Cyclophosphamide augments antitumor immunity: studies in an autochthonous prostate cancer model

Abstract

To study the immune response to prostate cancer, we developed an autochthonous animal model based on the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in which spontaneously developing tumors express influenza hemagglutinin as a unique, tumor-associated antigen. Our prior studies in these animals showed immunologic tolerance to hemagglutinin, mirroring the clinical situation in patients with cancer who are generally nonresponsive to their disease. We used this physiologically relevant animal model to assess the immunomodulatory effects of cyclophosphamide when administered in combination with an allogeneic, cell-based granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapy. Through adoptive transfer of prostate/prostate cancer-specific CD8 T cells as well as through studies of the endogenous T-cell repertoire, we found that cyclophosphamide induced a marked augmentation of the antitumor immune response. This effect was strongly dependent on both the dose and the timing of cyclophosphamide administration. Mechanistic studies showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory T cells in the tumor draining lymph nodes but not in the peripheral circulation. Interestingly, we also noted effects on dendritic cell phenotype; low-dose cyclophosphamide was associated with increased expression of dendritic cell maturation markers. Taken together, these data clarify the dose, timing, and mechanism of action by which immunomodulatory cyclophosphamide can be translated to a clinical setting in a combinatorial cancer treatment strategy.

Figure 1

Tolerance to GVAX in tumor-bearing ProHA × TRAMP mice. A, expression of HA by transfected TRAMP-C2 cells. Dotted line, isotype control. B, immunogenicity of T-GVAX. TRAMP-C2-HA cells were admixed with GM-CSF–secreting bystanders (T-GVAX) and administered intradermally 2 d after adoptive transfer of CFSE-labeled, HA-specific CD8⁺ T Cells. Peripheral blood cells from tail vein were harvested on indicated days postimmunization. Data are gated on HA-specific CD8⁺ Thy1.1⁺ lymphocytes that divided at least once. Mean ± SE. Three animals per group, representative of two experiments. C, T-GVAX-mediated proliferation. As above, CFSE plots gated on clonotypic (CD8⁺ Thy1.1⁺) T cells. D, tolerance to T-GVAX in tumor-bearing mice. CFSE-labeled HA-specific CD8⁺ T cells were adoptively transferred to indicated mice, and animals were treated 2 d posttransfer with T-GVAX immunotherapy. Seven days posttreatment, splenocytes were harvested and counted, and IFN-γ was quantified by intracellular staining. Three animals per group, representative of two experiments.

Figure 2

Timing of cyclophosphamide + T-GVAX. A, timing of immunomodulatory cyclophosphamide with fixed in vivo rest. Top, experimental design. Treatment initiated 24 h post–adoptive transfer in all groups. Bottom, tumor-bearing animals were sacrificed on day +7 postimmunotherapy, with indicated cyclophosphamide timing. HA-specific CD8⁺ T cells were quantified from indicated sites and evaluated for IFN-γ and tumor necrosis factor-α secretion by intracellular staining. Three animals per group, representative of two experiments. B, timing of cyclophosphamide with a fixed in vivo dwell time. Top, experimental design; bottom, HA-specific CD8⁺ T cells were quantified from indicated sites and evaluated for IFN-γ secretion by intracellular staining.

Figure 3

Dosage of immunomodulatory cyclophosphamide. A, experiment performed as in Fig. 2A, with intraperitoneal cyclophosphamide dose varied as indicated. Three animals per group, representative of two experiments. B, immunologic effects of combined cyclophosphamide (Cy)/T-GVAX treatment. Eighteen- to 20-week-old (tumor-bearing) ProHA × TRAMP mice were treated day −1 with 50 mg/kg cyclophosphamide intraperitoneally followed by T-GVAX on day 0. Animals were harvested on day +7 and analyzed as above. Three animals per group, representative of two experiments. C, representative dot plots for panel B. D, effects of cyclophosphamide on circulating lymphocyte numbers. Animals were treated on day 0 with indicated cyclophosphamide dose and analyzed for indicated lymphocyte populations using TruCount technology. Three animals per group, representative of two experiments.

Figure 4

Multidose studies. A, endogenous CTL function. Top, experimental design. Bottom, CTL function in B10.D2 (left) and ProHA × TRAMP (right) animals. Three animals per group, representative of two experiments. B, cyclophosphamide + T-GVAX in multidose regimen. HA-specific CD8⁺ T cells were adoptively transferred on day −2 to ProHA × TRAMP mice. Prostate glands were harvested on day +13 after initial treatment and evaluated for IFN-γ secretion by intracellular staining. Three animals per group, representative of two experiments. C, representative dot plots for panel B.

Figure 5

Antitumor effect of combined regimen. A, wet weight of the urogenital tract. Twelve animals per group. B, tumor score of treated animals. Tumor score computed by multiplying pathologic score × extent (see Materials and Methods). Twelve animals per group. C, histologic evaluation of treated animals. Representative H&E-stained sections.

Figure 6

Effects of low-dose cyclophosphamide + T-GVAX on Treg number and function. A, Treg depletion. Following low-dose cyclophosphamide administration, absolute number of CD4⁺ T cells = FoxP3⁺ quantified using intracellular staining on indicated days. n = 3 animals per group, repeated × 2. B, representative dot plots for panel A. C, Treg function. Treg (CD4⁺CD25⁺CD62L^high) isolated from DLN or spleens of indicated mice using magnetic bead sorting. In vitro suppression assay was done using 3.5 × 10⁴ suppressors and 3.5 × 10⁴ responders (ratio 1:1). Proliferation quantified by H3 incorporation. Three animals per group, representative of three experiments. D, ratio T_eff/Treg. CD4⁺ and CD8⁺ effector T cells (IFN-γ–positive) were quantified on day +14 following administration of T-GVAX + cyclophosphamide, and compared with Treg, quantified using FoxP3⁺ intracellular staining as above. Three animals per group, representative of two experiments.