Ariadna Mendoza-Naranjo

Prolonged Survival of Dendritic Cell–Vaccinated Melanoma Patients Correlates With Tumor-Specific Delayed Type IV Hypersensitivity Response and Reduction of Tumor Growth Factor β-Expressing T Cells

PURPOSE The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. PATIENTS AND METHODS Forty-three stage IV and seven stage III patients were vaccinated four times with TRIMEL/DC vaccine. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and regulatory T-cell populations were determined. Overall survival and disease progression rates were analyzed using Kaplan-Meier curves and compared with historical records. RESULTS The overall survival for stage IV patients was 15 months. More than 60% of patients showed DTH-positive reaction against the TRIMEL. Stage IV/DTH-positive patients displayed a median survival of 33 months compared with 11 months observed for DTH-negative patients (P = .0014). All stage III treated patients were DTH positive and remained alive and tumor free for a median follow-up period of 48 months (range, 33 to 64 months). DTH-positive patients showed a marked reduction in the proportion of CD4+ transforming growth factor (TGF) beta+ regulatory T cells compared to DTH-negative patients (1.54% v 5.78%; P < .0001). CONCLUSION Our findings strongly suggest that TRIMEL-pulsed DCs provide a standardized and widely applicable source of melanoma antigens, very effective in evoking antimelanoma immune response. To our knowledge, this is the first report describing a correlation between vaccine-induced reduction of CD4+TGFbeta+ regulatory T cells and in vivo antimelanoma immune response associated to improved patient survival and disease stability.

Cytoskeletal remodeling mediated by WASp in dendritic cells is necessary for normal immune synapse formation and T-cell priming

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

Targeting Cx43 and N-cadherin, which are abnormally upregulated in venous leg ulcers, influences migration, adhesion and activation of Rho GTPases.

Background Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available. Principal Findings In wound edge biopsies from human venous leg ulcers we found a striking upregulation of dermal N-cadherin, Zonula Occludens-1 and the gap junction protein Connexin43 (Cx43) compared to intact skin, and in stark contrast to the down-regulation of Cx43 expression seen in acute, healing wounds. We targeted the expression of these proteins in 3T3 fibroblasts to evaluate their role in venous leg ulcers healing. Knockdown of Cx43 and N-cadherin, but not Zonula Occludens-1, accelerated cell migration in a scratch wound-healing assay. Reducing Cx43 increased Golgi reorientation, whilst decreasing cell adhesion and proliferation. Furthermore, Connexin43 and N-cadherin knockdown led to profound effects on fibroblast cytoskeletal dynamics after scratch-wounding. The cells exhibited longer lamelipodial protrusions lacking the F-actin belt seen at the leading edge in wounded control cells. This phenotype was accompanied by augmented activation of Rac-1 and RhoA GTPases, as revealed by Förster Resonance Energy Transfer and pull down experiments. Conclusions Cx43 and N-cadherin are potential therapeutic targets in the promotion of healing of venous leg ulcers, by acting at least in part through distinct contributions of cell adhesion, migration, proliferation and cytoskeletal dynamics.

Functional Gap Junctions Accumulate at the Immunological Synapse and Contribute to T Cell Activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell–T cell signaling machinery that regulates T cell activation.

Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down‐regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10‐fold elevation of Cx43 protein, as well as a 6‐fold increase in N‐cadherin and a 2‐fold increase in ZO‐1 (zonular occludin‐1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up‐regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2‐fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound‐healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose‐induced Cx43 up‐regulation was prevented with Cx43shRNA (Cx43 short‐hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up‐regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural‐killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain‐related proteins (MIC) and UL16‐binding proteins (ULBP). It is regarded as a co‐activating receptor on NK cells, having an important role in the cell‐mediated immune response to tumours. We studied the influence of interleukin (IL)‐10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL‐10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL‐10‐treated FMS and IL‐10‐transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL‐10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL‐10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte‐activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL‐10‐treated FMS cells. Our results suggest a novel function for IL‐10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Gap Junction Intercellular Communications Regulate NK Cell Activation and Modulate NK Cytotoxic Capacity

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca2+ responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell–mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43–GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell–mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca2+ influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.