1223 Multiple MC1R variants associated with extensive freckles and red hair found in a Mongolian family T Suzuki 1 , Y Araki 1 , K Okamura 1 , B Munkhbat 2 , G Tamiya 3 and Y Hozumi 1 1 Department of Dermatology, Yamagata University Faculty of Medicine, Yamagata, Japan, 2 Central Sci- entific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar, Mongolia and 3 Tohoku Medical Megabank Organization, Tohoku University, Sendai, Miyagi, Japan Freckles are flat, tanned circular spots that typically are multiple and may develop on sun- exposed skin, especially on the face. In freckles, an increased number of melanocytes are not observed, but the melanocytes overproduce melanin granules changing the coloration of keratinocytes. The presence of freckles is related to variants of MC1R, which has been also reported an association with red hair more strongly. We present a very interesting case of a Mongolian boy with extensive freckles and harboring two MC1R variants as found by whole- exome sequencing (WES). The proband, born in a non-consanguineous nomadic family, was a 14-year-old Mongolian boy living in the northwestern part of Mongolia. He developed extensive freckles on the face, anterior chest, and the extensor surfaces of upper extremities (sun-exposed area) and showed lighter skin tone in comparison to other family members. The freckles became apparent when he was 5 years old, and the number and color intensity increased gradually ever since. WES was performed for the proband and his family to elucidate the genetic differences in the family. Three different variants in the MC1R were detected in the family, and two of them were evaluated as damaging. Furthermore, we confirmed these damaging variants were on different alleles respectively, indicating he was a compound heterozygote for both of the damaging variants. We concluded that the extensive freckles of the proband could be explained by the extensive sun exposure and his genetic makeup, compound heterozygosity for rare variants in MC1R. 1224 POU3F2 is a radioprotector and a tumor suppressor M Hamm, M Le Coz and L Larue Institut Curie, Orsay, France Melanoma is known as a radioresistant tumor. Melanoma is intra-tumorally highly hetero- geneous due to its genetic instability and plasticity. This heterogeneity may explain, at least in part, the natural neo and acquired resistance of melanoma against therapies. In a two dimensional biological world, cells may switch from proliferative to invasive, and vice versa. Two transcription factors, MITF and POU3F2, may be of great importance in this switch. POU3F2 is a transcription factor belonging to the Oct family. In vitro studies have shown that POU3F2 is transcriptionally controlled by the Lef/b-catenin complex and indirectly controlled by BRAF. Moreover, the level of POU3F2 mRNA is controlled by miR-211, which is directly induced by MITF. This switch would be modulated by the transcriptional activity of POU3F2 on MITF, but also on other crucial proteins of the melanocyte lineage such as PAX3. In a human melanoma metastasis, it appears that melanoma cells are mainly POU3F2-pos- itive or MITF-positive. However some cells express both or none of these two proteins. Here, we evaluated the importance of POU3F2 during the establishment, the renewal and trans- formation of melanocytes using genetically modified mouse models, human genetics and cell lines. It appears that POU3F2 is dispensable during the establishment and the renewal of melanocytes. The specific lack of POU3F2 in the melanocyte lineage reveals that this protein is important for melanoma resistance against ionizing irradiation, which consequently results in the disappearance of melanocyte stem cells over time. Moreover, mouse melanoma models relevant for humans were generated, showing that POU3F2 plays an important role during melanoma initiation and can be better used to improve melanoma therapies. 1225 Paving the road to explain melanocyte loss in vitiligo: An uncovered role of matrix metalloprotease MMP9 N Boukhedouni 1 , C Martins 1 , A Darrigade 1 , C Barrault 2 , J Garnier 2 , J Rambert 3 , A Taieb 1 , F Bernard 2 , J Seneschal 1 and K Boniface 1 1 University of Bordeaux, Bordeaux, France, 2 BIOalternatives, Genc ¸ ay, France and 3 Aquiderm, University of Bordeaux, Bordeaux, France Melanocyte loss is the pathological hallmark of vitiligo, the archetype of a chronic depig- menting inflammatory skin disorder. Yet, whether such disappearance results from melano- cyte death and/or detachment is still a matter of debate. We previously showed that vitiligo skin is imprinted with resident memory T cells producing elevated levels of IFNg and TNFa, while displaying moderate cytotoxicity. Therefore, we investigated the interplay between the inflammatory response characterizing vitiligo disease and melanocyte loss. We found that the combined activity of IFNg and TNFa induced melanocyte detachment rather than their death in reconstructed pigmented human epidermis through defect of E-cadherin expression, the major protein involved in melanocyte attachment to keratinocytes, and the release of soluble E-cadherin. Such phenomenon was undeniably observed in vitiligo patients skin and was further confirmed in vivo following dermal injection of both IFNg and TNFa. Additional experiments demonstrated that these two cytokines induced the production of active matrix metalloproteinase 9 (MMP9) by keratinocytes, leading to the cleavage of E-cadherin and instability of melanocytes. MMP9 levels were found increased in vitiligo skin and patients sera and positively correlated with the body surface area involved. Lastly, we showed that MMP9 inhibition downregulated melanocyte detachment in vitro and in vivo. These results emphasize a new mechanism to explain depigmentation associated with inflammation and highlight MMP9 as a new therapeutic target in vitiligo, a disease that still suffers from a lack of effective treatment. 1226 Role of CC-chemokine receptor 6 (CCR6) and CC-chemokine ligand 20 (CCL20) mediated immunosurveillance in malignant melanoma D Martı ´n-Garcı ´a 1 , A Enk 2 and AS Lonsdorf 1 1 University Hospital Heidelberg, Heidelberg, Germany and 2 Univ Hospital Heidelberg, Heidelberg, Germany Chemokine ligand 20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of subsets of B-cells, memory T cells and DCs expressing chemokine receptor 6 (CCR6), its exclusive receptor. CCL20 and a corresponding CCR6-expressing immune cell infiltrate have been detected in several malignancies, including melanoma. Yet, the func- tional contribution of the CCR6/CCL20 axis for the immune control of melanoma remains controversial. The characterization of CCR6-guided immune cell subsets and their functional contribution for the immune control of melanoma comprises the focus of this project. We evaluated the homeostatic and inducible secretion of CCL20 by different murine and human melanoma cell lines by ELISA. Both, murine (B16, Ret) and human (A375, C32) melanoma cell lines are capable of secreting CCL20 upon stimulation with pro-inflammatory cytokines in vitro. In order to determine the functional relevance of CCR6 on local tumor growth, B16 melanoma cells retrovirally transduced with a vector that constantly overexpresses CCL20 (B16-CCL20) were injected s.c. in wild type C57BL/6 (Wt) and congenic CCR6knockout (CCR6ko) mice. While differences in tumor growth between Wt and CCR6ko control groups (injected with a non-CCL20 expressing B16 line) were minimal (p<0.05), significantly reduced tumor growth was observed in B16-CCL20 injected CCR6ko mice when compared with Wt animals (p<0.005). Additionally, while tumors from both Wt experimental groups and B16-Control inoculated CCR6ko mice were characterized by an initial fast exponential growth that got stable over time, CCR6ko mice injected with B16-CCL20 showed constant exponential growth. Analysis of tumor infiltrating leucocytes revealed no differences in B- cells, CD4+ T-cells, T-regs, DCs or Macrophages between groups. While the precise mech- anisms require further investigation, our results suggest that CCL20 interactions in the microenvironment of cutaneous melanoma may be an essential factor for local tumor growth. 1227 Cold atmospheric plasma treatment of melanoma enhances immune response TA Freeman 1 , N Chernets 2 , V Alexeev 3 , C Snyder 4 and N Nikbakht 5 1 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 2 Thomas Jefferson University, Phila, PA, 3 Jefferson College Biomedical Science, Philadel- phia, PA, 4 Department of Biochemistry and Immunology, Thomas Jefferson University, Phila, PA and 5 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Phila, PA Our previous work has shown that nanosecond pulsed (nsP) plasma treatment of established B16 melanoma tumors resulted in complete remission greater than 60% of the time, and was dependent on reactive oxygen and nitrogen species (ROS and RNS). NsP-plasma is a cold atmospheric plasma (CAP) composed of ionized gases containing ions, radicals, ROS, RNS and nsP electric fields. Strikingly, within two hours of treatment both the vasculature and the tumor size were decreased by 50%. Histological examination revealed blood vessel break- down, areas of hemorrhage and cell lysis, increased cytochrome C release, a marker of mitochondrial death pathway, and H2AX, a marker for DNA damage/repair. Indirect immunofluorescent staining of control and nsP-plasma treated lesions at 48 h post treatment revealed a decrease in the number of blood vessels (decreased CD31 staining) and prolifer- ating cells (Ki-67+), in addition to increased infiltration of CD8+ T cells, CD4+ T cells and CD11c+ dendritic cells. Most importantly, treatment led to activation of B16-specific im- munity. Both cytotoxic T cell activity (CTL) and melanoma cell-reactive antibodies were increased in nsP-plasma-treated mice as compared to control naı ¨ve or tumor-bearing animals. As nsP-plasma treatment seemed to promote immune recognition of tumors, we hypothesized that animals in which tumors had been cleared after treatment would be resistant to a tumor rechallenge. To test this, 4 mice that had cleared their primary tumors were challenged with a new B16 tumor. In one of these mice, the challenge tumor completely failed to grow, while growth was severely delayed in the remaining 3 mice. Collectively these data suggest that nsP-plasma treatment of established tumors enhances the release of melanoma-derived an- tigens and activation of both humoral and cellular immunity specific for these targets. 1228 Actinic lentigines from European and Japanese volunteers are characterized by a molecular and cellular inflammatory micro-environment D Christine 1 , E Warrick 1 , E Bourreau 1 , S Nouveau 2 , P Bastien 1 , F Rousset 1 and F Bernerd 1 1 L’Oreal Research and Innovation, Aulnay sous Bois, France and 2 L’Ore´al Research and Innovation, Aulnay sous Bois, France Actinic lentigines (AL), also called age spots, are benign skin hyperpigmented lesions asso- ciated with age and chronic sun exposure. Despite their high prevalence in elderly people, the biological events underlying the development of these lesions remain unclear. In order to better understand the pathophysiological characteristics of AL, we characterized the inflammation response in AL from European and Japanese volunteers, by analyzing the gene expression profile associated with inflammation and by immunostaining of inflammatory/ immune cells in skin sections. Results showed that a large number of genes associated with inflammation/immune response were altered in AL versus adjacent non lesional skin (NL) representing w10% of total modulated genes. These genes were modulated in the same way in the two populations and indicate a proinflammatory process as attested by the activation of arachidonic acid pathway including PTGS2 (COX2). Furthermore, the overexpression of chemokines genes suggests that chemoattraction of inflammatory cells could take place in AL. To verify this, immunostaining of several cellular subsets was performed and revealed the increase of antigen-presenting cells (HLA-DR +), macrophages (CD68+) and CD4+ T-cells in the dermis of AL vs NL, which strengthens a chemoattraction role in the physiopathology of AL. Consequently, prostaglandins and chemokines release associated with the infiltration of inflammatory cells could contribute to the self-maintenance of an inflammatory microenvi- ronment in AL which may alter skin homeostasis and pigmentation. Normalizing skin by targeting this inflammatory loop could help for efficient and long-term treatment of age spots. ABSTRACTS | Pigmentation and Melanoma S208 Journal of Investigative Dermatology (2018), Volume 138