Search

[抄録] ヒトの1型糖尿病のモデルであるNODマウスにおいては糖尿病発症に性差があり, 過去のデータが示すようにcyclophosphamide(CY)が糖尿病発症を促進する. CY投与雌雄NODマウスにおける糖尿病発症とTh1サイトカインであるIL-2, IFN一γ およびTh2サイトカインであるIL-4, IL40産生能との関係を検討した.その結果, NODマウスではCY処理により上記4種サイトカインはいずれも産生能の増加を認めたが, その産生パター一ンには差があり, Th2/Th1サイトカイン比の不均衡を生じた. CY投与雌NODマウスでは投与1週後よりも2週後にIL-2(Th1サイトカイン)の産生能増加を示した. CY投与2週後の観察では, saline投与群と比較してIL-10/IL-2比の有意な低下を認め, 糖尿病発症をみた.本実験により, CYが雌NODマウスのTh1サイトカインとTh2サイトカインの不均衡を生じ, そのことが糖尿病発症を促進する一因となる可能性が示唆された。, 学位の種類：医学 学位授与年月日：1999/3/23 指導：青木, 矩彦 教授(Director: Prof. Aoki, Norihiko) 報告番号：甲第491号 学内授与番号：医641 NDL書誌ID：000000335691 実験協力者：大野, 恭裕(Ono, Yasuhiro) 今村, 稔(Imamura, Minoru)

Effects of ursodeoxycholic acid (UDCA) on antigen presentation were investigated using monoclonal antigen presenting cells (APCs) and T cells. A mouse B cell lymphoma line expressing immunoglobulin M (IgM) specific for trinitrphenyl (TNP), a T hybridoma cell line specific for chicken ovalbumin (OVA) and TNP conjugated OVA (TNP-OVA) were used as APCs, T cells and an antigen, respectively. The T cells are known to release interleukin 2 (IL-2) and to obtain cytotoxic activities when they recognize the particular sequence of 17 amino acids of ovalubmin (OVA peptide) in the context of major histocompatibility gene product I-Aᵈ molecules. UDCA suppressed IL-2 production and cytotoxic activities of T cells. These suppressed T cell functions were also observed when the OVA peptide was used instead of TNP-OVA. UDCA, however, did not reduce the spontaneous expression of either MHC class Ⅱ molecules or TNP-specific IgM on the surface of APC. The capping and internalization of surface IgM during antigen exposure were not affected by UDCA. Furthermore, antigen-pulsed B cells in the presence of UDCA retained the ability of stimulating T cells. These results suggest that UDCA does not impair the function of antigen presenting cells and that UDCA may interfere with the recongnition of processed antigen by T cells in antigen presentation.

The interleukin-2 receptors （IL-2Rs） take three forms : high-, intermediate-, and low-affinity receptors. IL-2Rs are made up of α., β and γ chains and other presently unidentified components which cross-link to IL-2. In this study, monoclonal antibodies termed MIRA-16 and MIRA-24 which were found to recognize IL-2R associated molecules were produced by immunizing a rat with ELT-5 cells. MIRA-16 antigen （64 kilodaltons） was down-regulated in ELT-5 cells upon stimulation with IL- 2. This was detected in lymphoid cells but not in non-lympoid cells. MIRA-24 antigen （40 kilodaltons） was coimmunoprecipitated with a 75 kilodalton molecule which is similar to the IL-2R β chain. In addition, the MIRA-24 antigen was found to possess IL-2 binding ability. These results demonstrated that the MIRA-16 and MIRA-24 antigens were IL-2R associated molecules.

Adoptive immunotherapy with lymphokine activated killer （LAK） cells and interleukin 2 （IL-2） is a cancer therapy that was originally reported by Rosenberg et al. In Japan, however, no promising efficacy has been obtained because of the limitation of dosage of IL-2 and LAK cells in the approved trials. For improving the efficacy of adoptive immunotherapy, we framed a new protocol for the combination of IL-2 and LAK cells, with cyclophosphamide （CY） because CY causes immunological activity to increase in small doses. This study compared the clinical efficacy of IL-2 plus CY plus LAK therapy （IL-2+CY+LAK）, IL-2 monotherapy, IL-2 plus LAK therapy and IL-2 plus CY therapy. There were 7, 6, 9 and 5 cases in the IL-2+CY+LAK group, IL-2 monotherapy group, IL-2 plus LAK therapy group and IL-2 plus CY therapy group, respectively. In terms of high clinical response, there was at least a partial response （PR） in the IL-2+CY+LAK group with 28.6%, as there was in the IL-2 plus CY therapy group with 20%, in comparison with the IL-2 monotherapy group with 0% and the IL-2 plus LAK therapy group with 0%. The rate for minor response （MR） or better was evidently high in the IL-2+CY+LAK group with 42.9% in comparison with other therapy groups. As for the changes of surface markers of lymphocytes in each therapy group, a significant increase of CD8 positive cells and a significant decrease of the CD 4/8 ratio were noted in the IL-2+CY+LAK group. In addition, significant cytotoxicities against Daudi cells both by LAK cells and by freshly-obtained lymphocytes were observed. These findings suggest that the 3-drug （IL-2, CY and LAK cells） combined therapy may have increased the effect of exogenous LAK cells and also induced endogenous LAK cells, resulting in the enhancement of antitumor efficacy. In conclusion, this 3-drug combined therapy would be useful in the treatment of malignant tumors.

学位の種類：医学 学位授与年月日：1990/9/15 指導：安富, 正幸 教授((Director: Prof. Masayuki, Yasutomi)) 報告番号：甲第154号 学内授与番号：医甲117 NDL書誌ID：000000236341 Ohnishi, Hiroaki

Interleukin 2（IL2） production and the nonspecific killer activities of peripheral blood mononuclear cells （PBMC） derived from cancer patients were investigated. IL2 activity of PHA stimulated lymphocytes derived from control subjects and patients with nontreated advanced carcinoma was 524.1±250.4 nano Jurkat Units （nJ. U.）/cell and 139.3±85.4 nJ. U./ cell, respectively. The mean value of IL2 activity in cancer patients was significantly lower than that of normal subjects and this phenomenon was observed in all the cancer patients having different primary lesions. IL1 release from bacterial lipopolysaccharide-stimulated monocytes in patients with carcinoma was significantly higher than that of normal subjects, suggesting that IL1 release was not responsible for the decrease of IL2 production in cancer bearers. Moreover, monocyte depletion augmented the IL2 release by PBMC of cancer patients and the monocytes derived from cancer patients possessed a higher suppressive activity on IL2 production than those from normal subjects. These results suggest that the increase of suppressive monocytes is responsible for the impairment of IL2 production. However, this suppression mediated by monocytes from cancer patients was not restored by a treatment with indomethacin, an inhibitor for synthesis of prostaglandin, which is known to be responsible for the decrease of IL2 in normal subjects, but was recovered by a treatment with phorbol myristate acetate （PMA）. By reconstitution of lymphocytes with PMA-treated monocytes, the inhibitory activity was diminished, confirming that PMA directly acted on the suppressive monocytes. Nonspecific killer activity of PBMC induced by PHA （PHA activated killer） revealed a significant decrease in cancer patients and a supplementation of recombinant IL2 （rIL2） restored its killing activity, suggesting that the relative decrease of endogenous IL2 is responsible for the impaired PHA activated killer induction. On the other hand, the killing activity of cancer PBMC induced by rIL2 （IL2 activated killer） was similar to that of normal subjects, indicating that the population of IL2 responding nonspecific killer cells in cancer patients is the same as that of normal subjects. Therefore, the supplementation of exogenous rIL2 for cancer patients may be a useful manner to restore the immune response in tumor bearers.

The AKR-derived T lymphoma cell line BW5147 was fused with a Mycobacterium tuberculosis-primed and boosted BALB/c T cell to produce a T cell hybridoma (A55-24) which continuously produces Interleukin 2 (IL2). A55-24 cells produce IL2 without stimulation by lectin or antigen. The hybridoma culture supernatant did not contain other lymphokine-activities, T cell-replacing factor (TRF), B cell growth factor (BCGF), or macrophage activating factor (MAF). The secreted IL2 possessed the following characteristics: 1) IL2 activity was precipitated at an ammonium sulphate saturation of 50-85% . 2) a molecular weight of 30 Kd 3) an isoelectric point of pH 5.3±0.2 4) IL2 activity was destroyed only partially by pH 2 treatment and heating (56℃, 30min), but eliminated by boiling (100℃, 10min). 5) IL2 activity was absorbed by an IL2-dependent cytotoxic T cell line. 6) Addition of IL1 and this IL2 could not generate any significant CTL responses from PNA(+)-thymocytes, whereas further addition of PPD-CFS obtained from PPD-stimulated Mycobacterium-primed T cells induced noticiable responses.

Normal activated T cells and Adult T cell leukemia cells express two classes of interleukin 2 receptors （IL-2R）, high and low affinity IL-2R. The growth signal seems to be delivered by IL-2R bound to the high affinity, but not the low affinity IL-2R. In this communication, we present a series of experiments we have conducted to find whether the high affinity IL-2R has an associated molecule. Cell surface antigens expressed on human and mouse cells were chemically cross-linked by DTSSP. Immunoprecipitation study with anti-Tac antibody demonstrated that a 75,000 dalton associated molecule as well as a 110,000 dalton molecule were detected on cells that express high affinity IL-2R, but not on cells that express only low affinity IL-2R. In order to confirm that the 75kd associated molecule plays an important role in the transmembrane signal transduction, cDNA coding for human p55 was transfected into mouse L cells and T cells. L cells only expressed p55 while IL-2R did not promote any growth. In contrast, mouse T cell expressed both p55 and the 75kd associated molecules. The proliferation of those T cells was inhibited by exogeneous recombinant IL-2, thus indicating that the 75kd associated molecule plays a crucial role in IL-2 mediated signal transduction. Furthermore, to discover which is the important part of p55 in its functional association with 75kd associated molecule, we have obtained cDNA encoding a chimeric receptor consisting of the extracellar portion of Tac antigen and the transmembrane as well as the cytoplasmic portion of the human insulin receptor. The cDNA was tronsfected into mouse T cells. These T cells expressed high and low affinity IL-2R, mediated IL-2 specific incibition of cell growth, and expressed associated molecules, suggesting that the extracellar region of p55 is important for the association between p55 and 75kd associated molecule. ?Recent studies suggest the presence of second IL-2 binding protein （β chain） which is present on the surface of cells that express high affinity IL-2R. In this communication we present a series of experiments we have also conducted to discover if the high affinity IL-2R has a β chain and relationship between the β chain and associated molecules. Chemical cross-linking study of 125I-labeled IL-2 demonstrated that the 75,000-80,000 dalton as well as 55,000 dalton （α chain） molecule was detected on the cells that expressed high affinity IL-2R, but not on the cells that expressed only low affinity IL-2R, and the Tac antibody blocks the binding of 125I-labeled IL-2 to p55 as well as p75 （β chain）. These results suggest the possibility that 75kd associated molecule and p75 （β chain） are the same molecule and that this molecule plays an important role in signal transduction

The present study was done to investigate the role of cell-mediated immunity in minimal change nephrotic syndrome (MCNS) by measuring interleukin 2 (IL-2) production and the response to IL-2 of peripheral blood lymphocytes (PBL). Moreover, the lymphocyte subpopulations capable of producing IL-2, and expressing IL-2 receptors (IL-2R), were assessed by flow cytometry. Subjects employed in the study were 41 patients with MCNS, 24 with membranous nephropathy (MN), 22 with IgA glomerulonephritis (IgA-GN), and 50 healthy volunteers as controls. Patients with MN and IgA-GN showed normal levels of IL-2 production by PBL. PBL of patients with MCNS, who were in the nephrotic stage prior to initiation of prednisolone (PSL) treatment or who were in remission less than one year, exhibited significantly lower levels of IL-2 production. In contrast, PBL of patients with MCNS, who were in remission more than 1 year or who could remit of PSL regimen, had normal IL-2 production. The IL-2 production by CD4＾+ cells from patients with MCNS at nephrotic stage was normal, but that production by CD8＾+ cells was markedly reduced, returning to normal when the disease was in remission. In patients with MCNS, the IL-2 production correlated positively with both the proportion of CD4＾+ cells and the ratio of CD4＾+/CD8＾+ and inversely with the proportion of CD8＾+ cells. The response to exogenous IL-2 of concanavalin A-induced lymphoblasts from patients with MCNS was significantly lower, although the proportion of IL-2R＾+ cells showed no difference from that of healthy volunteers. These findings suggest that defective IL-2 production and IL-2 response of PBL in patients with MCNS contribute to the pathogenesis of MCNS.

Spleen cells obtained from Lewis rats were cultured with 4μg/ml Con A for 96 hr and the activated cells were fused with BW5147, mouse T lymphoma cells. Three clones were obtained by fusion and expressed the rat interleukin 2 receptor （IL-2R）. The expression of rat IL-2R on those hybrid cells could be up-regulated by supernatants of Con A activated spleen cells （condition medium; CM）, IL-2 absorbed CM and IL-2 itself. IL-2R on hybrid cells could be down-regulated by murine monoclonal antibody, ART-18 that detects rat IL-2R. T cell hybridomas could express the rat IL-2R with high （Kd=109.6 pM） and low （Kd=5.35 nM） affinity for recombinant IL-2. Incubation of hybrid clones with IL-2 resulted in the proliferation of hybrid cells, whereas the proliferation of some clones was inhibited by IL-2, indicating that IL-2 had bifunctional properties on cell growth. All hybrid clones produced IL-2 constitutively. IL-2 produced by hybrid cells were bound to its receptor and promoted the proliferation of hybrid cells in some clones. The increased expression of IL-2R and secretion of IL-2 induced by the synergistic action of Ca++ inonophores and TPA indicated the importance of the Ca++ dependent protein kinase C in the T cell activation. Rat IL-2R from Con A activated rat T cells and T cell hybridomas were studied by both one-and two-dimensional SDS-PAGE with ART-18. The IL-2R derived from Con A activated rat T cells had 72-77 kd minor and 40-48 kd major components under non-reducing conditions and had 50-56 kd major and 35-38 kd minor components under reducing conditions. The 35-38 kd minor components in reducing conditions were derived from 40-48 kd major components in non-reducing conditions. The IL-2R derived from T cell hybridomas had 110 kd, 72-77 kd and 45-48 kd components under non-reducing conditions and had 110 kd, 50-56 kd and 35-38 kd components under reducing conditions. The 110 kd components, the third component of rat IL-2R was constantly observed in T cell ?hybridomas. The rat IL-2R derived from Con A activated normal rat T cells was found to have a pI between 5.0-5.6 and 6.0-6.4, whereas the rat IL-2R derived from T cell hybridomas was found to have a pI between 6.0-6.4. T cell hybridomas expressed an aberrant IL-2R on their cell surface. The aberrant IL-2R had a molecular weight of 40 kd under reducing conditions and a pI between 5.6-6.2.