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Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

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Abstract

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8+ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8+ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8+ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8+ T cells, which led to higher ratios of CD8+/Treg and CD8+/CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8+ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.

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Acknowledgments

This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) Head and Neck SPORE Program P50 DE019032 grant and the Milton J. Dance Jr. Head and Neck Center at Greater Baltimore Medical Center, Baltimore, MD.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Johns Hopkins Medical Institutions, 601 North Caroline Street, JHOC 6th floor, Baltimore, MD, 21287, USA

    Shiwen Peng, Sofia Lyford-Pike, Belinda Akpeng, Annie Wu, John R. Saunders & Sara I. Pai

  2. Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    Shiwen Peng, Chien-Fu Hung & T.-C. Wu

  3. Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    Chien-Fu Hung, T.-C. Wu & Sara I. Pai

  4. Ichor Medical Systems, Inc., San Diego, CA, USA

    Drew Hannaman

  5. Milton J. Dance Jr. Head and Neck Center, Greater Baltimore Medical Center, Baltimore, MD, USA

    John R. Saunders

  6. Department of Obstetrics and Gynecology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    T.-C. Wu

  7. Department of Molecular Microbiology and Immunology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    T.-C. Wu

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  1. Shiwen Peng
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Correspondence to Sara I. Pai.

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Shiwen Peng and Sofia Lyford-Pike are contributed equally to the work.

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262_2012_1322_MOESM1_ESM.tif

Supplemental Figure 1 High frequency of regulatory T cells (Treg) are present in HPV-related head and neck cancers. The frequency of Tregs was evaluated in the peripheral blood, contralateral normal tonsil tissue, and tumors of HPV-related head and neck cancer patients. Single-cell suspensions were prepared from tonsil tumor or contralateral normal tissue. PBMC was prepared by Ficoll density centrifugation. Cells were stained for surface CD4 and CD25, permeabilized, fixed, and stained for intracellular Foxp3 expression. a. Top panel: Representative flow cytometry data (based on gating of total CD4+ population). Bottom panel: Summary of the flow cytometry data demonstrating increased frequency of Tregs within the tumor microenvironment as compared to the contralateral normal tonsil and circulating peripheral blood (p = 0.01) of HPV-related head and neck cancer patients. b. A higher frequency of Tregs was found in the tumor microenvironment of HPV-related head and neck cancer patients (n = 5) compared to that of benign tonsil controls (n = 7) (p = 0.03). (TIFF 130 kb)

262_2012_1322_MOESM2_ESM.tif

Supplemental Figure 2 Daily administration of CTX can inhibit vaccine induced immunologic responses at a threshold dose. 1 × 105 TC-1 tumor cells per mouse were injected subcutaneously (s.c.) in the right flank of 5- to 8-wk-old C57BL/6 mice (five mice per group) and were treated with either pNGVL4a-CRT/E7(detox) DNA vaccine alone, daily dose of 10 mg/kg of CTX alone, daily dose of 20 mg/kg of CTX alone, daily dose of 10 mg/kg of CTX in combination with pNGVL4a-CRT/E7(detox) DNA vaccine, daily dose of 20 mg/kg of CTX in combination with pNGVL4a-CRT/E7(detox) DNA vaccine, or no treatment. 7 days after the last vaccination, single cells were prepared from spleen and tumors. To detect regulatory T cells, the lymphocytes were surface stained with anti-mouse CD4 followed by intracellular Foxp3 staining according to the manufacturer’s protocol. HPV-16 E7aa49-57 peptide-specific CD8+ T cells were evaluated with tetramer staining. Specifically, 1 × 105 tumor-infiltrating lymphocytes were stained with purified anti-mouse CD16/32, and then stained with FITC-conjugated anti-mouse CD8a, PE-conjugated HPV-16 E7aa49-57 peptide loaded H-2Db tetramer (provided by National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility), and APC-conjugated anti-mouse CD3. a Summary of the flow cytometry data of regulatory T cells from tumor-infiltrating lymphocytes. b Summary of the flow cytometry data of CD3+CD8+E7 tetramer+ T cells from tumor-infiltrating lymphocytes. (TIFF 67 kb)

262_2012_1322_MOESM3_ESM.tif

Supplemental Figure 3 Administration of CTX on TC-1 tumor growth in T cell deficient athymic nude mice has no anti-tumor effect. a Schedule of CTX treatment of TC-1 tumor-bearing athymic nude mice. b Tumor growth curve from TC-1 tumor-bearing athymic nude mice treated with CTX either daily or single dose. Briefly, 1 × 105 TC-1 tumor cells per mouse were injected into 5- to 8-week-old athymic nude mice (five mice per group) s.c. in the right flank. After 8 days, the mice were treated as indicated in the schematic diagram of the CTX treatment schedule (A). Tumor volume was calculated as described in the material and method section. (TIFF 64 kb)

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Peng, S., Lyford-Pike, S., Akpeng, B. et al. Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine. Cancer Immunol Immunother 62, 171–182 (2013). https://doi.org/10.1007/s00262-012-1322-5

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