In vivo sensitized and in vitro activated B cells mediate tumor regression in cancer adoptive immunotherapy

Abstract

Adoptive cellular immunotherapy utilizing tumor-reactive T cells has proven to be a promising strategy for cancer treatment. However, we hypothesize that successful treatment strategies will have to appropriately stimulate not only cellular immunity, but also humoral immunity. We previously reported that B cells in tumor-draining lymph nodes (TDLNs) may function as APCs. In this study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activated TDLN B cells alone mediated effective (p < 0.05) tumor regression after adoptive transfer into two histologically distinct murine pulmonary metastatic tumor models. Prior lymphodepletion of the host with either chemotherapy or whole-body irradiation augmented the therapeutic efficacy of the adoptively transferred TDLN B cells in the treatment of s.c. tumors as well as metastatic pulmonary tumors. Furthermore, B cell plus T cell transfers resulted in substantially more efficient antitumor responses than B cells or T cells alone (p < 0.05). Activated TDLN B cells conferred strong humoral responses to tumor. This was evident by the production of IgM, IgG, and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence of complement. Collectively, these data indicate that in vivo primed and in vitro activated B cells can be employed as effector cells for cancer therapy. The synergistic antitumor efficacy of cotransferred activated B effector cells and T effector cells represents a novel approach for cancer adoptive immunotherapy.

Fig 1

CD19⁺ B cells in the TDLN. TDLN cell suspension was prepared from freshly harvested TDLNs and stained with FITC-anti-CD3 (A), PE-anti-CD19 (B), or FITC-anti-B220 (C) respectively, or double-stained with PE-anti-CD19 and FITC-anti-B220 (D). Stained cells were then analyzed by flow cytometry using a FACScan flow microfluorometer as described in the Materials and Methods. Data are representative of five experiments performed.

Fig 2

Immunoglobulin production of TDLN B cells after activation. 2A. Purified and activated TDLN B cells produce IgG and IgM in response to tumor. B cells purified from MCA 205 TDLNs were activated with LPS or anti-CD40 respectively. Activated TDLN B cells (1×10⁶) were then re-stimulated with irradiated MCA 205 tumor cells for 24 hours. The culture supernatant with tumor re-stimulation (B cell + tumor) or without tumor re-stimulation (B cell alone) were collected and analyzed for the secretion of IgG and IgM by ELISA. 2B. LPS plus anti-CD40 activation significantly enhanced IgM production of TDLN B cells compared with LPS or anti-CD40 activation alone. Purified B cells from TDLN were activated with LPS and/or anti-CD40. Cultured supernatants at the end of cell activation were then collected and analyzed for immunoglobulin production using ELISA.

Fig 3

Activated TDLN B cells mediated effective tumor regression. 3A. Simultaneously activated T and B cells in unfractionated TDLN cells mediated significantly more efficient tumor regression than T cells alone. B6 mice were inoculated i.v. by tail vein with MCA 205 tumor cells to establish pulmonary metastases. Three days after tumor inoculation, the tumor-bearing mice were treated with tail vein injection of various doses of unfractionated MCA 205 TDLN cells activated with anti-CD3/anti-CD28/IL-2 or anti-CD3/anti-CD28/IL-2 + LPS/anti-CD40 respectively. Approximately 14 days after cell transfer, all mice were randomized and sacrificed, and lungs were harvested for enumeration of pulmonary metastatic nodules. There were 5 mice in each group, and all the animals formed tumor except for G2 when 3 × 10⁶ cells were infused. Data are representative of four experiments performed. 3B. Purified and activated TDLN B cells alone mediated tumor regression and the therapeutic efficacy can be significantly augmented by purified B cells plus T cells. Three-day pulmonary metastases were established as in 3A, but treated with purified and activated B cells alone, T cells alone or T cells plus B cells. Lungs were harvested for enumerations of pulmonary metastatic nodules as in 3A. There were 5 mice in each group, and all the animals formed tumor except for group 3 in both experiments. Data are representative of five experiments performed.

Fig 4

TBI or chemotherapy significantly augmented the therapeutic efficacy of adoptively transferred B cells. 4A. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c MCA 205 tumor model. Established (5-Day) s.c MCA 205 tumor-bearing mice were treated with TBI (5 Gy) followed by adoptive transfer of purified and activated MCA 205 TDLN B cells. Animals treated with TBI or B cells alone served as controls. Tumor size was then monitored. There were 6 mice in each group, and all the animals formed tumors. Data are representative of two independently conducted experiments. 4B. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c D5 tumor model. Established s.c D5 tumor-bearing mice were treated with TBI and/or adoptive transfer of purified and activated D5G6 TDLN B cells as in 4A. There were 6 mice in each group, and all the animals formed tumor. Data are representative of two experiments performed. 4C. The poorly immunogenic tumor, D5 melanoma, was used in this experiment. Five-day pulmonary metastases were established as in Fig. 3. In relevance to inoculation of D5 tumor cells on day 0, chemotherapy (1mg cyclophosphamide and 2 mg fludara) was administered i.p. on days 3 and 4 to induce lymphodelpletion. Various doses of purified and activated D5G6 TDLN B cells were given i.v. on day 5. Lungs were harvested for enumerations of pulmonary metastatic nodules as in Fig. 3. There were 6 mice in each group, and all the animals formed tumor.

Fig 4

TBI or chemotherapy significantly augmented the therapeutic efficacy of adoptively transferred B cells. 4A. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c MCA 205 tumor model. Established (5-Day) s.c MCA 205 tumor-bearing mice were treated with TBI (5 Gy) followed by adoptive transfer of purified and activated MCA 205 TDLN B cells. Animals treated with TBI or B cells alone served as controls. Tumor size was then monitored. There were 6 mice in each group, and all the animals formed tumors. Data are representative of two independently conducted experiments. 4B. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c D5 tumor model. Established s.c D5 tumor-bearing mice were treated with TBI and/or adoptive transfer of purified and activated D5G6 TDLN B cells as in 4A. There were 6 mice in each group, and all the animals formed tumor. Data are representative of two experiments performed. 4C. The poorly immunogenic tumor, D5 melanoma, was used in this experiment. Five-day pulmonary metastases were established as in Fig. 3. In relevance to inoculation of D5 tumor cells on day 0, chemotherapy (1mg cyclophosphamide and 2 mg fludara) was administered i.p. on days 3 and 4 to induce lymphodelpletion. Various doses of purified and activated D5G6 TDLN B cells were given i.v. on day 5. Lungs were harvested for enumerations of pulmonary metastatic nodules as in Fig. 3. There were 6 mice in each group, and all the animals formed tumor.

Fig 4

TBI or chemotherapy significantly augmented the therapeutic efficacy of adoptively transferred B cells. 4A. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c MCA 205 tumor model. Established (5-Day) s.c MCA 205 tumor-bearing mice were treated with TBI (5 Gy) followed by adoptive transfer of purified and activated MCA 205 TDLN B cells. Animals treated with TBI or B cells alone served as controls. Tumor size was then monitored. There were 6 mice in each group, and all the animals formed tumors. Data are representative of two independently conducted experiments. 4B. TBI significantly augmented the therapeutic efficacy of adoptively transferred B cells in the s.c D5 tumor model. Established s.c D5 tumor-bearing mice were treated with TBI and/or adoptive transfer of purified and activated D5G6 TDLN B cells as in 4A. There were 6 mice in each group, and all the animals formed tumor. Data are representative of two experiments performed. 4C. The poorly immunogenic tumor, D5 melanoma, was used in this experiment. Five-day pulmonary metastases were established as in Fig. 3. In relevance to inoculation of D5 tumor cells on day 0, chemotherapy (1mg cyclophosphamide and 2 mg fludara) was administered i.p. on days 3 and 4 to induce lymphodelpletion. Various doses of purified and activated D5G6 TDLN B cells were given i.v. on day 5. Lungs were harvested for enumerations of pulmonary metastatic nodules as in Fig. 3. There were 6 mice in each group, and all the animals formed tumor.

Fig 5

Significantly increased IgM, IgG and IgG2b production in the culture of T + B cells. Supernatant collected at the end of T and/or B cell activation with anti-CD3/anti-CD28/IL-2 and/or LPS/anti-CD40 were tested for IgM, IgG and IgG2b using ELISA. Data are representative of four independently performed experiments.

Fig 6

TDLN B cell-produced IgG2b binds specifically to tumor cells. D5, MCA205, and Pan-02 tumor cells were incubated with the culture supernatant (Sup) of MCA 205 TDLN B cells. Bound IgG2b onto the tumor cells was then detected by secondary antibody FITC-anti-mouse IgG2b. FITC-coupled secondary antibody isotype-matching control was also used. Numbers within each immunofluorescence dot plots indicate the percentage of viable cells stained positive with the FITC-coupled antibodies. Data are representative of three independently performed experiments.

Fig 7

Antibody and complement-mediated tumor cell lysis. 7A: IgG2b-enriched cell culture supernatant induces tumor cell cytotoxicity. Viable MCA 205 tumor cells (0.5 × 10⁶) were put in 4 mL test tubes in 450 µl CM plus 50µl cell culture supernatant from four cell groups respectively as indicated. Cells were incubated on ice for 1 hour. After centrifugation, culture supernatants were 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. Control groups included MCA 205 cells similarly cultured without cell culture supernatants or complement (MCA 205 only), or with complement alone (MCA 205 + C). 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. Data are representative of two independent experiments conducted. 7B: Antibody and complement-mediated cytotoxicity was 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. MCA 205 tumor cells were incubated with or without antibody-enriched cell culture supernatants and complement as in Fig. 7A. OD values were then measured via a multi-well spectrophotometer to evaluate cell lysis. Data are representative of three independent experiments conducted.

Fig 8

Tumor specific cytotoxicity mediated by antibodies and complement. Viable D5, MCA 205, or Pan-2 tumor cells (0.5 × 10⁶) were incubated with culture supernatants of D5G6 TDLN B cells from two separate experiments (Sup#1 and Sup#3) or with culture supernatant of MCA 205 TDLN B cells (Sup #2) in 4 mL test tubes as indicated. After that, rabbit complement was added for incubation for one hour. Control groups included tumor cells cultured with complete medium (Control), complement only (C), or antibodies without complement. 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.

Fig 9

Tumor specific cytotoxicity analyzed in 96-well culture plates and determined using the Quick Cell Proliferation Assay Kit. This assay was performed similarly as in Fig. 7B. D5 and MCA205 tumor cells were incubated with antibody-enriched cell culture supernatant of MCA 205 TDLN B cells (Sup#1) or D5G6 TDLN B cells (Sup#2) and complement. OD values were then measured via a multi-well spectrophotometer to evaluate cell lysis. Pan-02 tumor cells; tumor cells incubated with complete medium (Control), or complement only (C), or with antibody but no complement served as controls. Data were repeated in a second experiment.