Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8+ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8+ T cell clone. Confocal microscopy with Alexa Fluor®647-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8+ T cell cross-presentation thereafter.

Many approaches for cancer immunotherapy have targeted dendritic cells (DCs), directly or indirectly, for the induction of antitumor immune responses. DC-based vaccines have been developed using a wide variety of ex vivo DC culture conditions, antigen (Ag) source and loading strategies, maturation agents, and routes of vaccination. Adjuvants are used to activate innate immune cells at the vaccine injection site, to promote Ag transport to the draining lymph nodes (LNs) and to model adaptive immune responses. Despite years of effort, the effective induction of strong and durable antitumor T-cell responses in vaccinated patients remains a challenge. The study of vaccine interactions with other immune cells in the LNs and, more recently, in the injection site has opened new doors for understanding antitumor effector T-cell licensing and function. In this review, we will briefly discuss the relevant sites and up-to-date facts regarding possible targets for antitumor vaccine refinement. We will focus on the processes taking place at the injection site, adjuvant combinations and their role in DC-based vaccines, LN homing, and modeling vaccine-induced immune responses capable of controlling tumor growth and generating immune memory.

Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.

Tumor immune response is shaped by the tumor microenvironment (TME), which often evolves to be immunosuppressive, promoting disease progression and metastasis. An important example is melanoma tumors, which display high numbers of tumor-associated macrophages (TAMs) that are immunosuppressive but also have the potential to restore anti-tumor activity. However, to therapeutically target TAMs, there is a need to understand the early events that shape their tumor-promoting profile. To address this, we built and optimized 3D in vitro co-culture systems, composed of a collagen-I matrix scaffolding murine bone-marrow-derived macrophages (BMDMs), YUMM1.7 melanoma cells, and fibroblasts to recreate the early melanoma TME and study how interactions with fibroblasts and tumor cells modulate macrophage immune activity. We monitored BMDM behavior and interactions through time-lapse imaging and characterized their activation and secretion. We found that stromal cells induced a rapid functional activation, with increased motility and response from BMDMs. Over the course of seven days, BMDMs acquired a phenotype and secretion profile that resembled melanoma TAMs in established tumors. Overall, the direct cell–cell interactions with the stromal components in a 3D environment shape BMDM transition to a TAM-like immunosuppressive state. Our systems will enable future studies of changes in macrophage–stromal cross-talk in the melanoma TME