Radiotherapy combined with intratumoral dendritic cell vaccination enhances the therapeutic efficacy of adoptive T-cell transfer

Abstract

Treatment of C57BL/6 mice with cyclophosphamide (100 mg/kg) and fludarabine (200 mg/kg) induced nonmyeloablative lymphodepletion without inhibiting D5 melanoma tumor growth. Using this model, we found that induction of lymphopenia before adoptive transfer of ex vivo anti-CD3/CD28 activated and interleukin-2 expanded D5-G6 tumor draining lymph node cells enhanced the antitumor efficacy of the infused cells in both pulmonary metastases and subcutaneous D5 bearing mice. However, induction of lymphopenia did not promote intratumoral or extratumoral proliferation or accumulation of the infused cells. We have previously shown that radiotherapy enhances the therapeutic efficacy of intratumoral unpulsed dendritic cell vaccination in subcutaneous murine tumor models by augmenting the induction of antitumor cellular immune responses. Here, we confirmed this finding in a murine metastatic melanoma liver tumor model. Furthermore, local tumor irradiation combined with intratumoral dendritic cell administration significantly enhanced the therapeutic efficacy of tumor-reactive T cell adoptive transfer in this lymphodepleted liver tumor model. This was evident by reduced liver tumor size, decreased incidence of spontaneous intra-abdominal metastasis, and prolonged survival, resulting in 46% of mice cured. This enhanced antitumor activity was associated with a selective increase in proliferation, accumulation, and function of CD4+ rather than CD8+ infused cells. This multimodality regimen may have translational applications for the treatment of human cancers.

FIGURE 1

Treatment of mice with cyclophosphamide and fludarabine induces a profound, yet transient, reduction in the number of CD45⁺ and CD3⁺ cells in peripheral blood samples without inhibiting D5 tumor growth or prolonging mice survival. A, Naïve mice received chemotherapy on days 0 and 1. Blood samples, drawn at the designated time points, were analyzed for CD45⁺ and CD3⁺ cells. Data represents mean ± SE (n=5). *P<0.02. B and C, Mice were inoculated i.v. (B) or s.c. (C) with D5 cells on day 0. Chemotherapy was administered on days 3 and 4. Anti-tumor responses were evaluated by enumerating pulmonary metastases on day 15 (B left), measuring s.c. tumor size (C left), and monitoring mice survival (B and C right). (n=6). All experiments were repeated two times with similar results.

FIGURE 2

Induction of lymphopenia prior to adoptive transfer (AT) enhances the therapeutic efficacy of the infused cells. D5 pulmonary metastases (A) or s.c. tumors (B, C) were established in mice on day 0. Chemotherapy (chemo) was administered on days 3 and 4. AT (3×10⁷ cells, unless otherwise specified) was given on day 7, followed by IL-2. Anti-tumor responses were evaluated by enumerating lung metastases on day 16 (A), measuring tumor size (B), and monitoring mice survival (C). Data represents mean ± SE (n=5). A,*P<0.001 comparing equal number of cells transferred with or without chemotherapy. B and C,*P<0.005 versus all other groups. All experiments were repeated two times with similar results. NT, no treatment.

FIGURE 3

Induction of lymphopenia prior to AT does not enhance accumulation (A) or proliferation (B and C) of infused cells. Pulmonary metastases were induced in CD45.2 mice and treated as described in Fig. 2A. CFSE-labeled CD45.1 cells (3×10⁷) were used for AT. Spleens, blood samples, and lungs were collected 1, 3, and 5 days after AT. A, Tissue-derived single cell suspensions were analyzed for CD45.1⁺ cells. Data are presented as mean percentage (left panels) or mean absolute number (right panels) of CD45.1 T cells ± SE (n=5). *P<0.02. B, Dot plots of CD45.1 T cells with (a) or without (h) CFSE labeling before adoptive transfer. Representative dot plots (gated on CD45.1⁺ cells) of spleen cells obtained 1 (b and c), 3 (d and e), and 5 (f and g) days after AT from mice treated with AT+IL-2 (b, d, and f) or Chemo+AT+IL-2 (c, e, and g). Numbers in dot plots designate mean ± SE of the geometrical mean of CFSE intensity (n=5). Dotted lines indicate lower limit of detection. C, The geometrical mean of CFSE intensity of CD45.1⁺ cells in spleen samples was recorded. Data represent mean ± SE (n=5). Dotted line indicates lower limit of detection based on unstained cells.

FIGURE 4

Radiotherapy enhances the therapeutic efficacy of dendritic cell (DC) vaccination in a liver tumor model. D5 hepatic tumors were induced in mice and then treated with radiation (RT) plus i.t. DC injections. Anti-tumor responses were evaluated by measuring tumor size on day 14 (A), assessing for intra-abdominal metastatic spread on day 14, 19, and after death (B), and monitoring mice survival (C). Each group consisted of a total of 11 mice, treated in 2 independent experiments. A, Each data point represents an individual mouse; bars depict mean. B and C, Survival curves present cumulative data of 2 experiments. *P<0.02 versus no treatment (NT), **P<0.01 versus all other groups. Statistical analysis compared groups across the 2 experiments.

FIGURE 5

Radiotherapy (RT) plus dendritic cell (DC) vaccination enhances the therapeutic efficacy of adoptive transfer (AT). D5 hepatic tumors were induced in mice and then treated with RT+DC+Chemo+AT+IL-2. Anti-tumor responses were evaluated by measuring tumor size on day 14 via laparotomy (A), measuring tumor volume on day 19 via ultrasound imaging (B), assessing intra-abdominal metastatic spread on day 14 and after death (C), and monitoring mice survival (D). Each group consisted of a total of 11 mice, treated in 2 independent experiments. A, Each data point represents an individual mouse; bars depict mean. *P<0.0001 versus no treatment (NT) and chemotherapy plus interleukin-2 (Chemo+IL-2), **P<0.05 versus all other groups except RT+DC. Pictures of a representative D5 liver tumor from each group taken on day 14 are shown. B,*P<0.03. A sagittal (middle) and transverse (right) view of a representative ultrasound image of a D5 liver tumor from RT+DC are shown. C and D, Survival curves present cumulative data of 2 experiments. C,*P<0.02 versus NT and Chemo+IL-2, **P<0.002 versus all other groups. Representative pictures of disseminated intra-abdominal metastatic disease taken on day 14 from an untreated mouse (middle) and a mouse treated with Chemo+IL-2 (right) are shown. D,*P<0.0001 versus NT and Chemo+IL-2, **P<0.002 versus all other groups.

FIGURE 6

Radiotherapy (RT) plus dendritic cell (DC) vaccination promotes proliferation, accumulation, and IFNγ secretion of CD4⁺ cells. Liver tumors were induced in CD45.2 mice and then treated as described in Fig. 5. CFSE-labeled CD45.1 cells were used for adoptive transfer (AT). A, B, and C, All groups received chemotherapy plus IL-2 in addition to designated treatment. Erythrocyte-depleted peripheral blood cells, collected on days 2, 5, and 10 after AT, were analyzed for CD45.1⁺ (A), CD45.1⁺CD8⁺ (B), and CD45.1⁺CD8⁻ (B) cells. The geometrical mean of CFSE intensity (GMFI) of CD45.1⁺CD8⁺ and CD45.1⁺CD8⁻ cells was recorded (C). Dotted line indicates lower limit of detection based on mean value of unstained cells. Data on left represents mean ± SE (n=6). A, *P<0.02. B, *P<0.002. C, *P<0.01 versus CD4-RT+DC+AT, **P<0.02 versus CD8-RT+DC+AT. D, Blood samples were analyzed for IFNγ production of CD4⁺ and CD8⁺ cells in response to in vitro D5 tumor stimulation. Data points represent individual mice; bars depict mean. *P<0.05 versus no treatment (NT), ** P<0.02 versus all other groups. Representative dot plots or histograms acquired on day 5 after AT are shown in the right panels (A, no gate; B, gated on CD45.1⁺ cells; C, gated on CD45.1⁺CD8⁻ cells; D, gated on CD4⁺ cells).