8-Mop/uva Inhibits Maturation Of Extracorporeal Photochemotherapy (ecp) Generated Dendritic Cells

Extracorporeal photochemotherapy (ECP), a cellular therapy involving a light activated drug, is FDA approved for the treatment of Cutaneous T-cell lymphoma (CTCL) and used for reversal of Graft-versus-Host Disease (GHVD) and solid organ transplant rejection. The mechanism by which ECP functions as an immunomodulating treatment is unknown, although work by our lab and others suggests ECP generates functional Dendritic cells (DC) that may be involved in tolerization. Both radiation and chemotherapy have been shown to be involved in generation of tolerizing DC, and so we hypothesized that UVA activated chemotherapy 8-Methoxypsoralen (8MOP) exposure induces a tolerizing phenotype in ECP generated DC. In this study, normal donor peripheral blood mononuclear cells (PBMC) were treated in an in vitro model of the ECP apparatus, and collected (no8MOP/UVA sample) or treated with 8MOP (100ng/ml), UVA (2J/cm2) (8MOP/UVA sample). We measured phenotype and functionality of generated cells by two-color flow cytometry and functional assays. In all populations, we noted a statistically significant increase in percentage of cells staining double positive for DC markers (HLA-DR+/CD83+) 18 hours post treatment. 8MOP/UVA treatment reduced expression of mature DC markers (membrane HLA-DR/CD83) and co- stimulatory molecules (CD80/CD86) when compared to no8MOP/UVA and positive control DC, and failed to stimulate CD4+Tcells in antigen presenting assay. These data suggest 8MOP/UVA exposure during ECP inhibits DC maturation and induces a tolerizing phenotype. These tolerizing DC may play a role in the immunosuppressive effects of ECP in the treatment of GVHD and solid organ transplant rejection

A Critical Appraisal of Extracorporeal Photopheresis as a Treatment Modality for Acute and Chronic Graft-Versus-Host Disease

Although significant advances have been made in the biologic understanding of graft-versus-host disease (GVHD) and its treatment options, GVHD remains the single most challenging obstacle to the success of allogeneic hematopoietic cell transplantation (HCT) due to high risk of disabling morbidity and mortality. Extracorporeal photopheresis (ECP) has promising effects in controlling steroid-refractory GVHD, both acute and chronic, and it has been studied extensively. Its putative immunomodulatory mechanisms, while not immunosuppressive, position ECP as an attractive treatment strategy for GVHD patients who are already receiving global immunosuppression. However, ECP is relatively underutilized due in part to limited access and time commitment. Here, we review the recent findings on the ECP efficacy in both acute and chronic GVHD, primarily for steroid-refractory status, and we critically appraise its benefits. We also explore salient considerations on the optimal use of ECP in the treatment of refractory GVHD

Modulation Of Immunity By The Immunologic Zipper: The Role Of Glucocorticoid-Induced Leucine Zipper In Extracoporeal Photochemotherapy

Extracorporeal photochemotherapy (ECP) is a cellular therapy that is FDA approved for the treatment of a variety of T cell mediated diseases, including cutaneous T cell lymphoma, graft-versus-host disease, and solid organ transplantation rejection. Its ability to selectively suppress and stimulate immunity while causing minimal to no side effects in patients distinguishes it from other therapies including pharmacologic agents. Despite the clinical success of ECP, the exact mechanism by which ECP generates immunotolerance and immunogenicity in patients has been elusive. Since monocytes interact with platelets and serum proteins in the ECP chamber to undergo differentiation into dendritic cells (DCs), we examined whether 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) treatment modified the phenotypic and functional properties of these DCs in a laboratory model of ECP. In a dose-dependent fashion, 8-MOP/UVA treatment of ECP-induced antigen presenting cells induced the expression of glucocorticoid-induced leucine zipper (GILZ), a molecule shown to be both necessary and sufficient for tolerogenic DC phenotype and function. These GILZ expressing cells demonstrated a tolerogenic phenotype and down-regulated the expression of several co-stimulatory molecules including CD80, CD86, and ICAM1. Since the exposure of 8-MOP/UVA on ECP-induced dendritic antigen presenting cells is not uniform, a spectrum of DC is generated from the procedure. The dose-dependent induction of GILZ by 8-MOP/UVA may help to provide a molecular and mechanistic explanation of how ECP is capable of inducing immunosuppression and immunity with GILZ high DCs and GILZ low DCs respectively

The Glucocorticoid-Induced Leucine Zipper And Immunomodulation In Extracorporeal Photochemotherapy

Extracorporeal photochemotherapy (ECP) induces antigen-specific immune tolerance in graft-versus-host disease and solid-organ transplant rejection, and involves the ex vivo exposure of peripheral blood mononuclear cells to 8-methoxypsoralen plus ultraviolet A light (PUVA). In this study, we developed an in vitro model of ECP to decipher the immunomodulatory mechanisms of PUVA. The glucocorticoid-induced leucine zipper (GILZ) is both necessary and sufficient for the generation of dexamethasone-induced tolerogenic dendritic cells (DCs). We hypothesized that PUVA-induced activation of GILZ may contribute to the immune tolerance observed after ECP therapy in graft-versus-host disease and solid-organ transplantation. We report that PUVA acts via two pathways culminating in GILZ up-regulation in human monocyte-derived dendritic cells (MoDCs). Firstly, PUVA directly induces GILZ expression in MoDCs in a dose-dependent fashion (p \u3c 0.01). Secondly, PUVA acts indirectly through the generation of apoptotic lymphocytes to induce GILZ expression in an apoptotic cell dose-dependent fashion (p \u3c 0.01). MoDCs treated with PUVA, and/or exposed to lymphocytes rendered apoptotic by PUVA, up-regulated GILZ, down-regulated CD80, CD86 and CD83, became resistant to LPS-induced maturation, increased IL-10 production, and decreased production of pro-inflammatory cytokines (IL-12, IFN-gamma, IL-6, TNF-alpha), and chemokines (IL-8, MCP-1, MIP-1beta;, RANTES) (all p \u3c 0.05). Knockdown of GILZ via transient transfection with GILZ siRNA, compared to scramble siRNA, reduced IL-10 production and increased IL-12 production, demonstrating that GILZ is necessary for generating this tolerogenic cytokine profile. This study uncovers a potential molecular explanation for the immunomodulatory effects of PUVA, specifically through the induction of GILZ and polarization of immature MoDCs into tolerogenic DCs, and has implications for better understanding how ECP induces antigen-specific immunosuppression in vivo

The Application Of Extracorporeal Photochemotherapy To Head And Neck Squamous Cell Carcinoma

Extracorporeal Photochemotherapy (ECP) is an FDA-approved immunotherapy that has been treating cutaneous T cell lymphoma (CTCL) for over three decades. ECP’s antitumoral effect is a consequence of its generation of functional, physiologic, inflammatory monocyte-derived dendritic cells (MoDCs) and apoptotic, patient-derived tumor, which collectively, stimulate the adaptive immune system. Thus, in CTCL, ECP serves as a therapeutic dendritic cell vaccine against patient-specific neoantigens. This mechanism of action suggests ECP’s potential application to other solid tumors. We tested ECP’s applicability to head and neck squamous cell carcinoma (HNSCC) using a trackable antigen system involving the constitutively expressed HPV16 E7 oncoprotein. We hypothesized that ECP would successfully stimulate anti-epitope CD8 T cells, quantified by IFN-gamma ELISA, following processing and cross-presentation of HPV16 E7+ peptides and tumor cells by MoDCs. The trackable antigen system employed a commonly cited epitope, E7(11-19). E7+ short peptide and long peptide generated significant IFNg (p\u3c0.0001) relative to the null control group. Tumor cell line SCC61 T+ (E7hi) demonstrated significantly elevated IFNg production relative to SCC61 T- (non-E7 expressing tumor), but only in the presence of platelets, plate-passage, and overnight incubation (p\u3c0.0001). These results suggest an antigen-specific CD8 T cell response and reiterate critical ECP components that have previously been shown to facilitate immunogenic MoDC generation. Immunogenic MoDC phenotype was confirmed with flow cytometry of inflammatory surface markers and intracellular cytokines, all of which were generally upregulated following ECP. Overall, we have demonstrated a proof-of-principle for ECP’s therapeutic vaccination against HNSCC. This is particularly relevant because ECP offers unique synergistic potential with recently FDA-approved checkpoint inhibitors