Laia Grau-López

Comparative study of clinical grade human tolerogenic dendritic cells

BackgroundThe use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.MethodsTolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.ResultsDifferences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.ConclusionsOur results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.

Stable antigen-specific T-cell hyporesponsiveness induced by tolerogenic dendritic cells from multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease of the central nervous system. Current therapies decrease the frequency of relapses and limit, to some extent, but do not prevent disease progression. Hence, new therapeutic approaches that modify the natural course of MSneed to be identified. Tolerance induction to self‐antigens using monocyte‐derived dendritic cells (MDDCs) is a promising therapeutic strategy in autoimmunity. In this work, we sought to generate and characterize tolerogenic MDDCs (tolDCs) from relapsing‐remitting (RR) MSpatients, loaded with myelin peptides as specific antigen, with the aim of developing immunotherapeutics for MS. MDDCs were generated from both healthy‐blood donors and RR‐MSpatients, and MDDCmaturation was induced with a proinflammatory cytokine cocktail in the absence or presence of 1α,25‐dihydroxyvitamin‐D3, a tolerogenicity‐inducing agent. tolDCs were generated from monocytes of RR‐MSpatients as efficiently as from monocytes of healthy subjects. The RR‐MStolDCs expressed a stable semimature phenotype and an antiinflammatory profile as compared with untreated MDDCs. Importantly, myelin peptide‐loaded tolDCs induced stable antigen‐specific hyporesponsiveness in myelin‐reactive T cells from RR‐MS patients. These results suggest that myelin peptide‐loaded tolDCs may be a powerful tool for inducing myelin‐specific tolerance in RR‐MS patients.

Beneficial effect of tolerogenic dendritic cells pulsed with MOG autoantigen in experimental autoimmune encephalomyelitis.

Treatment with tolerogenic dendritic cells (TolDC) is a promising, cell‐based strategy to regulate autoimmune diseases such as multiple sclerosis (MS) in an antigen‐specific way. This technique involves the use of TolDC from MS patients cultured in the presence of vitamin D3 (VitD3) and pulsed with myelin peptides to induce a stable hyporesponsiveness in myelin‐specific autologous T cells.

Myelin peptides in multiple sclerosis.

The development of specific therapies for organ-specific autoimmune diseases requires the identification of relevant immunogenic epitopes, recognized by both pathogenic T cells and autoantibodies. Here, we review the most relevant studies focused in the identification of peptides in multiple sclerosis (MS) and the distinct T cell reactivity induced in patients compared to controls. Only a few studies reported significant differences in terms of T cell reactivity to them. The current knowledge on this issue, and the diagnostic and therapeutic possibilities opened by the identification of pathogenic MS epitopes are discussed in this paper.

Differential effects of monophosphoryl lipid A and cytokine cocktail as maturation stimuli of immunogenic and tolerogenic dendritic cells for immunotherapy

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1β, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy.

Regulatory role of vitamin D in T-cell reactivity against myelin peptides in relapsing-remitting multiple sclerosis patients

BackgroundLow levels of plasma 25-hydroxyvitaminD (25(OH)D) are associated with a higher incidence of multiple sclerosis (MS) due to the immune suppressive properties of vitamin D.The aim of this study was to determine the correlation between plasma 25(OH)D concentrations and clinical and immunological variables in a cohort of multiple sclerosis patients.MethodsPlasma 25(OH)D concentrations were evaluated in summer and winter in 15 primary progressive MS (PPMS) patients, 40 relapsing- remitting MS (RRMS) patients and 40 controls (HC). Protocol variables included demographic and clinical data, radiological findings and immunological variables (oligoclonal bands, HLADR15 and T-lymphocyte proliferation to a definite mix of 7 myelin peptides).ResultsDuring the winter, plasma concentrations were significantly lower in RRMS patients compared to HC, whereas no differences were found in summer. No relationships were found between plasma 25(OH)D concentrations and clinical or radiological variables. RRMS patients with a positive T-cell proliferation to a mix of myelin peptides (n = 31) had lower 25(OH)D concentrations.Conclusions25(OH)D is an immunomodulatory molecule that might have a regulatory role in T-cell proliferation to myelin peptides in RRMS patients.

Baseline Differences in Minor Lymphocyte Subpopulations may Predict Response to Fingolimod in Relapsing-Remitting Multiple Sclerosis Patients.

Fingolimod, oral treatment for relapsing–remitting multiple sclerosis (RRMS), is an agonist of sphingosine and its metabolite S1P that binds their receptors, blocking the egress of lymphocytes from lymph nodes. The aim of this study was immunomonitoring of minor peripheral lymphocyte subpopulations in RRMS patients under treatment with fingolimod and correlation with treatment response.

Similar biological effect of high-dose oral versus intravenous methylprednisolone in multiple sclerosis relapses.

Our aim was to investigate differences in immune mechanisms in multiple sclerosis (MS) relapse, after high-dose oral methylprednisolone (oMP) or intravenous methylprednisolone (ivMP). We measured serum cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ) in 39 of 49 MS patients with moderate-severe relapse, whom were treated with ivMP or oMP in a placebo-controlled, non-inferiority clinical trial. We assessed these cytokine levels at baseline and at 1 and 4 weeks post-treatment. The cytokine levels between oMP and ivMP were similar at any time. Proinflammatory cytokines (IL-6 and IFN-γ) were significantly decreased in both groups at week 1 (p = 0.05 / p = 0.03) and at week 4 (p = 0.04 / p = 0.05). This study provides further confirmatory evidence that oMP is not inferior to ivMP. Trial registration: clinicaltrials.gov identifier: NCT00753792

Baseline clinical status as a predictor of methylprednisolone response in multiple sclerosis relapses

Background: To date, there are no available factors to predict the outcome after multiple sclerosis relapse. Aim: To investigate factors that may be useful for predicting response to methylprednisolone treatment, following a relapse of multiple sclerosis (MS). Methods: The study included 48 MS patients enrolled in a double-blind multicenter trial to receive intravenous versus oral high-dose methylprednisolone treatment. Associations were sought between the disability status prior to relapse and the relapse severity, determined by changes in the Expanded Disability Status Scale (EDSS) score, as well as the improvements after treatment. We also analyzed the relationships between the number of magnetic resonance imaging (MRI) gadolinium-enhancing lesions (Gd+) and improvement. Results: A higher EDSS score before relapse was associated with more severe relapses (p = 0.04) and less marked improvement (odds ratio (OR) 1.8; 95% CI (1.2–2.2); p = 0.05) after methylprednisolone treatment. Relapse severity (p = 0.29) and the number of Gd+ lesions at relapse (p = 0.41) were not related with improvement. Conclusions: Clinical baseline status prior to MS relapse is a predictor of response to methylprednisolone treatment.

Characterization of recent thymic emigrants (RTEs), transitional B and Th17 cells in multiple sclerosis (MS).

The finding that APECED syndrome is due to a failure in negative thymic selection has suggested that central tolerance could play a role also in polygenic autoimmunity and that autoimmune patients should have an increase in RTEs. The aim of this work was to assess the frequency of RTEs, transitional B cells and Th17 lymphocytes in MS. PBLs from 55 MS patients [20 relapsing-remitting (RR) with disease modifying drugs (DMD), 20 RRMS without DMD, 10 progressive MS and 5 RRMS patients on relapse] and 32 healthy controls were analysed by FACS for RTEs, naive and memory T cells as well as Th17 cells and transitional B cells. No differences in the percentage of RTEs (CD45RA+CD31+PTK7+) (3.30±2.94 vs 3.05±1.54, 0.933) or B transitional cells (CD19+CD24hiCD38hiCD27-) (8.67±3.62 vs 8.59±3.8, p=0.45) were found between controls and MS. A significant decrease in the percentage of CD4 effector memory (CD45RA-CCR7-CD27+) (23.13±8.76 vs 20.8±6.1, p=0.043), as well as an increase in Th17 cells (6.9±3.2 vs5.49±2.33, p=0.007) was observed in MS vs controls. There were no differences in lymphocyte populations among the clinical forms of MS. RRMS under treatment had similar results to RRMS without DMD. Patients on relapse did show an increased proportion of CD4 effector memory cells, Th17 cells and RTEs and patients with higher EDSS had a higher percentage of Th17 cells (OR 2.3,p=0.045). The results of this preliminary study do not favour a failure in central tolerance in MS but confirm involvement of Th17 cells which are associated with a higher degree of disability.