Jingwu Z. Zhang

Role of IFN-γ in induction of Foxp3 and conversion of CD4+ CD25– T cells to CD4+ Tregs

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4(+)CD25(-) T cells to CD4(+) Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4(+)CD25(+)Foxp3(+) Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4(+)CD25(-) T cells led to conversion of CD4(+) Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4(+)CD25(-) T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.

Cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis.

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

Liver X receptor (LXR) mediates negative regulation of mouse and human Th17 differentiation

Th17 cells are a subset of CD4+ T cells with an important role in clearing certain bacterial and fungal pathogens. However, they have also been implicated in autoimmune diseases such as multiple sclerosis. Exposure of naive CD4+ T cells to IL-6 and TGF-β leads to Th17 cell differentiation through a process in which many proteins have been implicated. We report here that ectopic expression of liver X receptor (LXR) inhibits Th17 polarization of mouse CD4+ T cells, while LXR deficiency promotes Th17 differentiation in vitro. LXR activation in mice ameliorated disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, whereas LXR deficiency exacerbated disease. Further analysis revealed that Srebp-1, which is encoded by an LXR target gene, mediated the suppression of Th17 differentiation by binding to the E-box element on the Il17 promoter, physically interacting with aryl hydrocarbon receptor (Ahr) and inhibiting Ahr-controlled Il17 transcription. The putative active site (PAS) domain of Ahr and the N-terminal acidic region of Srebp-1 were essential for this interaction. Additional analyses suggested that similar LXR-dependent mechanisms were operational during human Th17 differentiation in vitro. This study reports what we believe to be a novel signaling pathway underlying LXR-mediated regulation of Th17 cell differentiation and autoimmunity.

Role of osteopontin in amplification and perpetuation of rheumatoid synovitis

Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. In this study, we demonstrate, for the first time to our knowledge, that overexpression of OPN in synovial T cells is associated with local inflammatory milieu and that OPN acts as an important mediator in amplification and perpetuation of rheumatoid synovitis. The study revealed that mRNA expression of OPN was highly elevated in CD4(+) synovial T cells derived from patients with RA, which correlated with increased OPN concentrations in synovial fluid (SF). The pattern of OPN overexpression was confined to rheumatoid synovium and correlated with coexpression of selected OPN receptors in synovial T cells, including integrins alphav and beta1 and CD44. RA-derived SF stimulated the expression of OPN in T cells, which was attributable to IL-10 present in SF and abrogated by anti-IL-10 antibody. Among the more than 300 autoimmune and inflammatory response genes examined, OPN selectively induced the expression of proinflammatory cytokines and chemokines known to promote migration and recruitment of inflammatory cells. Furthermore, it was evident that OPN activated transcription factor NF-kappaB in mononuclear cells. The study has important implications for understanding the role of OPN in rheumatoid synovitis and other inflammatory conditions.

Aberrant Regulation of Synovial T Cell Activation by Soluble Costimulatory Molecules in Rheumatoid Arthritis

T cell activation and function are critically regulated by positive and negative costimulatory molecules. Aberrant expression and function of costimulatory molecules have been associated with persistent activation of self-reactive T cells in autoimmune diseases such as rheumatoid arthritis (RA). In this study, initial analysis of costimulatory molecules led to the unexpected observation that, in addition to CD80, several negative regulators (e.g., CTLA-4, programmed death-1 (PD-1), and PD ligand-1) were overexpressed in synovial T cells and macrophages derived from RA patients as opposed to controls. The expression of CD80 and PD ligand-1 on monocytes could be induced in vitro by IFN-γ and TNF-α that were produced abundantly in RA-derived synovial fluid (SF). Furthermore, the soluble form of negative costimulatory molecules occurred at high concentrations in sera and SF of RA patients and correlated with titers of rheumatoid factor in RA patients. In particular, the levels of soluble PD-1 were found to correlate significantly with those of TNF-α in SF derived from RA patients. Detailed characterization of soluble PD-1 revealed that it corresponded to an alternative splice variant (PD-1Δex3) and could functionally block the regulatory effect of membrane-bound PD-1 on T cell activation. Our data indicate a novel pathogenic pathway in which overexpression of negative costimulatory molecules to restrict synovial inflammation in RA is overruled by the excessive production of soluble costimulatory molecules.

Increased CD8+ Cytotoxic T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

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.

Interferon beta induces T-helper 2 immune deviation in MS

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.

Naturally Processed HLA Class II Peptides Reveal Highly Conserved Immunogenic Flanking Region Sequence Preferences That Reflect Antigen Processing Rather Than Peptide-MHC Interactions

MHC class II heterodimers bind peptides 12–20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRs. We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

IMPAIRED APOPTOTIC DELETION OF MYELIN BASIC PROTEIN-REACTIVE T CELLS IN PATIENTS WITH MULTIPLE SCLEROSIS

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.

Regulation of chemokine receptor CCR5 and production of RANTES and MIP-1α by interferon-β

Abstract 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-β (IFN-β) 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-β-1a selectively inhibited mRNA expression for RANTES and MIP-1α and their receptor CCR5. T cell surface expression of CCR5 was significantly reduced in MS patients treated with IFN-β, correlating with decreased T cell transmigration toward RANTES and MIP-1α. The study provides new evidence suggesting that IFN-β treatment impairs chemokine-induced T cell trafficking by reducing the production of RANTES and MIP-1α and the expression of their receptors CCR5.