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Tissue sections of spleen and liver were stained with the F4/80 macrophage marker. These photos are representative of the whole organ of two tested mice.

Mice were inoculated on day 0 with 1 x 105 tumor cells. On day 9, when a palpable tumor was present, mice were treated with HPV16 E743-77 peptide in IFA. A) The frequency of myeloid cells in the live gate is indicated. B) Shown is a graph of tumor sizes in mm2 ten days after vaccination. Shown is the mean + SEM of eight mice per group.

The percentage of antigen specific and IFN-γ producing CD8 T cells in (A-B) blood and (C) lymph nodes of non-tumor-bearing mice on (A) day 7 and (B) day 15 after vaccination, as analysed by flow cytometry. Values of vaccinated groups with control- or PLX3397 chow were not significantly different (as analysed by Student T test, P > 0.05).

Mean tumor sizes and SEM of living animals are depicted for three groups: untreated (n=4), SLP vaccinated (peptide) (n=8) and SLP vaccination in the presence of PLX5622 (peptide + PLX) (n=8).

A) Wild-type C57BL/6 mice were injected s.c. with TC-1 tumor cells in the flank. Half of the mice received PLX chow, the other half control chow. Nine days later, when tumors were palpable, 50% of the mice in each group were treated with HPV16 E743-77 peptide in IFA in the opposing flank. Ten days later, on day 19 tumors were resected. Tumors were embedded in paraffin and stained for F4/80, as a marker for macrophages. Photos are representative for the whole tumor area and at least four samples per group.

Abundant macrophage infiltration of solid cancers commonly correlates with poor prognosis. Tumor-promoting functions of macrophages include angiogenesis, metastasis formation, and suppression of Th1-type immune responses. Here, we show that successful treatment of cervical carcinoma in mouse models with synthetic long peptide (SLP) vaccines induced influx of cytokine-producing CD8 T cells that strongly altered the numbers and phenotype of intratumoral macrophages. On the basis of the expression of CD11b, CD11c, F4/80, Ly6C, Ly6G, and MHC II, we identified four myeloid subpopulations that increased in numbers from 2.0-fold to 8.7-fold in regressing tumors. These changes of the intratumoral myeloid composition coincided with macrophage recruitment by chemokines, including CCL2 and CCL5, and were completely dependent on a vaccine-induced influx of tumor-specific CD8 T cells. CD4 T cells were dispensable. Incubation of tumor cells with T cell–derived IFNγ and TNFα recapitulated the chemokine profile observed in vivo, confirming the capacity of antitumor CD8 T cells to mediate macrophage infiltration of tumors. Strikingly, complete regressions of large established tumors depended on the tumor-infiltrating macrophages that were induced by this immunotherapy, because a small-molecule drug inhibitor targeting CSF-1R diminished the number of intratumoral macrophages and abrogated the complete remissions. Survival rates after therapeutic SLP vaccination deteriorated in the presence of CSF-1R blockers. Together, these results show that therapeutic peptide vaccination could induce cytokine-producing T cells with strong macrophage-skewing capacity necessary for tumor shrinkage, and suggest that the development of macrophage-polarizing, rather than macrophage-depleting, agents is warranted. Cancer Immunol Res; 3(9); 1042–51. ©2015 AACR.

Supplemental figure 3) Synergistic effects between cisplatin and peptide vaccination preserved with other treatment protocol

Induction of tumor-specific T-cell responses are dependent on cisplatin treated tumor cells.

Supplemental Video 2. TC-1 cells were in vitro cultured and treated with cisplatin (2 μg/ml). Cell death was followed for 22 hours using Annexin-V staining and automated imaging on a BD pathway 855 imager. Every hour a picture was taken.

Supplemental figure 2) Activation markers on T cells are not affected by cisplatin treatment.

Supplemental figure 1) Peptide vaccination synergizes with cisplatin, which allows lowering of the cisplatin dose.

Supplemental Video 4. TC-1 cells were in vitro cultured and treated with cisplatin (2 μg/ml) and TNFα (250iU). Cell death was followed for 22 hours using Annexin-V staining and automated imaging on a BD pathway 855 imager. Every hour a picture was taken.

Modulation of costimulatory molecule expression by HMGB1.

Purpose: Cancer immunotherapy, such as vaccination, is an increasingly successful treatment modality, but its interaction with chemotherapy remains largely undefined. Therefore, we explored the mechanism of synergy between vaccination with synthetic long peptides (SLP) of human papillomavirus type 16 (HPV16) and cisplatin in a preclinical tumor model for HPV16.Experimental Design: SLP vaccination in this preclinical tumor model allowed the elucidation of novel mechanisms of synergy between chemo- and immunotherapy. By analyzing the tumor immune infiltrate, we focused on the local intratumoral effects of chemotherapy, vaccination, or the combination.Results: Of several chemotherapeutic agents, cisplatin synergized best with SLP vaccination in tumor eradication, without requirement for the maximum-tolerated dose (MTD). Upon SLP vaccination, tumors were highly infiltrated with HPV-specific, tumor necrosis factor-α (TNFα)- and interferon-γ (IFNγ)–producing T cells. Upon combined treatment, tumor cell proliferation was significantly decreased compared with single treated and untreated tumors. Furthermore, we showed that TNFα strongly enhanced cisplatin-induced apoptotic tumor cell death in a JNK-dependent manner. This is consistent with upregulation of proapoptotic molecules and with enhanced cell death in vivo upon combined SLP vaccination and cisplatin treatment. In vivo neutralization of TNFα significantly reduced the antitumor responses induced by the combined treatment.Conclusion: Taken together, our data show that peptide vaccination with cisplatin treatment leads to decreased tumor cell proliferation and TNFα-induced enhanced cisplatin-mediated killing of tumor cells, together resulting in superior tumor eradication. Clin Cancer Res; 21(4); 781–94. ©2014 AACR.

Cisplatin-induced cell death enhances T-cell activation by upregulating CD70, CD80 and CD86 costimulatory molecules.

Categorizations of proteins released upon cisplatin treatment of tumor cells.

Supplemental Video 3. TC-1 cells were in vitro cultured wer treated with TNFα (250iU). Cell death was followed for 22 hours using Annexin-V staining and automated imaging on a BD pathway 855 imager. Every hour a picture was taken.

Supplemental Video 1. TC-1 cells were in vitro cultured and left untreated.Cell death was followed for 22 hours using Annexin-V staining and automated imaging on a BD pathway 855 imager. Every hour a picture was taken.

Cisplatin treatment does not affect the expression of costimulatory molecules on tumor cells, total myeloid cells of spleen and TDLN in vivo.

The maximum tolerated dose (MTD) of cisplatin does not induce liver or organ toxicity.

Effects of cisplatin on the tumor microenvironment.

Certain cytotoxic chemotherapeutic drugs are immunogenic, stimulating tumor immunity through mechanisms that are not completely understood. Here we show how the DNA-damaging drug cisplatin modulates tumor immunity. At the maximum tolerated dose (MTD), cisplatin cured 50% of mice with established murine TC-1 or C3 tumors, which are preclinical models of human papillomavirus (HPV)-associated cancer. Notably, the curative benefit of cisplatin relied entirely upon induction of tumor-specific CD8+ T cells. Mechanistic investigations showed that cisplatin stimulated tumor infiltration of inflammatory antigen-presenting cells (APC) expressing relatively higher levels of the T-cell costimulatory ligands CD70, CD80, and CD86. Cell death triggered by cisplatin was associated with the release of at least 19 proteins in the tumor environment that could act as damage-associated molecular patterns and upregulate costimulatory molecules, either alone or in concert, but the responsible proteins remain unknown. Essentially, the curative effect of cisplatin was abrogated in mice lacking expression of CD80 and CD86 on APCs. Furthermore, cisplatin treatment was improved by CTLA-4 blockade, which increases the availability of CD80/86 to bind to CD28. In contrast, there was no effect of CD27 stimulation, which replaces CD70 interaction. At the cisplatin MTD, cure rates could also be increased by vaccination with synthetic long peptides, whereas cures could also be achieved at similar rates at 80% of the MTD with reduced side effects. Our findings reveal an essential basis for the immunogenic properties of cisplatin, which are mediated by the induction of costimulatory signals for CD8+ T-cell–dependent tumor destruction. Cancer Res; 76(20); 6017–29. ©2016 AACR.

Supplementary Figure Legends and Supplementary Materials and Methods