Sho Matsushita

Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis.

Five types of dopamine receptors, termed D1 to D5, have been identified to date. The D1 and D5 receptors form the D1-like group that couples with the Galphas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Galphai class of G proteins. A D2-like-receptor (D2-like-R) antagonist L750667 induced dendritic cell (DC)-mediated Th17 differentiation. In contrast, a D1-like-R antagonist SCH23390 inhibited DC-mediated Th17 differentiation. The D1-like-Rs were expressed on both DCs and T cells, whereas D2-like-Rs were marginally expressed on CD4+CD45RA+ naïve T cells. In addition, SCH23390 had the ability to prevent experimental autoimmune encephalomyelitis (EAE) in mice. Spleen cells from EAE mice showed decreased IL-17 production, when SCH23390 was administered. Adoptive transfer of DCs treated with SCH23390 successfully prevented EAE. These findings indicate that antagonizing D1-like-Rs on DCs inhibits Th17 differentiation, thereby leading to an amelioration of EAE.

Single amino acid substitutions on a Japanese cedar pollen allergen (Cry j 1)-derived peptide induced alterations in human T cell responses and T cell receptor antagonism

We generated T cell clones specific to a Japanese cedar pollen allergen (Cry j 1) and investigated effects of altered T cell receptor (TCR) ligand on changes of T cell responses. One of these Cry j 1-specific T cell clones established from patients with Japanese cedar pollinosis, ST1.9, recognized an antigenic peptide Cry j 1 p335-346 in the context of HLA-DRA+DRB3*0301 molecules and secreted interleukin-4 dominantly, with a smaller amount of interferon-gamma. ST1.9 represented one of the major T cell clones specific to Cry j 1 in the donor, because a short-term cultured polyclonal T cell line specific to Cry j 1 exhibited the same character as the ST1.9. We synthesized various analog peptides derived from Cry j 1 p335-346 with single amino acid substitutions and determined key residues for interactions between TCR of ST1.9 and HLA-DR molecules. We also analyzed changes in the responses of ST1.9 to Cry j 1 p335-346-derived analog peptides. Of interest was that the substitution of 339threonine to valine resulted in a significant increase in interferon-gamma production, with no remarkable changes either in proliferative response or interleukin-4 production. Analog peptides carrying the substitutions of 339threonine to glycine or glutamine revealed TCR antagonism, without changes in their binding affinities to the DR molecule. Therefore single amino acid substitutions on an allergen peptide carrying the T cell epitope may suppress helper-T-dependent class switch pressure to IgE in B cells either by inducing increased interferon-gamma production or by inhibiting proliferative responses in helper-T cells.

Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis

BACKGROUND & AIMS The mechanism for development of primary biliary cirrhosis (PBC) remains enigmatic, but molecular mimicry has been implicated because of well-known cross-reactivity of human mitochondrial autoantigens and equivalent bacterial antigens. Virtually all patients with PBC have antimitochondrial autoantibodies (AMA), but, interestingly, approximately 50% also manifest antinuclear antibodies (ANA). METHODS To determine whether generation of ANA are due to molecular mimicry of mitochondrial peptides, we established 6 T-cell clones selected by a peptide corresponding to the E2 subunit of mitochondrial pyruvate dehydrogenase complex and analyzed for reactivity to mimicry peptides derived from mitochondrial and nuclear autoantigens, including control sequences. RESULTS For mitochondrial autoantigens, 1 peptide from the E2 subunit of the pyruvate dehydrogenase complex, 1 peptide from the E2 subunit of the oxo-glutarate dehydrogenase complex, 1 peptide from the E2 subunit of the branched-chain 2-oxoacid dehydrogenase complex, and 1 peptide from the E3-binding protein cross-reacted with these T-cell clones. For the nuclear autoantigens, 5 peptides from gp210 and 1 from Sp100 cross-reacted with these clones. Furthermore, 1 of 3 T-cell clones selected by recombinant gp210 protein reacted with a mimicry peptide corresponding to amino acids 188-201 of gp210, indicating that this part of the protein is a naturally processed immunodominant T-cell epitope. CONCLUSIONS These results demonstrate molecular mimicry between mitochondrial and nuclear autoantigens in PBC and that a mimicry peptide may become an immunodominant T-cell epitope. These data have significance not only for PBC but also for the production of ANA in other disease processes.

Neutrophilic Inflammation in Severe Asthma

Neutrophils may play an important role in the pathogenesis of severe asthma. Their infiltration into the airway is increased. Interleukin (IL)-8 is involved in this process, and is actually upregulated in the airways of patients. We have observed that in the absence of eosinophil chemoattractants, neutrophils stimulated by IL-8 augment eosinophil trans-basement membrane migration by releasing superoxide anion, matrix metalloproteinase, leukotriene B4 and platelet-activating factor. These findings suggest that IL-8-stimulated neutrophils could lead eosinophils to accumulate in the airways of asthmatic patients, which might be a mechanism for corticosteroid resistance in severe asthma. However, the mechanisms of IL-8 upregulation in the airway are not completely understood. Several studies suggest that IL-17 (or T helper 17 cells; Th17) is involved in the IL-8 upregulation observed in severe asthma. We clarified that dopamine induces Th17 differentiation through dopamine D1-like receptor (D1-like-R), and that the D1-like-R antagonist attenuates Th17-mediated diseases like experimental autoimmune encephalomyelitis. Furthermore, we demonstrated that a D1-like-R antagonist significantly suppressed ovalbumin (OVA)-induced neutrophilic airway inflammation in OVA T cell receptor-transgenic DO11.10 mice through inhibiting Th17-mediated immune responses. Therefore, dopamine D1-like-R antagonists could become useful for treating Th17-mediated neutrophil-dominant severe asthma. As inhaled corticosteroids are known to be less effective for controlling neutrophilic inflammation, a more effective therapeutic strategy for neutrophil-dominant asthma should still be elucidated.

Dopamine Induces IL-6–Dependent IL-17 Production via D1-Like Receptor on CD4 Naive T Cells and D1-Like Receptor Antagonist SCH-23390 Inhibits Cartilage Destruction in a Human Rheumatoid Arthritis/SCID Mouse Chimera Model

A major neurotransmitter dopamine transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1–D5. Several studies have shown that dopamine not only mediates interactions into the nervous system, but can contribute to the modulation of immunity via receptors expressed on immune cells. We have previously shown an autocrine/paracrine release of dopamine by dendritic cells (DCs) during Ag presentation to naive CD4+ T cells and found efficacious results of a D1-like receptor antagonist SCH-23390 in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis and in the NOD mouse model of type I diabetes, with inhibition of Th17 response. This study aimed to assess the role of dopaminergic signaling in Th17-mediated immune responses and in the pathogenesis of rheumatoid arthritis (RA). In human naive CD4+ T cells, dopamine increased IL-6–dependent IL-17 production via D1-like receptors, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, dopamine was localized with DCs in the synovial tissue of RA patients and significantly increased in RA synovial fluid. In the RA synovial/SCID mouse chimera model, although a selective D2-like receptor antagonist haloperidol significantly induced accumulation of IL-6+ and IL-17+ T cells with exacerbated cartilage destruction, SCH-23390 strongly suppressed these responses. Taken together, these findings indicate that dopamine released by DCs induces IL-6–Th17 axis and causes aggravation of synovial inflammation of RA, which is the first time, to our knowledge, that actual evidence has shown the pathological relevance of dopaminergic signaling with RA.

Monocytes Are Differentially Activated Through HLA-DR, -DQ, and -DP Molecules Via Mitogen-Activated Protein Kinases

When HLA-DR, -DQ, and -DP were cross-linked by solid-phase mAbs, monocytes produced monokines and only anti-DR markedly activated mitogen-activated protein (MAP) kinase extracellular signal-related kinase, whereas anti-DR, anti-DQ, and anti-DP all activated MAP kinase p38. Activation of extracellular signal-related kinase was not inhibited by neutralizing Ab to TNF-α. Anti-DR and DR-restricted T cells stimulated monocytes to produce relatively higher levels of proinflammatory monokines, such as IL-1β, whereas anti-DQ/DP and DQ-/DP-restricted T cells stimulated higher levels of anti-inflammatory monokine IL-10. IL-10 production was abrogated by the p38 inhibitor SB203580, but rather enhanced by the MAP/extracellular signal-related kinase kinase-I-specific inhibitor PD98059, whereas IL-1β was only partially abrogated by SB203580 and PD98059. Furthermore, DR-restricted T cells established from PBMC, which are reactive with mite Ags, purified protein derivative, and random 19-mer peptides, exhibited a higher IFN-γ:IL-4 ratio than did DQ- or DP-restricted T cells. These results indicate that HLA-DR, -DQ, and -DP molecules transmit distinct signals to monocytes via MAP kinases and lead to distinct monokine activation patterns, which may affect T cell responses in vivo. Thus, the need for generation of a multigene family of class II MHC seems apparent.

B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones

Th 17 cells represent a novel subset of CD4+ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ α β T cell clones and four lines that recognized wild‐type and mutant p53 proteins as well as p53 self peptides in an HLA class II‐restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153 – 166 and p108 – 122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193 – 204 in the context of DRB1*1401 and for p153 – 165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild‐type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401‐restricted T cell clone specific for p193 – 204 killed a B lymphoblastoid cell line homozygous for HLA‐DRB1*1401 when the cell line was pre‐pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti‐p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53‐reactive T cells in anti‐tumor immunity is discussed.

Characterization of self-glutamic acid decarboxylase 65-reactive CD4+ T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4+ T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4*0103); (b) one clone specific to GAD65 p413-433 + DR1 (DRB1*0101); (c) two clones specific to GAD65 p200-217 + either DR9 (DRB1*0901) or DR8 (DRB1*0802); and (d) two clones specific to GAD65 p368-388 + DP2 (DPA1*01 or 0201-DPB1*0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.

The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II to CD4+ T cells is crucial to initiate immune responses. We developed a new system for delivery of an antigenic peptide to the MHC class II pathway, using the invariant chain (Ii). We designed a mutated human p33-form Ii, CLIP-substituted Ii, in which streptococcal M12p55-68 (RDLEQAYNELSGEA) was substituted for CLIP (class II associated invariant chain peptide). We examined the peptide presenting function of this construct, in comparison with the previously reported C-terminal fused Ii, in which a cathepsin cleavage site and M12p54-68 was ligated to the C-terminus of Ii. Mouse L cell transfectants expressing either of these two mutated Ii along with HLA-DR4 could process and present M12p55-68 to the peptide specific and DR4-restricted CD4+ T cell clone. CLIP-substituted Ii was much more efficient in antigen presentation than was the C-terminal fused Ii. Similar to the wild-type Ii, the CLIP-substituted Ii was associated intracellularly with DR4 molecules. These results indicate that the peptide substituted for CLIP of Ii p33 bound to the groove of DR molecules in the same manner as CLIP and it was preferentially presented to the CD4+ T cell clone in the absence of HLA-DM molecules. This system may prove useful for immunotherapy with DNA vaccines or for construction of an antigen presenting cell library with diverse peptides.