Yoshikata Misaki

Generation of CD4+CD25+ Regulatory T Cells from Autoreactive T Cells Simultaneously with Their Negative Selection in the Thymus and from Nonautoreactive T Cells by Endogenous TCR Expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4+CD25+ T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4+CD25+ regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA × DO11.10 mice had increased numbers of CD4+CD25+ regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA × DO11.10 mice, T cells expressing endogenous TCR αβ chains were CD4+CD25− T cells, whereas T cells expressing autoreactive TCR were selected as CD4+CD25+ T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA × DO11.10 mice. In contrast, in DO11.10 mice, CD4+CD25+ T cells expressed endogenous TCR αβ chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4+CD25+ T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4+CD25+ T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4+CD25− T cells from autoreactive T cell repertoire.

Peroxisome proliferator-activated receptor-γ haploinsufficiency enhances B cell proliferative responses and exacerbates experimentally induced arthritis

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) controls adipogenesis and glucose metabolism. It was reported recently that PPAR gamma activation by its agonistic ligands modifies lymphocyte function. Since synthetic ligands are known to exert their effect via PPAR gamma-dependent and -independent pathways, we examined the physiological role of PPAR gamma in lymphocytes by using heterozygote mutant mice in which one allele of PPAR gamma is deleted (PPAR gamma(+/-)). In contrast to T cells, which did not exhibit a significant difference, B cells from PPAR gamma(+/-) showed an enhanced proliferative response to stimulation by either lipopolysaccharide or cross-linking of antigen receptors. Dysregulation of the NF-kappa B pathway in B cells from PPAR gamma(+/-) was indicated by spontaneous NF-kappa B activation, as well as increased I kappa B alpha phosphorylation and gel-shift activity following LPS stimulation. Mice primed with either ovalbumin or methylated BSA also showed enhanced antigen-specific immune response of both T and B cells, an immunological abnormality that exacerbated antigen-induced arthritis. These findings indicate that PPAR gamma plays a critical role in the control of B cell response and imply a role in diseases in which B cell hyperreactivity is involved, such as arthritis and autoimmunity.

Functional Reconstitution of Class II MHC-Restricted T Cell Immunity Mediated by Retroviral Transfer of the αβ TCR Complex

Transfer of the αβ TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient αβ TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted α or β TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323–339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323–339 challenge. These results indicate that αβ TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.

Systemic lupus erythematosus and thrombotic thrombocytopenic purpura: a case report and literature review.

AbstractWe describe a patient with systemic lupus erythematosus (SLE) who developed severe and acute thrombotic thrombocytopenic purpura (TTP). Detection of the fragmentation of peripheral red blood cells (RBC) helped the early diagnosis of TTP and the patient was rescued by extensive plasma exchange started promptly after the diagnosis. Because manifestations of TTP are similar to those in SLE, it is sometimes difficult to make an accurate diagnosis of TTP in SLE patients. We emphasise here the significance of the early diagnosis of TTP by the observation of fragmented RBC and the intensive therapy, including plasma exchange, for this very severe condition.

Glucocorticoids inhibit chemokine generation by human eosinophils

Recent identification of eosinophils as a cellular source of various cytokines suggests that eosinophil-derived cytokines contribute to allergic inflammation through either an autocrine or a paracrine fashion. The profound inhibitory effects of glucocorticoids (GCCs) on the production of various cytokines have been well recognized, however, there has been no definitive evidence that GCCs in fact inhibit cytokine generation by eosinophils. To verify the inhibitory ability of GCCs on eosinophil cytokine generation, we studied the effect of GCCs by determination of IL-8 and monocyte chemoattractant protein-1 (MCP-1) as parameters. Dexamethasone (DEX) inhibited both generation and secretion of IL-8 in a dose-dependent fashion. DEX also dampened formyl-methionyl-leucyl-phenylalanine-or ionomycin-induced eosinophil IL-8 production. Furthermore, MCP-1 production was also inhibited by DEX. The slope and the shape of the dose-response curve of DEX were similar irrespective of either the input stimuli or the output cytokines; half-maximal inhibition was observed at 10(-8) mol/L, and nearly complete abolishment was observed at 10(-7) mol/L. The competitive polymerase chain reaction for IL-8 mRNA and semiquantitative polymerase chain reaction for MCP-1 mRNA revealed that the inhibition occurred at a level of pretranslation. These results indicate that the beneficial effect of GCCs in allergic inflammation might be related, at least in part, to a direct effect of the drugs on eosinophil cytokine synthesis.

Antigen-Specific T Cells Transduced with IL-10 Ameliorate Experimentally Induced Arthritis Without Impairing the Systemic Immune Response to the Antigen

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 104. Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.

Reconstitution of CD8+ T Cells by Retroviral Transfer of the TCR αβ-Chain Genes Isolated from a Clonally Expanded P815-Infiltrating Lymphocyte

Gene transfer of TCR αβ-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8+ T cells by transfer of TCR αβ-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vβ subfamily usage of CD8+ TIL in the tumor sites demonstrated that T cells using Vβ10 efficiently infiltrated and expanded clonally. The TCR α- and β-chain sequences derived from a tumor-infiltrating CD8+/Vβ10+ single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR αβ-chain genes were retrovirally introduced into CD8+ T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2Ld mAb. Furthermore, T cells containing both TCR α- and β-chains, but not TCR β-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8+ T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8+ T cells by transfer of TCR αβ-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.

Clinical usefulness of anti-RNA polymerase III antibody measurement by enzyme-linked immunosorbent assay

OBJECTIVE To evaluate the clinical usefulness of measuring anti-RNA polymerase (RNAP) III antibody with a commercially available ELISA in Japanese patients with SSc. METHODS This multicentre study involved 354 patients with SSc, 245 with non-SSc CTDs and 102 healthy controls. ELISAs were used to detect anti-RNAP III antibody, anti-topo I antibody and ACA. The presence of anti-RNAP III antibody in selected serum samples was confirmed by immunoprecipitation (IP) assay. RESULTS By ELISA, anti-RNAP III antibody was detected in 38 (10.7%) patients with SSc, 3 (1.2%) with non-SSc CTD and no healthy controls. The clinical specificity for SSc was excellent (98.8%), although a small number of false positives occurred. The sensitivity of the anti-topo I and ACA ELISAs for SSc was 59.9%, which increased to 68.2% without a reduction in specificity when the anti-RNAP III measurement was added. Clinical features associated with positivity for the anti-RNAP III antibody include dcSSc, a high total skin score and a trend towards high prevalence of renal crisis, consistent with previous studies that used an IP assay. Furthermore, on clinical severity scales, SSc patients with anti-RNAP III antibody scored highest for skin and renal involvement among patients subgrouped by the presence of individual SSc-related antibodies. CONCLUSIONS The measurement of anti-RNAP III antibody by ELISA is useful in routine clinical practice, because it helps diagnose SSc and identify a disease subset with severe skin and renal involvement.

Regulation of Human Eotaxin Generation by Th1-/Th2-Derived Cytokines

Background: Accumulating evidence indicates that eotaxin is the primary mediator of tissue eosinophilia. In the present study, we analyzed the mechanisms of eotaxin generation by Th1-/Th2-derived cytokines in vitro. Methods: Human dermal fibroblasts, human umbilical vein endothelial cells and A549 human bronchial epithelial cell line cells were stimulated with TNF-α, IL-4, IFN-γ or TNF-α in combination with IL-4 or IFN-γ and the amount of eotaxin production was analyzed. Results: Fibroblasts produced nanogram/milliliter quantities of eotaxin. Proinflammatory cytokine TNF-α and Th2-type cytokine IL-4 each induced eotaxin production, and combination of them caused a marked synergistic increase in that production. On the other hand, Th1-type cytokine IFN-γ inhibited eotaxin generation at the protein/mRNA levels. Conclusion: The Th2-derived cytokine upregulated while the Th1-derived cytokine inhibited eotaxin production by fibroblasts. In view of the Th1/Th2 paradigm, these results indicate that (1) eotaxin regulates eosinophil accumulation in the Th2-dominant state such as allergic disease, and (2) direct suppression of eotaxin production by IFN-γ is one of the major mechanisms by which IFN-γ suppresses eosinophilic inflammation.

Polymyositis associated with focal mesangial proliferative glomerulonephritis with depositions of immune complexes

A 58-year-old man concurrently developed polymyositis (PM), interstitial lung disease, and nephrotic-range proteinuria. Renal biopsy revealed focal mesangial proliferative glomerulonephritis (mesPGN) with depositions of immunoglobulin and complements. A combination therapy of corticosteroid, intravenous immunoglobulin, and cyclosporine was found very effective for the patient. Glomerulonephritis associated with PM/dermatomyositis (DM) is rare. In our review of related literature, mesPGN was exclusively observed in polymyositis while membranous nephropathy in DM. The mechanism underlying the association between myositis and glomerulonephritis remains to be elucidated.