Eliciting protective immune responses against murine myeloma challenge in lymphopenia mice through adoptive transfer of tumor antigen-specific lymphocytes and immunization of tumor vaccine secreting mIL-21

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Abstract

Previous studies have indicated that the cytokine interleukin (IL)-21 may induce both innate and adaptive immune responses against tumors. The goal of this study was to evaluate a new adoptive immunotherapy strategy that combined lymphocytes from mice immunized with a murine myeloma vaccine secreting murine IL-21 (mIL-21-Sp2/0) in lymphopenic mice induced by cyclophosphamide. The data indicate that effective antitumor immunity was induced in mice receiving syngeneic murine lymphocytes from the mice immunized with the mIL-21-Sp2/0. More importantly, the efficacy against the Sp2/0 cell challenge was enhanced after the lymphocytes were activated and proliferated ex vivo before administration into the lymphopenic mice. We conclude that the adoptive transfer of tumor antigen-specific lymphocytes into mice immunized with mIL-21-Sp2/0 induced protective immune responses against myeloma challenge.

Introduction

A growing body of literature shows that the adoptive immunotherapy is an effective therapy method for treating malignant tumors. This immune strategy may deliver abundant tumor-specific immunocytes that are rapidly activated and proliferated in vitro.1, 2, 3 The transferred immunocytes complement endogenetic immunocytes and release cytokines to regulate immune responses and to enhance the effect of immunotherapy. In particular, the lymphocytes emerging from the lymphopenia-induced homeostatic proliferation acquire the characteristics of effector/memory cells.4, 5, 6, 7 However, there are some disadvantages of restricting the effects of this immunotherapy strategy. The disadvantages include no expansion of sufficient amounts of tumor antigen (Ag)-specific cells in vivo, lack of persistence of the transferred effector cells and less effective antitumor immunity in hosts.8, 9 Because of these disadvantages, considerable attention has been drawn to engineering cytokines with expansion of abundant tumor-specific cells in vitro to make up for the shortage in the adoptive immunotherapy in vivo. Recent studies indicate that interleukin (IL)-21 regulates innate and adaptive immune responses10 and has multiple immune functions, especially in promoting activation and proliferation of CD8+ T lymphocytes, in enhancing the cytotoxicity of cytotoxic T lymphocytes (CTLs), and in reducing the apoptosis of Ag-specific lymphocytes. Moreover, IL-21 functions as a co-stimulator for lymphocyte proliferation, for enhancing memory lymphocyte responses, and for modulating immunological homeostasis.7, 11, 12 Furthermore, the murine myeloma vaccine secreting murine IL-21 (mIL-21-Sp2/0) induced potent antitumor effects in mice.13 These studies support the use of IL-21 in the ex vivo generation of potent-specific CTLs for adoptive therapy or as an adjuvant cytokine during in vivo immunization against tumor Ag.14, 15, 16, 17, 18 In addition, combining immunotherapy and chemotherapy induces an effective antitumor response in a tumor-bearing body, such as the cyclophosphamide (Cy)-induced modulation of several cytokines, the homeostatic proliferation of transferred lymphocytes and homing of transferred lymphocytes to tumor mass for antitumor immunity.19, 20, 21 In this study, we adopted a new strategy of transferring tumor Ag-specific lymphocytes into syngeneic lymphopenia mice induced by Cy to explore if the strategy could stimulate a protective immune response against myeloma challenge. The study showed that syngeneic tumor Ag-specific lymphocytes, originated from the mice immunized with irradiated mIL-21-Sp2/0 and stimulated with the mIL-21-Sp2/0 ex vivo, were transferred into the Cy-induced lymphopenia mice immunized with mIL-21-Sp2/0 concurrently, and that this strategy may promote the proliferation of the adoptive lymphocytes and mice's lymphocytes in vivo, and assist in forming and sustaining special antitumor effects in the immune-reconstituted mice.

Material and methods

Mice, cell lines and transfection

BALB/c mice of 6–8 week of age were obtained from the University of Yangzhou of China. All mice were housed under the pathogen-free condition and the experiments were performed in compliance with the guidelines of the animal research ethics board of Southeast University. The Sp2/0 (BALB/c mice myeloma cells) and YAC-1 (Moloney leukemia-induced T-cell lymphoma of A/Sn mouse origin) cell lines were obtained from the Cellular Institute of China in Shanghai. The two cell lines were cultured at 37 °C in 5% CO2 atmosphere in RPMI 1640 supplemented with 10% fetal bovine serum that contained 100 U ml−1 penicillin G sodium and 100 μg ml−1 streptomycin sulfate. The SP2/0-mIL-21 cells that were transfected with pcDNA3.1-mIL-21 had been previously prepared by our laboratory.

Adoptive immunotherapy and Sp2/0 cell challenge

In the first experiments of the adoptive immunotherapy (Figure 1a), Balb/c mice were randomly divided into four groups. The control group mice were inoculated with phosphate-buffered saline (PBS). Three experimental groups were the naive lymphocyte group (mice inoculated with lymphocytes from the mice without any immunization), the Sp2/0-primed lymphocyte group (mice inoculated with lymphocytes from the mice immunized with Sp2/0 cells) and the mIL-21-Sp2/0-primed lymphocyte group (mice inoculated with lymphocytes from the mice immunized with mIL-21-Sp2/0 cells), respectively. In the secondary experiment of adoptive immunotherapy, Balb/c mice were randomly divided into control group, PBS-induced group, Sp2/0-cell-induced group and mIL-21-Sp2/0-cell-induced groups (Figure 1b). Control group mice were inoculated with lymphocytes from mice without any immunization, and PBS-induced group mice were inoculated with lymphocytes from mice just immunized with PBS. Sp2/0-cell-induced group and mIL-21-Sp2/0-cell-induced groups were the same as the Sp2/0-primed lymphocyte group and mIL-21-Sp2/0-primed lymphocyte group in the experiment described above. In the third experiment of the adoptive immunotherapy, control group mice were just injected with Cy and three experimental groups, one was the Cy-treated group, in which mice were induced with Cy and transferred with 2 × 105Ag-specific lymphocytes; another was Cy+Sp2/0 vaccine group, in which mice were induced with Cy and transferred with 2 × 105Ag-specific lymphocytes as well as immunized with the inactivated 1 × 106 Sp2/0 cell vaccine. The third group was Cy+Sp2/0-mIL-21 vaccine group, in which mice were induced with Cy and transferred with 2 × 105Ag-specific lymphocytes as well as immunized with the inactivated 1 × 106 Sp2/0-mIL-21 cell vaccine (Figure 1c). In the second and the third experiments, Balb/c mice were injected with Cy 100 mg per day per mouse for 2 days, and all isolated Ag-specific lymphocytes were incubated with the inactivated Sp2/0 vaccine or the inactivated mIL-21 tumor vaccine as the stimulators in the presence of IL-2 in vitro for 3 days, and then were transferred into lymphopenia mice induced by Cy. In these three-time sequential approach experiments, all mice were challenged with 1 × 105 wild-type Sp2/0 cells in the flanks of mice after mice received the immunotherapy, and tumor areas and mice survival rates were evaluated for each group. Mice were monitored for tumor outgrowth 2–3 times per week.7, 22 Five mice per group were used routinely and each experiment was repeated twice.

Figure 1
figure1

Effect of adoptive immunotherapy of myeloma in mice treated with different methods. (a) Three days after the lymphocytes were i.v. transferred into mice, the mice were challenged with 1 × 105 wild-type Sp2/0 cells. (b and c) All experimental groups are described in the Material and methods section. **P<0.01; *P<0.05.

In vivo tumor assays and preparation of lymphocytes from splenocytes and lymph nodes

Balb/c mice were inoculated s.c. in the flanks with 1 × 105 SP2/0 cells or 1 × 105 SP2/0-mIL-21 cells in the logarithmic grown phase. After about 12 days, the tumors were felt by touch in the mice and mice were killed after 5–7 weeks of observation. The lymphocytes were isolated from the mice as described in references.13, 23 Finally the harvested lymphocytes were used as effector cells of cytotoxic assay. Each experiment was repeated twice.

Proliferation experiments

In the in vitro proliferation experiment, 1 × 107 per ml lymphocytes were labeled with 0.5 μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Eugene, Eugene, OR) at 37 °C for 15 min. After the incubation, the lymphocytes were washed twice in PBS to sequester any free CFSE that had failed to diffuse into the cells. Then 1 × 106 lymphocytes were resuspended in 10% RPMI medium in 24-well plates and 1 × 105 irradiated mIL-21-Sp2/0 cells or irradiated Sp2/0 cells were added to plates as stimulator cells, respectively, supplemented with 10 ng IL-2. The cells were incubated at 37 °C for 3 days and were then analyzed by flow cytometry (FCM, BD Biosciences, San Jose, CA).24 In the ex vivo proliferation experiment, 2 × 105 tumor Ag-specific lymphocytes were labeled with CFSE and then injected into tail veins of the lymphopenia mice. After 3 days, the lymophocytes were collected from mice and then detected by FCM. The proliferative data were analyzed with CellQuest software, and proliferation activity index was expressed as the percentage.

ELISPOT assay for IFN-γ

Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay was performed with Kit according to manufacturer's protocol (BD eBioscience, San Jose, CA) and as was described in reference.25, 26

Cell staining and flow cytometry analysis

In analyzing the forkhead box protein P3 (FOXP3)-expressing T cells according to manufacturer's protocol (eBioscience, San Jose, CA). Briefly, the mononuclear cells were stained with the surface molecule antibody (Ab) and washed with cold PBS. The cells were resuspended in the fix/perm buffer and incubated at 4 °C for 3 h in dark, and then washed twice in a permeabilization buffer. Anti-mouse/rat Foxp3-phycoerythrin was then added. The cells were incubated for 30 min and washed twice in PBS, and were then analyzed in FCM.20, 27 In analyzing the CD4+, CD8+ or CD25+-expressing T cells, all lymphocytes from the spleens and lymph nodes suspensions were co-stained with fluorescein isothiocyanate-conjugated anti-CD4+ Ab, phycoerythrin-conjugated anti-CD8+ or anti-CD25+, which were purchased from eBioscience. These conjugated cells were kept in light for 30 min at 4 °C and the molecular expression was analyzed by FCM according to the protocol described by the manufacturer.28

Cytotoxicity assays

The CFSE/7-amino actinomycin D (7-AAD) cytotoxicity assay was performed as described in previous reports.24, 29

Tumor histopathology

Mice were killed 5–7 weeks after Sp2/0 cell challenge. The tumor tissues in mice were removed and fixed in 10% formalin. The formalin-fixed tumors were embedded in paraffin. Serial thin tumor tissue sections (5 μm) were cut and mounted on SuperFrost Plus glass slides, fixed in methanol and stained with hematoxylin and eosin. The slides were viewed under a Zeiss Axioplan light microscope (San Diego, CA) at a magnification of × 200.23, 26

Statistical analysis

The data from individual mouse experiments were maintained in a Paradox database. Statistical comparisons were performed using the Student's t-test for the differences between the experimental groups and the control group. The results were expressed as means ± s.d. A P-value less than 0.05 was considered statistically significant.

Results

Adoptive transfer of lymphocytes enhanced antitumor immunity in the lymphopenia mice immunized with Sp2/0-mIL-21 tumor vaccine

To the best of our knowledge, the ability of raising immune memory from SP2/0-mIL21 tumor vaccine may help with protecting against tumor cell challenge in mouse model. Therefore, we designed the first experiment of adoptive immunotherapy of myeloma in mice. However, the results showed that mice were not capable of effectively suppressing the tumor growth (Figure 1a). Then, we adopted the idea of homeostatic-driven proliferation and droved mice into lymphopenia by using Cy and it may have effectively enhanced adoptive lymphocyte homeostatic proliferation and improve antitumor immunity.19, 20 The secondary experiment results showed that, from day 12 to day 16, the tumors were generated in control group, but this tumor generation was not found until days 19–23 in Sp2/0-cell-induced group or not found until day 19–29 in Sp2/0-mIL-21-cell-induced group. The tumor regression and longevity effects were obviously seen in Sp2/0-mIL-21-cell-induced group. However, the results were still not satisfactory as all mice had tumor outgrowth in Sp2/0-mIL-21-cell-induced group in 29 days (Figure 1b). Therefore, we further designed the third experiment of adoptive immunotherapy for mice injected with Cy and transferred with Ag-specific lymphocytes from the mice immunized with Sp2/0 cells or mIL-21-Sp2/0 cells, and again immunized with the inactivated Sp2/0 or Sp2/0-mIL-21 cell vaccine. As shown in Figure 1c, all mice had tumor outgrowth on days 14–26 in control and Cy-treated groups. In contrast, 20% mice (2 of 10) treated with Cy+Sp2/0 vaccine and 40% mice (4 of 10) treated with Cy+Sp2/0-mIL-21 vaccine resulted in significant protection against Sp2/0 cell challenge and tumor was not found until day 75. In particular, in the mice treated with Cy+Sp2/0-mIL-21 vaccine, the tumor regression and longevity effects were markedly different compared with the mice treated with Cy+Sp2/0 vaccine (P<0.05) or the control group (P<0.01).

Analyzing lymphocyte proliferative activity

We designed the lymphocyte proliferative experiment to find out whether the lymphocytes were sensitized in vivo and proliferated ex vivo after mice were inoculated with the inactivated SP2/0 cells or after lymphopenia mice were treated with the different methods. The FCM analysis results suggested that the lymphocytes were actually sensitized in vivo because they proliferated more powerfully to inactivated SP2/0 cells or inactivated SP2/0-mIL21 cells in vitro compared with control group. The lymphocyte proliferative activity was 81.04±3.68% in Sp2/0-mIL-21-cell-induced group, 69.82±2.95% in Sp2/0-cell-induced group and 8.16±1.38% in control group. There were statistically significant differences between control group and each experimental group (P<0.01; Figure 2a). In Figure 2b, lymphocyte proliferative activity ex vivo was 75.06±7.86% in Cy+Sp2/0-mIL-21 vaccine group, 59.04±6.68% in Cy+Sp2/0 vaccine group, 55.82±5.95% in Cy-treated group and 2.16±1.38% in control group. The lymphocyte proliferative activity in Cy+Sp2/0-mIL-21 vaccine group showed statistically significant differences compared with Cy+Sp2/0 vaccine group (P<0.05), Cy-treated group (P<0.01) and control group (P<0.001).

Figure 2
figure2

Detecting lymphocyte proliferative responses to sp2/0 cells by FCM. Lymphocyte proliferative activities in vitro in the different groups are shown in (a) AI–AIII. (b) BI–BIII indicate lymphocyte proliferative activities ex vivo in the different groups.

Detecting IFN-γ-producing effective cells by ELISPOT assay

Figure 3a shows that the background level was lower than 10 spot-forming cells (SFC) per 105 lymphocytes but the number of SFC reached up to 59–76/105cells in mice immunized with the inactivated Sp2/0-mIL-21 cells. There were more IFN-γ-producing lymphocytes (36–45 per 105 cells) in mice immunized with inactivated Sp2/0 cells than in mice immunized with lymphocyte culture media or control group (0–4 per 105 cells) after lymphocytes were incubated with inactivated Sp2/0 cells for 24 h. It was also encouraging to see that lymphocyte-producing IFN-γ was the highest in quantity in adoptive immunotherapy of lymphopenia mice that were simultaneously immunized with inactivated Sp2/0-mIL-21 cells for 7 days. The number of SFC reached up to 263–288 per 105 cells. In contrast, there were only 161–182 per 105 cells in adoptive immunotherapy of lymphopenia mice immunized with the inactivated Sp2/0 cells, 116–128 per 105 cells in the lymphopenia mice and 2–6 per 105 cells in PBS control group.

Figure 3
figure3

Comparison of IFN-γ-producing effective cells by ELISPOT. Number of lymphocyte-producing IFN-γ in vitro is shown. The highest SFCs per 105 lymphocytes were found in mice immunized with the inactivated Sp2/0-mIL-21 cell vaccine (a) or in the adoptive immunotherapy of lymphopenia mice induced by Cy and immunized with the inactivated Sp2/0-mIL-21 cells concurrently (b). **P<0.001, *P<0.01.

FCM analyzing cytotoxicity

IL-21 can promote the expansion of CD8+ T cells and show potential activities as an antitumor agent.30, 31 The lymphocytic cytotoxicity is important in augmenting cell-mediated antitumor immunity. Therefore, we analyzed the lymphocytic cytotoxicity in mice treated with the different methods. The results showed that Cy-treated mice not only promoted homeostatic proliferation of transferred lymphocytes and the number of lymphocyte-producing IFN-γ (Figure 3) but also enhanced lymphocytic cytotoxicity (Figure 4). In contrast, although the lymphocytic cytotoxicity was also augmented in mice inoculated with Sp2/0-mIL-21 cells or Sp2/0 cells (Figure 4a), the magnitude of augmentation was weaker than that of Cy+Sp2/0-mIL-21 vaccine group (Figure 4b).

Figure 4
figure4

Cytotoxicity of lymphocytes to Sp2/0 cells tested by 7-AAD assay. In (a) the cytotoxicity of lymphocytes to Sp2/0 cell was 44.57±7.72% in Sp2/0-mIL-21-cell-induced group and it was enhanced significantly compared with Sp2/0-cell-induced group (28.22±6.78%) or control group (22.38±4.94%). (b) The cytotoxicity of lymphocytes was increased more markedly in Cy+Sp2/0-mIL-21 vaccine group (55.68±7.27%) compared with Cy+Sp2/0 vaccine group (40.12±9.82), or Cy-treated group (31.92±8.62%). For control group, the Sp2/0 cell was incubated with RPMI 1640 medium alone to measure basal cell apoptosis. **P<0.01, *P<0.05.

CD4+ and CD8+ T lymphocyte phenotype change

Both IL-2 and IL-21 cytokines promote the function of effector CD8+ T cells, but their distinctive effects on the Ag-mediated differentiation of naive CD8+ T cells into effector CD8+ T cells are not clearly understood.32 In this study, we wanted to know if Ag-specific lymphocytes were mainly CD8+ T cell phenotype. The results indicated that lymphocytes from the mice immunized with inactivated Sp2/0 cells were mostly CD8+ T cells when the lymphocytes were cultured with inactivated Sp2/0-mIL-21 cells for 5 days ex vivo, and the ratio of CD4+ T/CD8+ T cells was reversed (Figure 5a). Figure 5b also shows that major lymphocyte subtype from the mice induced by Cy was still CD8+ T cell phenotype after the lymphocytes were stimulated with inactivated Sp2/0-mIL-21 cells ex vivo. The ratio of the CD4+ T/ CD8+ T cells was also reversed and CD4+ T cell phenotype was also decreased markedly.

Figure 5
figure5

FCM analysis of CD4+ and CD8+ T cell phenotypes. The CD8+ T cell phenotypes were changed obviously: 64.49±4.64% Sp2/0-mIL-21-cell-induced group, 45.14±3.82% in Sp2/0-cell-induced group, 28.42±2.98% in lymphocytes alone group and 19.08±3.68% in control group (a). The CD8+ T cells were also increased markedly (57.88±4.62%) after the mice were treated with Cy and immunized with Sp2/0-mIL-21 vaccine group. The CD8+ T cells in other groups were 45.52±5.26% in Cy+Sp2/0 vaccine group, 36.54±4.87% in Cy-treated group and 27.67±4.32% in control group (b). The ratio of CD4+ lymphocytes was decreased in all experimental groups.

FCM analysis of CD4+CD25+ Foxp3+regulatory T cells

Recent studies on graft-versus-host reaction and antitumor immunity have provided proofs that natural CD4+CD25+Foxp3+ regulatory T (nTreg) cells inhibit protective immunity and antitumor effectiveness of adoptive immunotherapy, and that elimination of CD25+cells in vivo restores protective immunity in mice.33, 34 In observing the effect of Sp2/0-mIL-21 tumor vaccine on the nTreg cells in adoptive immunotherapy of tumor mice, we detected the change of CD4+CD25+ Foxp3+T lymphocytes from the spleens and lymph nodes. It was found that the number of CD4+CD25+ lymphocytes (Figure 6a) was gradually decreased in mice treated with the different methods. The observed decreases were about 1.52% in the mice treated with Cy, 1.33% in the mice treated with Cy+Sp2/0 vaccine and 0.86% in the mice treated with Cy+Sp2/0-mIL-21 tumor vaccine. The change in the Foxp3+ lymphocytes (Figure 6b) coincided with that of CD4+CD25+ lymphocytes. The results showed that the CD4+CD25+Foxp3+ lymphocytes (Figures 6a and b) were notably reduced after the adoptive immunotherapy for the mice that were treated with Cy and immunized with Sp2/0-mIL-21 cells, and that this strategy could assist in antitumor immunity in mice.

Figure 6
figure6

Detection of CD4+ CD25+ Foxp3+ T cells by FCM. CD4+ CD25+T lymphocytes and Foxp3+ T lymphocytes from the spleens and lymph nodes are shown in a and b, respectively. Remarkable decrease of the CD4+ CD25+ Foxp3+ T lymphocytes was found in the mice treated with Cy+Sp2/0-mIL-21 vaccine group, and the change was statistically significant compared with Cy+Sp2/0 vaccine group (P<0.05) or Cy-treated group (P<0.01) or control group (P<0.001).

Histological analysis of tumor tissues in mice treated with different methods

Histological analyses were performed on the tumor sites to investigate the cellular immune mechanisms of tumor rejection in the lymphopenia mice immunized with the IL-21-secreted tumor vaccine. The results revealed that some necrotic or apoptotic tumor cells were attributable to increased tumor-infiltrating lymphocytes (Figures 7e and f). In contrast, the active growth of tumor cells and obvious nucleic division or diverse nucleic types were found in both the mice without any treatment (Figure 7a) and the mice induced by Cy alone (Figure 7b). Although only a few tumor-infiltrating lymphocytes and some apoptosis tumor cells were visible in tumor tissues (Figures 7c and d), the tumor cell necrosis, apoptosis and vascular bleeding were more obvious in Figures 7e and f. Histopathological analyses suggest that the tumor-infiltrating lymphocytes have a biological role in this.

Figure 7
figure7

Histopathology of tumor tissues after adoptive immunotherapy of tumor mice induced by Cy and challenged with Sp2/0 cells (HE × 200). (a) Control group; (b) Cy-induced group; (c and d) adoptive immunotherapy of lymphopenia mice immunized with Sp2/0 cells. (e and f) Adoptive immunotherapy of lymphopenia mice immunized with Sp2/0-mIL-21 cells.

Discussion

In this study, we transferred the lymophocytes isolated from the spleens and lymph nodes of mice immunized with IL-21-secreted tumor vaccine into mice that had been previously been challenged with the Sp2/0 tumor cells. The findings suggested that the tumor Ag-specific lymophocytes that were transferred into the syngeneic mice generated a weak antitumor effect, indicated by an inability to inhibit tumor outgrowth when the mice were challenged with the Sp2/0 cells (Figure 1a). Obviously, this adoptive immunotherapy was not an effective way to suppress tumor growth in the mice. Possible reasons for this observation were that the low immunogenicity of tumor cells did not elicit Ag-specific lymphocytes, and that the number of transfers of the Ag-specific lymophocytes and proliferative ability in vivo were limited. Therefore, we used inactivated mIL-21 tumor vaccine as the stimulators and incubated Ag-specific lymphocytes for 3 days in the presence of IL-2 ex vivo instead of the Sp2/0 cells in an attempt to boost the immunogenicity of tumor cells. After the lymphocytes were treated ex vivo and then transferred into the lymphopenia mice challenged with the Sp2/0 cells, the efficacy of the adoptive immunotherapy was, however, still not satisfactory notwithstanding that the strategy of the adoptive immunotherapy used was better than that of the transfer of tumor Ag-specific lymophocytes to the mice alone (Figure 1b).

Cyclophosphamide can induce modulation of several cytokines and promote homeostatic activation of the adoptively transferred lymphocytes, and can induce an effective antitumor response in cancer patients. Depletion of the immune elements before the adoptive cell transfer can dramatically improve the antitumor efficacy of transferred T cells.19, 35 These studies have suggested to us that the activated lymphocytes proliferated in vitro and transferred to lymphopenia mice could enhance the adoptive transfer of the lymphocytes homeostatic proliferation and improve the immune protection against tumor cell challenge.

Finally, we adopted a novel experiment approach that involved transferring homeostasis-promoted lymphocytes and tumor Ag-specific lymphocytes to lymphopenia mice that were immunized concurrently with the secreting IL-21 tumor vaccine to augment the efficacy of the adoptive immunotherapy. The satisfactory results suggested that, under the Cy-inducing lymphopenia state, the mice that received the tumor Ag-specific lymphocytes and mIL-21-Sp2/0 vaccine, worked for the effective proliferation of transferred lymphocytes and the mice's immune cells in vivo, and assisted in forming and sustaining special antitumor effects in the tumor-bearing mice. The surprising findings showed that two out of the ten mice treated with the Cy and Sp2/0 vaccine markedly developed the protection against the Sp2/0 cell challenge, and so did four out of the ten mice treated with the Cy and Sp2/0-mIL-21 vaccine. Particularly, the mice treated with the Cy and Sp2/0-mIL-21 vaccine showed better results than any other experimental groups in terms of the tumor outgrowth, the regression and the longevity effects. There were still 60% of the mice that developed tumors, however, even if their tumor outgrowth, regression and longevity effects were markedly different from the mice treated with the other vaccination strategy. Although the mice acquired strong antitumor immunity from the optimum vaccination strategy, the numbers of tumor Ag-specific lymphocytes were limited and the Sp2/0 cells grew fast. Consequently, the protection effects did not reach 100% in the study.

To understand the mechanism for the elicited protective immune responses against the Sp2/0 cell challenge, we evaluated the lymphocyte proliferative responses to the Sp2/0 cells, the IFN-γ-producing effective cells, the lymphocytic cytotoxicity, and CD4+, CD8+ and the CD4+CD25+ Foxp3+ T cell phenotypes. The results indicated that the adoptive immunotherapy of myeloma in the lymphopenia mice inoculated with the Sp2/0-mIL-21 vaccine showed statistically significant differences from the other groups in their lymphocyte proliferative activity in vitro and ex vivo, in the number of lymphocyte-producing IFN-γ, and in the lymphocytic cytotoxicity to the Sp2/0 cells. The T-cell phenotypes of the Ag-induced lymphocytes were largely CD8+ T cells, whereas the CD4+ and CD8+ T cell phenotype changes implied that the lymphocytic cytotoxicity to the Sp2/0 cells was mostly mediated by the CD8+ T cells. Besides, spleen also have natural killer cells, and we hypothesize that the activity of natural killer cells can partly augment the cell-mediated antitumor immunity.

Owing to the Treg populations having a central role in limiting the efficacy of the tumor vaccine,36 the data in this study indicated that the CD4+CD25+ Foxp3+ lymphocytes from the spleens and the lymph nodes were obviously decreased after the adoptive immunotherapy of myeloma in the lymphopenia mice. This result coincided with a recent reported study that local IL-21 promoted the therapeutic activity of the effector T cells by decreasing Treg cells within the tumor microenvironment.37

Our previous study suggested that the mIL-21 secreted locally by tumor cell vaccines may induce dendritic cells, natural killer cells and CTL accumulation at the tumor sites. This would let the natural killer cells directly attack the tumor cells, allow dendritic cells to present tumor Ag to CTL and activate the CTL activity effectively.13, 14 Accordingly, the histopathological analyses indicated that the lymphocytes, the monocytes and the neutrophils infiltrated into the tumor tissues and adhered to the vascular walls, and that the tumor cell necrosis and apoptosis were clearly shown in the optimal therapeutic group (Figures 7e and f).

In conclusion, our study data showed that the adoptive tumor Ag-specific lymphocytes transferred into the lymphopenia mice immunized with the mIL-21-secreting tumor vaccine elicited protective immune responses against the Sp2/0 cell challenge through the Cy-induced homeostatic proliferation of the transferred lymphocytes and increased the biological activities of the transferred lymphocytes. The local IL-21-enhancing therapeutic effects in the tumor-bearing mice were also associated with the decreased Treg cells. This study created a new strategy of combining immunotherapy and chemotherapy to elicit an effective antitumor response in the mice model.

References

  1. 1

    Butler MO, Lee JS, Ansen S, Neuberg D, Hodi FS, Murray AP et al. Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell. Clin Cancer Res 2007; 13: 1857–1867.

  2. 2

    Li J, Mookerjee B, Wagner J . Purification of melanoma reactive T cell by using a monocyte-based solid phase T-cell selection system for adoptive therapy. J Immunother 2008; 31: 81–88.

  3. 3

    Powell DJ, Dudley ME, Hogan KA, Wunderlich JR, Rosenberg SA . Adoptive transfer of vaccine-induced peripheral blood mononuclear cells to patients with metastatic melanoma following lymphodepletion. J Immunol 2006; 177: 6527–6539.

  4. 4

    Goldrath AW, Bogatzki LY, Bevan MJ . Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J Exp Med 2000; 192: 557–564.

  5. 5

    Cho BK, Rao VP, Ge Q, Eisen HN, Chen J . Homeostasis stimulated proliferation drives naive T cells to differentiate directly into memory T cells. J Exp Med 2000; 192: 549–556.

  6. 6

    Prlic M, Blazar BR, Khoruts A, Zell T, Jameson SC . Homeostatic expansion occurs independently of costimulatory signals. J Immunol 2001; 167: 5664–5668.

  7. 7

    Wang LX, Jorgen K, Peter A, Shu S, Plautz GE . Memory T cells originate from adoptively transferred effectors and reconstituting host cells after sequential lymphodepletion and adoptive immunotherapy. J Immunol 2004; 172: 3462–3468.

  8. 8

    Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314: 126–129.

  9. 9

    Wilde VD, Benghiat FS, Novalrivas M, Lebrun JF, Kubjak C, Oldenhove G et al. Endotoxin hyperresponsiveness upon CD4+ T cell reconstitution in lymphopenic mice: control by natural regulatory T cells. Eur J Immunol 2008; 38: 48–53.

  10. 10

    Parrish-Novak J, Dillon SR, Nelson A, Hammond A, Sprecher C, Gross JA et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 2000; 408: 57–63.

  11. 11

    Ettinger R, Sims GP, Fairhurst AM, Robbins R, da Silva YS, Spolski R et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J Immunol 2005; 175: 7867–7879.

  12. 12

    Nutt SL, Brady J, Hayakawa Y, Smyth MJ . Interleukin 21: a key player in lymphocyte maturation. Crit Rev Immunol 2004; 24: 239–245.

  13. 13

    Dou J, Lili C, Fengshu Z, Tang Q, Zhang AF, Zhang LF et al. Study of immunotherapy of murine myeloma by an IL-21-based tumor vaccine in BALB/C mice. Cancer Biol Ther 2007; 6: 1871–1879.

  14. 14

    Dou J, Guobin C, Jing W, Fengshu Z, Junsong C, Xuesong F et al. Preliminary study on mouse interleukin 21 application in tumor gene therapy. Cellular Mol Immunol 2004; 1: 388–396.

  15. 15

    Li Y, Yee C . IL-21 mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8+ cytotoxic T lymphocytes. Blood 2004; 111: 229–235.

  16. 16

    Iuchi T, Teitz-Tennenbaum S, Huang J, Redman BG, Hughes SD, Li M et al. Interleukin-21 augments the efficacy of T-cell therapy by eliciting concurrent cellular and humoral responses. Cancer Res 2008; 68: 4431–4441.

  17. 17

    Li Y, Bleakley M, Yee C . IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8T cell response. J Immunol 2005; 175: 2261–2269.

  18. 18

    Fina D, Fantini MC, Pallon F, Monteleone G . Role of interleukin-21 in inflammation and allergy. Inflamm Allergy Drug Targets 2007; 6: 63–68.

  19. 19

    Bracci L, Moschella F, Sestili P, La Sorsa V, Valentini M, Canini I et al. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clin Cancer Res 2007; 13: 644–653.

  20. 20

    Julien T, Nathalie C, Noel S, Roux S, Novault S, Menard C et al. Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines. J Immunol 2006; 176: 2722–2729.

  21. 21

    Pawel M, Andrea B, Claudia W, Citrin DE, Rosenberg SA, Childs R et al. Increased intensity lymphodepletion and adoptive immunotherapy—how far can we go? Nat Clin Pract Oncol 2006; 3: 668–681.

  22. 22

    Sara EH, Monika CW, Stephen P, Jameson SC . The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells. Nat Immunol 2006; 7: 475–481.

  23. 23

    Dou J, Xiaowu H, Fengshu Z, Wang J, Chen JS, Chen GB . Investigation of GM-CSF immune accessory effects in tumor-bearing mice by direct gene immunization. Immunol Invest 2006; 35: 227–237.

  24. 24

    Rutigliano JA, Johnson TR, Hollinger TN, Fischer JE, Aung S, Graham BS . Treatment with anti-LFA-1 delays the CD8+ cytotoxic T-lymphocyte response and viral clearance in mice with primary respiratory syncytial virus infection. J Virol 2004; 78: 3014–3023.

  25. 25

    Naeem K, Donna B, Rachel B, Nayak L, Rickinson AB, Moss PA . T cell recognition patterns of immunodominant cytomegalovirus antigens in primary and persistent infection. J Immunol 2007; 178: 4455–4465.

  26. 26

    Rosenberg SA, Sherry RM, Morton KE, Scharfman WJ, Yang JC, Topalian SL et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J Immunol 2005; 175: 6169–6176.

  27. 27

    Martin B, Fae T, Jin L, Hesse M . Naturally occurring CD4+Foxp3+ regulatory T cells are an essential, IL-10-independent part of the immunoregulatory network in Schistosoma mansoni egg-induced inflammation. J Immunol 2006; 176: 5374–5387.

  28. 28

    Plas E, Carroll VA, Jilch R, Simak R, Mihaly J, Melchior S et al. Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer. Cytometry 2001; 46: 184–189.

  29. 29

    Herve L, Michele F, Riviere Y, Gougeon ML . A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity. J Immunol Methods 2001; 253: 177–187.

  30. 30

    Turk MJ, Guevara-Patino JA, Rizzuto GA, Engelhorn ME, Sakaguchi S, Houghton AN et al. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J Exp Med 2004; 20: 771–782.

  31. 31

    Smyth MJ, Teng MW, Sharkey J, Westwood JA, Haynes NM, Yagita H et al. Interleukin 21 enhances antibody-mediated tumor rejection. Cancer Res 2008; 68: 3019–3025.

  32. 32

    Hinrichs CS, Spolski R, Paulous CM, Gattinoni L, Kerstann KW, Palmer DC et al. IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood 2008; 111: 5326–5333.

  33. 33

    Deepe GS, Gibbons RS . TNF-alpha antagonism generates a population of antigen-specific CD4+CD25+ T cells that inhibit protective immunity in murine histoplasmosis. J mmunol 2008; 180: 1088–1097.

  34. 34

    Ohmura Y, Yoshikawa K, Saga S, Ueda R, Kazaoka Y, Yamada S et al. Combinations of tumor-specific CD8+ CTLs and anti-CD25mAb provide improved immunotherapy. Oncol Rep 2008; 19: 1265–1270.

  35. 35

    Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+T cells. JEM 2005; 202: 907–912.

  36. 36

    Tuettenberg A, Schmitt E, Knop J, Jonuleit H . Dendritic cell-based immunotherapy of malignant melanoma: success and limitations. J Dtsch Dermatol Ges 2007; 3: 190–196.

  37. 37

    Hill JA, Hall JA, Sun CM, Cai Q, Ghyselinck N, Chambon P et al. Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4CD44hi cells. Immunity 2008; 29: 758–770.

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Acknowledgements

We thank Dr Aifeng Zhang for the assistance in histological section and analysis, and Dr Rod Donlan (Centers for Disease Control and Prevention in Atlanta, GA, USA) for kindly reviewing the article. This work was supported in part by the Program for the Top Researchers in Six Fields of Jiangsu Province, China (no. D14) and in part by the 973 Program of China (no. 2006CB933206).

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Correspondence to J Dou or N Gu.

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Keywords

  • adoptive immunotherapy
  • mIL-21
  • tumor vaccine
  • lymphopenia

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