice. Five Balb/c mice were grafted in each of the treated groups. The phenotypes of bone marrow, thymus, and spleen cells from inoculated Balb/c mice were analyzed 16 days postgrafting (Table 6). No difference in the percentage of cells displaying the various markers could be detected between treated and nontreated mice, including the cells displaying Desire It and CD4+ (also in Balb/c mice treated with G7A4 cells). The Desire 1 antibody stains 9-10% of bone marrow cells from Balb/c mice treated or not treated with the transfected or the parental T cell hybridoma, revealing a background level. In the spleen we noted a high percentage (25%) of cells expressing KB5C20 TCR in control or mice treated with DO11107 cells, due to nonspecific staining of immunoglobulins carried by B lymphocyte membrane. After 9 days in culture, mice grafted with the DO11107 clone have a large population of CD3+CD4- and CD4-CDS- T cells in bone marrow, thymus, and spleen (Table 7). However, the cells in culture from mice grafted with transfected G7A4 clone have a majority of T cells bearing the antigens CD3+CD4+, CDS+Thy 1.2+, and Desire 1 in all tissues tested. This population also appears in culture from thymocytes. DISCUSSION The mouse hybridoma DO1110 was originally obtained by the fusion of AKR thymoma BW5147 to chicken ovalbumin-specific T cell blasts from Balb/c mice. This hybridoma secretesIL-2 when challenged with ovalbumin in the context of IAd- or IAb-presenting cells. Clone DO11107 is a derivative of DO1110 characterized by the nonexpression of the parental LY/@ heterodimer. Consequently, this hybridoma expresses only the BW5 147 TCR and does not respond to ovalbumin in the context of restricted IAd or IAb. KB5C20 is a cytotoxic clone from the BlO.BR mouse which recognizes Class I H2Kb molecules. Transfection of both cvand /I genes from a cytotoxic clone does not necessarily lead to CD8 expression, nor does it suffice to retarget the recipient toward a MHC Class I specificity as described by Ansell and Bancroft (12). Transfection techniques which utilize chemical agents (such as DEAE dextran and calcium phosphate) do not give high transfection efficiencies with T cell hybridoma (21). In the present study, two other techniques were employed: electroporation (22, 23) and protoplast fusion (19). Electroporation was not very efficient (one Desire l+ clone for 8 X 1O7transfected cells) compared to protoplast fusion (one Desire 1+ clone for 5 X lo5 transfected cells). However, both techniques gave rise to clones displaying similar levels of surface antigens. Transfection of TCR (Yand p chain genes from a cytotoxic clone into T DO1I 107 hybridoma induces CD4 and CD5 antigen reexpression. Moreover, transfected T cell hybridoma has particular cytotoxic reactions and induces spleen cell proliferation, whatever the haplotype. It seemsthat KB5C20 TCR a//3 was not implicated in this phenomenon but the role of the CD4 molecule has been questioned. Incubation of the stimulator G7A4 hybridoma with 1 pg mAb Desire I/ 1O6cells for 1 hr did not reveal any effect on the murine spleen cell response (24). Transfected T hybridoma treatment with 1 pg of L3T4 mAb/106 cells resulted in the inhibition
4 9.3 5 24.9 0.04 69.9 0.4
0.9
3.6 95.1
Thyl.Z+ Desire 1+ CD3+CD4+ CD3+CD4CD3-CD4+ CD3-CD4CD4+CD8+
CD4+CD8-
CD4-CD8+ CD4-CD8-
I.1 94.7
2.2
8.8 II 9.1 16.8 0.08 74 1.9
4.5
Grafted with DOl11107”
0.7 96.1
2
2.8 12.6 4.5 9.4 0.02 86.2 1.2
5.8
Grafted with G7A4b
12.3 21.1
19.6
94.6 5.2 54.5 15.7 10.9 18.9 46.3
26.7
Untreated
Note. NT, not tested. a Day = 0, engraftment of Balb/c mice with 20 million DO11107 cells. ’ Day = 0, engraftment of Balb/c mice with 20 million transfected G7A4 cells. ’ Mean of five mice.
4’
Untreated
CD5+
Balb/c surface Ag
Bone marrow cells
10.2 I2
14.4
97.5 1.9 66.4 11.3 IO 12.3 63.5
25.1
Grafted with D0111107”
Thymus cells
7.2 12.9
9.2
90. I 1.2 40.8 5.7 34 19.6 70.7
19.1
Grafted with G7A4’
Tissue cells (% of positive cells)
NT
NT
45.1 25 25.6 53.9 1.5 19.1 NT
30
Untreated
23.1 58.8
15.4
25.5 24.3 33.6 51.5 0.04 14.9 2.8
16.4
Grafted with D0111107”
Spleen cells
Cell Surface Antigens on Bone Marrow Cells, Thymocytes, and Splenocytes from Grafted and Ungrafted Balb/c Mice (at Day 16)
TABLE 6
25.2 59
12.1
25.3 23.3 22 50.5 0.08 21.4 3.7
14.4
Grafted with G7A4b
w
z
3
3 -i F z 4
F R s 0
2 % 5
-l
114
BRUNAUD,
CAROSELLA, AND CHARPENTIER TABLE 7
Surface Antigens on Bone Marrow, Thyrnocytes, and Spleen on Cultured Cells from Balb/c Mice Grafted with DO11107 Parental Cells or with G7A4-Transfected Cells (Tested 9 Davs after Culture) Tissue cells (W of positive cells) Bone marrow cells
Thymus cells
Spleen cells
Balb/c surface Ag
Grafted with DO11107”
Grafted with G7A4 b
Grafted with D011107”
Grafted with G7A4b
Grafted with D011107”
Grafted with G7A4 b
CD5+ Thyl.2+ Desire l+ CD3+CD4+ CD3+CD4CD3-CD4+ CD3-CD4CD4+CD8+ CD4+CD8CD4-CD8+ CD4-CD8-
0.6 99.1’ 1.5 0.5 90.4 0.02 9.1 0.02 0.02 0.02 99.9
63.4 99.4 89.7 92.4 4.5 0.02 3.1 0.02 99.7 0.08 0.2
1.1 99.5 1.1 0.6 92.5 0.02 6.9 0.02 0.02 0.03 99.9
80.9 97 92.6 99.5 0.02 0.42 0.02 0.02 98.5 0.02 1.4
1.4 97.4 0.3 9.3 76.6 0.02 14.1 0.02 0.8 1.8 97.3
32.6 98.7 84.6 74.1 21.4 0.3 4.3 6.5 65.2 1.2 27.1
u 16 days after engraftment of parental T cell DOI 1107. b 16 days after engraftment of transfected T cell G7A4. ’ Mean of five mice.
of >97% of spleen cell proliferation. In vitro CD4 molecule expression on transfected T cells leads to allogeneic proliferation, and we observed a CD4+ T cell hybridoma with an unusual cytotoxic reactivity. The activities of the parental or transfected hybridoma cells were tested in vivo in SCID and Balb/c mice. SCID mice that received transfected G7A4 cells present in the spleen, a large T cell population expressing CD3, CD4, CD5, Thy1.2, and Desire 1 antigens. We observed that 15% of the spleen cells from SCID mice express CD3 antigen, and 27% express Thy 1.2 and CD4 molecules. There is no corresponding CD3+ and Thy1.2’ population in mice administered with the parental DO11107 hybridoma becausethe percentage of spleen cells carrying these corresponding antigens was similar to that of the control. In vivo, we observed CD4’ T cell proliferation. In vitro, both DO11107 and G7A4 display similar percentagesof CD3 and Thy 1.2 cells (97 and 98%, respectively), as well as similar doubling times (data not shown). In vivo, we studied the phenotype of splenic cells from SCID mice 9 weeks after inoculation with parental and transfected hybridoma T cells. We observed similar Thy 1.2,CD4, and Desire 1 antigen expressionfound on cells from SCID mice engrafted with either the G7A4 or the DO11107 clone. This fact confirms that transfected cells do not grow more rapidly in vivo than parental tumor cell lines. We noted that spleen cells rescued from SCID mice grafted with G7A4 cells present a lower percentageof Desire 1 antigens (8.48%) compared to Thyl.2 or CD4 expression
T HYBRIDOMA
a/P GENE
TRANSFECTION
115
(27%) while these three markers are similarly expressedon G7A4 cells. This difference in percentagesof splenocytes could be explained by the following: 1. The exogenous tumor cells have undergone a change in vivo such as a relative instability in the expression of transfected genes. After 24 or 48 hr of culture without the presenceof G4 18 (geniticin), cultured cells from bone marrow, thymus, and spleen from SCID mice inoculated with G7A4 cells expressed a stable phenotype virtually identical to that of cell populations initially administered. Surface antigen expression was not modified during the in vivo state (Table 5 vs Table 1). The CD4+, Thy1.2+, and Desire l+ cell percentages show that it is not only due to a particular growth of this tumor T cell line. 2. T cells of SCID origin are elicited by infusion of the transfected T cell hybridoma. Expression of CD4 on the surface of the transfected clone could be accompanied by the appearanceof a T helper population in vivo and possibly by the secretion of various cytokines capable of inducing T cell maturation in these mice. SCID mice which received either the parental or the transfected clones did not display T and B cell reconstitution as could be observed after PBL inoculation of Balb/c mice (data not shown). The number of cells injected in SCID mice (20 X lo6 cells/SCID mice) induced splenomegaly and hepatomegaly. In Balb/c mice, whether engrafted or not, we could not detect any difference among the cell populations expressing surface markers CD3, CD4, CD5, Thy 1.2, and Desire 1. No T cell differentiation could be observed but we did observe a tolerance state which seemsto appear particularly in the thymus from grafted Balb/c mice (Table 7 vs Table 6). We observed CD4 T cell proliferation but no cytotoxic reactions in vivo after T cell hybridoma transfection in SCID or Balb/c mice, which means that. in viva, another factor could modify the activities revealed in vitro. Madsen et ul. (25) have recently reported heart allograft survival in Balb/c (H-2d) mice inoculated with L cells (H-2d) transfected with ClassI chain genesbearing different haplotypes. We were interested in evaluating the role of TCR cu/fl in skin allograft survival using transfected T cell hybridoma inoculation before skin graft. We were not able (data not shown) to decreaseskin allograft rejection. These data indicate that, in rive, the role of various components must be shown to allow the development of an animal model for transplantation. In conclusion, we have demonstrated that transfection of T cell hybridoma with TCR cu/pchain genesfrom a cytotoxic clone induces the helper effect (in vitro and in viva) and unusual cytotoxic activity seen (in vitro) due to CD4 molecule expression. In these studies, SCID mice may prove to be a good model for use in the testing of transfected T cell clones. ACKNOWLEDGMENTS This work was supported by grants from INSERM-CNAMTS, D&legation B la recherche clinique AP/HP and ARC. C. Brunaud received grants from Institut Pasteur MCrieux Strums et Vaccins.
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