Effective anti-tumor adoptive immunotherapy: utilization of exogenous IL-2-independent cytotoxic T lymphocyte clones

Michihiro Iwashiro1,2, Wang Jinyan3, Masaaki Toda3, Wang Linan3, Takuma Kato3 and Kagemasa Kuribayashi2,3

1 Department of Oral and Maxillofacial Surgery, and 2 Institute of Immunology, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan 3 Department of Bioregulation, Mie University School of Medicine, Mie 514-8507, Japan

The first two authors contributed equally to this work
Correspondence to: K. Kuribayashi, Department of Bioregulation, Mie University School of Medicine, 2-174 Edobashi, Tsu 514-8507, Japan. E-mail: keiyo{at}doc.medic.mie-u.ac.jp
Transmitting editor: M. Miyasaka


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To attain one of the final goals for cancer immunotherapy, cytotoxic T lymphocyte (CTL) clones were selected on the basis of exogenous IL-2 independence after limiting dilution culture from mixed lymphocyte tumor cell culture cells of FBL-3 tumor-immune spleens. About 10% of the clones could be propagated up to >5 times by weekly passages in the presence of splenic feeder and stimulating tumor cells. Two of the representative FBL-3-specific CTL clones that were able to undergo the fifth passage were expanded in large numbers for adoptive transfer by two rounds of a weekly passage with medium containing IL-2. FBL-3-specific CTL clones thus obtained showed a strong ability to eliminate the established tumors when transferred into tumor-bearing nude mice. In addition, the cells were recovered from the mouse spleen even 8 months after the transfer. The most striking differences between the CTL clones used in this experiment and those maintained conventionally in the presence of IL-2 were the abilities to produce IL-2 by themselves and the high expression level of the integrin molecule, VLA-4, that disappeared when cultured completely in the continuous presence of IL-2 in vitro during 12 weeks. In addition, concomitant with the disappearance of exogenous IL-2 independence and VLA-4 expression, the CTL clones lost their capacity to eradicate the tumor in vivo. Thus, the higher capacity of CTL clones to produce IL-2 on their own seemed to be correlated with the in vivo efficacy for tumor eradication and the long-term maintenance of their physiological profiles typical of memory T cells.

Keywords: adoptive transfer, IL-2-independent cytotoxic T lymphocyte clone, memory cytotoxic T lymphocyte, tumor eradication, VLA-4


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A variety of immunotherapy modalities are currently being exploited for the eradication of tumors in both human and murine systems. In most cases, the aim of the therapy is to induce tumor-specific cytotoxic T lymphocytes (CTL) that have the potential to eradicate tumors in vivo (13). In order to accomplish this, numerous approaches have been utilized for the enhancement of CTL generation. For instance, tumor cells can be engineered to produce cytokines such as IL-2 or IFN-{gamma}, which augment the immune responses against tumor cells either by bypassing Th cells or by increasing the MHC class I density on the tumor cell surface through the activation of an antigen-processing pathway respectively (4,5). In addition, various efforts are being made to produce better results through active immunization, i.e. utilizing a tumor vaccine in the form of tumor antigenic peptides, and their combination with dendritic cells (professional antigen-presenting cells) (6,7).

While the development of such tumor vaccines is indispensable, adoptive immunization has kept its advantageous aspects because tumor-specific effector T cells could be expanded in vitro almost indefinitely (8,9). Adoptive transfer of tumor-immune lymphocytes has extensively been utilized not only for human cancer therapy, but also as a model system for the characterization of effector cell populations in tumor-bearing mice (1012).

CTL lines or clones maintained for a long period in IL-2-containing medium, in many cases, showed little effect on tumor eradication in vivo despite the fact that their cytotoxic activities in vitro are much higher than that of spleen cells from tumor-bearing mice (13,14). This is true for both murine and human systems. Such ineffectiveness of the CTL lines or clones is thought to occur in the course of long-term culture in IL-2-containing medium, resulting from the changes in IL-2 requirements and of the surface molecules which cause the loss of normal migratory functions (15,16). Some studies, although few in number, have reported that CTL lines or clones which did not require the exogenous addition of IL-2 for their long-term maintenance in vitro were remarkably effective in eradicating tumor cells by adoptive transfer in vivo (11,17). However, in such studies, the characteristics of CTL clones were not described in detail.

In this study, we established CTL clones with no requirement for exogenous IL-2, expecting such CTL clones to exert in vivo efficacy for tumor rejection as reported earlier by us and others (11,17). Furthermore, the in vivo efficacy of CTL clones which were maintained for >5 weeks without exogenous IL-2 was tested by adoptive transfer into tumor-bearing nude mice. Consequently, CTL clones selected in such a manner were found to be remarkably effective for tumor rejection. They survived, and were recovered from spleen and peripheral blood several months after the adoptive transfer.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Breeding pairs of C57BL/6 (B6) nude (nu/nu) and their heterozygous (nu/+) euthymic mice were originally purchased from Clea Japan (Tokyo, Japan). The nude or their heterozygous mice were raised and maintained under specific pathogen-free conditions in the Facilities of Experimental Animals, Faculty of Medicine, Kyoto University.

Tumors
FBL-3 is a Friend murine leukemia virus-induced erythroleukemia cell line of B6 origin. The tumor was maintained in B6 nu/nu mice in their ascitic forms. FBL-3 tumor cells were highly immunogenic in that 10 x 106 cells were rejected in 4 weeks in syngeneic B6 mice (18,19). An unrelated tumor EL-4 (H-2b) was used in this study as a negative control in in vivo tumor inoculation experiments.

In vivo immunization with tumor cells
Six- to 12-week-old heterozygous B6 mice were s.c. inoculated on their backs with 5–10 x 106 FBL-3 cells in 0.1 ml HBSS. The mice were given up to four i.p. booster doses of 5 x 106 tumor cells before use.

Generation of FBL-3-specific CTL from spleen or peripheral blood mononuclear cells
One week after the complete regression of the tumor, mixed lymphocyte tumor cell culture (MLTC) was performed between FBL-3-immune B6-nu/+ spleen cells and 100 Gy-irradiated FBL-3 tumor cells in 2 ml of complete medium in 24-well multidish culture plates (Corning Glass Works, Corning, NY). The complete medium (CM) used in this experiment was composed of RPMI 1640 supplemented with 10% FCS (Gibco, Grand Island, NY) and 5 x 10–5 M 2-mercaptoethanol. The cultures were incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2 in air. After the incubation, the cells were harvested, washed and resuspended. When the adoptive transferred cells were recovered by MLTC from peripheral blood, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Lymphocyte Separation Solution (Nachalai Tesque, Kyoto, Japan) density gradient. PBMC (2 x 105 cells/well) were cultured in the presence of IL-2 (1 ng/ml) together with irradiated spleen cells (2.5 x 105 cells) and FBL-3 tumor cells (2 x 104 cells) for 7 days.

Limiting dilution (LD) cultures followed by expansion of CTL clones without IL-2
A LD culture of MLTC cells was carried out on a 1 cell/well basis in the presence of 2.5 ng/ml of human rIL-2 (TGP-3; Takeda, Osaka, Japan) together with irradiated (20 Gy) B6 spleen cells (5 x 105 cells) and irradiated (100 Gy) FBL-3 tumor cells (5 x 104 cells) in a final volume of 0.2 ml/well of a flat-bottomed 96-well microculture plate (Corning Glass Works). After the replenishment on day 7 with fresh medium containing IL-2, the cell number of growing cells derived from a single colony was counted on day 10. Cells (1 x 105) from each well were transferred to a well of 2-ml cultures, which consisted of 5 x 106 irradiated B6 splenic feeder cells and 2 x 105 irradiated FBL-3 tumor cells in CM not containing IL-2. At the second passage of CTL clones, aliquots of each cloned cells that yielded >106 cells/well were frozen for future use after culturing in IL-2 together with splenic feeder and FBL-3 tumor cells. IL-2-dependent CTL clones BFF9 and B413-4 were established and maintained as described elsewhere (18).

Cell-mediated cytotoxicity assay
The cytotoxic activity of the MLTC cells or CTL clones was determined using a standard 4-h 51Cr-release assay, as described elsewhere (19). The percentage of specific 51Cr release was calculated according to the following formula: (c.p.m. experiment – c.p.m. spontaneous)/c.p.m. total – c.p.m. spontaneous) x 100 (%).

Analysis of cell surface phenotype by flow cytometry
Cultured CTL and spleen cells were harvested, washed and suspended in PBS containing 2% FCS and 0.1% NaN3. Cells were stained with the following mAb. Anti-CD8{alpha}, anti-TCR{alpha}ß, anti-rat IgG (all conjugated with FITC), anti-V{alpha}3 (phycoerythrin-conjugated), anti-CD44 and anti-ICAM-1 mAb were purchased from PharMingen (San Diego, CA). N9-127 (anti-TCR idiotype) mAb was prepared as reported previously (18,19). Anti-CD2 (20), anti-LFA-1 (KBA) (21) and anti-VLA-4 (PS/2) mAb (22) were kindly donated by Dr Yagita and Dr Miyake respectively. Cells were analyzed on a FACScan flow cytometer.

Semiquantitative measurement of cytokine mRNA
Cytokine mRNA levels were determined by semiquantitative RT-PCR techniques as described elsewhere (23). On day 7 after the previous seeding, CTL clones were harvested and 1 x 106 cells in CM were plated in a well precoated overnight with anti-CD3 mAb (2 µg/ml), followed by incubation at 37°C for 12 h. Anti-CD3 mAb-producing hybridoma 145-2C11 was a gift from Dr Bluestone (24). The mAb was prepared by precipitation with ammonium sulfate from ascites and used for CTL activation after coating the wells of a 24-well multidish at a concentration of 2 µg/ml in PBS at 4°C for 16 h.

Adoptive transfer
Various doses of CTL clones in 1 ml of sterile HBSS were injected i.v. into the tail vein of B6 nude mice, onto the backs of which 1 x 106 FBL-3 tumor cells had been inoculated s.c. 1 day before unless specified. The mean perpendicular diameter of tumors was measured every 3–4 days to assess the tumor growth.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Selection of CTL clones
MLTC cells were cloned in the presence of IL-2 on 1 cell/well basis for 7–10 days. Growing cells were used only from wells originating from a single colony as determined from microscopic observations during days 4–7. Subsequently, 1 x 105 cells were transferred to each well of a 24-well multiplate in the absence of IL-2 as described in Methods. Cells (1 x 105) were seeded at a weekly interval together with irradiated FBL-3 tumor and syngeneic splenic feeder cells. At every passage, recovered cell number was counted to determine duration of cell survival without the addition of IL-2. At the second passage, the cloned cells from a well showing >10-fold (1 x 106 cells/well) increase in cell number were frozen for future use. One of the representative experiments is shown in Table 1. Up to 4 weeks after LD, ~30% (7/22) of the CTL clones survived, but following incubation for a further week, only two clones (9%) remained. A similar pattern of cell survival was observed in the other two experiments (data not shown). Among seven CTL clones (out of 78 clones examined) which had survived for >5 weeks in three experiments, we selected two CTL clones referred to as BL5-9 and BLM2-8 (clone nos 12 and 14 in Table 1 respectively). BL5-9 and BLM2-8 were Thy-1.2+, CD4, CD8+ and expressed TCR composed of V{alpha}3Vß5 and V{alpha}1Vß10 [(127 idiotype (Id)+] (18,19) respectively (data not shown). The in vitro cytotoxic activities of these clones were rather low (35–50% specific cytotoxicity at an E:T ratio of 40 for both CTL clones). The two CTL clones are denoted hereafter as ‘IL-2-independent’ in this report for convenience. V{alpha}3+ and 127Id+ CTL clones BFF9 and B413-4 respectively were used as controls for exogenous IL-2-dependent CTL clones (18). In addition, all these CTL clones recognized Friend leukemia virus gag-encoded sequence as reported previously (2527).


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Table 1. Cell numbers recovered on weekly passages of CTL clones without addition of exogenous IL-2
 
The growth of CTL clones BL5-9 and BLM2-8 in the presence or absence of exogenous IL-2
The proliferative capacities of CTL clones BL5-9 and BLM2-8 were examined on the third passage in the presence or absence of exogenous IL-2. Both CTL clones proliferated more vigorously than the IL-2-dependent CTL clones in the presence of exogenous IL-2 (Fig. 1A). In addition, BL5-9 showed faster proliferation in 1 day than BLM2-8 and IL-2-dependent CTL clones. While the conventional IL-2-dependent CTL clones were unable to grow at all without IL-2, CTL clones BL5-9 and BLM2-8 showed ~8-fold increase in cell number around day 5–6 respectively after seeding at 2 x 105 cells/well (Fig. 1B). The proliferation was inhibited by the simultaneous addition of anti-IL-2R{alpha} (PC61) and -IL-2Rß (TMß1) mAb (data not shown). Both of the IL-2-dependent CTL clones required at least 250 pg/ml of exogenous IL-2 to survive at most for 5 days without any increase in their cell number (data not shown). These results suggested that the two IL-2-independent clones had the capacity to secrete IL-2 and stimulate their proliferation in autocrine and paracrine fashions. In fact, the mRNA level of IL-2, not IL-4, was increased 12 h after anti-CD3 mAb stimulation of day 7 CTL clones. BL5-9 exhibited much higher levels than BLM2-8 (Fig. 2).



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Fig. 1. Time course of recovered cell numbers of CTL clones in the presence (A) and absence (B) of any exogenous IL-2 (2.5 ng/ml) supplement. Cells (2 x 105) of each CTL clone (open squares: BL5-9; open circles: BLM2-8; solid circles: BFF9 and solid squares: B413-4) were seeded in a well of 24-well multidish together with irradiated splenic feeder (5 x 106 cells/well) and FBL-3 tumor cells (2 x 105 cells/ well). Recovered cell numbers were counted on the days indicated.

 


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Fig. 2. Changes of IL-2 and IFN-{gamma} mRNA levels before and after immobilized anti-CD3 mAb stimulation. mRNA levels of day 7 CTL clones BL5-9 (lanes 1 and 3) and BLM2-8 (lanes 2 and 4) were examined by RT-PCR before (lanes 1 and 2) and 12 h after (lanes 3 and 4) the stimulation with immobilized anti-CD3 mAb. Lane 5 represents the levels of IL-4 expression of Th2 clone 24-2 (19) which was activated in the same manner. The Th2 clone was used as positive control for IL-4.

 
In vivo efficacy of CTL clones BL5-9 and BLM2-8 by adoptive transfer
It is generally known that conventional CTL clones which required exogenous IL-2 for their maintenance showed conspicuous tumor-specific cytotoxic activities in vitro, whereas only marginal prolongation of survival of the tumor-bearing mice was observed when transferred adoptively (Fig. 3A versus B) (13,14). On the other hand, both of the IL-2-independent CTL clones, BL5-9 and BLM2-8, exerted remarkable dose-dependent effects that led to tumor eradication when transferred adoptively into tumor-bearing B6 nude mice on day 1 of tumor inoculation (Fig. 3C and D). However, CTL clone BLM2-8 required 10 times more cells (3 x 107 cells) than BL5-9 (3 x 106 cells) to eradicate the FBL-3 tumor by adoptive transfer (Fig. 3C versus D). The rejection of the tumor was specific in that an unrelated tumor EL-4 grew progressively in all the mice that were treated in a similar fashion (data not shown). Therefore, CTL clones BL5-9 were used in most of the following experiments. In addition, the other five IL-2-independent CTL clones showed similar characteristics in tumor rejection by adoptive transfer (data not shown).



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Fig. 3. FBL-3 tumor eradication by CTL clones in B6 nude mice by adoptive transfer. B6 nude mice were inoculated s.c. with 1 x 106 FBL-3 tumor cells. Mice were subsequently treated on day 1 without CTL clone (A), with exogenous IL-2-dependent CTL clone BFF9 (B) or with IL-2-independent BLM2-8 (C) and BL5-9 (D). The numbers above each panel represent the numbers of each CTL clone transferred to each recipient. The indicated number of cells in 1 ml HBSS was injected i.v. as indicated by an arrow. Each line represented the tumor growth in a B6 nude mouse of each group. A representative of two experiments is shown.

 
Complete regression of established tumors by IL-2-independent CTL clone BL5-9
Next we tested the extent of BL5-9 potency in eradication of the FBL-3 tumor. B6 nude mice were left untreated until the 1 x 106 FBL-3 cells inoculated s.c. grew to ~1 cm in diameter around day 12. Subsequently, 1 x 107 BL5-9 cells were adoptively transferred into the tumor-bearing nude mice. As shown in Fig. 4(A and B), following the central necrosis around 12 days post-transfer (on day 24 after tumor inoculation), rapid disappearance of the tumors was observed in all the mice tested. Another 12 mice treated similarly showed the same results (data not shown).




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Fig. 4. Established FBL-3 tumor was rejected by adoptive transfer of CTL clone BL5-9. (A) The results of tumor rejection by adoptive transfer of CTL clone BL5-9 (1 x 107 cells) on day 12 when the inoculated tumor (1 x 106 cells) reached ~1 cm in diameter. The days in (A) indicate those after the tumor inoculation. (B) The tumor growth of untreated B6 nude mice (a) and adoptively transferred mice with CTL clone BL5-9 (1 x 107 cells) on day 1 (b). (c) Tumor growth in the same group as shown in Fig. 4(A).

 
Long-term survival and maintenance of tumor-rejection capacity of CTL clones transferred into B6 nude mice
Adoptive transfer of 1 x 107 cells of IL-2-independent CTL clone BL5-9 into tumor-bearing B6 nude mice endowed the mice with the ability to reject FBL-3 tumors as was shown in Fig. 3. The transferred CTL clones survived for a long time, maintaining their capacity to reject the second challenge of the same but not unrelated tumor. An example is demonstrated in Fig. 5. Even 5 months after the adoptive transfer, all the mice rejected the second inoculation of the FBL-3 tumor in a similar pattern to that of the first rejection (Fig. 5B). However, an unrelated EL-4 tumor was never rejected (Fig. 5A). Consequently, we examined the duration that the transferred cells remained in spleens of normal B6 nude mice, using the anti-TCR mAb as markers. CTL clones (30 x 106 cells) were injected into normal B6 nude mice. Figure 6 shows that about half of the BL5-9 (V{alpha}3+) and one-fourth of BLM2-8 (127Id+) of the adoptively transferred IL-2-independent, but not IL-2-dependent CTL clones were found in normal B6 nude mice on day 7, in which CTL clone BL5-9 was detected even 60 days after the transfer (18). In accordance with these results, in the spleen of a mouse that had rejected the established tumors as shown in Fig. 4, ~2.7% of the spleen cells expressed the same V{alpha}3+ TCR as the transferred CTL clone BL5-9 even 8 months after the rejection (Fig. 7A versus B). In such mice, the mean percentage of CD8+V{alpha}3+ T cells in total spleen cells was 1.2 ± 0.9% (n = 4). Such cells were expanded by MLTC cultures for 5 days in CM without the addition of exogenous IL-2 (Fig. 7C) and were demonstrated to express Vß5 similar to BL5-9 (data not shown). Not only using those from the spleen cells but also the peripheral blood lymphocytes of the same mice, the transferred CTL were detected and propagated by two rounds of MLTC, in this case in the presence of exogenous IL-2 (1 ng/ml) (Fig. 7D). In addition, the CTL clone BL5-9 thus expanded from the spleen by up to 2–3 weekly passages in the presence of IL-2 (2.5 ng/ml) was again effective in vivo to the same degree as the parental CTL when injected i.v. into FBL-3 tumor-bearing B6 nude mice (data not shown). Such characteristics of a long-term lifespan accompanying in vivo efficacy was also the case, but less effectively, for another CTL clone, BLM2-8 (data not shown). Neither normal spleen cells nor peripheral blood lymphocytes led to the expansion of CD8+V{alpha}3+ T cells (data not shown).



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Fig. 5. Long-term maintenance of the specific tumor rejection capacity of tumor-rejecting B6 nude mice. The B6 nude mice which had rejected the FBL-3 tumor as shown in Fig. 3(D) (1 x 107 cells on day 1) had the capability to reject the second challenge of the same FBL-3 tumor even at 150 days (B), but not unrelated tumor EL-4 (A). In a similar experiment for BLM2-8 (3 x 107 cells on day 1), about half were unable to reject the second challenge of FBL-3 tumors at 150 days (data not shown).

 


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Fig. 6. The existence of large numbers of IL-2-independent CTL clones in B6 nude mouse spleens. Thirty million IL-2-dependent [BFF9 (A) and B413-4 (B)] and IL-2-independent [BL5-9 (C) and BLM2-8 (D)] CTL clones were injected i.v. into normal B6 nude mice (nine mice per group). The percentages of TCR V{alpha}3+ (A and C) and 127Id+ (B and D) cells were determined in CD8+ spleen cells by flow cytometry. Subsequently, actual numbers of transferred CTL clones were calculated on the basis of the number of spleen cells from the respective mice. Each point indicates the mean of three mice ± SD.

 


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Fig. 7. Recovery of the transferred CTL clones from the tumor-rejecting B6 nude mice. No significant V{alpha}3+ cells were found in normal B6 nude mice (A). From the spleen of a mouse that was shown in Fig. 4, a significant number of V{alpha}3+ cells was detected 8 months after the adoptive transfer. (B). The cells could be expanded by MLTC for 5 days without the addition of exogenous IL-2 (C). In the peripheral blood of the same mouse as in (C), <1% but a significant number of CD8+V{alpha}3+ cells was found. These cells could be expanded by two rounds of 5-day MLTC only by IL-2-containing media (1 ng/ml) as shown in (D).

 
Correlation of the in vivo efficacy of CTL clones with the expression level of VLA-4 and its down-regulation by long-term culture in IL-2
Expression levels of certain cell adhesion molecules have been shown to correlate intimately with the in vivo migration of the transferred T cells (15,16). Therefore, we compared the expression level of several cell surface molecules between IL-2-dependent and -independent CTL clones (Fig. 8). Although CD44 expression was minimally reduced in the IL-2-dependent CTL clone, the most conspicuous difference was noted for the expression level of VLA-4 between the two types of CTL clones. Both of the IL-2-independent CTL clones exhibited significant levels of VLA-4 molecules on their surfaces, whereas no or little VLA-4 molecules were observed on all of the IL-2-dependent CTL clones that we examined in addition to B413-4 and BFF9 (data not shown). Expression of VLA-4 was down-regulated almost completely during 3–4 months by culture with IL-2 at the concentration of 2.5 ng/ml, which was normally used for CTL maintenance (Fig. 9A). Together with the loss of VLA-4 expression, these cells became unable to grow without the addition of exogenous IL-2 (data not shown). Simultaneously, they were deprived of the ability to eradicate tumors in vivo (Fig. 9B).



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Fig. 8. Comparison of cell surface markers between IL-2-dependent and IL-2-independent CTL clones. Cell surface markers as indicated above each panel were stained by the corresponding mAb and analyzed by FACScan. Expression levels of the surface molecules were compared between IL-2-dependent (B413-4 and BFF9) and IL-2-independent CTL clones (BL5-9 and BLM2-8).

 


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Fig. 9. Loss of VLA-4 expression in IL-2-independent CTL clones incubated with IL-2. IL-2-independent CTL clones (BL5-9 and BLM2-8) completely lost their VLA-4 expression by culturing in the presence of IL-2 (2.5 ng/ml) after 12 weeks together with irradiated splenic feeder and FBL-3 tumor cells (A). In the course of this culture, both CTL clones became IL-2 dependent (data not shown) and unable to exert anti-FBL-3 tumor activity in vivo (B). VLA-4+ (middle panel) and VLA-4 (right panel) CTL clones of BL5-9 (1 x 107) were injected i.v. into FBL-3 tumor-bearing B6 nude mice on day 1 post-tumor cell inoculation. Arrows indicate the day of adoptive transfer.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cancer therapy by adoptive transfer has been widely used in experimental models as well as in clinical situations such as melanoma and solid cancer treatment (10,28,29). Judging from the experimental models, which resulted in successful tumor rejection, a common feature emerged that no exogenous IL-2 was required to maintain the CTL for adoptive transfer. Klarnet et al. used a CTL clone that secreted IL-2 on their own and was maintained for a long time without exogenous IL-2 (17,30). Previously, we established CTL lines that consisted of two populations, T cell lymphoma RL1-specific CTL and autoreactive Th cells (11). In the latter cell line, the Th population responded to self-MHC class II of the splenic feeder cells and secreted IL-2 to assist in the maintenance of long-term survival of the tumor-specific CTL population in concert with Th cells themselves. These cell lines were effective for tumor eradication in vivo and were maintained for a long time in the absence of exogenous IL-2. Taking these studies into consideration, exogenous IL-2 independence seemed to be one of the main prerequisites for the tumor-specific CTL clones to function in vivo physiologically. Thus, we attempted to isolate ‘IL-2-independent’ CTL clones and demonstrated in this study that CTL clones that survived without IL-2 addition for >5 weeks after LD were effective in tumor eradication by adoptive transfer. These CTL clones secreted IL-2 by themselves. This study suggested that the effectiveness in vivo seemed to be correlated with two characteristics of the CTL clones. One was the ability to produce IL-2 on their own. The other was the expression of the ß1 integrin VLA-4.

Both BL5-9 and BLM2-8 CTL clones secreted IL-2 as was evidenced by RT-PCR and proliferation inhibition with anti-IL-2R{alpha} and -IL-2Rß mAb. Although one cannot say that in vivo activity by adoptive transfer is correlated to memory phenotypes, the CTL clones that we isolated exhibit certain characteristics for memory CTL in that they survived and recirculated systemically for a long period after adoptive transfer, and expressed high levels of memory marker CD44 on their surfaces. In addition, higher and faster proliferation in response to the antigen was thought to be linked to memory function (3133). With regard to this, the degree of exogenous IL-2-independent proliferative capacity in vitro, IL-2-producing capability and in vivo antitumor activity after adoptive transfer might be mutually correlated with each other as was observed for CTL clones BL5-9 and BLM2-8. Accordingly, studies to confirm the correlation are now in progress.

Concerning the second characteristic, the expression level of VLA-4 on the cell surface was quite different between IL-2-independent CTL clones and exogenous IL-2-dependent CTL clones that were ineffective for tumor eradication by adoptive transfer. VLA-4 is thought to play an essential role in migration of lymphocytes to the site where target molecules such as viruses and tumors are expressed (34,35). Although this molecule is also utilized as a memory cell marker, to our knowledge it has not been reported so far that VLA-4 expression on CTL was down-regulated in the course of culture with IL-2. In this study, we showed that the loss of VLA-4 molecules in the presence of exogenous IL-2 occurred concomitantly with the IL-2 dependency in vitro and loss of in vivo efficacy of CTL clones. All the exogenous IL-2-dependent CTL clones that we examined completely lost VLA-4 expression and effectiveness for tumor rejection in vivo (Fig. 8 and unpublished data). Thus, both properties of CTL clones were suggested for CTL clones to play pivotal roles in self-maintenance and physiological migration in vivo (3639).

The two IL-2-independent CTL clones in this study occupied about half to one-fourth of the normal nude mouse spleen cells on day 7 after the adoptive transfer of 3 x 107 cells. Surprisingly, in addition, even 8 months after the adoptive transfer into tumor-bearing mice, significant numbers of the spleen cells consisted of the CTL clones that had been transferred (Fig. 7). Such a long lifespan might be inherent in the CTL clones such as BL5-9 and BLM2-8. However, most of the transferred CTL underwent a demise after rejecting the tumor. This might be due to the heterogeneity in their survival capacity, despite their clonal origin. The heterogeneity would be generated during their clonal expansion both in vitro and in vivo in the capacity of IL-2 secretion, the expression levels of cell surface molecules required for physiological lymphocyte migration and in the functional level of cell survival-determining machinery such as Bcl-2 versus Bad (33,40).

Most of the heterogeneities generated in such ways would be disadvantageous to the survival of transferred cells. However, a small size population might enjoy benevolent influences from the signals that they were given from the outside milieu (41,42). For example, many cytokines such as IL-4, IL-7 and IL-15 have been reported to function as T cell survival-promoting factors. Such cytokines are ubiquitous in the lymphoid organs. Despite the demise of most transferred cells, a limited population that was receptive to such survival-promoting cytokines might survive as memory cells (42).

In contrast to the systemic circulation ability of the IL-2-independent CTL clones, exogenous IL-2-dependent CTL clones did not migrate to spleen and peripheral blood. Most of the cells were trapped in the liver and destroyed within a few days (37,38 and our unpublished data).

Taking all these results into consideration, this study suggested that it is possible to select CTL clones effective for tumor eradication in vivo on the basis of the degree of their IL-2 secretion. Since the down-regulated expression of VLA-4 seemed to be the secondary event following culture of CTL clones with IL-2, the capacity to secrete IL-2 on their own seemed to be of prime importance in such selection. However, the incidence of establishing IL-2-independent CTL clones might be much lower when tumors with an average immunogenicity were used, instead of highly immunogenic FBL-3. The model in this report might provide us with the system to analyze the fate and characteristics of memory CTL in vivo at a clonal level. In fact, the mice that were transferred with CTL clone BL5-9 could be utilized as long-term providers of in vivo effective CTL clones for tumor eradication. In addition, the higher capacity of IL-2 secretion by CTL clones or lines might be predictive for judging whether the CTL would be effective in vivo for human cancer treatment by adoptive transfer.


    Acknowledgements
 
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.


    Abbreviations
 
CM—complete medium

Id—idiotype

LD—limiting dilution

MLTC—mixed lymphocyte tumor cell culture


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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