Committed memory effector type 2 cytotoxic T (Tc2) cells are ineffective in protective anti-tumor immunity

Immunology Letters 95 (2004) 77–84

Committed memory effector type 2 cytotoxic T (Tc2) cells are ineffective in protective anti-tumor immunity Jeong-Su Doa , Youn-Hwa Choia , Sung-Hye Shinb , Ho Keun Yic , Pyung Han Hwangc , Sang-Yun Nama,∗ a

Department of Biological Science, School of Science and Technology, Jeonju University, Jeonju 560-759, South Korea b Christian Medical Research Center, Presbyterian Medical Center, Jeonju 560-750, South Korea c Department of Pediatrics, Jeonbuk National University Medical School, Jeonju 560-712, South Korea Received 19 March 2004; received in revised form 16 June 2004; accepted 17 June 2004 Available online 14 July 2004

Abstract Cytotoxic CD8+ T cells (Tc) are a major effector cell population in protection against tumor growth and classified into Tc1 or Tc2 based on their cytokine-secreting profiles. However, their relative tumor protective roles remain undefined. In the present study, CD8+ memory T cells were obtained from mice given with CT26-IL 12 and tumor-specific Tc1 and Tc2 cells were induced by in vitro primary stimulation (1◦ ). In vivo anti-tumor immunity and in vitro cytotoxicity of 1◦ Tc2 memory effector cells were highly protective comparably to 1◦ Tc1, but they secreted high level of IFN␥ as well as IL 4 and IL 5. Moreover, memory cells obtained again from tumor-protected mice by either 1◦ Tc1 or Tc2 transfer showed indistinguishable, Tc1-like, cytokine profiles. These results strongly suggest that 1◦ Tc2 cells are insufficiently polarized. Tc2 memory effector cells were therefore examined for their transitional anti-tumor activity during consecutive stimulation until Th2 commitment. Secondary stimulation (2◦ ) markedly reduced secretion of IFN␥ (by 94%) and in vivo tumor protection (by 83%). Tertiary (3◦ ) and further stimulation completely abrogated both of tumor protective activity and IFN␥ secretion of Tc2 cells. This progressive loss of activity following repeated stimulation was accompanied by a reduction of in vitro cytotoxicity to CT26 tumor cells. In addition, when 1◦ Tc2 cells were trans-differentiated to Tc1 during secondary stimulation, 2 of 6 cultures recovered tumor protective activity concomitantly with IFN␥ secretion, indicating that repeated stimulation does not deteriorate tumor protective activity of 2◦ Tc2 cells. Collectively, these data demonstrate that highly committed Tc2 cells are ineffective in tumor protection. © 2004 Elsevier B.V. All rights reserved. Keywords: CD8; T cells; Tc1/Tc2 cells; Memory cells; Anti-tumor activity

1. Introduction Although tumor protection can be successfully mediated by a variety of immune mechanisms, it has become apparent that CD8+ T cells are a major immunological effector cell population mediating resistance to cancer. In support of this concept, the adoptive transfer of anti-tumor CD8+ T cells results in delayed tumor growth [1–3], whereas deletion of CD8+ T cells exacerbates tumor growth [4]. In addition, strategies to activate CD8+ T cell immune responses,

∗ Corresponding author. Tel.: +82 632202518; fax: +82 632202054. E-mail address: [email protected] (S.-Y. Nam).

0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.06.006

e.g., vaccination with dendritic cells, also induce protection against tumor growth [5–7]. Analogous to the common terminology of type 1 and 2 helper T (Th1 and Th2) cells for the CD4+ compartment [8], T cytotoxic (Tc)1 and Tc2 cells can be distinguished within CD8+ cells in vitro [9,10] and in vivo [11,12] by their characteristic cytokine secretion profiles. Tc1 cells secrete IFN␥ and TNF␣, but Tc2 cells, in contrast, produce IL 4, IL 5 and IL 10 [9,10]. Cytokines secreted by CD8 subpopulations can contribute to anti-tumor reactivity by direct and indirect mechanisms. For instance, IFN␥ and TNF␣, and IL 4 can modulate surface antigen expression, which may lead to enhanced immune response to cancer [12–15] and/or altered growth kinetics of tumor cells [16–18]. As main effector

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cells, both of CD8+ Tc1 and Tc2 can kill tumor cells comparably via perforin-dependent pathway [19,20], although Fas-mediated cytotoxicity is exerted only by Tc1 [19,20]. Several reports have shown that adoptive transfer of either Tc1 or Tc2 effector cells induce tumor protection and increase survival times in tumor-bearing mice via distinct mechanism [21–23]. However, other reports argue that tumor-specific Tc2 cells have little or no effect on tumor growth, contrary to Tc1 which exerts strong anti-tumor activity [24–26] and type 2 cytokine inhibits CD8+ effector cell activity [15,27]. Consequently, it still remains uncertain whether only CD8+ Tc1 or both type cells are effective in in vivo anti-tumor protection. Given that vaccination strategies have become a promising immunological approach to cancer therapy, relative in vivo activity of memory Tc1 and Tc2 requires more precise definition in order to design a vaccine with the highest efficacy. We considered that most studies deal with in vitro activated Tc1 and Tc2 cells for comparison of their activity, and the resulting cells are virtually polarized via a single in vitro stimulation. In the present study, using memory cells obtained from mice in vivo immunized with IL 12-transfected live tumor cells, it was found that CD8+ T cells stimulated primarily under Th2-favoring conditions secreted high level of type 1 cytokine, IFN␥. We therefore sought to determine whether memory Tc2 cells have potential to protect against tumor growth prior to their final commitment and show here that committed memory effector Tc2 cells no longer exhibit in vitro and in vivo anti-tumor activity in our CT26-IL 12 tumor model. This understanding of Tc1 and Tc2 cell profiles should support the optimization of vaccine strategies.

2. Materials and methods 2.1. Animals Male BALB/c 5–6-week-old mice were purchased from Korean Research Institute for Chemical Technology (KRICK). The mice were maintained in an environmentally controlled chamber and used when 7–8 weeks old. 2.2. Tumor cell lines and in vitro culture CT26 colon adenocarcinoma cells were maintained in culture in IMDM medium supplemented with 10% heat inactivated FBS (Moregate, Melbourne, Australia), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 250 ng/ml amphotericin B (Sigma, St. Louis, MO). 2.3. Generation of IL 12 expressing tumor (CT26-IL 12) cells Construction of pAIL 12-p35 and pAIL 12-p40 and transfection was performed as previously described [28].

Briefly, fusion plasmids, pAIL 12-p35 and pAIL 12-p40 were constructed by inserting the 0.7 kb SalI–EcoRI fragment (p35) or 1.06 kb SalI–BamHI fragment (p40) encoding the murine IL 12 subunit p35 and p40, respectively, into the Sal I site of the pH␤-actin-1-Neo vector DNA. Electroporation was initiated by resuspending cells at a concentration of 3 × 106 cells/0.8 ml in DMEM containing 10% FBS at RT. Plasmid DNAs, 15 ␮g each, were added to the cell suspension, incubated at RT for 10 min, and followed by electroporation (Bio-Rad, Richmond, CA) and incubation at RT for 10 min. The cell suspension was transferred to 10 ml DMEM. Electroporated cells were incubated at 37 ◦ C for 48 h and washed twice with PBS and then seeded in 24-well chambers at a density of 5 × 103 cells/well in the presence of G418 (400 ␮g/ml). Only clones from wells containing single clone were selected for further analysis and individual transfectant clones were tested for expression of IL 12 using ELISA. Selected clones usually secreted 1.8–3.7 ng/ml of IL 12 during the course of 24 h. 2.4. Tumor immunization and tumor lysate BALB/c mice were injected i.p. with CT26-IL 12 and 4 weeks later, surviving mice were challenged with wild type (WT) CT26 cells by s.c. at footpads and tail base. Ten days after challenge, mice were sacrificed for CD8+ T cell preparation. Tumor lysate was used for in vitro antigenic stimulation of memory Tc cells. Briefly, tumor cells obtained by mechanical detachment were lysed by three freeze–thaw cycles in sterile phosphate-buffered saline (PBS; pH 7.4; 1 × 107 cells/ml). Lysate was homogenized for 10 s three times using an ultrasonic homogenizer and cell debris was removed by centrifugation and membrane filtration. The protein concentration of lysate was assessed using a BCA protein assay kit (Pierce, Rockford, IL) and concentrated to 2 ␮g/ml. Aliquots of this lysate mixture were used for stimulation of CD8+ T cells at 1/100 dilution. 2.5. In vitro generation of memory effector Tc1 and Tc2 cells Draining, inguinal and popliteal lymph node cells were pooled from immunized mice and incubated at 37 ◦ C for 1 h in culture dishes to eliminate adherent cells before purification. CD8+ T cells were purified using the MACS magnetic separation system according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were resuspended at a concentration of 107 cells/90 ␮l in PBS containing 0.5% FBS. CD4+ and B cells were depleted by incubating with 10 ␮l of anti-CD4 (clone GK1.5) and anti-B220 Ab-conjugated microbeads/107 cells (clone M5/114.15.2) at 4 ◦ C for 15 min and separation in a magnetic column. Remaining CD4+ and MHC class II+ cells were further depleted by incubation of cells with anti-CD4 (L3T4) and anti-MHC

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class II (M5114) antibodies plus complement at 37 ◦ C. The freshly isolated CD8+ T cells were typically 95% pure as demonstrated by immunofluorescence. For Tc1 cell generation, CD8+ T cells (1 × 106 cells/ml) were stimulated with CT26 lysate (1/100) in the presence of IL 2 (20 U/ml; supernatant from the X63Ag.IL 2 murine cell line), mouse recombinant IL 12 (5 ng/ml; R & D Systems, Minneapolis, MN) and anti-IL 4 mAb (1 ␮g/ml; 11B11). For Tc2 cell generation, IL 2 (20 U/ml), IL 4 (5 ng/ml; PeproTech, Rocky Hill, NJ) and rat anti-IFN␥ mAb (1 ␮g/ml; CalTag Laboratories, Burlingame, CA) were added. B cell-enriched spleen cells following plastic adherence were used for antigen presenting cells after treatment with 50 ␮g/ml of mitomycin C for 15 min and washing four times with IMDM containing 5% FBS. Those cells were designated as primary stimulated (1◦ ) Tc1 or Tc2 cells. Tc2 cells were rested for 2 days and then restimulated (2◦ ) in the presence of IL 4 and antiIFN␥. For further stimulation (3◦ or 4◦ ), cytokines and antibodies were no longer included, but relied on CT26 lysate exposure. 2.6. Adoptive transfer and immunotherapy model BALB/c mice received up to 5 × 106 in vitro generated memory effector Tc1 or Tc2 cells through the lateral tail vein. Twenty-four hours later, mice were challenged with 5 × 105 CT26 cells injected s.c. into the left flank and tumor growth was measured every other day for 12 days. Mean tumor size was calculated as the mean product of bisecting tumor diameters. 2.7. Cytokine secretion assay Culture supernatants were collected for determine cytokine secretion. Levels of IL 2, IL 4, IL 5, IL 12 and IFN␥ were assayed by ELISA according to the manufacture’s instructions. Antibody pairs and standards were purchased from Biosource (Camarillo, CA, USA) (for IL 4, IL 12 and IFN␥) or BD Pharmingen (San Diego, CA) (for IL 5 and IL 12). 2.8. In vitro CTL assay Cytotoxicity against targets was quantified using a 4 hrelease assay in triplicate. Target cells were labeled with Na2 51 CrO4 (NEN, Boston, CA) for 90min at 37 ◦ C. After washing, 51 Cr-labeled target cells were incubated with cultured CD8+ effector cells at different E:T ratios in 96 well round-bottom plates. After 4 h incubation at 37 ◦ C with 5% CO2 , culture supernatants were harvested and counted in a gamma-counter. Spontaneous release of 51 Cr was determined by incubation of targets in the absence of effectors, whereas maximum release of 51 Cr was determined by incubation of targets in 1% Triton X-100. Results are expressed as the percent specific release and were calculated as follows: 51 Cr

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Table 1 Tumor protection and cytokine secretion by primary stimulated (1◦ ) memory effector Tc1 and Tc2 cells Type Tumor protection (%) Tc1 Tc2 a b c

(5/5)b

100 100 (5/5)

Cytokine secretion (pg/ml)a IL 2

IFN␥

IL 4

IL 5

1283 ± 121 15,267 ± 933 NDc 1220 ± 121 13,172 ± 1,253 946 ± 71 1113 ± 202 NDc

Mean ± S.E. from 4 to 6 mice. Number of mice protected/ number of total mice tested. Not detectable.

% specific lysis = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. For tumor-specific CTL and non-specific NK cell activity assay, wild type CT26 and Yac-1 cells were used for target cells, respectively.

3. Results Table 1 shows in vivo tumor protective activity of transferred 1◦ Tc1 and Tc2 memory effector cells and their in vitro cytokine secretion. Both of in vitro generated 1◦ Tc1 and Tc2 memory effector cells are highly effective for tumor protection. Tc1 cells secrete high amounts of IFN␥ (15.3 ng/ml), but non-detectable amounts of neither IL 4 nor IL 5. However, Tc2 cells also secrete comparably high level of IFN␥ (13.2 ng/ml) as well as IL 4 (946 pg/ml) and IL 5 (1.1 ng/ml). Matching production levels of IL 2 (1.28 versus 1.22 ng/ml) indicates that 1◦ Tc1 and Tc2 cells are basically comparable in their biological activities. These results suggest that Tc2 cell polarization was insufficient and prompted us to confirm Tc2 cell polarization after adoptive transfer. Based on the previous finding that polarized CD8 T cells can persist in producing type 1 and type 2 cytokines in vivo [29], we analyzed cytokine profiles of memory effector Tc1 and Tc2 cells, which were transferred and then harvested from the adoptive hosts 1 month later. As shown in Table 2, in vivo persisting memory Tc cells produced high levels of IFN␥ (5.5–5.8 ng/ml) and low level of type 2 cytokines regardless of the transferred Tc cell type. Their indistinguishable cytokine profiles suggest that both types of persisting memory Table 2 Cytokine secretion by in vivo persisting memory Tc1 and Tc2 cellsa obtained from adoptive hosts Type of transferred cells

Cytokine production (pg/ml)b IL 2

IFN␥

IL 4

IL 5

Tc1 Tc2

670 ± 86 790 ± 88

5856 ± 682 5582 ± 605

23 ± 4 24 ± 3

66 ± 7 83 ± 8

a CD8+ T cells were obtained from the mice protected by transfer of memory effector Tc1 and Tc2 cells 1 month after tumor challenge and in vitro stimulated with tumor antigen in the absence of cytokine and antibody addition for 3 days. b Mean ± S.E. from five mice.

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cells were Tc1-like, because production of IL 4 and IL 5 were much lower than those prior to transfer (24/946 and 83/1113 pg/ml, respectively), when compared to decrease in IL 2 and IFN␥ production (790/1220 and 5582/13172 pg/ml, respectively) (Tables 1 and 2). The above results indicated that 1◦ Tc2 cells were not fully polarized and determination of precise anti-tumor activity of Tc2 cells requires evaluation after commitment. Tc2 cells were therefore investigated for their in vivo anti-tumor activity during consecutive stimulations with tumor antigen during this commitment. In addition, their cytokine secretion profiles were also assessed in parallel. As shown in Fig. 1A, secondary stimulation (2◦ ) of Tc2 cells markedly reduced IFN␥ production by 93% (Fig. 1A) and protective anti-tumor activity by 84% (Fig. 1B), whereas IL 2 and type 2 cytokine secretion was not significantly altered. Moreover, tertiary (3◦ ) and further stimulation of Tc2 cells completely abrogated tumor protective function (Fig. 1B) and concomitantly reduced IFN␥ secretion to the basal level (Fig. 1A). This progressive loss of tumor protection following consecutive stimulation of Tc2 cells was also regained by measuring of tumor size in unprotected mice. As shown in Fig. 1C, inhibition of tumor growth by Tc2 cells was gradually dampened during repetitive stimulations. Such progressive loss of in vivo anti-tumor activity of Tc2 cells was also directly related with in vitro cytotoxicity to WT CT26, as shown in Fig. 2A. To dissect the involvement of nonspecific NK cells or LAK cells in this cytotoxicity, we performed the same procedure against Yac-1 targets in parallel. As shown in Fig. 2B, cytotoxic activity of Tc1 and Tc2 to Yac-1 cells was minimal, demonstrating that our data reflect cytotoxic activity of highly antigen-specific CTL, but not non-specific NK cells or LAK cells. Our above results raise a question whether prolonged in vitro stimulation can reduce anti-tumor activity of Tc2 cells. To examine this possibility, 1◦ Tc2 cells were transdifferentiated to Tc1 (1◦ Tc2 → 2◦ Tc1) and then compared to 2◦ Tc2 cells for their in vivo tumor protection and cytokine secretion. As shown in Fig. 3, trans-differentiated (1◦ Tc2 → 2◦ Tc1) cells produced relatively higher in vitro IFN␥ secretion when compared to 2◦ Tc2 cells but secreted high levels of IL 4 and IL 5 (Fig. 3A). Their in vivo antitumor activity (33%) (Fig. 3B) and in vitro cytotoxicity (Fig. 3C) was sustained only partially, but not completely, when compared to 1◦ Tc1 cells. These data suggest that loss of IFN␥ productivity renders Tc2 cells unprotective, regardless of repetition of stimulation procedure. Thus, we finally assessed coupling of Tc1 polarity and in vivo antitumor activity. As shown in Fig. 4, two 1◦ Tc2 → 2◦ Tc1 cells protective from tumor growth were high IFN␥ producers (14–15 ng/ml), while four unprotective cells were low IFN␥ producers (7–8 ng/ml). However, they secreted similar levels of IL 2, IL 4 and IL 5. These data strongly suggest that in vivo anti-tumor activity of 1◦ Tc2 → 2◦ Tc1 cells are closely linked to IFN␥ productivity, i.e., the polarity to Tc1.

Fig. 1. Cytokine secretion and anti-tumor activity of memory effector Tc2 during repeated stimulation. Cytokine secretion (A) by in vitro stimulation with tumor lysate (1/100) was determined by ELISA. Tumor protection (B) was evaluated for 12 days after adoptive transfer of 5 × 106 Tc2 cells followed by challenge of 5 × 105 CT26 tumor cells. Inhibition of tumor growth was examined for unprotective Tc2 cells (n = 4–5) except 1◦ Tc1 and 1◦ Tc2 cells which were all protective.

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Fig. 2. In vitro cytotoxicity of memory effector Tc2 along repeated stimulation. Cytotoxicity against wild type CT26 (A) or NK-sensitive Yac-1 (B) targets was quantified using a 4 h 51 Cr release assay in triplicate at indicated E/T ratios.

4. Discussion Active vaccination is the most promising strategy in cancer immunotherapy, in which tumor associated antigen [30,31], viral protein [32,33], and intrinsic [34,35] or genetically modified tumor cells [36,37] are employed. In these protocols, CD8+ cytotoxic T lymphocyte response is a crucial component required for eradication of tumors [1–4], and vaccine efficacy is entirely dependent on successful development of memory cells. Thus, induction of highly effective memory CTL has been a goal of most of current experimental studies in this field. However, it is unclear whether both of memory Tc1 and Tc2 cells have equivalent potential for tumor protection. For an insight into this issue and its application in vivo, we considered two points for this study; in vivo immunization and using memory cells from in vivo primed mice instead of in vitro stimulated effector cells. Our first finding was that primary antigenic stimulation (1◦ ) resulted in an insufficient polarization of Tc2 cells. In vivo anti-tumor activity of both 1◦ Tc1 and Tc2 memory ef-

Fig. 3. Tumor protection and cytokine secretion of trans-differentiated memory effector Tc cells. 1◦ Tc2 cells were trans-differentiated to Tc1 (1◦ Tc2 → 2◦ Tc1) during secondary stimulation and their tumor protection (A), cytokine production (B) and in vitro cytotoxicity (C) was compared to 2◦ Tc2 (1◦ Tc2 → 2◦ Tc2) cells.

fector cells were perfectly protective and 1◦ Tc1 cells secreted no type 2 cytokines, indicating that they were polarized. However, 1◦ Tc2 cells secreted high levels of IL 4 and IL 5 and also high levels of IFN␥, which was comparable to Tc1 (Table 1), suggesting that they were not fully differentiated. Incomplete

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Fig. 4. Comparison of cytokine production of protective (2 of 6) and unprotective (4 of 6) trans-differentiated (1◦ Tc2 → 2◦ Tc1) memory effector cells.

polarization of 1◦ Tc2 memory effector cells was supported by cytokine profiles of previously transferred 1◦ Tc1 and Tc2 cells which were harvested 1 month later from adoptive hosts and then restimulated with tumor antigen. They showed indistinguishable cytokine secretion profiles regardless of either 1◦ Tc1 or Tc2 transfer (Table 2). In addition, although cytokine secretion of 1◦ Tc2 memory effector cells was attenuated (Table 2) (to 33% in IL 2 and 58% in IFN␥ production) when compared to those prior to transfer (Table 1), comparison revealed a preferential decrease of type 2 cytokines such as IL 4 and IL 5 (to 97.4% and 92.5%, respectively). Cerwenka et al. [29] have observed that memory Tc can persist in vivo for at least 13 weeks without change of cytokine profile. Accordingly, it is presumed that 1◦ Tc2 memory effector cells lost Tc2-like activity during in vivo stay after adoptive transfer and they had not been fully polarized yet at the time point of transfer. These data prompted us to evaluate anti-tumor activity of Tc2 memory cells over the course of polarization until commitment. Evidence of insufficient polarization of 1◦ Tc2 memory effector cells was provided by subsequent data of cytokine secretion with repeated stimulation. Second (2◦ ) and tertiary (3◦ ) stimulation of memory Tc2 cells resulted in a gradual reduction in IFN␥ secretion (Fig. 1A), showing an advance to Tc2 commitment. No significant changes in IL 2, IL 4 and IL 5 secretion indicate that repetitive stimulation does not affect default activity of those cells. Concomitantly with Tc2 polarization, repeated stimulation resulted in a great impairment of tumor protective function of Tc2 cells (83.3% reduction by 2◦ ) and tertiary stimulation completely abrogated anti-tumor activity (Fig. 1B). Data of tumor growth in the mice transferred with unprotective 2◦ , 3◦ and 4◦ Tc2 memory effectors (Fig. 1C) clearly supported direct relationship between loss of protective activity of Tc2 cells and polarization to Tc2 following successive stimulation. Moreover, such gradual loss of protective activity of Tc2 cells was also accompanied with their in vitro cytotoxicity against CT26 tumor cells (Fig. 2A). Additional evidence is that tumor pro-

tective 1◦ Tc2 → 2◦ Tc1 cells are all high IFN␥ producers, Tc1-like, whereas unprotective cells are poor IFN␥ producers, Tc2-like (Fig. 4). These results thus clearly demonstrate an overall impairment in anti-tumor activity of committed Tc2 cells. Progressive loss of anti-tumor activity observed in this study may not result from Tc2 polarization, but from prolonged in vitro stimulation. We could not clarify this issue because repeated stimulation of Tc1 cells was unsuccessful. This result was already described by other investigators [38–40], showing that Th1 and Tc1 cells are much more sensitive to Fas- or granzyme B-mediated apoptosis than Th2 and Tc2 cells. To exclude this possibility, we trans-differentiated 1◦ Tc2 to 2◦ Tc1 cells (1◦ Tc2 → 2◦ Tc1) and compared to 2◦ Tc2 cells. Two of 6 cultures were trans-differentiated Tc1-like cells, which all showed tumor protective functions identical to 1◦ Tc1 cells, suggesting that impaired anti-tumor activity of 2◦ Tc2 cells is unrelated to double stimulation. The data from the this work suggest that skewing of CD8+ cells toward Tc2 can lead to the impairment of overall in vivo and in vitro anti-tumor functions of cytotoxic T lymphocytes. However, other investigators showed opposing data that Tc2 as well as Tc1 cells are effective in tumor protection through distinct mechanisms [21–23]. Although this contradiction was not addressed in this study, several explanations are possible. First, we used memory cells rather than na¨ıve cells. It has been described that Th2 cells reject tumors through inflammatory cell recruitment and immunological memory was not generated [41,42]. Therefore, roles of na¨ıve and memory cells may not be identical in inflammatory responses, such that effector Tc2 and memory effector Tc2 cells can function differently. Secondly, immunization with different kinds of tumor cells will result in a distinctly biased response. Additionally, there is likely an immunogenetic difference between intrinsic tumor cells and OVA-transfected tumor cells. Lastly, we used IL 12-transfected tumor cells whereas other studies used OVA-transfected cells. It is well documented [43,44] that IL 12 is a critical cytokine for driving T cells toward type 1 and thus memory cells generated by under Th1-favoring conditions may affect type 1 and 2 differentiation. It is another intriguing issue whether failure of committed Tc2 memory effector cells in tumor protection is due to inability to secrete IFN␥. Although IFN␥ is the main effector of IL 12 anti-tumor activities, exogenous IFN␥ cannot completely substitute for in situ IL 12 activity [18]. For instance, other cytokine such as IL 15 can mediate anti-tumor activity of IL 12 independently from IFN␥ [45]. In summary, the data obtained in our mouse tumor model demonstrate that committed Tc2 memory effector cells no longer exhibit in vitro and in vivo anti-tumor activity. This finding provides a foundation on which rational and effective modalities can be approached in current experimental immunotherapy of cancer including active vaccination and adoptive transfer of cytotoxic T lymphocytes.

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