Effective Melanoma Immunotherapy in Mice by the Skin-Depigmenting Agent Monobenzone and the Adjuvants Imiquimod and CpG

Background: Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity. Methodology and Principal Findings: We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16. F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation. Conclusions: MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B-and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clini

Autoimmune Destruction of Skin Melanocytes by Perilesional T Cells from Vitiligo Patients

In vitiligo, cytotoxic T cells infiltrating the perilesional margin are suspected to be involved in the pathogenesis of the disease. However, it remains to be elucidated whether these T cells are a cause or a consequence of the depigmentation process. T cells we obtained from perilesional skin biopsies, were significantly enriched for melanocyte antigen recognition, compared with healthy skin-infiltrating T cells, and were reactive to melanocyte antigen-specific stimulation. Using a skin explant model, we were able to dissect the in situ activities of perilesional T cells in the effector phase of depigmentation. We show that these T cells could infiltrate autologous normally pigmented skin explants and efficiently kill melanocytes within this microenvironment. Interestingly, melanocyte apoptosis was accompanied by suprabasal keratinocyte apoptosis. Perilesional T cells did, however, not induce apoptosis in lesional skin, which is devoid of melanocytes, indicating the melanocyte-specific cytotoxic activity of these cells. Melanocyte killing correlated to local infiltration of perilesional T cells. Our data show that perilesional cytotoxic T cells eradicate pigment cells, the characteristic hallmark of vitiligo, thereby providing evidence of T cells being able to mediate targeted autoimmune tissue destruction

Skin-Depigmenting Agent Monobenzone Induces Potent T-Cell Autoimmunity toward Pigmented Cells by Tyrosinase Haptenation and Melanosome Autophagy

In this study, we report the previously unknown mechanism of inducing robust anti-melanoma immunity by the vitiligo-inducing compound monobenzone. We show monobenzone to increase melanocyte and melanoma cell immunogenicity by forming quinone-haptens to the tyrosinase protein and by inducing the release of tyrosinase-and melanoma antigen recognized by T cells-1 (MART-1)-containing CD63+ exosomes following melanosome oxidative stress induction. Monobenzone further augments the processing and shedding of melanocyte-differentiation antigens by inducing melanosome autophagy and enhanced tyrosinase ubiquitination, ultimately activating dendritic cells, which induced cytotoxic human melanoma-reactive T cells. These T cells effectively eradicate melanoma in vivo, as we have reported previously. Monobenzone thereby represents a promising and readily applicable compound for immunotherapy in melanoma patient

Amplification of N-Myc is associated with a T-cell-poor microenvironment in metastatic neuroblastoma restraining interferon pathway activity and chemokine expression

Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN-amplified neuroblastomas

Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma

Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8(+) T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8(+) T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4(R24C )(Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8(+) T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4(R24C), promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4(R24C) antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies

MIC-treated mice show a sustained NK cell expansion and circulating melanocyte antigen-specific CD8+ T cells.

<p><b>A</b>, Peripheral blood was collected from the tailvein of treated mice on day 8 and 23 of treatment, and average ratios between T cells (CD3+, black sections), B cells (CD19+, white sections), NK cells (NK1.1+, CD3-, grey sections) and other peripheral blood leukocytes (PBL; dashed sections) were determined for the PBL (n = 7 mice per group). Interestingly, MIC-treated mice showed a significant NK cell expansion on both day 8 and 23 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-t002" target="_blank">table 2</a>, “Exp. 4”). This expansion of NK cells was also found in CpG-, imiquimod- and CI-treated mice, although for these animals this reaction was only found on day 8. Monobenzone alone did not influence PBL ratios on either time point, comparable to untreated mice. Depicted data is representative of three independent <i>in vivo</i> mouse experiments. For the statistical analysis of the <i>in vivo</i> differences in NK cell counts in these experiments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-t002" target="_blank">table 2</a>. <b>B</b>, Peripheral blood CD8+ T cells were tested for binding to H2-K_b/TRP-2_180-188-tetramers at day 120 following tumor inoculation (day 85 after treatment cessation, n = 4). TRP-2 represents one of the immunodominant epitopes of B16.F10 melanoma. Long-term surviving, MIC-treated mice showed a significant population of TRP-2-specific CD8+ T cells circulating in their peripheral blood at day 120, as compared to untreated mice 10 days after tumor inoculation (n = 7). Binding to control H2-K_b/OVA_257-264-tetramer by the tested PBL was negative (data not shown).</p

Statistics of <i>in vivo</i> tumor experiments.

<p>ns: not significant (considered if p>0.05). Exp.: experiment.</p><p>nt: not tested. TFS: tumor-free survival.</p><p>*: Unpaired <i>t</i>-test. CI: CpG & imiquimod.</p><p>**: Logrank test for survival (endpoint tumor size max 200 mm²). MIC: monobenzone, imiquimod & CpG.</p>1<p>:Day of tumor size comparison (last day on which experimental animals were all alive).</p><p>For Exp. 2 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-g001" target="_blank">Fig. 1A/B</a>, for Exp. 3 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-g003" target="_blank">Fig. 3C</a> (upper panel), for Exp. 4 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-g003" target="_blank">Fig. 3A/B and C</a> (lower panel),</p

Statistics of <i>in vivo</i> peripheral blood NK cell counts.

<p>Exp.: experiment. CI: CpG, imiquimod.</p><p>nt: not tested. MIC: monobenzone, imiquimod & CpG.</p><p>ns: not significant (considered if p>0.05). For Exp. 4 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-g004" target="_blank">Fig. 4A</a>.</p><p>*: Unpaired t-test.</p>1<p>:Day after tumor inoculation on which PBL were tested.</p