The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

Purpose: The success of immunotherapy with dendritic cells (DC) to treat cancer is dependent on effective migration to the lymph nodes and subsequent activation of antigen-specificTcells. In this study, we investigated the fate of DC after intradermal (i.d.) or intranodal (i.n.) administration and the consequences for the immune activating potential of DC vaccines in melanoma patients. Experimental Design: DC were i.d. or i.n. administered to 25 patients with metastatic melanoma scheduled for regional lymph node resection. To track DC in vivo with scintigraphic imaging and in lymph nodes by immunohistochemistry, cells were labeled with both [ 111 In]-indium and superparamagnetic iron oxide. Results: After i.d. injection, maximally 4% of the DC reached the draining lymph nodes. When correctly delivered, all DC were delivered to one or more lymph nodes after i.n. injection. Independent of the route of administration, large numbers of DC remained at the injection site, lost viability, and were cleared by infiltrating CD163+ macrophages within 48 hours. Interestingly, 87 F 10% of the surviving DC preferentially migrated into the T-cell areas, where they induced antigen-specificT-cell responses. Even though more DC reached theT-cell areas, i.n. injection of DC induced similar antigen-specific immune responses as i.d. injection. Immune responses were already induced with <5 Â 10 5 DC migrating into theT-cell areas. Conclusions: Monocyte-derived DC have high immune activating potential irrespective of the route of vaccination. Limited numbers of DC in the draining lymph nodes are sufficient to induce antigen-specific immunologic responses.

The chemokines CXCL9, 10, and 11 exert their action via CXC chemokine receptor-3 (CXCR3), a receptor highly expressed on activated T cells. These interferon ␥ (IFN␥)-induced chemokines are thought to be crucial in directing activated T cells to sites of inflammation. As such, they play an important role in several chronic inflammatory diseases including ulcerative colitis, multiple sclerosis, artherosclerosis, and delayed-type hypersensitivity reactions of the skin. In this study, we first demonstrate that in COS-7 cells heterologously expressing CXCR3, CXCL11 is a potent activator of the pertussis toxin (PTX)-sensitive p44/ p42 mitogen-activated protein kinase (MAPK) and Akt/phosphatidylinositol 3 kinase (PI3K) pathways. Next, we show that these signal transduction pathways are also operative and PTX sensitive in primary human T cells expressing CXCR3. Importantly, abrogation of these signaling cascades by specific inhibitors did not block the migration of T cells toward CXCR3 ligands, suggesting that MAPK and Akt activation is not crucial for CXCR3-mediated chemotaxis of T cells. Finally, we demonstrate that CXCR3-targeting chemokines control T-cell migration via PTX-sensitive, phospholipase C pathways and phosphatidylinositol kinases other than class I PI3K␥. (Blood.

Dendritic cells (DC) are professional antigenpresenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting proWle, DC are able to skew the immune response in a speciWc direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the eVects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/ 8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E 2 (PGE 2 ) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE 2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.

Success of immunotherapy with dendritic cells (DC) to treat cancer is highly dependent on their interaction with and activation of antigen specific T cells. To maximize DC-T cell contact accurate delivery of the therapeutic cells into the lymph node, or efficient trafficking of DC to the lymph nodes of the patient is essential. Since responses are seen in some patients but not in others, monitoring of the injected cells may be of major importance. Tracking of cells with magnetic resonance (MR) imaging is a non-invasive method that provides detailed anatomical information and is therefore more informative for the evaluation of the localization of therapeutic cells after injection than e.g. scintigraphic imaging. To challenge the sensitivity of this novel technique, we investigated the minimum amount of label and the number of cells required for MR imaging and the effect of labeling on DC function. DC were labeled with different concentrations of a clinically approved MR contrast agent consisting of superparamagnetic iron oxide particles and were imaged at both 3 and 7 T. Our results demonstrate the following: (i) When loaded with 30 (64) pg Fe/cell, cell numbers as low as 1,000 cells/mm 3 at 3 T and 500 cells/mm 3 at 7 T could be readily imaged; (ii) Labeling does not affect cell viability and function; (iii) Because of its high spatial resolution and sensitivity, MRI is ideally suited to track therapeutic cells in vivo. ' 2006 Wiley-Liss, Inc.

Upon activation, tissue residing immature dendritic cells (DC) start to migrate towards the draining lymph node and mature into efficient antigen-presenting cells. During maturation DC loose their capacity to endocytose antigens, change their surface expression of adhesion molecules, chemokine receptors, and costimulatory molecules, and change morphology. We employed 2D-PAGE and mass spectrometry to identify additional differentially expressed proteins in immature and mature DC. Human monocyte-derived DC were matured with LPS and protein expression profiles were compared before and after maturation. One of the proteins differentially expressed between immature and mature DC was identified as the actin-binding protein cofilin. We show here that cofilin is dephosphorylated in response to several maturation stimuli (i.e. CD40 ligand, LPS or a combination of TNF- § and prostaglandin E 2 ). Moreover, dephosphorylated cofilin translocated towards the plasma membrane during maturation. Importantly, this correlated with an increase in filamentous actin and the appearance of veils, suggesting a role for cofilin in cytoskeletal rearrangements during maturation.

Purpose: Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investigated DC migration in vivo in humans under different conditions. Experimental Design: HLA-A*02:01 patients with melanoma were vaccinated with mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (NCT00243594). For this study, patients received an additional intradermal vaccination with 111In-labeled mature DCs. The injection site was pretreated with nonloaded, activated DCs, TNFα, or Imiquimod; granulocyte macrophage colony-stimulating factor was coinjected or smaller numbers of DCs were injected. Migration was measured by scintigraphy and compared with an intrapatient control vaccination. In an ex vivo tissue model, we measured CCL21-directed migration of 19F-labeled DCs over a period of up to 12 hours using 19F MRI to supplement our patient data. Results: Pretreatment of the injection site induced local inflammatory reactions but did not improve migration rates. Both in vitro and in vivo, reduction of cell numbers to 5 × 106 or less cells per injection improved migration. Furthermore, scintigraphy is insufficient to study migration of such small numbers of 111In-labeled DCs in vivo. Conclusion: Reduction of cell density, not pretreatment of the injection site, is crucial for improved migration of DCs to LNs in vivo. Clin Cancer Res; 19(6); 1525–33. ©2013 AACR.