Journal of Immunological Methods, 154 (1992) 235-243
235
© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.(10
JIM 06939
In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells Yosbimasa W a d a a Hideyuki Ikeda a, Daisuke U e d a ~, Masahiko O h t a a, Shuji Takahashi a, Koichi H i r a t a b, Noriyuki Sato a and Kokichi Kikuehi a a Department of Pathology and h Department of Surgery, Sapporo Medical College, 060 Sapporo, Japan
(Received27 February 1992,revisedreceived21 April 1992,accepted4 May 1992)
Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at - 180°C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U / m l ) of rlL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rlL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells. Key words: CytotoxicT lymphocyte;Human autologous tumor; Cryopreservation;Lymphocyte
Introduction The cytotoxic mechanism of human autologous tumor cells by CTL is still largely unknown. In order to investigate this mechanism, we have attempted to establish pairs of human CTL and
Correspondence to: N. Sato, Department of Pathology, Sapporo Medical College,060 Sapporo, Japan. Abbreviations: CTL, eytotoxic T lymphocytes; DMSO, dimethyl sulfoxide: FCS, fetal calf serum; (r)lL-2, (recombinant) interleukin-2;mAb, monoclonalantibody; MLTC,mixed lymphocyte autologous tumor cell culture; MMC, mitomycin C; PBL, peripheral blood lymphocytes;TCR, T cell antigen receptors; TIL, tumor-infiltratinglymphocytes.
autologous tumor cells from various tissue origins (T. Sato et al., 1986, 1988, 1989; Okubo et al., 1989). If successful, tumor cell lines could be established within 2-3 months after beginning the primary culture of the malignant effusions. On the other hand, normal T cells from peripheral blood could usually be maintained only for several months. This is especially true for (~D8( + ) CTL. These T cells could not produce T cell growth factors such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12 and INF3,, which might be required for their own cell proliferation. It may be possible to culture TIL from the malignant effusions for a longer period of time if frequent antigenic stimulation is performed (T. Sato et al., 1986). How-
236 ever, it is generally difficult to culture T cells for long periods. [:urthermore, from a practical standpoint, a continuous culture of cells is not always required. Accordingly, we must use cryopreserved CTL. However, we have often encountered difficulties in recovering cryopreserved CTL well enough for experimental use. To resolve this problem, we established a new method to proliferate CTL that had been thawed from a frozen stock. Using CTL and autologous tumor cells (N. Sate et al., 1992), it was demonstrated that not only rlL-2, but also supernatants from the primary culture containing TIL and autologous tumor cells, would be required for proliferating CTL. The presence of MMC-treated autologous tumor cells was beneficial in the proliferation of eryopreserved CTL. However, to obtain the specificity of CTL cytotoxicity, we found that withdrawal of rlL-2 from the culture 24 h before the cytotoxic assays might be critical. This suggests that continuous culture of CTL in the presence of a high dose of rlL-2 may result in a non-physiological cytotoxicity conditions.
Materials and methods
Cell culture, MLTC and CTL cloning The establishment of autologous tumor lines and functional CTL clones has been described previously (T. Sate et al., 1986, 1989; N. Sate et al, 1992). In this study we used pairs of autologous tumor lines, HST-2 and PUN, and CTL clones, TcHST-2 and TePUN. Reverse PCR analysis of TCR variable (V) gene usage showed that the TcHST-2 clone could produce mRNA encoding the V/~ 20 gene (manuscript in preparation). HST-2 gastric tumor and PUN pancreatic tumor lines were obtained from the malignant ascitic effusion by culturing for approximately 2 months after the initiation of primary cell culture. Subcutaneous inoculation of 1 × 106 of these tumor cells into nude mice resulted in a 100% tumor incidence. TcHST-2 was derived from PBL, and TcPUN from TIL. PBL and TIL were separated by Ficoll-Conray density gradient. Lymphocytes collected from the interface were washed twice in PBS and cultured in AIM-V (GIBCO, Grand Island, NY) medium containing 350 U / m l of
rlL-2 (Shionogi Pharmaceutical Co. Ltd., Tokyo, Japan). in the mixed lymphocyte autologous tumor cell cultures (MLTC), 5 × 106 iymphocytes were cocultured with 1 x 105 MMC-treated HST-2 cells for 2 days at 37°C in a 5% CO 2 incubator. Cultures were grown in culture flasks (Coming No. 25100) in AIM-V without rIL-2. These activated lymphoeytes were separated by a FicolI-Conray density gradient centrifugation at 1000 x g for 30 rain, and then cultured in AIM-V medium containing 350 U / m i of rlL-2. MLTC was done at least twice at 2-week intervals prior to the eytotoxicity assays. Before the eytotoxieity assays, these T cells were cultured in the absence of rlL-2 for 24 h, since this treatment usually conferred the specific eytotoxicity on CTL as described below.
mAs and phenotype analysis by FACS We employed the following mAbs. Antibodies reacting to CD2, CD3, CD4, CD8 and CD56 were obtained from the Ortho Diagnostic Systems Co. (Tokyo, Japan), and C D l l a (LFAla) (SPV-L7), CD16 (MG38) and CD45RO (UCHL1) from Nichirei Co. (Tokyo), respectively. An antibody to a complex of CD3-TCRa//3 (antiTCR, WT31) antigens was purchased from Becton Dickinson (Mountain View, CA). Using these mAbs, the surface phenotype of T cells were detected by FACS as described elsewhere (Konno et al., 1989).
Influence of FCS concentration for cryopreservation, and requirements for the conditioned medium on the efficient recovery of functional CTL clone In order to assess whether FCS content at cryopreservation of CTL could affect the proliferation potential of thawed CI'L, 1 × 107 per sample of T cells were cryopreserved for 1 year at - 1 8 0 ° C in RPMI 1640 containing 10% DMSO and various concentrations (10, 20 and 50%) of FCS. Cells were quic~y thawed in a water bath at approximately 37°C, washed twice in PBS, and the cell viability was determined by trypan blue dye exclusion. Cells were then cultured for 1 week with AIM-V medium containing 350 U / m l of rIL-2. We also studied the proliferative capability of cryopreserved CTL cultured for 1 week
237 with AIM-V medium containing (1) only 350 U / m l of rlL-2, (2) 50% of the supernatant of primary cell culture with TIL and autologous tumors, and 350 U / m l rlL-2, and (3) 50% of the supernatant, 350 U / m l rlL-2 and the presence of MMC-treated autologous tumor cells, adjusted to a ratio of 50 lymphocytes/tumor cell, as a feeder as well as an antigenic stimulant. The proliferating capability of these cells was determined by counting the viable cell number under the phase contrast microscope. The cell number was expressed as mean + SE.
Cytotoxicity assays and influence of rlL-2 content on the specificity of the thawed CTL clone The conventional 5tCr-release cytotoxicity assay has been described previously (T. Sato et ai., 1986, 1989). Briefly, 1 × 106 target cells were labeled with 100/zCi sodium chromate and incubated in FCS-free RPMI 1640 for 3 h at 37°C. These cells were washed three times with FCSfree RPMI 1640, and 1 × 104 target cells in 100 /zl RPMI 1640 containing 10% FCS were seeded into round-bottomed 96-weU plates (Coming No. 25850). Thereafter, 100/~1 of effector cell suspension at 25 E / T ratio was added. After 10-12 h incubation at 37°C, 100/~1 of culture supernatant was harvested, and counted with a liquid scintillation counter. The percent iysis was calculated as described elsewhere (T. Sato et al., 1986). In this study, two separate experiments were done, and all determinations were made in triplicate. The data are presented as the mean + SE of % cytotoxicity. To determine the influence of rIL-2 concentration on the specificity of cytotoxicity by thawed and proliferated CTL, we employed CTL in two different culture conditions. TcHST-2 cells were continuous cultured in the p r c s e , c e of 350 U / m l rIL-2 in one culture, and without rIL-2 for 24 h in the other, before being used for cytotoxicity assays. Inhibition assays with mAbs and cold targets For determination of cytotoxic specificity, we performed two kinds of experiments, in addition to the conventional cytotoxicity assay. The first was an inhibition study of cytotoxicity by using mAbs reacting against T cell surface antigens.
CTL were treated for 60 min at 4°C with a saturating amount 3f mAbs reacting to CD2, 3, 4, 8, l l a , 45RO, and a complex of C D 3 - T C R a / ~ antigens. Cells were washed in PBS and used in the cytotoxicity assays against a SmCr-labeled autologous tumor target. The second was the cold (SmCr-unlabeled) target inhibition study. 104 5'Cr-labeled (hot) target cells were admixed with 104 cold target cells and 2.5 × 105 CTL, and the inhibition of cytotoxicity was determined. As cold target cells we used autologous tumor cells and allogeneic tumor lines, including Daudi (B lymphoma), K562 (erythroleukemi~0, U937 ( m o n a cytic leukemia), HeLa (cervical carcinoma) and SSTW (signet ring cell stomach carcinoma).
Results Relationship between cell recovery and different FCS concentrations on cryopreserving the CTL clone We first examined whether the concentration of FCS when CTL clones are cryopreserved influences cell recovery. As shown in Table l, there was no significant difference in viability when
TABLE I RELATIONSHIP BETWEEN CELL RECOVERY AND DIFFERENT FCS CONCENTRATIONS AT CRYOPRESERVING CTL CLONES FCS (%)
10 20 50
CTL
Viability a at the time of thawing(%) TcHST-2 66.2_+8.8 TcPUN 55.8_+6.2 TcHST-2 72.6_+7.5 TcPUN 63.8_+4.7 TcHST-2 74.3+5.8 TcPUN 59.1 -+3.8
Numberof proliferated cells a
1.0_+0.2×105 0.6_+0.2×105 1.0_+0.2×106 0.8_+0.1x 106 2.0_+0.1× 107 1.1 -+0.2 × 107
a Approximately 1 x 10 7 TcHST-2 and TcPUN were c~opreserved for 1 year at -180~C in RPMI 1640 containing 10% DMSO and 10, 20 and 50% FCS. Cells were thawed quickly, and viabilitywas counted by trypan blue dye exclusion. Then cells were cultured with AIM-V medium containing350 U/ml rlL-2. After 1 week cultivationat 37°C, the number of proliferated cells was counted. Results are from two experiments; data are expressed as means_+SE.
238 thawing the cryopreserved CTL with 10 20 and 50% FCS supplements. However, when CTL was cryoprcservcd in a medium containing 50% FCS, thawed cells d e m o n s t r a t e d good proliferation. Cells cryopr,;servcd with 20% FCS grew slightly, but cells with 10% FCS hardly proliferated at all. It should be noted that TcHST-2 and T c P U N showed almost the same viability and proliferation in each cryopreselving condition.
bzfluence of the conditioned medium on recot'ety of the thawed CTL clone To determine the proliferating and cytotoxic potential of CTL cryopreserved with 5(1% FCS at - 1 8 0 ° C , we cultured thawed CTL according to three different methods. As shown in Table !1, the data indicate that the addition of the primary culture supernatant plus M M C - t r e a t e d autologous cells was most effective for the proliferation of TcHST-2 and TcPUN. The culture supernatant alone seemed to work quite well. How-
TABLE !! INFLUENCE OF TIlE CONDITIONED MEDIUM ON THE PROLIFERATION AND CYTOTOXIC FUNCTION OF THAWED CTL CLONES Conditioned medium ~ Number of % cytotoxicity ~ CTL proliferated cells b (1) TcHST-2
12.11+11.31×Ill7
TcPUN ( 1 . 4 + 0 . 2 ) X 1117 (2150e~ of primary culture supernatant TcHST-2 (I.I _+11.2)>"Ill~: TcPUN (11.8_+0.1))
235
© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.(10
JIM 06939
In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells Yosbimasa W a d a a Hideyuki Ikeda a, Daisuke U e d a ~, Masahiko O h t a a, Shuji Takahashi a, Koichi H i r a t a b, Noriyuki Sato a and Kokichi Kikuehi a a Department of Pathology and h Department of Surgery, Sapporo Medical College, 060 Sapporo, Japan
(Received27 February 1992,revisedreceived21 April 1992,accepted4 May 1992)
Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at - 180°C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U / m l ) of rlL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rlL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells. Key words: CytotoxicT lymphocyte;Human autologous tumor; Cryopreservation;Lymphocyte
Introduction The cytotoxic mechanism of human autologous tumor cells by CTL is still largely unknown. In order to investigate this mechanism, we have attempted to establish pairs of human CTL and
Correspondence to: N. Sato, Department of Pathology, Sapporo Medical College,060 Sapporo, Japan. Abbreviations: CTL, eytotoxic T lymphocytes; DMSO, dimethyl sulfoxide: FCS, fetal calf serum; (r)lL-2, (recombinant) interleukin-2;mAb, monoclonalantibody; MLTC,mixed lymphocyte autologous tumor cell culture; MMC, mitomycin C; PBL, peripheral blood lymphocytes;TCR, T cell antigen receptors; TIL, tumor-infiltratinglymphocytes.
autologous tumor cells from various tissue origins (T. Sato et al., 1986, 1988, 1989; Okubo et al., 1989). If successful, tumor cell lines could be established within 2-3 months after beginning the primary culture of the malignant effusions. On the other hand, normal T cells from peripheral blood could usually be maintained only for several months. This is especially true for (~D8( + ) CTL. These T cells could not produce T cell growth factors such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12 and INF3,, which might be required for their own cell proliferation. It may be possible to culture TIL from the malignant effusions for a longer period of time if frequent antigenic stimulation is performed (T. Sato et al., 1986). How-
236 ever, it is generally difficult to culture T cells for long periods. [:urthermore, from a practical standpoint, a continuous culture of cells is not always required. Accordingly, we must use cryopreserved CTL. However, we have often encountered difficulties in recovering cryopreserved CTL well enough for experimental use. To resolve this problem, we established a new method to proliferate CTL that had been thawed from a frozen stock. Using CTL and autologous tumor cells (N. Sate et al., 1992), it was demonstrated that not only rlL-2, but also supernatants from the primary culture containing TIL and autologous tumor cells, would be required for proliferating CTL. The presence of MMC-treated autologous tumor cells was beneficial in the proliferation of eryopreserved CTL. However, to obtain the specificity of CTL cytotoxicity, we found that withdrawal of rlL-2 from the culture 24 h before the cytotoxic assays might be critical. This suggests that continuous culture of CTL in the presence of a high dose of rlL-2 may result in a non-physiological cytotoxicity conditions.
Materials and methods
Cell culture, MLTC and CTL cloning The establishment of autologous tumor lines and functional CTL clones has been described previously (T. Sate et al., 1986, 1989; N. Sate et al, 1992). In this study we used pairs of autologous tumor lines, HST-2 and PUN, and CTL clones, TcHST-2 and TePUN. Reverse PCR analysis of TCR variable (V) gene usage showed that the TcHST-2 clone could produce mRNA encoding the V/~ 20 gene (manuscript in preparation). HST-2 gastric tumor and PUN pancreatic tumor lines were obtained from the malignant ascitic effusion by culturing for approximately 2 months after the initiation of primary cell culture. Subcutaneous inoculation of 1 × 106 of these tumor cells into nude mice resulted in a 100% tumor incidence. TcHST-2 was derived from PBL, and TcPUN from TIL. PBL and TIL were separated by Ficoll-Conray density gradient. Lymphocytes collected from the interface were washed twice in PBS and cultured in AIM-V (GIBCO, Grand Island, NY) medium containing 350 U / m l of
rlL-2 (Shionogi Pharmaceutical Co. Ltd., Tokyo, Japan). in the mixed lymphocyte autologous tumor cell cultures (MLTC), 5 × 106 iymphocytes were cocultured with 1 x 105 MMC-treated HST-2 cells for 2 days at 37°C in a 5% CO 2 incubator. Cultures were grown in culture flasks (Coming No. 25100) in AIM-V without rIL-2. These activated lymphoeytes were separated by a FicolI-Conray density gradient centrifugation at 1000 x g for 30 rain, and then cultured in AIM-V medium containing 350 U / m i of rlL-2. MLTC was done at least twice at 2-week intervals prior to the eytotoxicity assays. Before the eytotoxieity assays, these T cells were cultured in the absence of rlL-2 for 24 h, since this treatment usually conferred the specific eytotoxicity on CTL as described below.
mAs and phenotype analysis by FACS We employed the following mAbs. Antibodies reacting to CD2, CD3, CD4, CD8 and CD56 were obtained from the Ortho Diagnostic Systems Co. (Tokyo, Japan), and C D l l a (LFAla) (SPV-L7), CD16 (MG38) and CD45RO (UCHL1) from Nichirei Co. (Tokyo), respectively. An antibody to a complex of CD3-TCRa//3 (antiTCR, WT31) antigens was purchased from Becton Dickinson (Mountain View, CA). Using these mAbs, the surface phenotype of T cells were detected by FACS as described elsewhere (Konno et al., 1989).
Influence of FCS concentration for cryopreservation, and requirements for the conditioned medium on the efficient recovery of functional CTL clone In order to assess whether FCS content at cryopreservation of CTL could affect the proliferation potential of thawed CI'L, 1 × 107 per sample of T cells were cryopreserved for 1 year at - 1 8 0 ° C in RPMI 1640 containing 10% DMSO and various concentrations (10, 20 and 50%) of FCS. Cells were quic~y thawed in a water bath at approximately 37°C, washed twice in PBS, and the cell viability was determined by trypan blue dye exclusion. Cells were then cultured for 1 week with AIM-V medium containing 350 U / m l of rIL-2. We also studied the proliferative capability of cryopreserved CTL cultured for 1 week
237 with AIM-V medium containing (1) only 350 U / m l of rlL-2, (2) 50% of the supernatant of primary cell culture with TIL and autologous tumors, and 350 U / m l rlL-2, and (3) 50% of the supernatant, 350 U / m l rlL-2 and the presence of MMC-treated autologous tumor cells, adjusted to a ratio of 50 lymphocytes/tumor cell, as a feeder as well as an antigenic stimulant. The proliferating capability of these cells was determined by counting the viable cell number under the phase contrast microscope. The cell number was expressed as mean + SE.
Cytotoxicity assays and influence of rlL-2 content on the specificity of the thawed CTL clone The conventional 5tCr-release cytotoxicity assay has been described previously (T. Sato et ai., 1986, 1989). Briefly, 1 × 106 target cells were labeled with 100/zCi sodium chromate and incubated in FCS-free RPMI 1640 for 3 h at 37°C. These cells were washed three times with FCSfree RPMI 1640, and 1 × 104 target cells in 100 /zl RPMI 1640 containing 10% FCS were seeded into round-bottomed 96-weU plates (Coming No. 25850). Thereafter, 100/~1 of effector cell suspension at 25 E / T ratio was added. After 10-12 h incubation at 37°C, 100/~1 of culture supernatant was harvested, and counted with a liquid scintillation counter. The percent iysis was calculated as described elsewhere (T. Sato et al., 1986). In this study, two separate experiments were done, and all determinations were made in triplicate. The data are presented as the mean + SE of % cytotoxicity. To determine the influence of rIL-2 concentration on the specificity of cytotoxicity by thawed and proliferated CTL, we employed CTL in two different culture conditions. TcHST-2 cells were continuous cultured in the p r c s e , c e of 350 U / m l rIL-2 in one culture, and without rIL-2 for 24 h in the other, before being used for cytotoxicity assays. Inhibition assays with mAbs and cold targets For determination of cytotoxic specificity, we performed two kinds of experiments, in addition to the conventional cytotoxicity assay. The first was an inhibition study of cytotoxicity by using mAbs reacting against T cell surface antigens.
CTL were treated for 60 min at 4°C with a saturating amount 3f mAbs reacting to CD2, 3, 4, 8, l l a , 45RO, and a complex of C D 3 - T C R a / ~ antigens. Cells were washed in PBS and used in the cytotoxicity assays against a SmCr-labeled autologous tumor target. The second was the cold (SmCr-unlabeled) target inhibition study. 104 5'Cr-labeled (hot) target cells were admixed with 104 cold target cells and 2.5 × 105 CTL, and the inhibition of cytotoxicity was determined. As cold target cells we used autologous tumor cells and allogeneic tumor lines, including Daudi (B lymphoma), K562 (erythroleukemi~0, U937 ( m o n a cytic leukemia), HeLa (cervical carcinoma) and SSTW (signet ring cell stomach carcinoma).
Results Relationship between cell recovery and different FCS concentrations on cryopreserving the CTL clone We first examined whether the concentration of FCS when CTL clones are cryopreserved influences cell recovery. As shown in Table l, there was no significant difference in viability when
TABLE I RELATIONSHIP BETWEEN CELL RECOVERY AND DIFFERENT FCS CONCENTRATIONS AT CRYOPRESERVING CTL CLONES FCS (%)
10 20 50
CTL
Viability a at the time of thawing(%) TcHST-2 66.2_+8.8 TcPUN 55.8_+6.2 TcHST-2 72.6_+7.5 TcPUN 63.8_+4.7 TcHST-2 74.3+5.8 TcPUN 59.1 -+3.8
Numberof proliferated cells a
1.0_+0.2×105 0.6_+0.2×105 1.0_+0.2×106 0.8_+0.1x 106 2.0_+0.1× 107 1.1 -+0.2 × 107
a Approximately 1 x 10 7 TcHST-2 and TcPUN were c~opreserved for 1 year at -180~C in RPMI 1640 containing 10% DMSO and 10, 20 and 50% FCS. Cells were thawed quickly, and viabilitywas counted by trypan blue dye exclusion. Then cells were cultured with AIM-V medium containing350 U/ml rlL-2. After 1 week cultivationat 37°C, the number of proliferated cells was counted. Results are from two experiments; data are expressed as means_+SE.
238 thawing the cryopreserved CTL with 10 20 and 50% FCS supplements. However, when CTL was cryoprcservcd in a medium containing 50% FCS, thawed cells d e m o n s t r a t e d good proliferation. Cells cryopr,;servcd with 20% FCS grew slightly, but cells with 10% FCS hardly proliferated at all. It should be noted that TcHST-2 and T c P U N showed almost the same viability and proliferation in each cryopreselving condition.
bzfluence of the conditioned medium on recot'ety of the thawed CTL clone To determine the proliferating and cytotoxic potential of CTL cryopreserved with 5(1% FCS at - 1 8 0 ° C , we cultured thawed CTL according to three different methods. As shown in Table !1, the data indicate that the addition of the primary culture supernatant plus M M C - t r e a t e d autologous cells was most effective for the proliferation of TcHST-2 and TcPUN. The culture supernatant alone seemed to work quite well. How-
TABLE !! INFLUENCE OF TIlE CONDITIONED MEDIUM ON THE PROLIFERATION AND CYTOTOXIC FUNCTION OF THAWED CTL CLONES Conditioned medium ~ Number of % cytotoxicity ~ CTL proliferated cells b (1) TcHST-2
12.11+11.31×Ill7
TcPUN ( 1 . 4 + 0 . 2 ) X 1117 (2150e~ of primary culture supernatant TcHST-2 (I.I _+11.2)>"Ill~: TcPUN (11.8_+0.1))
