Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B, and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, from immunotypes A to C, there were increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with natural killer and mature dendritic cells only rarely present. Whereas higher densities of CD8⁺ T cells correlated best with survival, a higher density of CD45⁺ leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma. Cancer Res; 72(5); 1070–80. ©2012 AACR.

Peptide vaccines incorporate one or more short or long amino acid sequences as tumor antigens, combined with a vaccine adjuvant. Thus, they fall broadly into the category of defined antigen vaccines, along with vaccines using protein, protein subunits, DNA, or RNA. They remain one of the most immunogenic approaches, based on measures of T-cell response in the blood or in draining lymph nodes. However, existing peptide vaccines have had limited success at inducing clinical tumor regressions, despite reliable induction of T-cell responses. Several new developments offer promise for improving peptide vaccines, including use of long peptides, optimization of adjuvants including toll-like receptor agonists, and combination with systemic therapies that may reduce tumor-associated immune dysfunction, such as blockade of PD-1/PD-L1 interactions. To apply these new approaches optimally, it will be critical to study their effects in the context of defined antigens, for which peptide vaccines are optimal.

Purpose: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed.Patients and Methods: Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]).Results: In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P < .02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm.Conclusion: The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.

In 2012, it was estimated that 9180 people in the United States would die from melanoma and that more than 76,000 new cases would be diagnosed. Surgical resection is effective for early-stage melanoma, but outcomes are poor for patients with advanced disease. Expression of tumor-associated antigens by melanoma cells makes the disease a promising candidate for immunotherapy. The hematopoietic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) has a variety of effects on the immune system including activation of T cells and maturation of dendritic cells, as well as an ability to promote humoral and cell-mediated responses. Given its immunobiology, there has been interest in strategies incorporating GM-CSF in the treatment of melanoma. Preclinical studies with GM-CSF have suggested that it has antitumor activity against melanoma and can enhance the activity of anti-melanoma vaccines. Numerous clinical studies have evaluated recombinant GM-CSF as a monotherapy, as adjuvant with or without cancer vaccines, or in combination with chemotherapy. Although there have been suggestions of clinical benefit in some studies, results have been inconsistent. More recently, novel approaches incorporating GM-CSF in the treatment of melanoma have been evaluated. These have included oncolytic immunotherapy with the GM-CSF–expressing engineered herpes simplex virus talimogene laherparepvec and administration of GM-CSF in combination with ipilimumab, both of which have improved patient outcomes in phase 3 studies. This review describes the diverse body of preclinical and clinical evidence regarding use of GM-CSF in the treatment of melanoma.

Increased immune cell infiltration into tumors is associated with improved patient survival and predicts response to immune therapies. Thus, identification of factors that determine the extent of immune infiltration is crucial, so that methods to intervene on these targets can be developed. T cells enter tumor tissues through the vasculature, and under control of interactions between homing receptors on the T cells and homing receptor ligands (HRLs) expressed by tumor vascular endothelium and tumor cell nests. HRLs are often deficient in tumors, and there also may be active barriers to infiltration. These remain understudied but may be crucial for enhancing immune-mediated cancer control. Multiple intratumoral and systemic therapeutic approaches show promise to enhance T cell infiltration, including both approved therapies and experimental therapies. This review highlights the intracellular and extracellular determinants of immune cell infiltration into tumors, barriers to infiltration, and approaches for intervention to enhance infiltration and response to immune therapies.