Raquel Cabezón

Intraperitoneal Administration of Autologous Tolerogenic Dendritic Cells for Refractory Crohn's Disease: A Phase I Study.

BACKGROUND AND AIMS Ex vivo-generated autologous tolerogenic dendritic cells [tolDCs] can restore immune tolerance in experimental colitis. The aim of this study was to determine the safety and tolerability of administration of autologous tolDCs in refractory Crohns disease [CD] patients. METHODS A phase-I, single-centre, sequential-cohorts, dose-range study was designed. Stable tolDCs were generated ex vivo from monocytes following a previously developed protocol, and administered by sonography-guided intraperitoneal injection. Six sequential refractory-CD cohorts were established: the first three cohorts received a single intraperitoneal injection of tolDCs at escalating doses [2 x 10(6)/5 x 10(6)/10 x 10(6)]; and the last three cohorts received three biweekly intraperitoneal injections at same escalating doses. Safety was sequentially evaluated. Patients were assessed from week 0 to 12 and followed up for 1-year period for safety. RESULTS Nine patients were included. No adverse effects were detected during tolDC injection or follow-up. Three patients withdrew from the study due to CD worsening. Crohns Disease Activity Index [CDAI] decreased from 274 [60] {mean (standard deviation [SD])} to 222 [113] [p = 0.3]; one [11%] patient reached clinical remission [CDAI < 150] and two [22%] clinical response [CDAI decrease ≥ 100]. Crohns Disease Endoscopic Index of Severity [CDEIS] decreased from 18 [5] to 13 [8] [p = 0.4]; lesions improved markedly in three patients [33%]. Quality of life (inflammatory bowel disease questionnaire [IBDQ]) changed from 125 [27] to 131 [38] [p = 0.7]; remission [IBDQ at Week 12 ≥ 170] was reached in one [11%] case and response [IBDQ score increase ≥ 16] in two [22%]. CONCLUSIONS Intraperitoneal administration of autologous tolDCs appears safe and feasible in refractory CD patients. Further studies should be developed to test clinical benefit, determine the optimal administration route and dose, and monitor the immune responses; See [www.eudract.ema.europa.eu, EudraCT number 2007-003469-42; www.aemps.gob.es number PEI 08-049].

Therapeutic Potential of Tolerogenic Dendritic Cells in IBD: From Animal Models to Clinical Application

The gut mucosa undergoes continuous antigenic exposure from food antigens, commensal flora derived ligands, and pathogens. This constant stimulation results in controlled inflammatory responses that are effectively suppressed by multiple factors. This tight regulation, necessary to maintain intestinal homeostasis, is affected during inflammatory bowel diseases (IBD) resulting in altered immune responses to harmless microorganisms. Dendritic cells (DCs) are sentinels of immunity, located in peripheral and lymphoid tissues, which are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of pathogen recognition receptors allows DCs to initiate immune responses. However, in the absence of danger signals, different DC subsets can induce active tolerance by inducing regulatory T cells (Treg), inhibiting inflammatory T helper cell responses, or both. Interestingly, several protocols to generate clinical grade tolerogenic DC (tol-DCs) in vitro have been described, opening the possibility to restore the intestinal homeostasis to bacterial flora by cellular therapy. In this review, we discuss different DC subsets and their role in IBD. Additionally, we will review preclinical studies performed in animal models while describing recent characterization of tol-DCs from Crohns disease patients for clinical application.

Gram-Negative Enterobacteria Induce Tolerogenic Maturation in Dexamethasone Conditioned Dendritic Cells

Dendritic cells have been investigated in clinical trials, predominantly with the aim of stimulating immune responses against tumours or infectious diseases. Thus far, however, no clinical studies have taken advantage of their specific immunosuppressive potential. Tolerogenic DCs may represent a new therapeutic strategy for human immune-based diseases, such as Crohn’s disease, where the perturbations of the finely tuned balance between the immune system and the microflora result in disease. In the present report, we describe the generation of tolerogenic DCs from healthy donors and Crohn’s disease patients using clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterize their response to maturation stimuli. Interestingly, we found out that dexamethasone-conditioned DCs keep their tolerogenic properties to Gram-negative bacteria. Other findings included in this study demonstrate that the combination of dexamethasone with a specific cytokine cocktail yielded clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. Importantly, in regard to their clinical application, the tolerogenic phenotype of DCs remained stable after the elimination of dexamethasone and after a second stimulation with LPS or bacteria. All these properties make this cell product suitable to be tested in clinical trials of inflammatory conditions including Crohn’s disease.

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.

Analysis of HLDA9 mAbs on plasmacytoid dendritic cells

Dendritic cells are a heterogeneous population of bone marrow derived leucocytes that play a crucial role in both pathogen recognition and the initiation of primary T cell immune responses. Plasmacytoid dendritic cells (pDCs), also known as natural interferon-producing cells, comprise one of two major human dendritic cell subsets that strongly influences immune balance. pDCs remain a poorly characterized subset. Several studies have suggested the existence of a close phenotypic and functional relationship between B-cells and pDCs. The surface reactivity of a panel of 96 monoclonal antibodies submitted to the ninth Human Leukocyte Differentiation Antigens workshop (HLDA9) B cell section was analyzed using pDCs as target cells. The results showed that eight of the mAbs reacted positively on pDCs: CD86, CD229, CD319, CD305, CD184, CD84, CD85g and FcɛRIa, confirming previously published reports. Interestingly, this study also revealed the expression of eight surface molecules not previously described on pDCs, including CD352(NTBA), CD272(BTLA), CD357(GITR), CD48, CD270(HVEM), Galectin-3, CD148, and CD361. The present report summarizes the expression of these molecules on freshly isolated pDCs. Significantly, we have identified several new molecules expressed by these intriguing cells, ones we believe will open new avenues for the study of pDC functionality and their role in human health and diseases.

New insights into the phenotype of human dendritic cell populations

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin‐derived DCs, tonsil leukocytes, monocyte‐derived DCs, CD34‐derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell‐surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte‐surface molecules.

In vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells

Cellular therapies using immune cells and in particular dendritic cells (DCs) are being increasingly applied in clinical trials and vaccines. Their success partially depends on accurate delivery of cells to target organs or migration to lymph nodes. Delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. Thus, the design of an optimal DC therapy would be improved by optimizing technologies for monitoring DC trafficking. Magnetic resonance imaging (MRI) represents a powerful tool for non-invasive imaging of DC migration in vivo. Domestic pigs share similarities with humans and represent an excellent animal model for immunological studies. The aim of this study was to investigate the possibility using pigs as models for DC tracking in vivo. Porcine monocyte derived DC (MoDC) culture with superparamagnetic iron oxide (SPIO) particles was standardized on the basis of SPIO concentration and culture viability. Phenotype, cytokine production and mixed lymphocyte reaction assay confirmed that porcine SPIO-MoDC culture were similar to mock MoDCs and fully functional in vivo. Alike, similar patterns were obtained in human MoDCs. After subcutaneous inoculation in pigs, porcine SPIO-MoDC migration to regional lymph nodes was detected by MRI and confirmed by Perls staining of draining lymph nodes. Moreover, after one dose of virus-like particles-pulsed MoDCs specific local and systemic responses were confirmed using ELISPOT IFN-γ in pigs. In summary, the results in this work showed that after one single subcutaneous dose of pulsed MoDCs, pigs were able to elicit specific local and systemic immune responses. Additionally, the dynamic imaging of MRI-based DC tracking was shown using SPIO particles. This proof-of-principle study shows the potential of using pigs as a suitable animal model to test DC trafficking with the aim of improving cellular therapies.

Up‐regulation of EP2 and EP3 receptors in human tolerogenic dendritic cells boosts the immunosuppressive activity of PGE2

Dendritic cells (DCs) are APCs essential in regulating the immune response. PGE2, produced during inflammation, has a pivotal role in the maturation of DCs and, therefore, is vital for the immune response. The large variety of biologic functions governed by PGE2 is mediated by its signaling through 4 distinct E‐type prostanoid (EP) receptors. Immunogenic DCs express EP2 and EP4, which mediate the PGE2 signaling. However, the expression and function of EP receptors in human tolerogenic DCs (tol‐DCs), which present an inhibitory phenotype, have not yet, to our knowledge, been assessed. To clarify the role of EP receptors in tol‐DCs, we examined the expression of different EP receptors and their effect using selective agonists in human cells. We find that EP2 and EP3 expression are up‐regulated in in vitro–generated tol‐DCs compared with mature DCs (mDCs). Activation of EP2–EP4 has a direct effect on the surface expression of costimulatory molecules and maturation receptors, such as CD80, CD83, and CD86 or MHCII and CCR7 in tol‐DCs, the latter being exclusively modulated by PGE2–EP4 signaling. Importantly, we find that EP2 and EP3 receptors are involved in tolerance induction through IL‐10 production by tol‐DCs. These results are in sharp contrast with the inflammatory role of EP4. Moreover, we show that DCs generated in the presence of agonists for EP receptors, induce naive T cell differentiation toward polarized Th1/Th17 cells. Given the differential effects of EP receptors, our results suggest that EP receptor agonist/antagonists might become relevant novel drug templates to modulate immune response.

Tolerogenic Dendritic Cells as a Promising Antigen-Specific Therapy in the Treatment of Multiple Sclerosis and Neuromyelitis Optica From Preclinical to Clinical Trials

The identification of activated T-lymphocytes restricted to myelin-derived immunogenic peptides in multiple sclerosis (MS) and aquaporin-4 water channel in neuromyelitis optica (NMO) in the blood of patients opened the possibility for developing highly selective and disease-specific therapeutic approaches. Antigen presenting cells and in particular dendritic cells (DCs) represent a strategy to inhibit pro-inflammatory T helper cells. DCs are located in peripheral and lymphoid tissues and are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of receptors involved in pathogen recognition confers to DCs the property to initiate immune responses. However, in the absence of danger signals different DC subsets have been revealed to induce active tolerance by inducing regulatory T cells, inhibiting pro-inflammatory T helper cells responses or both. Interestingly, several protocols to generate clinical-grade tolerogenic DC (Tol-DC) in vitro have been described, offering the possibility to restore the homeostasis to central nervous system-related antigens. In this review, we discuss about different DC subsets and their role in tolerance induction, the different protocols to generate Tol-DCs and preclinical studies in animal models as well as describe recent characterization of Tol-DCs for clinical application in autoimmune diseases and in particular in MS and NMO patients. In addition, we discuss the clinical trials ongoing based on Tol-DCs to treat different autoimmune diseases.

Nanoencapsulated budesonide in self-stratified polyurethane-polyurea nanoparticles is highly effective in inducing human tolerogenic dendritic cells.

The design of innovative strategies to selectively target cells, such antigen-presenting cells and dendritic cells, in vivo to induce immune tolerance is gaining interest and relevance for the treatment of immune-mediated diseases. A novel loaded-nanosystem strategy to generate tolerogenic dendritic cells (tol-DCs) was evaluated. Hence budesonide (BDS) was encapsulated in multiwalled polyurethane-polyurea nanoparticles (PUUa NPs-BDS) based on self-stratified polymers by hydrophobic interactions at the oil-water interface. DCs treated with encapsulated BDS presented a prominent downregulation of costimulatory molecules (CD80, CD83 and MHCII) and upregulation of inhibitory receptors. Moreover, DCs treated with these PUUa NPs-BDS also secreted large amounts of IL-10, a crucial anti-inflammatory cytokine to induce tolerance, and inhibited T lymphocyte activation in a specific manner compared to those cells generated with free BDS. These results demonstrate that PUUa NPs-BDS are a highly specific and efficient system through which to induce DCs with a tolerogenic profile. Given the capacity of PUUa NPs-BDS, this delivery system has a clear advantage for translation to in vivo studies.