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We studied the degradation of vanillic acid in soil was studied by high performance liquid chromatography and gas chromatography. Illumina MISEQ high-throughput sequencing was used to identify the abundance of bacteria and fungi in soil. The results showed that when the concentration of vanillic acid decreased to a certain level in the soil, its degradation rate became slow. The application of vanillic acid gradually decreased the total number of bacterial OTUs in soil, while the total number of fungal OTUs increased. At the same time, the richness and diversity of bacteria were always higher than fungi. Key microbial taxa at different degradation stages of vanillic acid were identified at the phylum and genus level, including bacteria (Proteobacteria, Firmicutes, Actinobacteria, Chloroflexi, Methylibium, Methylobacillus, Aeromicrobium, Pseudonocardia) and Fungi (Ascomycota, Zygomycota, Basidiomycota, Rhodotorula, Mortierella

Objective This pilot clinical study was undertaken to investigate the role of T cell vaccination in the induction of regulatory immune responses in patients with rheumatoid arthritis (RA). Methods Autologous synovial T cells were selected for pathologic relevance, rendered inactive by irradiation, and used for vaccination. Fifteen patients received T cell vaccination via 6 subcutaneous inoculations over a period of 12 months. Results T cell vaccination led to induction of CD4+ Tregs and CD8+ cytotoxic T cells specific for T cell vaccine. There was selective expansion of CD4+,Vβ2+ Tregs that produced interleukin-10 (IL-10) and expressed a high level of transcription factor Foxp3, which coincided with depletion of overexpressed BV14+ T cells in treated patients. CD4+ IL-10–secreting Tregs induced by T cell vaccination were found to react specifically with peptides derived from IL-2 receptor α-chain. The expression level of Foxp3 in CD4+ T cells and increased inhibitory activity of CD4+,CD25+ Tregs were significantly elevated following T cell vaccination. The observed regulatory immune responses collectively correlated with clinical improvement in treated patients. In an intent-to-treat analysis, a substantial response, defined as meeting the American College of Rheumatology 50% improvement criteria, was shown in 10 of the 15 patients (66.7%) and was accompanied by a marked improvement in RA-related laboratory parameters. Conclusion These findings suggest that T cell vaccination induces regulatory immune responses that are associated with improved clinical and laboratory variables in RA patients.

Interferon beta (IFN beta) has complex immune regulatory properties that contribute to its treatment effect on multiple sclerosis (MS). In this study, we investigated the role of IFN beta in differentiation and functional properties of monocytes and monocyte-derived dendritic cells that are critical to the inflammatory process in MS. The results revealed that IFN beta inhibited intracellular production of interleukin (IL)-1b (PB/0.01) in both monocytes exposed toin vitro treatment of IFN beta and monocytes analysedex vivo from MS patients treated with IFN beta. IFN beta was shown to modulate differentiation of monocytes into dendritic cells in the presence of IL-4 and GM-CSF, which resulted in a delayed differentiation process. Furthermore, it characteristically altered the phenotypic features of differentiated dendritic cells by inhibiting the expression of CD1a, CD11b, CD11c, CD123 and CD209 while upregulating costimulatory molecules, such as CD86. The selective regulatory properties of IFN beta appeared to render the function of differentiated dendritic cells to produce an increased amount (PB/0.01) while their ability to secrete proinflammatory IL-12 and TGF beta was significantly reduced. The observed collective effects of IFN beta seemed to correlate with Th2 immune deviation. The study has provided new insights into the regulatory mechanisms of IFN beta in the treatment of MS.

Multiple sclerosis (MS) is thought to correlate with an array of clinically relevant biomarkers produced during inflammatory process. In this study, a novel gene expression profiling technology was developed and characterized to quantitatively measure the expression profiles of 34 genes selected based on their role in inflammation and their susceptibility to regulation by current MS treatment agents, beta-interferon (IFN) and glatiramer acetate (GA). Potential clinical applications of the technology were evaluated by in vitro and ex vivo analyses in peripheral blood mononuclear cells (PBMC) obtained from MS patients and controls. Interferon-inducible genes were universally up-regulated after in vitro treatment with beta-IFN while the expression of other selected genes encoding cytokines and molecules related to T cell trafficking, activation and apoptosis was variably affected. Beta-IFN and GA exhibited distinctive and characteristic regulatory effects on the expression of the selected genes. Similar regulatory properties of beta-IFN and GA were seen by ex vivo analysis of PBMC specimens in a self-paired study by comparing specific changes induced by beta-IFN or GA treatment in the same patients as well as in a group study by measuring specific profiles in treatment groups compared with an untreated group. Furthermore, the technology served as a simple and sensitive assay for detection of beta-IFN neutralizing antibody based on the blocking effect of serum antibodies on the known regulatory properties of beta-IFN on PBMC. The findings provide important information on the immunoregulatory properties of beta-IFN and GA and support potential clinical applications of this technology in detection of neutralizing antibody (NAB) and evaluation of treatment responses in MS patients.

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8+ T cell responses and their functional properties in patients with MS. There were significantly increased CD8+ T cell responses to 9-mer MBP peptides, in particular MBP111–119 and MBP87–95 peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8+ T cell lines were of the Th1 phenotype, producing TNF-α and IFN-γ and belonged to a CD45RA−/CD45RO+ memory T cell subset. Further characterization indicated that the CD8+ T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP111–119) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8+ T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8+ CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.

T cell reactivity to candidate myelin autoantigens, such as myelin basic protein (MBP), may play an important role in the pathogenesis of multiple sclerosis (MS). Although MBP-reactive T cells have been found to undergo in vivo activation in patients with MS, their true precursor frequency in MS is unknown as current frequency analysis is commonly based on the T cell functional responses to MBP. In this study, we developed a TCR sequence-based ex vivo detection system using colony hybridization with oligonucleotide probes specific for CDR3 of selected T cell clones for the analysis of true T cell precursor frequency in PBMC. The results revealed that the precursor frequency of five independent T cell clones recognizing the immunodominant MBP(83-99) region was found to be in the range of 1.6 x 10(-4) in total T cells in three HLA-DR2 patients with MS compared to that of 0.25 x 10(-4) in HLA-DR2 healthy individuals. The observed frequency of MBP(83-99)-reactive T cells in MS patients was considerably higher than those measured in parallel by cell culture-based analysis (2.3 x 10(-6)) or by enzyme-linked immunospot assay (3.9 x 10(-5)) in the same peripheral blood mononuclear cell specimens. Furthermore, the study showed that MBP(83-99)-reactive T cells detected ex vivo belonged to CD45RA+, CD25+ and CD95- T cell subsets as evidenced by preferential expression of specific TCR transcripts in these cell fractions.

Glatiramer acetate (GA) is a treatment option for multiple sclerosis. Although its mechanism of action remains unclear, evidence has emerged supporting the role of GA as an immunomodulatory drug that regulates T‐cell function. It has been demonstrated that long‐term GA treatment induces a serum antibody response; however, the functional properties of these ‘reactive antibodies’ are unknown. It has been speculated that GA‐induced antibodies may have a blocking effect that can inhibit the immunologic activity of GA. This study was conducted to determine whether serum antibodies induced by GA treatment can block the in vitro immunoregulatory effects of GA on T‐cell proliferation and cytokine production. Forty‐two patients with relapsing‐remitting multiple sclerosis who were treated with GA for 1–5 years were examined for GA antibody titres using enzyme‐linked immunoabsorbent assay (ELISA). Thirty‐three percent of patients developed high antibody titres [antibody binding index (ABI) = 16–64] and 14% had low antibody titres (ABI = 4) after 1 year on treatment. Results showed that purified GA antibodies blocked the stimulatory effects of GA on GA‐specific T‐cell lines of Th0 cytokine profile. The increase in interleukin‐10 (IL‐10) and IL‐4 levels and the decrease in IL‐12 and tumour necrosis factor‐α levels, normally seen with GA stimulation, were reversed in the presence of GA antibodies. The study has important implications in our understanding of the potential role of high‐titre GA antibodies in the treatment of multiple sclerosis. Received December 2, 2002. Revised May 28, 2003. Second revision June 20, 2003. Accepted June 23, 2003

T cells recognizing myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In vivo clonal expansion of MBP-reactive T cells in MS may relate in part to dysfunction of peripheral regulatory mechanisms, including the anti-idiotypic immune network. In this study, we examined anti-idiotypic immune responses and the functional properties of anti-idiotypic T cells in patients with MS and healthy controls using TCR peptides corresponding to a CDR3 sequence motif preferentially expressed among T cells recognizing the 83-99 immunodominant peptide of MBP in some patients with MS. The study demonstrated that anti-idiotypic T cells could be induced in vitro by 8mer and 15mer peptides containing the CDR3 motif in MS patients and healthy controls respectively. The estimated precursor frequency of the anti-idiotypic T cells was slightly reduced in MS patients compared to control subjects. The obtained anti-idiotypic T cells recognizing the 15mer TCR peptide were found to express the CD4 phenotype, produce predominantly IL-10 and inhibit the proliferation of autologous T cells recognizing the immunodominant peptide of MBP. Anti-idiotypic T cells induced by the 8mer TCR peptide were predominantly CD8+ cytotoxic T cells and exhibited cytotoxic activity against autologous MBP-specific T cells expressing the CDR3 sequence. When added in primary culture, both TCR peptides had a significant inhibitory effect on the T cell responses to the immunodominant peptide of MBP. The findings suggest that anti-idiotypic immune responses can be activated by selected TCR peptides and may play an important role in the in vivo regulation of MBP-reactive T cells.

Beta-interferon (beta-IFN) has a proven treatment effect on relapsing-remitting multiple sclerosis (MS), presumably through its regulatory properties on T-cell activation and cytokine production. This paper examines whether combination therapy of beta-IFN with prednisone would enhance immunoregulatory effects of beta-IFN by measuring serum levels of selected proinflammatory cytokines and soluble T-cell activation markers associated with MS. The selected markers were analyzed in MS patients treated with beta-IFN alone (n-22) and beta-IFN combined with a low daily dose of prednisone (n-33), as compared with those in 27 healthy controls at baseline and at a three-month interval for one year. The study confirmed that beta-IFN treatment inhibited serum levels of tumor necro sis factor-alpha (TNFa) and intracellular adhesion molecule-1 (IC A M-1) in patients with MS. However, combination therapy did not significantly enhance the inhibitory effect of beta-IFN treatment on the production of TNFa, interleukin (IL)-12, IL-2R, and IC A M-1, while the addition of prednisone antagonized the effect of beta-IFN on up-regulation of IL-10 and soluble C D95. No difference in the occurrence of binding antibodies to beta-IFN was found between the two treatment groups. The findings are important for the understanding of the role of combination therapy in the treatment of MS.

Viral infections are though to play an important role in the pathogenesis of multiple sclerosis (MS) potentially through molecular mimicry. An identical sequence was found in both myelin basic protein (MBP, residues 96–102), a candidate autoantigen for MS, and human herpesvirus-6 (HHV-6 U24, residues 4–10) that is a suspected viral agent associated with MS. In this study, we showed that greater than 50% of T cells recognizing MBP93-105 cross-reacted with and could be activated by a synthetic peptide corresponding to residues 1 to 13 of HHV-6 U24 in MS patients. The estimated precursor frequency of these cross-reactive T cells recognizing both peptides, MBP93-105 and HHV-6 (U24)1-13, was significantly elevated in MS patients compared with that in healthy controls. These cross-reactive CD4+ T cells represented the same Th1 phenotype as that of monospecific T cells recognizing MBP93-105. There were increased antibody titers for both peptide HHV-6 (U24)1-13 and peptide MBP93-105 in the same patients with MS compared with those in healthy controls, suggesting B-cell sensitization to the antigens in MS patients. The study provides important evidence in the understanding of the potential role of HHV-6 infection/reactivation in the activation of autoimmune reactivity to MBP and its implication in the pathogenesis of MS. Ann Neurol 2003

Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.

Trafficking of inflammatory T cells into the central nervous system (CNS) plays an important role in the pathogenesis of multiple sclerosis. The directional migratory ability of peripheral T cells is associated with interactions of chemokines with their receptors expressed on T cells. In this study, transmigration of peripheral T cells toward a panel of chemokines was examined in patients with multiple sclerosis and healthy individuals using Boyden chemotactic transwells. A significantly increased migratory rate preferentially toward RANTES and MIP-1alpha, but not other chemokines, was found in T cells obtained from multiple sclerosis patients as opposed to healthy individuals (P: < 0.001). The migratory T-cell populations represented predominantly Th1/Th0 cells while non-migratory T cells were enriched for Th2-like cells. The study demonstrated further that aberrant migration of multiple sclerosis-derived T cells toward RANTES and MIP-1 alpha resulted from overexpression of their receptors (CCR5) and could be blocked by anti-CCR5 antibodies. These findings have important implications for our understanding of the mechanism underlying aberrant T cell trafficking in multiple sclerosis.

Objective: To examine the in vivo immunoregulatory properties of interferon beta-1a (IFN beta-1a) on the T cell responses to myelin basic protein (MBP) and to evaluate the occurrence of the blocking antibodies characterized by the ability to reverse the effects of IFN beta on T cells in MS patients treated with IFN beta. Methods: The precursor frequency of T cells recognizing MBP and control antigens was estimated in a microwell culture system. The cytokine profile of T cell lines was measured in ELISA. The binding antibodies were determined in ELISA and Western blot. Cytopathic test and the T cell functional assays were used to determine the blocking effects of the binding antibodies. Results: Treatment with IFN beta resulted in a substantial reduction in the precursor frequency of MBP-reactive T cells in MS patients. The cytokine profile of MBP-reactive T cells that sustained the treatment was altered toward an increased production of interleukin (IL)-10 and decreased production of tumor necrosis factor (TNF)α and IFN-γ. The immunoregulatory properties of IFN beta on T cells could be blocked by the binding antibodies derived from a proportion of patients treated with IFN beta (4 of 64, 6.25%). The blocking antibodies also neutralized anti-viral activity of IFN beta in cytopathic assays, corresponding to previously described neutralizing antibodies. Conclusions: Treatment with IFN beta alters the cytokine profile by enhancing the production of IL-10 and downregulating Th1 cytokines, which may contribute to clinical benefit in MS. The treatment also induces blocking antibodies that impair the immunoregulatory properties of IFN beta in some individuals.

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In this study, we demonstrated that subcutaneous inoculations with irradiated autologous MBP-reactive T cell clones (T cell vaccination) elicited CD8 + antiidiotypic T cell responses and CD4 + Th2 cell responses in patients with MS. Both regulatory cell types induced by T cell vaccination contributed to the inhibition of MBP-reactive T cells while they differed in the recognition pattern and functional properties. We describe for the first time that the Th2 regulatory cells reacted with activated but not resting T cells in the context of MHC class II molecules and inhibited the proliferation of MBP-reactive T cells through the secretion of IL-4 and IL-10. The T-T cell interaction mediated by Th2 regulatory cells was independent of the antigen specificity of activated T cells. The findings have important implications for our understanding of the regulatory mechanism induced by T cell vaccination.

Th0 clones recognizing an immunodominant peptide of myelin basic protein (residues 83–99) were derived from patients with multiple sclerosis. We demonstrate that analogue peptides with alanine substitution at Val86 and His88 had a unique partial agonistic property in inducing Th0 →Th1 and Th0 →Th2 deviation of the myelin basic protein-reactive T cell clones, respectively. Th0 to Th1 deviation induced by peptide 86V→A correlated with up-regulation of Fyn and ZAP-70 kinase activities. Conversely, Th0 to Th2 deviation induced by peptide 88H→A was associated with complete failure to activate Fyn and ZAP-70 kinases. The observed Th1 and Th2 shift also correlated, to a lesser extent, with Lck kinase activity that was down-regulated with Th1 deviation and increased with Th2 deviation in some T cell clones. We demonstrated that the Th1 and Th2 shift induced by the analogue peptides was a reversible process, as the T cell clones previously exposed to either 86V→A or 88H→A peptide could revert to an opposite phenotype when rechallenged reciprocally with a different analogue peptide. The study has important implications in our understanding of regulation of TCR-associated tyrosine kinases by altered peptide ligands and its role in cytokine regulation of autoreactive T cells.

T cell responses to the immunodominant peptide (residues 83–99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83–99 peptide. Twenty MBP83–99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some Vβ13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, Vβ13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83–99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83–99 peptide, as evident by its expression in the majority of MBP83–99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83–99-reactive T cells. The findings have important implications in monitoring and targeting MBP83–99-reactive T cells in MS.

T cell responses to myelin basic protein (MBP) may play an important role in the pathogenesis of multiple sclerosis (MS). If MBP-reactive T cells are involved in the disease processes and undergo clonal activation and expansion, their precursor frequency would be increased in patients with MS. The frequency of MBP-reactive T cells is also influenced by regulatory mechanisms in vivo, including apoptotic deletion. In this study, we examined changes in the frequency of MBP-reactive T cells in patients with MS as a function of the apoptotic deletional mechanism in vivo, using a cell culture-based assay. A significantly increased frequency of MBP-reactive T cells was found in patients with MS relative to healthy individuals only when Fas-ligand antibody was used to block apoptosis. This result indicates that a significant proportion of MBP-reactive T cells are sensitive to apoptosis and are not deleted in vivo in patients with MS, as opposed to healthy individuals, thus suggesting a functional deficit in apoptotic deletional mechanism. Surviving Fas-sensitive MBP-reactive T cell lines represent distinct subpopulations preferentially recognizing the 111-139 region of MBP and exhibiting a Th2 cytokine profile. The findings are relevant to our understanding of regulation of MBP-reactive T cells in vivo in MS.

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83-99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83-99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83-99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81-95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83-99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.

MBP-specific autoreactive T cells are considered pro-inflammatory T cells and thought to play an important role in the pathogenesis of multiple sclerosis (MS). Here, we report that MBP(83-99)-specific T cells generated from MS patients (n = 7) were comprised of pro-inflammatory and regulatory subsets of distinct phenotypes. The pro-inflammatory phenotype was characterized by high production of IFN-gamma, IL-6, IL-21 and IL-17 and low expression of FOXP3, whereas the regulatory subset expressed high levels of FOXP3 and exhibited potent regulatory functions. The regulatory subset of MBP-specific T cells appeared to expand from the CD4(+)CD25(-) T-cell pool. Their FOXP3 expression was stable, independent of the activation state and it correlated with suppressive function and inversely with the production of IFN-gamma, IL-6, IL-21 and IL-17. In contrast, the phenotype and function of FOXP3(low) MBP-specific T cells were adaptive and dependent on IL-6. The higher frequency of FOXP3(high) MBP-specific T cells was observed when IL-6 was neutralized in the culture of PBMC with MBP. The study provides new evidence that MBP-specific T cells are susceptible to pro-inflammatory cytokine milieu and act as either pro-inflammatory or regulatory T cells.

IFN-beta currently serves as one of the major treatments for MS. Its anti-inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T-cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro-inflammatory cytokines, IL-17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN-beta on the regulation of OPN and IL-17 in MS patients. We found that IFN-beta used in vitro at 0.5-3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4(+) T-cell level. In addition, IFN-beta inhibited the production of IL-17 and IL-21 in CD4(+) T cells. It has been described that IFN-beta suppresses IL-17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN-beta also acted directly on the CD4(+) T cells to regulate OPN and IL-17 expression through the type I IFN receptor-mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN-beta to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN(+) and IL-17(+) cells decreased in IFN-beta-treated EAE mice along with decreases in serum levels of OPN and IL-21. Importantly, decreased OPN production by IFN-beta treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN-beta treatment can down-regulate the OPN and IL-17 production in MS. This study provides new insights into the mechanism of action of IFN-beta in the treatment of MS.

Vasoactive intestinal peptide (VIP) has a unique property of regulating T(h)1 and T(h)2 immunity of CD4+ T cells. In this study, we demonstrated, for the first time, that differential expression of VIP receptors and a compensatory mechanism directly affect the responsiveness of CD4+ T cells and their T(h)1 and T(h)2 properties to VIP. The expression of VIP receptor-1 (VPAC1) and VPAC2 in CD4+ T cells changed reciprocally in the context of the activation state. In activated CD4+ T cells of healthy individuals, markedly decreased VPAC1 expression was compensated for by increased expression of VPAC2 induced by T cell activation. In contrast, there was altered expression of VPAC2 in activated CD4+ T cells derived from multiple sclerosis (MS) patients, which rendered CD4+ T cells less responsive to VIP and skewed the system to a predominantly in a T(h)1 direction. Detailed characterization with agonist peptides of VIP showed that residues Met and Ser at positions 17 and 25 of VIP were critical to its regulatory properties through interaction with VAPC2. Furthermore, altered levels of VPAC2 expression in T cells of MS patients were not associated with single-nucleotide polymorphism in the encoding region of the VPAC2 gene but with gene regulation as characterized by a distinct DNA footprinting pattern in the promoter region of the VPAC2 gene in MS as compared with controls. This study has provided new evidence for an intrinsic mechanism associated with an aberrant, pro-inflammatory state of CD4+ T cells in MS.

Immunization with irradiated autologous T cells (T cell vaccination) is shown to induce regulatory T cell responses that are poorly understood. In this study, CD4+regulatory T cell lines were generated from patients with multiple sclerosis that received immunization with irradiated autologous myelin basic protein-reactive T cells. The resulting CD4+regulatory T cell lines had marked inhibition on autologous myelin basic protein-reactive T cells and displayed two distinctive patterns distinguishable by the expression of transcription factor Foxp3 and cytokine profile. The majority of the T cell lines had high Foxp3 expression and secreted both IFN-γ and IL-10 as compared with the other pattern characteristic of low Foxp3 expression and predominant production of IL-10 but not IFN-γ. CD4+regulatory T cell lines of both patterns expressed CD25 and reacted with activated autologous T cells but not resting T cells, irrespective of antigen specificity of the target T cells. It was evident that they recognized preferentially a synthetic peptide corresponding to residues 61–73 of the IL-2 receptor α chain. T cell vaccination correlated with increased Foxp3 expression and T cell reactivity to peptide 61–73. The findings have important implications in the understanding of the role of CD4+regulatory T cell response induced by T cell vaccination.

T-lymphocytes play an important role in rheumatoid arthritis (RA). In this study, we evaluated the hypothesis that common T-cell receptor (TCR) structural features may exist among infiltrating T cells of different RA patients, if the TCR repertoire is shaped by interaction with common self or microbial antigens in the context of susceptible HLA genes in RA. Synovial lesion tissue (ST), synovial fluid (SF) and blood specimens from RA patients and controls were analyzed for TCR V gene repertoire by real-time PCR. There was highly skewed BV14 and BV16 usage in synovial T cells of RA as opposed to those of controls, which was accompanied with a trend for correlation between skewed BV16 and DRB1(*)0405. Immunoscope analysis of the V-D-J region of ST-derived T cells demonstrated oligoclonal and polyclonal expansion of BV14(+) and BV16(+) T cells. Detailed characterization using specific BV and BJ primers further revealed common clonotypes combining the same BV14/BV16, BJ and CDR3 length. DNA cloning and sequence analysis of the clonotypes confirmed identical CDR3 sequences and common CDR3 sequence motifs among different RA patients. The findings are important in the understanding of BV gene skewing and CDR3 structural characteristics among synovial infiltrating T cells of RA.

Multiple sclerosis is thought to involve aberrant immune responses to myelin autoantigens. Haematopoietic stem-cell transplantation (HSCT) is in clinical trials for progressive multiple sclerosis based on the rationale that it destroys aberrant immune system, while recapitulation of lymphocyte ontogeny might alter the immune system and slow down disease progression. This study was undertaken to analyse characteristics of the T-cell receptor (TCR) repertoire, serum cytokine profile and the T-cell responses to myelin basic protein (MBP) in the reconstituted immune system in progressive multiple sclerosis. The study revealed that, following autologous HSCT, the T-cell immunity recovered in two distinctive phases. The first phase was characterized by limited T-cell immunity as a result of selective expansion of pre-existing T cells commonly expressing the TCR beta chain variable region (TCR BV) 20 and increased serum cytokine production during the first several months. The second phase of T-cell reconstitution coincided with increased thymic T-cell output 9-12 months after HSCT. T cells reconstituted from stem-cell grafts had the distinctive properties of comprehensive T-cell immunity and a broad TCR repertoire. T cells recognizing MBP were initially depleted by immunoablation and rapidly expanded from the reconstituted T-cell repertoire in 12 months. The reconstituted MBP-reactive T cells exhibited a broader epitope recognition repertoire while maintaining the same skewed reactivity pattern compared with that seen at baseline. The findings have important implications in the understanding of the role of HSCT as a potential treatment for multiple sclerosis.

Myelin basic protein (MBP)-reactive T cells are potentially involved in the pathogenesis of multiple sclerosis (MS), and can be depleted by subcutaneous inoculations with irradiated autologous MBP-reactive T cells (T cell vaccination). This preliminary open label study was undertaken to evaluate whether depletion of MBP-reactive T cells would be clinically beneficial to patients with MS. Fifty-four patients with relapsing-remitting (RR) MS (n=28) or secondary progressive (SP) MS (n=26) were immunized with irradiated autologous MBP-reactive T cells and monitored for changes in rate of relapse, expanded disability scale score (EDSS) and MRI lesion activity over a period of 24 months. Depletion of MBP-reactive T cells correlated with a reduction (40%) in rate of relapse in RR-MS patients as compared with the pre-treatment rate in the same cohort. However, the reduction in EDSS was minimal in RR-MS patients while the EDSS was slightly increased in SP-MS patients over a period of 24 months. Serial semi-quantitative MRI examinations suggest stabilization in lesion activity as compared with baseline MRI. The findings suggest some potential clinical benefit of T cell vaccination in MS and encourage further investigations to evaluate the treatment efficacy of T cell vaccination in controlled trials.

The protective role of pregnancy in autoimmune conditions, such as multiple sclerosis (MS), is potentially associated with immune regulation by sex hormones produced during pregnancy. This study was undertaken to examine the regulatory effects of estriol on the T cell functions, including transmigration and the cytokine production. The results revealed for the first time that estriol significantly inhibited T cell transmigration at a concentration range typical of pregnancy, which correlated with decreased T cell expression of matrix metalloproteinase-9. Estriol was also found to alter the cytokine profile of T cells toward Th2 phenotype by up-regulating the production of IL-10 and inhibiting TNFalpha secretion of T cells. However, the inhibitory effects of estriol on T cells were not antigen-dependent. Further characterization indicated that estriol inhibited nuclear transcription factor kappa B (NF-kappa B), which controls a variety of immune-related genes. This study provides new evidence that estriol is a potent regulator for the T cell functions potentially through its interaction with the NF-kappa B signaling pathway.

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). Immunization with irradiated MBP-reactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that suppress circulating MBP-reactive T cells. This T cell-T cell interaction is thought to involve the recognition of TCR expressed on target T cells. The study was undertaken to define the idiotypic determinants responsible for triggering CD8+ cytotoxic anti-idiotypic T cell responses by T cell vaccination in patients with MS. A panel of 9-mer synthetic TCR peptides corresponding to complementarity-determining region 2 (CDR2) and CDR3 of the immunizing MBP-reactive T cell clones were used to isolate anti-idiotypic T cell lines from immunized MS patients. The resulting TCR-specific T cell lines expressed exclusively the CD8 phenotype and recognized preferentially the CDR3 peptides. CDR3-specific T cell lines were found to lyze specifically autologous immunizing MBP-reactive T cell clones. The findings suggest that CDR3-specific T cells represented anti-idiotypic T cell population induced by T cell vaccination. In contrast, the CDR2 peptides were less immunogenic and contained cryptic determinants as the CDR2-specific T cell lines did not recognize autologous immunizing T cell clones from which the peptide sequence was derived. The study has important implications in our understanding of in vivo idiotypic regulation of autoimmune T cells and the regulatory mechanism underlying T cell vaccination.

Immunization with irradiated autoreactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that preferentially recognize complementarity-determining region 3 sequences, contributing to clonal depletion of autoreactive T cells. However, it remains unknown whether T cell vaccination elicits anti-idiotypic humoral responses and whether the anti-idiotypic Abs play a similar role in the regulatory mechanism induced by T cell vaccination. In this study we examined the occurrence, the reactivity pattern, and the regulatory role of anti-idiotypic Abs elicited by T cell vaccination in patients with multiple sclerosis. We demonstrated for the first time that B cells producing anti-idiotypic Abs could be isolated from vaccinated patients. These EBV-transformed B cell lines were selected for specific reactivity to a 20-mer TCR peptide incorporating a common complementarity-determining region 3 sequence of the immunizing T cell clones. The resulting anti-idiotypic Abs were found to react with the original immunizing T cell clones and exhibit an inhibitory effect on their proliferation. The findings suggest that anti-idiotypic Ab responses can be induced by T cell vaccination in humans and that their regulatory properties are likely to contribute to the suppression of myelin basic protein-reactive T cells in vaccinated patients. The study has important implications in our understanding of the regulatory role of the anti-idiotypic humoral responses induced by T cell vaccination.

Trafficking of inflammatory T cells into the brain is associated with interactions of certain chemokines with their receptors, which plays an important role in the pathogenesis of multiple sclerosis (MS). We examined whether interferon-beta (IFN-beta) had the ability to regulate the production of chemokines and the expression of their receptors in T cells derived from patients with MS. It was demonstrated for the first time that in vitro exposure of T cells to IFN-beta-1a selectively inhibited mRNA expression for RANTES and MIP-1alpha and their receptor CCR5. T cell surface expression of CCR5 was significantly reduced in MS patients treated with IFN-beta, correlating with decreased T cell transmigration toward RANTES and MIP-1alpha. The study provides new evidence suggesting that IFN-beta treatment impairs chemokine-induced T cell trafficking by reducing the production of RANTES and MIP-1alpha and the expression of their receptors CCR5.

Multiple sclerosis (MS) is a demyelinating disease of presumed T cell autoimmunity against self myelin. We hypothesized that if myelin-reactive T cells are associated with the disease processes, they may undergo activation and expansion during acute exacerbation. In this study, we examined the precursor frequency, epitope recognition and cytokine profile of myelin-reactive T cells in 14 relapsing/remitting MS patients during exacerbation and remission. The study revealed that T cells recognizing the immunodominant peptides of candidate myelin antigens, including myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein, occurred at increased precursor frequency during acute exacerbation. The T cell responses to MBP focused on the immunodominant regions (residues 83-99 and 151-170) during exacerbation and shifted toward other epitopes of MBP at the time of remission. Furthermore, there was a marked increase in the production of T(h)1 cytokines among T cell lines obtained during exacerbation compared to those obtained during remission. The study demonstrated that myelin-reactive T cells underwent selective activation and expansion during acute MS exacerbation. In contrast, myelin-reactive T cells found during remission in the same patients generally resembled those identified in healthy controls with some discrepancies. The findings suggest potential association of aberrant myelin-reactive T cell responses with acute exacerbation in MS, which may reflect transient activation of myelin-reactive T cell populations of pathogenic potential.

Objective: To define the in vitro effects of interferon beta 1a (IFN-β1a) on myelin basic protein (MBP)-reactive T cells and to determine its regulatory mechanism on cytokine networks in patients with MS. Methods: The proliferation and cytokine production of MBP-reactive T-cell clones were measured in thymidine uptake assays and ELISA respectively. The precursor frequency of MBP-reactive T cells was estimated in a microwell culture system. Results: IFN-β inhibited the proliferation of established MBP-reactive T-cell clones, which correlated with enhanced production of anti-inflammatory interleukin (IL)-4 and IL-10, and a decrease in tumor necrosis factor alpha (TNF-α) and IFN-γ. When examined with peripheral blood mononuclear cells (PBMCs), IFN-β was found to reduce the in vitro T-cell responses to MBP, as indicated by the significantly decreased frequency of MBP-reactive T cells. The decreased frequency of MBP-reactive T cells corresponded to an augmented production of IL-4 and IL-10. Although the level of TNF-α and IFN-γ was generally unaltered or decreased, IFN-β appeared to enhance the production of IFN-γ in PBMCs derived from some individuals with MS. Conclusion: Interferon beta 1a (IFN-β) suppresses myelin basic protein (MBP)-reactive T cells and induces immune deviation toward the production of T-helper 2 cytokines, which may contribute to its therapeutic benefit in MS. The study also suggests some heterogeneity in MBP-reactive T-cell responses to IFN-β in different individuals with MS.