Lisa M. Ebert

Regulatory T-Cell–Mediated Attenuation of T-Cell Responses to the NY-ESO-1 ISCOMATRIX Vaccine in Patients with Advanced Malignant Melanoma

Purpose: NY-ESO-1 is a highly immunogenic antigen expressed in a variety of malignancies, making it an excellent target for cancer vaccination. We recently developed a vaccine consisting of full-length recombinant NY-ESO-1 protein formulated with ISCOMATRIX adjuvant, which generated strong humoral and T-cell–mediated immune responses and seemed to reduce the risk of disease relapse in patients with fully resected melanoma. This study examines the clinical and immunologic efficacy of the same vaccine in patients with advanced metastatic melanoma. Experimental Design: Delayed-type hypersensitivity responses, circulating NY-ESO-1–specific CD4+ and CD8+ T cells, and proportions of regulatory T cells (Treg) were assessed in patients. Results: In contrast to patients with minimal residual disease, advanced melanoma patients showed no clinical responses to vaccination. Although strong antibody responses were mounted, the generation of delayed-type hypersensitivity responses was significantly impaired. The proportion of patients with circulating NY-ESO-1–specific CD4+ T cells was also reduced, and although many patients had CD8+ T cells specific to a broad range of NY-ESO-1 epitopes, the majority of these responses were preexisting. Tregs were enumerated in the blood by flow cytometric detection of cells with a CD4+CD25+FoxP3+ and CD4+CD25+CD127− phenotype. Patients with advanced melanoma had a significantly higher proportion of circulating Treg compared with those with minimal residual disease. Conclusions: Our results point to a tumor-induced systemic immune suppression, showing a clear association between the stage of melanoma progression, the number of Treg in the blood, and the clinical and immunologic efficacy of the NY-ESO-1 ISCOMATRIX cancer vaccine.

Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties

Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133(+) EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133(+) cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.

Desmoglein 2 promotes vasculogenic mimicry in melanoma and is associated with poor clinical outcome

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread.

A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34+CD45dim hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34+CD45dimDSG2+ progenitor cells are multi-potent and pro-angiogenic in vitro. Using a ‘knockout-first’ approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2lo/lo) and observed that, in response to reduced levels of Dsg2: (i) CD31+ ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell–cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.

Control of immune cell entry through the tumour vasculature: a missing link in optimising melanoma immunotherapy|[quest]|

Metastatic melanoma remains a fatal disease to many worldwide, even after the breakthrough introduction of targeted therapies such as BRAF inhibitors and immune checkpoint blockade therapies such as CTLA‐4 and PD‐1 inhibitors. With advances in our understanding of this disease, as well as the increasing data gathered from patient studies, the significance of the host immune response to cancer progression and response to treatment is becoming clear. More specifically, the presence of intratumoral CD8+ cytotoxic T‐cells correlates with better prognosis whereas the accumulation of monocytes/macrophages and neutrophils in the tumour is often associated with worse prognosis. Access and infiltration of circulating leukocytes into the tumour is governed by adhesion molecules and chemokines expressed by the endothelial cells of the vasculature. This review focuses on the adhesion molecules and chemokines which control the homing of CD8+ cytotoxic T‐cells, monocytes and neutrophils to peripheral tissues, including tumours. We discuss the role of these leukocyte subsets in regulating melanoma growth, and detail the mechanisms used by tumours to selectively recruit or exclude these leukocytes for their own advantage. In doing so, we bring to light an underappreciated component of tumour biology which should be considered in combination with current treatments to selectively alter the leukocyte composition of tumours and ultimately enhance treatment outcome.

Fos-icking for control of angiogenesis: increasing the longevity of peritoneal dialysis

Recurring peritonitis reduces the effectiveness of peritoneal dialysis by increasing fibrosis and angiogenesis, ultimately causing ultrafiltration failure (UFF). Identifying the processes underlying UFF will provide new hope for patients with chronic kidney disease. Catar and colleagues demonstrate that transforming growth factor-β, tumor necrosis factor-α, and interleukin-1 synergize to significantly increase the production and release of vascular endothelial growth factor by mesothelial cells, which, if untreated, will promote peritoneal angiogenesis, leading to UFF.

Logic-gated approaches to extend the utility of chimeric antigen receptor T-cell technology

Chimeric antigen receptor (CAR)-T cell therapy has been clinically validated as a curative treatment for the difficult to treat malignancies of relapsed/refractory B-cell acute lymphoblastic leukaemia and lymphoma. Here, the CAR-T cells are re-directed towards a single antigen, CD19, which is recognised as a virtually ideal CAR target antigen because it has strong, uniform expression on cancer cells, and is otherwise expressed only on healthy B cells, which are dispensable. Notwithstanding the clinical success of CD19-CAR-T cell therapy, its single specificity has driven therapeutic resistance in 30% or more of cases with CD19-negative leukaemic relapses. Immune checkpoint blockade is also a highly successful cancer immunotherapeutic approach, but it will be less useful for many patients whose malignancies either lack a substantial somatic mutation load or whose tumours are intrinsically resistant. Although CAR-T cell therapy could serve this unmet medical need, it is beset by several major limitations. There is a lack of candidate antigens that would satisfy the requirements for ideal CAR targets. Biological properties such as clonal heterogeneity and micro-environmental conditions hostile to T cells are inherent to many solid tumours. Past clinical studies indicate that on-target, off-tumour toxicities of CAR-T cell therapy may severely hamper its application. Therefore, re-designing CARs to increase the number of antigen specificities recognised by CAR-T cells will broaden tumour antigen coverage, potentially overcoming tumour heterogeneity and limiting tumour antigen escape. Tuning the balance of signalling within bi-specific CAR-T cells may enable tumour targeting while sparing normal tissues, and thus minimise on-target, off-tumour toxicities.