IL-21 plus IL-2-cultured TDLN cells mediate effective tumor regression. Anti-CD3/anti-CD28-activated MCA 205 TDLN cells were cultured in IL-2, IL-21, or IL-2 plus IL-21. Mice with 3-day established MCA 205 pulmonary metastases were treated by the adoptive transfer of activated and expanded TDLN cells (3 × 10⁶ cells/mouse) along with the administration of IL-2 (4 × 10⁴IU twice daily for 4 days). Fifteen days after tumor injection, all mice were euthanized for enumeration of pulmonary metastasis nodules. *p<0.05 compared with control. **p<0.05 compared with any other groups. The data were reproduced in a second experiment.

Humoral responses contribute significantly to T cell+IL-2+IL-21-elicited antitumor immunity. A and B: Significantly stimulated antibody production by IL-21 and IL-2 administration during T cell therapy. Blood samples were collected at the end of T cell plus IL-2 and/or IL-21 therapy either from pulmonary metastasis-bearing mice (A) or from subcutaneous tumor-bearing mice (B) and assessed for serum total IgG and IgM as well as tumor-specific IgG production using ELISA as described in the Materials and Methods. C: Requirement for B cells in T cell+IL-2+IL-21-elicited antitumor immunity. Subcutaneous tumor models were established in wt mice or in B^−/− mice and were then treated with T cell adoptive transfer plus IL-2 and IL-21 administration as in Fig.4. Similar data were generated in a second experiment.

IL-21 administration augments the antitumor efficacy of adoptively transferred TDLN cells in the pulmonary metastatic and subcutaneous tumor models. MCA 205 TDLN cells were activated with anti-CD3/anti-CD28 followed by expansion in IL-2. Mice wit h 3-day established MCA 205 pulmonary metastases were treated by adoptive transfer of these effector TDLN cells at the indicated doses accomplished by IL-21 administration. IL-21 was administrated i.p. at the indicated doses for 4 days starting on the day when adoptive T cell transfer was performed without (A) or with (B, C) IL-2 administration. Lungs were harvested and the pulmonary metastases were enumerated 15 days after tumor inoculation. D: IL-21 administration during TDLN cell transfer suppresses the growth of subcutaneous tumor in synergy with IL-2. Three days after MCA 205 tumor injection subcutaneously on the right flank, mice were exposed to whole body irradiation at 5Gy. Immediately after irradiation, antibody-activated and IL-2-cultured MCA 205 TDLN cells were transferred into the tumor-bearing mice with IL-2 (4×10⁴ IU twice a day) and/or IL-21 (20 µg daily) i.p. administration for 4 days. P<0.05 on day 17 comparing IL-2 plus IL-21 with IL-2 or IL-21 alone. Data are representative of two independently performed experiments.

IL-21 in synergy with IL-2 modulates both type 1 and type 2 cytokine secretion of TDLN cells. 1A: TDLN cells were activated with anti-CD3 and anti-CD28 for two days followed by culture in IL-2 (60 IU/ mL), IL-21 (1 µg/mL), or IL-2 plus IL-21 for 3 days. Unfractionated TDLN cells, purified CD4 or CD8 TDLN cells were used in these assays. Culture supernatants were collected at the end of cell expansion in order to detect the released cytokines by ELISA. *p<0.05 comparing IL-2 plus IL-21 cultured cells with any other groups in the same chart. Antibody-activated and IL-2/IL-21-cultured TDLN cells were stained for intracellular IFNγ (1B) and IL-10 (1C) and analyzed by FACS. For intracellular staining of cytokines, PMA (10ng/mL), ionomycin (500ng/mL) and GolgiStop (1µl/mL) or GolgiPlug (1 µl/mL) were added to cell culture in CM and incubated for 4 to 5 hours before staining. 1D: Cytokine secretion of IL-2/IL-21-cultured TDLN cells in response to tumor stimulation. Antibody-activated and IL-2 and/or IL-21-cultured TDLN cells were co-cultured with irradiated autologous MCA 205 tumor cells or EL-4 tumor cells (specificity control). Twenty four hours later, the supernatants were collected and analyzed for cytokine production. *p<0.01 compared with IL-2 cultured TDLN cells. **p<0.001 compared with any other groups in IL-10 secretion. Data are representative of two independent experiments.

T cell plus IL-21 and IL-2 therapy confers systemic immunity to the treated hosts. 4A: Protective antitumor immunity granted by the T cell plus IL-21 and IL-2 therapy. Subcutaneous MCA 205 tumors in the mice treated with adoptively transferred TDLN cells plus IL-2, IL-21, or IL-2 plus IL-21 administration were surgically resected on day 17 after tumor injection. These mice were then re-challenged with MCA205 or EL-4 tumor cells (specificity control) to the opposite (left) flank on day 17. Naïve mice were used as control. Tumor size was measured every 2 or 3 days. Data are representative of two independent experiments. 4B: Generation of memory T cells post T cell+IL-2+IL-21 combined therapy. S.c. tumor-bearing animals were treated with transferred T cells plus administrated IL-2 and IL-21 as in Fig. 4. PBMCs isolated from the non-treated (control) group or from the T cell+IL-2+IL-21-treated group at the end of therapy (day 17) were stained with FITC anti-CD3 and PE anti-CD44, PE anti-Ly-6G/Ly-6C antibodies. Analysis of the stained cells was performed using the CellQuest software. Dot plots were gated on CD3⁺ cells, and the percentage of double positive cells was recorded. Data were repeated in a second experiment. 4C: Elevated serum levels of IFNγ and IL-10 in the animals subjected to T cell plus IL-21 and T cell plus IL-21 and IL-2 therapy. Blood samples were collected at the end of T cell plus IL-2 and/or IL-21 therapy from the subcutaneous tumor-bearing mice. Serum IFNγ and IL-10 concentrations were then assessed using ELISA.

IgG2b increases in the sera of T cell+IL-2+IL-21-treated animals, binds specifically to MCA205 tumor cells, and such immune sera mediates tumor cell lysis in the presence of complement. 6A: Obviously increased IgG2b in the sera of animals subjected to T cell+IL-2+IL-21 therapy. Sera collected from the animals at the end of therapy in Fig.5C were tested for IgG2b using ELISA. Two mice from each experiment group were examined in each test. Two tests were performed. Therefore, data of the two T cell+IL-2+IL-21-treated wt mice are representative of four mice tested in this group of total 5–6 mice. 6B: Serum IgG2b from immune mice specifically binds to MCA205 tumor cells. MCA205 tumor cells were incubated with the sera from each of the 4 mice (indicated by 1, 2, 3, 4) subjected to T cell+IL-2+IL-21 therapy in Fig. 5C. Bound serum IgG2b onto the tumor cells were then detected by secondary antibody FITC-anti-mouse IgG2b. FITC-coupled secondary antibody isotype-matching control was also used. Use of secondary antibody alone without pre-incubation with the sera revealed no non-specific binging to tumor cells. Direct staining using FITC-anti-mouse MHCI served as positive control in the system. Furthermore, an irrelevant tumor cell line Pan-02 was used to verify that binging of IgG2b from immune sera to MCA205 cells was tumor specific. Number within each immunofluorescence histogram indicates the percentage of viable cells stained positive with the FITC-coupled antibodies. 6C: IgG2b-enriched immune sera induce tumor-specific cytotoxicity. Viable MCA205 tumor cells (0.5 × 10⁶) were put in 4 ml test tubes in 450 µl CM plus 50µl sera from two non-treated control mice respectively as indicated or 50µl sera from three immune mice subjected to T cell+IL-2+IL-21 therapy. Cells were incubated on ice for 1 hour. After spin, culture supernatant was discarded and 450 µl fresh CM plus 50µl rabbit complement was added to each tube followed by incubation in a 37°C water bath for another one hour. Viable cells were then counted under the microscope after trypan blue staining. Percentage (%) of viable cells was calculated by dividing the remaining viable cells with 0.5 × 10⁶. Some groups were left untreated (CM only), or treated with complement (C) alone. Pan-02 tumor cells were used for specificity control. Data are representative of two independent experiments conducted. 6D: Immune serum and complement-mediated cytotoxicity analyzed in 96-well culture plates and determined using the Quick Cell Proliferation Assay Kit. This assay was performed according to the manufacturer’s instructions. MCA205 tumor cells were cultured with the sera of six T cell+IL-2+IL-21-treated mice respectively (mice 1, 2 were used in Expt.1 and mice 3, 4, 5, 6 were used in Expt.2), followed by culture in complement. Some groups were cultured in CM without sera and complement or with complement alone. OD values were then measured via a multi-well spectrophotometer to evaluate cell lysis.