Tim3-hIg expressed in tumor cells inhibits in vivo tumor growth

Recent studies revealed that administration of Tim3-hIg protein blocks Tim3 and Tim3 ligand, suggesting the up-regulation of Th1-driven immune response. Th1-driven immune response is involved in cytotoxic T lymphocyte activation in tumor immunity. But the involvement of Tim3 protein in tumor protection has not been well studied. Therefore, I examined whether the inhibition of Tim3 pathway affect tumor suppression. First of all, the stable cell clones secreting Tim3-hIg were established. 3LL cells, Lewis lung cancer cell line, were transfected with eukaryotic Tim3-hIg- or hIg- and bicistronic enhanced green fluorescent protein (EGFP) expression plasmid and then the clones were selected by limiting dilution. The Tim3-hIg or hIg expression was confirmed indirectly as detecting EGFP expression by flow cytometry (Fig. 9A) and mRNA transcripts by RT-PCR method (Fig. 9B).

To assess the blockage effects of Tim3-hIg in in vivo tumor growth, each cell clones were inoculated subcutaneously into the lesion of C57BL/6 mice and then the tumor growth was measured for 3 weeks. The tumor growth rate of clones secreting Tim3-hIg (T1 and T2) was lower than control clones (G1and G2: clone expressing EGFP, H2 and H3: clone secreting hIg) (Fig. 9C). Although, the in vitro cell growth of H2 and H3 was faster than G1 and G2 (Fig. 9D), the tumor growth of H2 and H3 in mice was behind of G1 and G2, which imply that tumor growth was not directly determined by in vitro cell growth rate. Therefore, these results suggest that reduced tumor growth rate of clones T1 and T2 could be due to the expression of Tim3 pathway inhibitor, Tim3-hIg.

46 A

C D

Fig. 9. Tim3-hIg inhibits in vivo tumor growth. For establishment of 3LL cells secreting either Tim3-hIg or hIg, 3LL cells were transfected with either pIRES2-Tim3-hIg or pIRES2-hIg plasmid and selected by treatment of neomycin (2 μg/ml). 3LL-derived cell lines were confirmed by GFP expression by flow cytometry. CON: parental 3LL cells; G1 and G2: 3LL cells expressing GFP; H1, H2 and H3: 3LL cells secreting hIg protein; T1, T2: 3LL cells secreting Tim3-hIg (A). Tim3-hIg or hIg transcript was analyzed by RT-PCR.

Specific primers for Tim3-hIg and hIg, respectively, were used (B). The mice (4 heads per each cell line) were injected subcutaneously with 5 X 10⁵ of the indicated stable cell lines in 100 μl of PBS. Every two days after injection, tumor size was measured by caliper gauge (C). The growth rate of each clone was measured by MTT assay in vitro culture system. Total cell number was calculated using standard curve with known cell numbers (D). Data is shown as mean±SD. Representative data from two independent experiments are shown.

H. Tim3-hIg increases the efficacy of prophylactic tumor vaccine

I next examined whether prophylactic tumor vaccination with cells expressing Tim3-hIg suppress tumor growth. First, 3LL cells were transfected with plasmid expressing Tim3-hIg or hIg, treated with mitomycin (50 μg/ml) to prevent proliferation with G1 cells.

After treatment of mitomycin, cell viability was verified by trypan blue staining and cultivation for 2 days. In capability of mitomycin-treated cells to develop a tumor was also verified. The transfection efficiency was monitored by flow cytometry for detecting EGFP expression (Fig. 10A). The secretion of Tim3-hIg and hIg was demonstrated by IP-WB (Fig. 10B) and then used as prophylactic tumor vaccine.

Ten days after immunization, the mice were challenged with G1 cells and then, tumor growth was measured for 3 weeks. The tumor growth was reduced significantly in the mice immunized with 3LL transfectants secreting Tim3-hIg compared with mice immunized with 3LL transfectants secreting hIg or control mice from 14 days after tumor challenge (p< 0.005, Fig. 10C). These results suggest that Tim3-hIg may enhance the efficacy of prophylacitic tumor vaccine and the blockage of Tim3 and Tim3 ligand may promote antitumor memory response.

49 A B

Fig. 10. Tim3-hIg increases the efficacy of prophylactic tumor vaccine. 3LL cells were

transfected with plasmid expressing either Tim3-hIg or hIg together with EGFP. Transfection efficiencies of cells were determined by detection of EGFP using flow cytometry (A). The Tim3-hIg and hIg protein, respectively in culture supernatant of transfected cells were detected by Western blotting using anti-human Ig Ab (B). The mice were vaccinated s.c.

with 6 x 10⁵3LL cells transfected with plasmid expressing either Tim3-hIg or hIg 36 hr before vaccination and treated with mitomycin for prevention of proliferation. The mice were inoculated with G1 cells in 100 μl PBS 10 day after vaccination Every two days after injection, tumor size was measured (C). PBS: mice injected with PBS; hIg: mice vaccinated with 3LL cells secreting hIg; Tim3-hIg: mice vaccinated with 3LL cells secreting Tim3-hIg.

Data is shown as mean±SD from two independent experiments. *, P <0.002 between hIg and Tim3-hIg.

I. The frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in mice injected with Tim3-hIg expressing tumor cells

The regulatory T cells (Tregs) expressing a CD4⁺CD25⁺Foxp3⁺ phenotype have been associated with suppression of antitumor immune responses. Several groups have reported that a large number of Tregs are present in tumors and draining lymph nodes of mice as well as in patients with a poor prognosis (Bromwich et al., 2003; Webster et al, 2006). To monitor whether Tregs frequency is affected by the blockage of Tim3 pathway, in tumor-draining lymph nodes of mice, the frequency of Tregs was detected using 3-color flow cytometry. The proportion of Tregs in draining lymph node of mice bearing tumor expressing Tim3-hIg was significantly descended, comparing with G1- or G2-bearing mice 3 weeks after tumor-challenge (Fig. 11A). To determine whether the decreased frequency of Tregs was the result of direct modulation by Tim3-hIg or a reflection of the reduced tumor mass, I analyzed the frequency of Tregs in LNs of mice 10 day after tumor challenge when tumor growth was vaccinated with tumor cells expressing either Tim3-hIg or hIg (Fig. 11B). The draining lymph node of mice vaccinated with Tim3-hIg secreting Tim3-hIg harbored similar level of Tregs compared to control mice vaccinated with 3LL secreting hIg or injected with PBS. However, the frequency of Tregs was significantly increased in mice bearing a tumor compared to the tumor-free normal mice (p < 0.05).

These results suggest that Tim3-hIg may reduce the frequency of Tregs indirectly through the inhibition of tumor growth.

51 A B

Fig. 11. The frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in mice given Tim3-hIg expressing tumor cells. The frequenccies of Tregs were assessed in lymph-node of mice (23 day) after s.c. injection of the indicated tumor cells. The cells isolated from draining LN (dLN) and non-draining LN (dLNs) were labeled with APC-anti-CD4, PE-anti-CD25 and FITC-anti-Foxp3 antibody and analyzed with flow cytometry. G1, G2: 3LL cells stably expressing GFP protein; T1, T2: 3LL cells stably expressing Tim3-hIg and GFP protein (A).

The mice were vaccinated s.c. with PBS only, mitomycin-treated cells secreting either hIg or Tim3-Ig cells. Ten days after the mice were injected s.c. with G1 tumor cells. The lymph-nodes were isolated on 10 day after tumor challenge (B). CON: normal mice; PBS: mice injected with 100 μl of PBS; hIg: mice injected 3LL cells transiently expressing hIg protein;

Tim3-hIg: mice injected with 3LL cells transiently expressing Tim3-Ig protein. Each symbol represents each mouse (6-12 heads per group). Data is shown as mean±SD.

J. Tim3-hIg expression marginally enhances the efficacy of the therapeutic tumor vaccine but not of the chemotherapy

Prophylactic tumor vaccine expressing Tim3-hIg was shown to be effective to suppression of tumor progression. Therefore I examined whether therapeutic tumor vaccine expressing Tim3-hIg would be an therapeutic modality. Tumor-bearing mice were injected with 3LL cells expressing which were mitomycin-treated and expressing either Tim3-hIg or hIg day 7 and day 16 after tumor challenge. Although tumor progression was similar i mice vaccinated with cells expressing Tim3-hIg or hIg, the tumor size of mice vaccinated with 3LL cells expressing Tim3-hIg was significantly smaller than control mice (p < 0.05 on day 8, 14, 20 and 21, Fig. 12A).

To evaluate whether a therapeutic tumor vaccine expressing Tim3-hIg might enhance the efficacy of chemotherapy, the tumor-bearing mice were administered with 5-FU on day 9 and day 11 after tumor challenge, and then with mitomycin-treated cells expressing Tim3-hIg on day 11 and on day 13. Tumor growth was markedly reduced in mice administered with 5-FU, however, there was no significant difference between mice administered with 5-FU alone and administered subsequently with cells expressing Tum3-hIg in addition on 5-FU. Using this protocol, there was a tendency for decreased tumor growth in mice vaccinated with cells expressing Tim3-hIg compared to control mice, however, the difference in growth was not statistically significant (Fig. 12B). These results suggest that Tim3 pathway inhibition can enhance the efficacy of therapeutic tumor vaccine depending on the administration protocol.

53 A B

Fig. 12. Tim3-hIg expression marginally enhances the efficacy of the therapeutic tumor vaccine but not of the chemotherapy. Mice were injected with G1 tumor cells. For construction of tumor vaccine, 3LL cells transfected with either pIRES2-Tim3-hIg or pIRES2-hIg and treated with mitomycin (5 μg/ml). Mice with G1 tumors were treated with either Tim3-hIg secreting 3LL-tumor vaccine (Tim3-hIg), hIg (hIg) or left without tumor vaccine on 7 and 16 day (A). Mice with G1 tumors were injected with either 5-FU (70 μg/kg) on 9 and 11 day, or Tim3-hIg secreting 3LL cells (6 x 10⁵) on day 11 and day 13.

Another mice were injected with Tim3-hIg combined with 5-Fu on same date. PBS: mice were not vaccinated, injected only PBS; hIg: mice injected with 3LL cells secreting hIg;

Tim3-hIg: mice injected with 3LL cells secreting Tim3-hIg. Every 2 days after injection, tumor size was measured by caliper gauge. Data is shown as mean±SD. *, P <0.002 between PBS and Tim3-Ig. Representative data from two independent experiments are shown.

54 and IFN-γ than TIM3^low cells 4h of stimulation with PMA and A23187. These results are compatible with previous studies that TIM3⁺ CD4⁺ T cells sorted from spleen cells of healthy human subject shows reduced expression of the IL-2 and IFN-γ compared to TIM3^- CD4⁺ T cells 12 h after stimulation with anti-CD3 and anti-CD28 (Hastings et al., 2009) and that TIM3-expressing T cells of HIV-1 infected patients fail to produce cytokine or proliferate 6h after stimulation with gag peptide or superantigen (Jones et al., 2008).

I found the molecular basis for deficient IL-2 transcription in Jurkat T cells-over expressing TIM3. In these cells, the activities of AP-1 and NFAT activity was reduced.

The AP-1 activity was reduced by suppression of c-Jun transcription without suppression of c-Fos transcription whereas the reduction of NFAT activity was partly due to insufficient NFAT1 dephosphorylation. It is already well-demonstrated that AP-1 and NFATs induce IL-2 production together after T cell activation (Hermann-Kleiter and Baier, 2010; Nguyen et al., 2010). Thus, it is conceivable that defect of c-Jun expression and NFAT1 to change an active form in a timely fashion results in deficient IL-2 transcription in Jurkat T cells-over expressing TIM3.

In anergy T cell, defective transcription of IL-2 is associated with impaired AP-1 transcription through poor expression of the AP-1 family members, c-Fos, FosB whereas NFAT is not involved in defective transcription of IL-2 (Mondino et al., 1996). In addition, CTLA-4, inhibitory receptor of T cell activation, results in reduced production IL-2 which is accompanied by reduced activation of the transcription factors NF-κB, NFAT and AP-1 (Krummel and Allison, 1995; Brunner et al., 1999; Olsson et al., 1999). Furthermore, recent one report demonstrates that CTLA-4 inhibits IL-2 and IFN-γ production by activating the ubiquitin ligase, Itch, which probably ubiquitinates JunB (Hoff et al., 2010).

Thus the mechanisms underlying regulation of IL-2 production in T cells expressing TIM3 appear to be different from those in anergic T cells and cells expressing CTLA-4.

This study raises a question how c-Jun expression is reduced in Jurkat T cells-over expressing TIM3. In the c-Jun promoter, two AP-1 sites, a proximal AP-1 site located between bp -71 and -64 and a distal AP-1 sites located between bp -190 and -183 has been identified. C-Jun is autoregulated by binding to AP-1 binding sites of the c-jun enhancer (Tornaletti et al., 1995; Unlap et al., 1992). In addition, ATF-2 together with c-Jun bind to a TRE site in the c-jun promoter and induce c-jun transcription (Gupta et al., 1994). Thus, a potential explanation for defective c-Jun expression in Jurkat T cells-over expressing TIM3 would be poor transcriptional activity of transcriptional factors binding to c-Jun promoter. In the other hands, the transcriptional activities of Jun and Fos are posttranslationally regulated by a mitogen-activated protein (MAP) kinase. The phosphorylation of c-Jun and c-Fos have been shown to be regulated by Jun N-terminal kinses (JNK) and Fos-regulating kinase (FRK), respectively (Macian et al., 2001; Morton

et al., 2003). Thus, it would be interesting to measure the extent of phosphorylated c-Jun and c-Fos in cells expressing TIM3 in further study.

This study shows the insufficient NFAT1 dephosphorylation in Jurkat T cells-over expressing TIM3 when these cells were stimulated by calcium ionophore, A23187.

Activation of the NFAT pathway occurs by calcium-signaling pathway after T cell activation. Released calcium binds clamodulin, which in turn activates the calmodulin-dependent phosphatase, calcineurin. NFAT proteins are dephosphorylated by activated calcineurin, leading to their nuclear translocation and the induction of IL-2 gene transcription (Aramburu et al., 1999; Gwack et al., 2007). The activity of calcineurin is controlled not only by calcium and calmodulin but also by several calcineurin inhibitors.

These include calcineurin-binding protein 1 (CABIN1; also known as CAIN), the A-kinase anchor protein AKA79 (also known as AKAP5) and members of the Down’s syndrome critical region (DSCR)/modulatory calcineurin-interacting protein (MCIP) family of calcineurin inhibitors, which are known as calcipressins (Coghlan et al., 1995;

Sun et al., 1998; Kashishian et al., 1998; Klauck et al., 1996; Rothermel et al., 2000). In this sense, because calcium influx could not be shown to be aberrant in Jurkat T cells-over expressing TIM3, calcineurin activity would suppress calcineurin-inhibitors in these cells.

The importance of cytoplasmic tail of TIM3 in regulated in this study was demonstrated in this study using a series of deletion mutants. I found that Jurkat T cells expressing c-terminal region from E²⁶¹ to P³⁰¹ of TIM3 cytoplasmic tail containing 5 tyrosines was required for transcriptional reduction of IL-2 and IFN-γ and for suppression of activity of NFAT as well as AP-1. The Y²⁶⁵ within a tyrosine phosphorylation motif,

RSEENIY is phosphorylated by ligation of galectin-9 (Mooney et al., 2002; van-de Wayer et al., 2006). Its phosphorylation is suggested to be mediated by the Tec family tyrosine kinase Itk, at least in HEK293 cells and the binding site of protein containing SH2 domain (van-de Wayer et al., 2006). It is not known how tyrosine phosphorylation of TIM3 regulates its function in T cells or other cell types.

Recent findings regarding the role of Tim3 in tumor development suggest that Tim3 is a promising target for anti-tumor immunotherapy. The interaction of Tim3 with galectin-9 enhances anti-tumor immunity and prolongs the survival of tumor-bearing mice (Nagahara et al., 2008). Nasopharyngeal tumors secrete exosomes containing galectin-9, which have been implicated in immune escape by the tumor through the induction of tumor-specific Tim3⁺ T-cell death (Klibi et al., 2009). Furthermore, Tim3 expressed in lymphoma-associated endothelium leads to tumor progression through the inhibition of CD4⁺ T-cell activation and Th1 polarization (Huang et al., 2010). In addition, the interaction of Tim3 expressed by endothelial cells with melanoma cells facilitates metastasis through the activation of NF-kB in the melanoma cells (Wu et al., 2010). In this study, I demonstrated the tumor-suppressive effect of Tim3 pathway inhibition by subcutaneous administration of tumor cells transiently expressing Tim3-hIg. The expression of Tim3-hIg by tumor cells delayed tumor growth in mice even when only around 30% of tumor cells were transiently expressing Tim3-hIg. Therapeutic vaccination with tumor cells expressing Tim3-hIg also showed a transient inhibitory effect on tumor growth and, compared to the report of Dardalhon and colleagues (Ju et al., 2010), in which 100 μg of inhibitory anti-Tim3 Ab was given intraperitoneally, the quantity of Tim3-hIg administered in our study was

decreased by 1000-fold. An increased amount and frequency of Tim3-hIg expressing tumor vaccine may further enhance the tumor-suppressive effect.

TIM3 modulates innate and adaptive immune cell function. It up-regulates cross-presentation of antigen by dendritic cells through increased phagocytosis of apoptotic cells, whereas it down-regulates Th1, CD8 T-, and NK-cell activation (Sanchez-Fueyo, 2003;

Golden-Mason et al., 2009; Nakayama et al., 2009; Dekruyff et al., 2010; Ju et al., 2010).

The Tim3 pathway also regulates the generation and function of immunosuppressive cells such as CD11b⁺Ly-6G⁺ myeloid cells (MDSC) and CD4⁺CD25⁺Foxp3⁺ Tregs in a Tim3 transgenic mouse and an autoimmune murine model, respectively (Sanchez-Fueyo et al., 2003; Dardalhon et al., 2010). An elevated proportion of Tregs in the total CD4⁺ T-cell population have been detected in several different human cancers, including lymphoma, lung, and ovarian tumors (Woo et al., 2001; Yang et al., 2006). I also observed an increased frequency of Tregs in tumor-draining LNs and a lower frequency of Tregs in mice bearing Tim3-hIg expressing tumors than in control mice; however, I could not conclude that Tim3-hIg expression directly regulated Treg generation since decreased frequency could be due to lower tumor burden rather than Tim3 pathway inhibition. This findings show that Tim3-hIg expressing prophylactic tumor vaccine suppressed tumor growth through the inhibition of the Tim3 pathway, independent of the Treg cell frequency.

Although I could not dissect the underlying mechanisms of the tumor-suppressive effect of Tim3 pathway inhibition, two possibilities are conceivable. First, Tim3 pathway inhibition may promote anti-tumor immunity by suppressing the generation of MDSCs.

The frequency of MDSCs is increased in various tumors, the depletion of which leads to

anti-tumor immunity and tumor regression (Almand et al., 2001; Suzuki et al., 2005;

Gabrilovich et al., 2009; Yan et al., 2010). In a Tim3 transgenic mouse model and a galectin-9 (Tim3 ligand) transgenic mouse model, an increase in MDSCs and inhibition of immune responses are observed (Dardalhon et al., 2010). Interestingly, I found that Tim3-hIg expressing therapeutic tumor vaccine did not exhibit an additive inhibitory effect on tumor growth when given in combination with 5-FU, which has recently been reported to selectively kill MDSCs and thus enhance anti-tumor immunity (Vincent et al., 2010).

Second, Tim3 pathway inhibition may enhance anti-tumor immunity by antagonizing phagocytosis of apoptotic cells and thereby circumventing the immunosuppressive effect of apoptosis. Tim3 binds phosphatidylserine on the apoptotic cell surface and facilitates phagocytosis of apoptotic cells. Tim3 pathway inhibition reduces phagocytosis of apoptotic cells and induces autoantibody production, indicating the break of peripheral tolerance (Nakayama et al., 2009). In concordance with this, the efficacy of a therapeutic vaccine for melanoma is significantly augmented when the phagocytosis of apoptotic cells is inhibited by the binding of milk fat globule epidermal growth factor 8 to phosphatidylserine (Jinushi et al., 2007). Conclusively, these results suggest that Tim3 pathway inhibition can enhance the efficacy of tumor vaccination and anti-tumor immunity.

V. CONCLUSION

In this study, the molecular mechanisms for the inhibitory function of TIM3 in Th1-mediated cytokine production was investigated and reduced activities of AP-1 and NFAT through defect in c-Jun expression and NFAT dephosphorylation, were found in TIM3 expressing cells. Further, the C-terminal region was shown to be important in suppression of IL-2 and IFN-γ expression as well as in inhibition of activities of AP-1 and NFAT. In the evaluation of the effect of Tim3-hIg expression on tumor progression, I found that Tim3-hIg expression in tumor cells decreased tumor growth and that Tim3-hIg expression increased efficacy of the whole cell tumor vaccine. Taken together, these results expand

문서에서 The Suppressive Mechanisms of TIM3 on T Cell Cytokine Production and The Anti-Tumor Effect of TIM3-Pathway Inhibition (페이지 55-0)