Challenges in tolerogenic dendritic cell therapy for autoimmune diseases: the route of administration.

Tolerogenic dendritic cells (tolDCs) are a promising strategy to treat autoimmune diseases since they have the potential to re-educate and modulate pathological immune responses in an antigen-specific manner and, therefore, have minimal adverse effects on the immune system compared to conventional immunosuppressive treatments. TolDC therapy has demonstrated safety and efficacy in different experimental models of autoimmune disease, such as multiple sclerosis (MS), type 1 diabetes (T1D), and rheumatoid arthritis (RA). Moreover, data from phase I clinical trials have shown that therapy with tolDCs is safe and well tolerated by MS, T1D, and RA patients. Nevertheless, various parameters need to be optimized to increase tolDC efficacy. In this regard, one important parameter to be determined is the most appropriate route of administration. Several delivery routes, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranodal, and intraarticular routes, have been used in experimental models as well as in phase I clinical trials. This review summarizes data obtained from preclinical and clinical studies of tolDC therapy in the treatment of MS, T1D, and RA and their animal models, as well as data from the context of cancer immunotherapy using mature peptide-loaded DC, and data from in vivo cell tracking experiments, to define the most appropriate route of tolDC administration in relation to the most feasible, safest, and effective therapeutic use.

Assessing Blood-Based Biomarkers to Define a Therapeutic Window for Natalizumab.

Natalizumab is a monoclonal antibody that binds CD49d. Although it is one of the most effective treatments for Relapsing-Remitting Multiple Sclerosis (RRMS), a dosing regimen has not been optimized for safety and efficacy in individual patients. We aimed to identify biomarkers to monitor Natalizumab treatment and to establish a personalized dose utilizing an ongoing longitudinal study in 29 RRMS patients under Natalizumab with standard interval dose (SD) of 300 mg/4wks or extended interval dose (EID) of 300 mg/6wks. Blood samples were analyzed by flow cytometry to determine CD49d saturation and expression in several T and B lymphocytes subpopulations. Each patient was analyzed at two different timepoints separated by 3 Natalizumab administrations. Natalizumab and sVCAM-1 levels in serum were also analyzed using ELISA. To determine the reproducibility of various markers, two different timepoints were compared and no significant differences were observed for CD49d expression nor for saturation; SD patients had higher saturation levels (~80%) than EID patients (~60%). A positive correlation exists between CD49d saturation and Natalizumab serum levels. CD49d expression and saturation are stable parameters that could be used as biomarkers in the immunomonitoring of Natalizumab treatment. Moreover, Natalizumab and sVCAM-1 serum levels could be used to optimize an individual's dosing schedule.

Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-ß in a Preclinical Model of Multiple Sclerosis.

Autologous antigen-specific therapies based on tolerogenic dendritic cells (tolDC) offer the possibility to treat autoimmune diseases by restoring homeostasis and targeting specifically autoreactive responses. Here, we explore the hypothesis that systemic inflammation occurring in autoimmune diseases, such as multiple sclerosis (MS), can generate a disease-specific environment able to alter the functionality of tolDC. In this context in fact, a combined therapy of tolDC with an immunomodulatory treatment could potentiate the beneficial effect of this antigen-specific cell therapy. For this purpose, we analyzed the efficacy of a combined therapy based on the use of vitamin D3 (VitD3)-tolDC plus interferon beta (IFN-beta) in MS. VitD3-tolDC were generated from healthy donors and MS patients and co-cultured with allogeneic peripheral blood mononuclear cells, in the presence or absence of IFN-beta. In vitro, VitD3-tolDC treatment reduced the percentage of activated T cells and allogeneic proliferation, whereas VitD3-tolDC+IFN-beta treatment enhanced the suppressive ability of VitD3-tolDC and, additionally, induced a shift towards a Th2 profile. To determine the clinical benefit of the combined therapy, C57BL/6-experimental autoimmune encephalomyelitis (EAE)-induced mice were treated with antigen-specific VitD3-tolDC and/or IFN-beta. Treatment of EAE mice with combined therapy ameliorated the disease course compared to each monotherapy. These results suggest that a combined therapy based on antigen-specific VitD3-tolDC and IFN-beta may represent a promising strategy for MS patients.

Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).

Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC trigger for the induction of tolerance remain elusive. For this reason, we performed a full phenotypical, functional, and transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong antigen-specific reduction of T cell proliferation, combined with a decrease in the relative prevalence of TH1 subpopulations and IFN-γ production. The analysis of the transcriptomic profile of T CD4+ cells evidenced a significant down-modulation of genes involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, highlighting the role of JUNB gene as a potential biomarker of these processes. Consequently, our results show the induction of a strong antigen-specific hyporesponsiveness combined with a reduction on the TH1 immune profile of T cells upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these cells and, therefore, their therapeutic potential in the clinic.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients.

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.

Th1Th17CM Lymphocyte Subpopulation as a Predictive Biomarker of Disease Activity in Multiple Sclerosis Patients under Dimethyl Fumarate or Fingolimod Treatment.

Peripheral blood biomarkers able to predict disease activity in multiple sclerosis (MS) patients have not been identified yet. Here, we analyzed the immune phenotype of T lymphocyte subpopulations in peripheral blood samples from 66 RRMS patients under DMF (n = 22) or fingolimod (n = 44) treatment, by flow cytometry. A correlation study between the percentage and absolute cell number of each lymphocyte subpopulation with the presence of relapses or new MRI lesions during 12-month follow-up was performed. Patients who had undergone relapses showed at baseline higher percentage of Th1CM cells (relapsed: 11.60 ± 4.17%vs. nonrelapsed: 9.25 ± 3.17%, p < 0.05) and Th1Th17CM cells (relapsed: 15.65 ± 6.15%vs. nonrelapsed: 10.14 ± 4.05%, p < 0.01) before initiating DMF or fingolimod treatment. Kaplan-Meier analysis revealed that patients with Th1Th17CM (CD4+CCR7+CD45RA-CCR6+CXCR3+) cells > 11.48% had a 50% relapse-free survival compared to patients with Th1Th17CMcells < 11.48% whose relapse-free survival was 88% (p = 0.013, log-rank test). Additionally, a high percentage of Th1Th17CM cells was also found in patients with MRI activity (MRI activity: 14.02 ± 5.87%vs. no MRI activity: 9.82 ± 4.06%, p < 0.01). Our results suggest that the percentage of Th1Th17CM lymphocytes at baseline is a predictive biomarker of activity during the first 12 months of treatment, regardless of the treatment.

Optimal response to dimethyl fumarate is mediated by a reduction of Th1-like Th17 cells after 3 months of treatment.

AIM: Dimethyl fumarate (DMF) is one of the most promising therapies for relapsing-remitting multiple sclerosis (RRMS) patients since it has shown immunomodulatory and neuroprotective effects. However, a percentage of RRMS patients do not exhibit an optimal response to DMF. The objective of this study was to identify early biomarkers of treatment response by analyzing changes in peripheral leukocyte subpopulations directly in whole blood samples. METHODS: A longitudinal and prospective study analyzing peripheral blood leukocyte subpopulations in 22 RRMS patients before initiating DMF treatment (baseline) and at 1, 3, 6, and 12 months of follow-up was performed. Differences between no evidence of disease activity (NEDA) and ongoing disease activity (ODA) patients were analyzed. RESULTS: The beneficial effect of DMF was associated with a specific depletion of memory CD4+ and CD8+ T lymphocytes and B cells. Importantly, only NEDA patients showed (a) a shift from a pro- to an antiinflammatory profile, with an increase of Th2 cells and a decrease of Th1-like Th17 lymphocytes; and (b) an increase of regulatory CD56bright NK cells. CONCLUSION: The optimal response to DMF is mediated by a shift to antiinflammatory and immunoregulatory profile, which puts forward Th1-like Th17 lymphocytes as a potential early biomarker of treatment response.

Ethyl Pyruvate Induces Tolerogenic Dendritic Cells.

Dendritic cells (DC) are professional antigen presenting cells that have a key role in shaping the immune response. Tolerogenic DC (tolDC) have immuno-regulatory properties and they are a promising prospective therapy for multiple sclerosis and other autoimmune diseases. Ethyl pyruvate (EP) is a redox analog of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that EP ameliorates experimental autoimmune encephalomyelitis, a multiple sclerosis murine model. Here, we expanded our study to its tolerogenic effects on DC. Phenotypic analysis has shown that DC obtained from mice or humans reduce expression of molecules required for T cell activation such as CD86, CD83, and HLA-DR under the influence of EP, while CD11c expression and viability of DC are not affected. Furthermore, EP-treated DC restrain proliferation and modulate cytokine production of allogeneic lymphocytes. These results demonstrate that EP has the ability to direct DC toward tolDC.

Comparative transcriptomic profile of tolerogenic dendritic cells differentiated with vitamin D3, dexamethasone and rapamycin.

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However, the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic, as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim, in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC), dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However, individually, CYP24A1, MUCL1 and MAP7 for vitD3-tolDC; CD163, CCL18, C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC, constituted good candidate biomarkers for each respective cellular product. In addition, a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways, while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes.

Searching for the Transcriptomic Signature of Immune Tolerance Induction-Biomarkers of Safety and Functionality for Tolerogenic Dendritic Cells and Regulatory Macrophages.

The last years have witnessed a breakthrough in the development of cell-based tolerance-inducing cell therapies for the treatment of autoimmune diseases and solid-organ transplantation. Indeed, the use of tolerogenic dendritic cells (tolDC) and regulatory macrophages (Mreg) is currently being tested in Phase I and Phase II clinical trials worldwide, with the aim of finding an effective therapy able to abrogate the inflammatory processes causing these pathologies without compromising the protective immunity of the patients. However, there exists a wide variety of different protocols to generate human tolDC and Mreg and, consequently, the characteristics of each product are heterogeneous. For this reason, the identification of biomarkers able to define their functionality (tolerogenicity) is of great relevance, on the one hand, to guarantee the safety of tolDC and Mreg before administration and, on the other hand, to compare the results between different cell products and laboratories. In this article, we perform an exhaustive review of protocols generating human tolDC and Mreg in the literature, aiming to elucidate if there are any common transcriptomic signature or potential biomarkers of tolerogenicity among the different approaches. However, and although several effectors seem to be induced in common in some of the most reported protocols to generate both tolDC or Mreg, the transcriptomic profile of these cellular products strongly varies depending on the approach used to generate them.

Monitoring CD49d Receptor Occupancy: A Method to Optimize and Personalize Natalizumab Therapy in Multiple Sclerosis Patients.

BACKGROUND: In natalizumab-treated relapsing-remitting MS (RRMS) patients, various extended interval dosing strategies are under evaluation to minimize severe treatment-associated side effects, mainly progressive multifocal leukoencephalopathy development. Up to now, it has not been presented any approach, even in form of assay design, to determine the optimal percentage of CD49d receptor occupancy (RO) associated with a favorable clinical, radiological, and immunological response. METHODS: A multiparametric quantitative flow cytometry method was settled to measure CD49d RO on peripheral blood lymphocytes. The analytical protocol was tested in a 6-month follow-up from 19 RRMS patients treated with the natalizumab standard dosing of every 4 weeks or an extended-interval dosing of every 6 weeks. RESULTS: Extended natalizumab dose schedule promoted an increase of CD49d molecules per cell surface and a reduction of CD49d RO levels. The reduction observed on CD49d RO was not only depending on dose schedule but also on individual parameters such as body mass. Interestingly, individual clinical outcome was apparently the same between the different dose schedules or even better with the extended interval dosing. CONCLUSIONS: Following up CD49d RO levels with a well-regulated monitoring work scheme is crucial to further identify over-/under-treated patients and to define a safe, personalized natalizumab regimen. © 2017 International Clinical Cytometry Society.

Liposome-based immunotherapy against autoimmune diseases: therapeutic effect on multiple sclerosis.

AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.

Cell-based Tolerogenic Therapy, Experience from Animal Models of Multiple Sclerosis, Type 1 Diabetes and Rheumatoid Arthritis.

Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.

Myeloid-derived suppressor cells expressing a self-antigen ameliorate experimental autoimmune encephalomyelitis.

Previous work by our group showed that transferring bone marrow cells transduced with a self-antigen induced immune tolerance and ameliorated experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We also found that following retroviral transduction of murine bone marrow (BM) cells, the majority of cells generated and transduced were myeloid-derived suppressor cells (MDSCs). Here, we aimed to determine whether purified antigen-expressing MDSCs have similar therapeutic effects than those of unfractionated BM, and to investigate their potential mechanisms. We performed phenotypic and functional analyses in these cells using the same animal model, and we used purified antigen-expressing MDSCs in preventive and therapeutic approaches. These cells exerted therapeutic effects similar to those of BM cells, which depended upon self-antigen expression. The majority of monocytic (M)-MDSCs expressed the immunosuppressive molecule programmed death ligand-1 (PD-L1), CD80, CD86 and MHC class II molecules. Additionally, the animals infused with antigen-expressing cells exhibited lower percentages of activated T cells and higher percentages of B cells with a regulatory phenotype (B220+CD1dhigh CD5+) in the spleen than their respective controls. MDSCs expressing self-antigens, alloantigens or therapeutic transgenes are tolerogenic and can be exploited therapeutically in autoimmune diseases, transplantation and in gene therapy, respectively.

Cryopreserved vitamin D3-tolerogenic dendritic cells pulsed with autoantigens as a potential therapy for multiple sclerosis patients.

BACKGROUND: Tolerogenic dendritic cells (tolDC) have been postulated as a potent immunoregulatory therapy for autoimmune diseases such as multiple sclerosis (MS). In a previous study, we demonstrated that the administration of antigen-specific vitamin D3 (vitD3) tolDC in mice showing clinical signs of experimental autoimmune encephalomyelitis (EAE; the animal model of MS) resulted in abrogation of disease progression. With the purpose to translate this beneficial therapy to the clinics, we have investigated the effectivity of vitD3-frozen antigen-specific tolDC pulsed with myelin oligodendrocyte glycoprotein 40-55 peptide (f-tolDC-MOG) since it would reduce the cost, functional variability and number of leukapheresis to perform to the patients. METHODS: Mice showing EAE clinical signs were treated with repetitive doses of f-tolDC-MOG. Tolerogenic mechanisms induced by the therapy were analysed by flow cytometry and T cell proliferation assays. RESULTS: Treatment with f-tolDC-MOG was effective in ameliorating clinical signs of mice with EAE, inhibiting antigen-specific reactivity and inducing Treg. In addition, the long-term treatment was well tolerated and leading to a prolonged maintenance of tolerogenicity mediated by induction of Breg, reduction of NK cells and activation of immunoregulatory NKT cells. CONCLUSIONS: The outcomes of this study show that the use of antigen-specific f-tolDC promotes multiple and potent tolerogenic mechanisms. Moreover, these cells can be kept frozen maintaining their tolerogenic properties, which is a relevant step for their translation to the clinic. Altogether, vitD3 f-tolDC-MOG is a potential strategy to arrest the autoimmune destruction in MS patients.

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

AIMS: 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. METHODS: Prospective study. T- and B-cell subpopulations were analyzed using multiparametric flow cytometry in peripheral blood from 14 RRMS patients under treatment with fingolimod at baseline, +1, +3, +6, +9, and +12 months of follow-up. Response to therapy was assessed at month +12. RESULTS: Most changes in minor lymphocyte subpopulations occurred in the first month of treatment and were maintained until the end of follow-up. The basal percentages of recent thymic emigrants (RTEs) and transitional B cells were lower in responder patients than in nonresponders. After 1 month of follow-up, the percentages of late effector memory CD4(+) T cells in peripheral blood were higher in responder patients. CONCLUSION: If confirmed in a bigger cohort of patients, analysis of percentages of minor lymphocyte subpopulations in peripheral blood of patients with RRMS prior and after +1 month of treatment might predict clinical response to fingolimod.

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

BACKGROUND: 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 D(3) (VitD3) and pulsed with myelin peptides to induce a stable hyporesponsiveness in myelin-specific autologous T cells. AIM: The purpose of this study was to analyze the in vivo effect of VitD3-TolDC treatment on experimental autoimmune encephalomyelitis, an animal model of MS. METHODS: Bone marrow-derived TolDC cultured in the presence of VitD3 and pulsed with peptide 40-55 of the myelin oligodendrocyte glycoprotein (MOG(40-55)) were administrated preventively, preclinically, and therapeutically to EAE-induced mice. RESULTS: We found that VitD3-TolDC-MOG treatment showed a beneficial effect, not only decreasing the incidence of the disease but also reducing the severity of the clinical signs mediated by induction of regulatory T cells (Treg), as well as IL-10 production and reduction of Ag-specific lymphoproliferation. Our results support VitD3-TolDC-peptide(s) treatment as a potential strategy to restore tolerance in autoimmune diseases such as MS.

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients.

Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.