Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 α-chain (CD11a) in MS patients.

OBJECTIVE: To determine the long-term effect of natalizumab (NTZ) treatment on the expression of integrins and chemokine receptors involved in the migration of T cells towards the central nervous system (CNS). METHODS: We drew the blood of 23 patients just before starting NTZ therapy and every 12 months thereafter, for up to 48 months of treatment. We assessed the ex-vivo expression of phenotype markers (CCR7 and CD45RA), CNS-addressing integrins (CD11a, CD49d and CD29) and chemokine receptors (CXCR3 and CCR6) in CD4+ or CD8+ T-cell subsets by flow cytometry. RESULTS: As compared to the pre-NTZ values, there was a marked increase in central memory (CCR7+/CD45RA-) CD4+ T cells and in effector memory (CCR7-/CD45RA-) CD8+ T cells at 12 and 24 months. In addition to an expected downregulation of both VLA-4 subunits (CD49d/CD29), we also found decreased T-cell expression of CXCR3 at 12 months, and of CD11a (LFA-1 αL subunit) at 12 months, but mostly at 24 months of NTZ treatment. CONCLUSION: Our data show a nadir of CD11a expression at 2 years of NTZ treatment, at the peak of incidence of progressive multifocal leukoencephalopathy (PML), indirectly suggesting that a lack of these molecules may play a role in the onset of PML in NTZ-treated patients.

Rivastigmine for HIV-associated neurocognitive disorders: a randomized crossover pilot study.

OBJECTIVE: To assess the efficacy and safety of rivastigmine for the treatment of HIV-associated neurocognitive disorders (HAND) in a cohort of long-lasting aviremic HIV+ patients. METHODS: Seventeen aviremic HIV+ patients with HAND were enrolled in a randomized, double-blind, placebo-controlled, crossover study to receive either oral rivastigmine (up to 12 mg/day for 20 weeks) followed by placebo (20 weeks) or placebo followed by rivastigmine. Efficacy endpoints were improvement on rivastigmine in the Alzheimer's disease assessment scale-cognitive subscale (ADAS-Cog) and individual neuropsychological scores of information processing speed, attention/working memory, executive functioning, and motor skills. Measures of safety included frequency and nature of adverse events and abnormalities on laboratory tests and on plasma concentrations of antiretroviral drugs. Analyses of variance with repeated measures were computed to look for treatment effects. RESULTS: There was no change on the primary outcome ADAS-Cog on drug. For secondary outcomes, processing speed improved on rivastigmine (trail making test A: F(1,13) = 5.57, p = 0.03). One measure of executive functioning just failed to reach significance (CANTAB spatial working memory [strategy]: F(1,13) = 3.94, p = 0.069). No other change was observed. Adverse events were frequent, but not different from those observed in other populations treated with rivastigmine. No safety issues were recorded. CONCLUSIONS: Rivastigmine in aviremic HIV+ patients with HAND seemed to improve psychomotor speed. A larger trial with the better tolerated transdermal form of rivastigmine is warranted. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that rivastigmine is ineffective for improving ADAS-Cog scores, but is effective in improving some secondary outcome measures in aviremic HIV+ patients with HAND.

MOBP-specific cellular immune responses are weaker than MOG-specific cellular immune responses in patients with multiple sclerosis and healthy subjects.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Myelin oligodendrocyte glycoprotein (MOG) and myelin oligodendrocyte basic protein (MOBP) were both shown to be highly encephalitogenic in animal models of MS. In contrast, the association of MOG- and MOBP-specific humoral or cellular immune responses and MS in humans is far less established. In this study, we sought to analyse MOG- and MOBP-specific T-cell responses in a large cohort of patients with various stages of the disease. Patients with other neurological diseases and healthy subjects were enrolled to serve as control study subjects. We determined the proliferation and the secretion of IFN-γ secretion in our cohort. We found that MOG-specific T-cell responses were higher and more frequent as compared to MOBP-specific ones. However, both MS patients and control study subjects had similar myelin-specific T-cell responses at the periphery, thus calling for more precise studies at CNS level.

Marked increase of the astrocytic marker S100B in the cerebrospinal fluid of HIV-infected patients on LPV/r-monotherapy.

OBJECTIVE: To determine changes of cerebrospinal fluid (CSF) biomarkers of patients on monotherapy with lopinavir/ritonavir. DESIGN: The Monotherapy Switzerland/Thailand study (MOST) trial compared monotherapy with ritonavir-boosted lopinavir with continued therapy. The trial was prematurely stopped due to virological failure in six patients on monotherapy. It, thus, offers a unique opportunity to assess brain markers in the early stage of HIV virological escape. METHODS: : Sixty-five CSF samples (34 on continued therapy and 31 on monotherapy) from 49 HIV-positive patients enrolled in MOST. Using enzyme-linked immunosorbent assay, we determined the CSF concentration of S100B (astrocytosis), neopterin (inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-ß 1-42 (Aß), the latter three indicating neuronal damage. Controls were CSF samples of 29 HIV-negative patients with Alzheimer dementia. RESULTS: In the CSF of monotherapy, concentrations of S100B and neopterin were significantly higher than in continued therapy (P = 0.006 and P = 0.013, respectively) and Alzheimer dementia patients (P < 0.0001 and P = 0.0005, respectively). In Alzheimer dementia, concentration of Aß was lower than in monotherapy (P = 0.005) and continued therapy (P = 0.016) and concentrations of tTau were higher than in monotherapy (P = 0.019) and continued therapy (P = 0.001). There was no difference in pTau among the three groups. After removal of the 16 CSF with detectable viral load in the blood and/or CSF, only S100B remained significantly higher in monotherapy than in the two other groups. CONCLUSION: Despite full viral load-suppression in blood and CSF, antiretroviral monotherapy with lopinavir/ritonavir can raise CSF levels of S100B, suggesting astrocytic damage.

Impairment of JCV-specific T-cell response by corticotherapy: effect on PML-IRIS management?

OBJECTIVE: To investigate the impact of corticosteroids (CS) on the viral-specific T-cell response, in particular the JC virus (JCV)-specific one, in an attempt to determine the optimal timing of CS in the management of progressive multifocal leukoencephalopathy-immune reconstitution inflammatory syndrome (PML-IRIS). METHODS: A blood draw was performed before and 7 days after the administration of IV CS to 24 patients with relapsing multiple sclerosis (MS). The phenotypic pattern of T cells was determined by CCR7 and CD45RA. To assess the impact of CS treatment on proliferative response of JCV-, influenza-, and Epstein-Barr virus (EBV)-specific T cells, a thymidine incorporation proliferation assay was performed. An intracellular cytokine staining assay was performed to determine the effect of CS treatment on the production of cytokine by virus-specific T cells. JCV T-cell assays were performed only in JCV-infected patients with MS as detected by serologies (Stratify) or detection of JCV DNA in the urine by PCR. RESULTS: CS led T cells, CD4+ and CD8+, toward a less differentiated phenotype. There was a significant decrease of EBV-, influenza-, and JCV-specific T-cell proliferative response upon CS treatment. There was a significant decrease in the frequency of interferon (IFN) γ- and tumor necrosis factor (TNF) α-producing JCV-specific CD8+ T cells, but not EBV- or influenza-specific CD4+ or CD8+ T cells. CONCLUSIONS: CS have a profound impact on the virus-specific T-cell response, especially on JCV, suggesting that when CS are considered, they should not be given before the onset of clinical or radiologic signs of IRIS. Studies addressing directly patients with MS with natalizumab-caused PML are warranted. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that methylprednisolone treatment decreases the frequency of JCV-specific CD8+ T cells producing IFN-γ and TNFα, impairing control of JCV, suggesting this should be used to treat but not to prevent PML-IRIS. No clinical outcomes were measured.

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.

Immunological and clinical consequences of treating a patient with natalizumab.

BACKGROUND: Long-term therapy with natalizumab increases the risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVES: We present a patient study through therapy, the diagnosis of PML (after 29 infusions), plasma exchange (PE) and development of immune reconstitution inflammatory syndrome (IRIS). METHODS: Routine diagnostics, magnetic resonance imaging (MRI), immunological status (flow cytometry, T-cell migration assays and T-cell repertoire analysis), and brain biopsy with immunohistological analysis. RESULTS: CD49d decreased after 12 months of treatment. At PML diagnosis, CD49d expression and migratory capacity of T cells was low and peripheral T-cell receptor (TCR) complexity showed severe perturbations. The distribution of peripheral monocytes changed from CCR5+ to CCR7+. After PE some changes reverted: CD49d increased and overshot earliest levels, migratory capacities of T cells recovered and peripheral TCR complexity increased. With no clinical, routine laboratory or cerebrospinal fluid (CSF) changes, MRI 2 months after PE demonstrated progressive lesion development. Brain histopathology confirmed the presence of infiltrates indicative of IRIS without clinical signs, immunologically accompanied by CCR7/CCR5 recovery of peripheral monocytes. CONCLUSION: Natalizumab-associated immunological changes accompanying PML were reversible after PE; IRIS can occur very late, remain asymptomatic and be elusive to CSF analysis. Our study may provide insights into the changes under treatment with natalizumab associated with JC virus control.

Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects.

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-ß. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

Cytokine mRNA profile of Epstein-Barr virus-stimulated highly differentiated T cells in multiple sclerosis: a pilot study.

The reason why EBV-specific cellular immune responses are abnormal in multiple sclerosis (MS) patients is still missing. In this exploratory pilot study, we assessed IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-gamma, TGF-beta1 and FOXP3 mRNA expression in EBV-stimulated highly differentiated T cells (T(HD)) of MS patients and healthy controls (HC). We found increased levels of IFN-gamma and IL-4 mRNA in CD8+ T(HD) cells of MS patients. All the other tested molecules were expressed similarly in MS patients and HC. Interestingly, increased IFN-gamma and IL-4 suggest that the control of EBV replication may be insufficient in MS patients.

Immune responses to JC virus in patients with multiple sclerosis treated with natalizumab: a cross-sectional and longitudinal study.

BACKGROUND: Natalizumab is used to prevent relapses and progression of disability in patients with multiple sclerosis but has been associated with progressive multifocal leukoencephalopathy (PML). We aimed to better understand the associations between JC virus, which causes PML, and natalizumab treatment. METHODS: We prospectively assessed patients with multiple sclerosis who started treatment with natalizumab. Blood and urine samples were tested for the presence of JC virus DNA with quantitative real-time PCR before treatment and at regular intervals after treatment onset for up to 18 months. At the same timepoints, by use of proliferation and enzyme-linked immunospot assays, the cellular immune responses against JC virus, Epstein-Barr virus, cytomegalovirus, myelin oligodendrocyte glycoprotein, and myelin oligodendrocyte basic protein (MOBP) were assessed. Humoral immune response specific to JC virus was assessed with an enzyme immunoassay. The same experiments were done on blood samples from patients with multiple sclerosis before and 10 months after the start of interferon beta treatment. FINDINGS: We assessed 24 patients with multiple sclerosis who received natalizumab and 16 who received interferon beta. In patients treated with natalizumab, JC virus DNA was not detected in the blood at any timepoint. However, JC virus DNA was present in the urine of six patients and in most of these patients the concentrations of JC virus DNA were stable over time. Compared with pretreatment values, the cellular immune response was increased to cytomegalovirus at 6 months, to JC virus at 1, 9, and 12 months, and to Epstein-Barr virus and MOBP at 12 months. Humoral responses remained stable. There were no increases in cellular immune responses specific to the viruses or myelin proteins in the 16 patients treated with interferon beta. INTERPRETATION: Natalizumab increases cellular immune responses specific to viruses and myelin proteins in the peripheral blood after 1 year, without evidence of viral reactivation. FUNDING: Swiss National Foundation, Swiss Society for Multiple Sclerosis, and Biogen Dompé.

Intrathecal immune responses to EBV in early MS.

EBV has been consistently associated with MS, but its signature in the CNS has rarely been examined. In this study, we assessed EBV-specific humoral and cellular immune responses in the cerebrospinal fluid (CSF) of patients with early MS, other inflammatory neurological diseases (OIND) and non-inflammatory neurological diseases (NIND). The neurotropic herpesvirus CMV served as a control. Virus-specific humoral immune responses were assessed in 123 consecutive patients and the intrathecal recruitment of virus-specific antibodies was expressed as antibody indexes. Cellular immune responses tested in the blood of 55/123 patients were positive in 46/55. The CD8(+) CTL responses of these 46 patients were assessed in the blood and CSF using a CFSE-based CTL assay. We found that viral capsid antigen and EBV-encoded nuclear antigen-1, but not CMV IgG antibody indexes, were increased in early MS as compared with OIND and NIND patients. There was also intrathecal enrichment in EBV-, but not CMV-specific, CD8(+) CTL in early MS patients. By contrast, OIND and NIND patients did not recruit EBV- nor CMV-specific CD8(+) CTL in the CSF. Our data, showing a high EBV-, but not CMV-specific intrathecal immune response, strengthen the association between EBV and MS, in particular at the onset of the disease.

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis.

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.

Severe post-EBV encephalopathy associated with myelin oligodendrocyte glycoprotein-specific immune response.

The mechanisms leading to CNS disorders after EBV infections are unclear. We report the case of a patient who developed a severe, but reversible, encephalopathy following an infectious mononucleosis. We detected no EBV DNA in the blood or in the cerebrospinal fluid (CSF) and no EBV-specific antibodies in the CSF. However, we found a potent MOG-specific cellular and humoral immune response. Interestingly, MOG-specific cellular immune response rapidly decreased, paralleling the improvement of clinical condition. In conclusion, this detailed study shows that acute EBV infection can trigger a potent auto-inflammatory response in the CNS, without evidence of an overt infection.

CSF enrichment of highly differentiated CD8+ T cells in early multiple sclerosis.

CD8+ T cells may play an important role in multiple sclerosis (MS). Whether these cells would be involved in early stages of MS is unclear. We enrolled 52 patients with suspected MS, determined the recruitment of their highly differentiated (CCR7-/CD45RA+ or -) T cells (T(HD)) in the CSF as compared to peripheral blood and followed them for 12+/-7.3 months. A ROC curve showed that a CD8+/CD4+ T(HD) cells ratio of 0.94 helped to distinguish relapsing-remitting (RR-MS) and possible MS (Po-MS) from primary-progressive MS (PP-MS) and other neurological diseases patients (OND) patients (p=0.039), risk ratio of 2.29 (95% CI: 1.13-4.66; p=0.006). The CSF enrichment in CD8+ T(HD) cells was greater than in CD4+ T(HD) cells in RR/Po-MS patients (p=0.024) and than in CD8+ T(HD) cells in PP-MS/OND patients (p=0.006). These data suggest that CD8+ T(HD) cells play a role in the early stages of RR-MS.

DNA-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic cells.

This paper provides a review of the role of dendritic cells (DC) in microparticle-mediated immune response and the advantages of associating DNA to microparticles in order to increase the potency of DNA vaccination in vivo. To begin with, different methods for the preparation of DNA-loaded microparticle with poly(lactide) (PLA)/poly(lactide-co-glycolide) (PLGA) polymers are presented. Further, the effects of DNA-loaded microparticles on DC in vitro are extensively examined including transfection and stimulation of DC, a key feature of the immune response. Finally, in vivo tracking of DNA-loaded microparticles and induction of immune responses upon DNA-loaded microparticle administration in different animal models and with various routes of administration are reviewed.

Modulation of allergic responses in mice by using biodegradable poly(lactide-co-glycolide) microspheres.

BACKGROUND: Biodegradable poly(lactide- co -glycolide) (PLGA) microspheres are a promising carrier for vaccine delivery capable of maturing antigen-presenting cells to stimulate T-cell-mediated immune responses. However, the potential of microspheres to downregulate an allergic response in vivo is unknown. OBJECTIVE: The aim of this study was to determine whether microspheres could potentiate DNA vaccination against allergy and to evaluate the immunomodulatory properties of microspheres alone. METHODS: Mice were treated prophylactically with DNA-loaded plain PLGA microspheres before sensitization with phospholipase A2 (PLA2), the major allergen of bee venom. PLA2-specific IgG1, IgG2a, IgE in serum were measured for 8.5 months, and splenocyte proliferative responses and cytokine profiles were determined. Protection against anaphylaxis was evaluated after injection of an otherwise lethal dose of PLA2. RESULTS: Phospholipase A2-specific IgG1 and IgG2a production turned out to be 2 times higher using cationic microspheres compared with anionic microspheres, but was not influenced by the presence of DNA. In contrast, reduction in IgE production and T-cell hyporesponsiveness were observed with all microsphere formulations. Recall challenge with PLA2 triggered combined expression of both IL-4 and IFN-gamma, together with sustained expression of IL-10 that can explain the protective effect against anaphylaxis. CONCLUSION: Our data suggest a dual mechanism that does initially rely on a TH2 to TH1 immune deviation and then on IL-10-mediated suppression. This is the first physiological demonstration that plain PLGA microspheres can induce tolerance in mice for as long as 6 months postsensitization.

Transfection of a mouse dendritic cell line by plasmid DNA-loaded PLGA microparticles in vitro.

Targeting of DC for DNA vaccination may be achieved by DNA-loaded poly(lactide-co-glycolide) (PLGA) biodegradable microparticles, since DC efficiently capture these microparticles in vitro and in vivo. DNA was encapsulated in PLGA microparticles by spray-drying. Various additives were tested and process parameters adjusted in order to prevent degradation of the DNA during encapsulation. The highest degree of supercoiled DNA was maintained by adding a strong buffering agent, such as PBS or NaHCO(3), whereas the cryoprotective lactose did not show a significant protective effect. DNA-containing PLGA microparticles were administered to a mouse DC line. Transfection efficacy was compared with commonly employed cationic transfectants and was visually assessed by green fluorescent protein expression. Transfection rate was very low in DC for all microparticle formulations and was comparable with commonly used cationic transfectants. It is concluded that the transfection of DC using PLGA microparticles is feasible, but efforts need to be undertaken to improve transfection efficiency in vitro, which may in addition lead to improved immune responses in vivo.

Composition and surface charge of DNA-loaded microparticles determine maturation and cytokine secretion in human dendritic cells.

PURPOSE: Biodegradable microparticles prepared from poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) have been shown to be promising carrier systems for vaccine delivery. Here, we have investigated the capacity of different PLA and PLGA microparticle formulations to induce stimulation of human blood monocyte-derived dendritic cells (DCs). METHODS: Stimulation of human derived dendritic cells by plain microparticles were compared with microparticles loaded with plasmid DNA or double-stranded salmon DNA either by encapsulation or adsorption to the surface of cationic microparticles. Stimulation of DCs was monitored by the up-regulation of surface maturation markers CD83 and CD86 and the secretion of IL-12 and TNF-alpha. RESULTS: Slowly degrading PLA microparticles did not induce any detectable stimulation or activation of DCs. In contrast, fast degrading PLGA microparticles were able to influence DC maturation and cytokine secretion dependent on their surface charge. Anionic PLGA microparticles induced an up-regulation of CD83 and high TNF-alpha secretion, which was further enhanced up to the level of the potent stimulator lipopolysaccharide (LPS) when plasmid DNA was encapsulated. Moreover, the secretion of significant amounts of IL-12 was observed. Cationic PLGA microparticles induced an up-regulation of CD86 and moderate TNF-alpha secretion, but no IL-12 secretion, with no additional effects in the presence of plasmid DNA. CONCLUSIONS: The data suggest that the composition and charge of biodegradable DNA-loaded microparticles profoundly influences maturation and cytokine secretion in DCs. Thus, the individual formulation of microparticles used as a vaccine carrier system might considerably influence the profile of the immune response.

Improved stimulation of human dendritic cells by receptor engagement with surface-modified microparticles.

Dendritic cells (DC) need to be stimulated before they can function to initiate immune responses. This study investigates whether microparticles loaded with antibodies specific for selected receptors expressed by DC can induce stimulation of these cells. Plain microparticles were compared with microparticles which were surface-loaded with specific antibodies for human CD40, Fc(gamma), alpha(v)beta3 and alpha(v)beta5 integrin receptors. The antibodies were either physically adsorbed or covalently linked to the microparticle surface. Anti-CD40 antibody and human IgG immobilised on the surface of microparticles induced enhanced DC maturation and activation as expressed by CD83 and CD86 upregulation. IL-12 secretion was induced at a detectable but relatively low level. Both anti-integrin antibodies (anti-alpha(v)beta3 and anti-alpha(v)beta5) induced comparable and considerable maturation of DC, but only anti-alpha(v)beta3 antibody induced significant activation of DC, whereas anti-alpha(v)beta5 did not. The stimulatory effects were most pronounced by employing microparticles with covalently linked antibodies, but were also observed to a minor extent when the antibodies were physically adsorbed to polystyrene and biodegradable poly(lactide-co-glycolide) microparticles. Engineering of microparticles by surface conjugation of specific ligands to stimulate DC may increase the effectiveness of microparticulate vaccine delivery systems.