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Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of β-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2Rγnull mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h−ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10–metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response.

CD8陽性の細胞傷害性T細胞(CTL)は,ウイルス感染細胞や腫瘍細胞の排除などの免疫応答において重要な役割を演じる,しかしその機能的な成熟に関しては,不明である.我々は,以前in vitro培養系において,細菌性スーパー抗原,毒素性ショック症候群毒素-1(TSST-1)の一次刺激で得られた成人末梢血由来CD4陽性T細胞芽球が,TSST-1の再刺激に対し強い増殖とサイトカイン産生を示すのに対し,臍帯血および胸腺由来のCD4陽性T細胞芽球は,両反応において無反応性(アナジー)を示し機能的に未熟であることを見出した.本研究は,同様の実験系を用いてヒトCD8陽性T細胞の機能的成熟について明らかにすることを目的とした.TSST-1刺激で得られた成人末梢血CD8陽性T細胞芽球は,TSST-1再刺激に対する増殖およびサイトカイン(TNF-α,IFN-γ)産生,TSST-1特異的なヒトB細胞(Daudi細胞)傷害において強い反応を示した.一方,臍帯血および胸腺CD8陽性T細胞芽球は,増殖でアナジーを認め,臍帯血CD8陽性T細胞芽球は,低サイトカイン産生を示した.細胞傷害活性は両者とも,成人末梢血CD8陽性T細胞芽球と同レベルの誘導を認めた.また,早産の臍帯血CD8陽性T細胞芽球も成人末梢血CD8陽性T細胞芽球と同レベルの細胞傷害活性を認めた.以上の結果から,ヒト臍帯血および胸腺CD8陽性T細胞は,CD4陽性T細胞と同様な機能的未熟性を有することが明らかになった.しかし,異物の排除に係る細胞傷害活性は,成人末梢血CD8陽性T細胞と同レベルに誘導されることを見出した.一般に末梢リンパ組織においてT細胞が機能的に成熟するためには出生後ある程度の時間を要することが示唆されている.したがって,本研究の結果は,新生児CD8陽性T細胞の免疫応答を理解するための有用な情報になると考えられる., Cytotoxic T lymphocytes (CTLs) play an important role in immune responses, and provide potent defenses against virus infection and intracellular pathogens. However, as compared with helper T lymphocytes, CTL development remains to be clarified. In this study we tried to find out the differences in maturity of human thymus, cord blood (CB), and adult peripheral blood (APB)-derived CD8^+ T cells, and examined whether these T cells are vulnerable or not to anergy induction. Using toxic shock syndrome toxin-1 (TSST-1), CD8^+ T cell blasts with different origins were prepared and re-stimulated to estimate their immunological responses. The results showed that the proliferative response and TNF-α production upon re-stimulation with TSST-1 in thymic and CB CD8^+ T cell blasts were much lower than those of APB, although the capacity for cytotoxic activity was comparable at all three different stages of T cell development. Therefore, thymic and CB CD8^+ T cells obviously had immature traits, but appeared to have the capacity to eliminate pathogenic factors in terms of cytotoxic activity.

本研究でわれわれは細菌性スーパー抗原の黄色ブドウ球菌腸管毒素A (staphylococcal enterotoxin A, SEA)応答性のC57BL/6マウス脾臓Vβ3^+CD4^+, Vβ3^+CD8^+, Vβ11^+CD4^+,およびVβ11^+CD8^+T細胞のSEAによるアナジー誘導感受性を解析した.CD4^+T細胞はSEA刺激に対して,CD8^+T細胞と比べて,高レベルの増殖反応とIL-2産生反応を示した.CD4^+T細胞をSEAとインターロイキン-2で刺激して得られたCD4^+芽球化T細胞はSEA再刺激に対して高レベルの増殖反応とIL-2産生を示したが,同様な操作により得られたCD8^+芽球化T細胞はほとんど応答性を示さなかった.SEA誘導CD4^+芽球化T細胞から得られたVβ3^+CD4^+芽球化T細胞はSEA刺激に対して高レベルの増殖反応とIL-2産生を示したが,同様にして得られたVβ11^+CD4^+芽球化T細胞は無反応であった.本研究によって,Vβ3^+CD4^+T細胞はSEAによるアナジー誘導に抵抗性であり,他の3種のSEA応答性T細胞画分は高感受性であることが判明した.SEA応答性マウスT細胞画分のSEAによるアナジー誘導の感受性はレセプターVβ表現とCD4/CD8サブセットによって大きく影響を受けると考えられた., In the present study, we analyzed susceptibility to the in vitro induction of anergy by a superantigen (SAG), staphylococcal enterotoxin A (SEA), in SEA-reactive murine Vβ3^+CD4^+, Vβ3^+CD8^+, Vβ11^+CD4^+, and Vβ11^+CD8^+ T cells. CD4^+ T cells exhibited a high proliferative response and IL-2 production upon primary stimulation with SEA, while CD8^+ T cells exhibited only low responses. CDC T cell blasts prepared by stimulating CD4^+ T cells with a combination of SEA and IL-2 were highly responsive to restimulation with SEA, while CD8^+ T cell blasts prepared in the same way were unresponsive. Vβ3^+CD4^+ T cell blasts prepared from SEA-induced CD4^+ T cell blasts were highly responsive to restimulation with SEA, while Vβ11^+CD4^+ T cell blasts prepared in the same manner were unresponsive. These results indicate that Vβ3^+CD4^+ T cells have a low susceptibility while other three SEA-reactive T cell fractions have a high susceptibility to anergy induction with SEA. The susceptibility to anergy induction by a certain SAG in several distinct T cell fractions reactive with that SAG is probably influenced largely by the expression of Vβ elements and the CD4/CD8 subsets.

我々はスーパー抗原,黄色ブドウ球菌腸管毒素A(SEA)2回投与の影響をC57BL/6,β^+ミクログロブリンノックアウトおよびCD4ノックアウトマウスを用いて検討した.1回目のSEA投与では,SEA応答性T細胞4画分(Vβ3^+D4^+,Vβ3^+D8^+,Vβ11^+CD4^+,Vβ11^+CD8^+)は共に投与2日目をピークに増幅し,その後対照のレベルまで減少する.しかし,1回目の投与後,2日目にSEAを再投与するとSEA応答性T細胞画分はそれぞれ異なった反応を示した.増幅したVβ3^+CD4^+細胞はさらに投与後2日目まで増幅した.増幅したVβ3^+CD8^+およびVβ11^+CD4^+T細胞は再投与後2日目まで増幅した状態を維持した.一方,増幅したVβ11^+CD8^+T細胞は再投与後,すぐに減少した.SEA2回目投与後,3時間目の各細胞集団のIL-2レセプターα鎖の発現量はVβ3^+CD4^+およびVβ11^+CD4^+T細胞で90%以上,Vβ3^+CD8^+T細胞では約80%,Vβ11^+CD8^+T細胞では約70%であり,これらの細胞集団の大多数はSEA認識能力を保持していた.以上の結果より,Vβ11^+CD8^+T細胞のアナジー感受注は著しく高く,Vβ3^+CD4^+T細胞は低く,Vβ3^+CD8^+およびVβ11^+CD4^+T細胞はその中間であろうと推測した., We examined the immunologic behaviors of a superantigen Staphylococcal enterotoxin A (SEA)-reactive four distinct T cell populations (Vβ3^+CD4^+, Vβ3^+CD8^+, Vβ11^+CD4^+ and Vβ11^+CD8^+ T cells) in mice injected with SEA twice in a 2-day interval. The four T cell populations increased equally at 2 days after the first injection and thereafter decreased equally to the control level. However, these four T cell populations expanded by the first SEA injection exhibited different behaviors upon the second SEA injection, depending on the T cell receptor Vβ elements expressed and the T cell subsets. Vβ3^+CD4^+ T cells expanded exhibited further expansion, and Vβ11^+CD8^+ T cells expanded decreased rapidly. Vβ3^+CD8^+ and Vβ11^+CD4^+ T cells expanded were sustained in similar levels for 2 days after the second injection. Peak of serum IL-2 activity was seen in a higher level and at earlier hours after the second SEA injection than the first injection. Levels of IL-2 receptor a chain expression were more than 90% in Vβ3^+CD4^+ T cells and Vβ11^+CD4^+ T cells, about 80% in Vβ3^+CD8^+ T cells, and about 70% in Vβ11^+CD8^+ T cells, suggesting that large parts of these T cell populations can recognized SEA. These results suggest that Vβ3^+CD4^+ T cells are low and Vβ11^+CD8^+ T cells are high in the susceptibility to anergic induction with SEA. Vβ3^+CD8^+ and Vβ11^+CD4^+ T cells may be sustained at intermediate level.

スーパー抗原は通常のタンパク抗原とは異なり,アクセサリー細胞(A細胞)上のMHCクラスII分子に直接結合し,特定のT細胞レセプターβ鎖V領域(Vβ)を表現する大きなT細胞レパートリーと反応する.したがって,細菌性スーパー抗原は,ヒトおよびマウス末梢リンパ球に同様な強い活性化を惹き起こす.しかし,ある種の細菌性スーパー抗原staphylococcal enterotoxin B (SEB),SEC等では,マウス末梢リンパ球は,ヒト末梢リンパ球と比較して著しく低応答性である.この反応性の差が,T細胞自体の反応性の差によるものか,A細胞のアクセサリー活性の差によるものかは末だ明らかではない.細菌性スーパー抗原によるT細胞反応の強さを決定する因子を解明するため,同一T細胞を用いて,ヒトおよびマウスMHCクラスII分子陽性細胞の存在下でSEAおよびSEBに対する反応性を比較,検討した.その結果,SEBに対するヒトとマウス末梢リンパ球の反応性の差は,A細胞のアクセサリー活性の差に基づくことが示唆された.また,アクセサリー活性の差は,反応丁細胞内Ca^濃度にも差を惹き起こすことから,T細胞活性化の初期から影響を与えているものと考えられた.細菌性スーパー抗原による生体異常反応は,異常に強いT細胞反応に起因すると考えられている.したがって,T細胞反応の強さがA細胞のレベルで決定することが明らかとなったことは,細菌感染症の制御の上で有用であると思われる., Bacterial superantigens (SAGs) bind to major histocompatibility complex (MHC) class II molecules on accessory cells (AC) and stimulate T cells upon interaction with the Vβ portion of the T cell receptor (TCR). We have recently shown that bacterial SAGs produced by Staphylococcus aureus, Staphylococcal enterotoxin A (SEA), SEB, SEC, and toxic shock syndrome toxin-1 (TSST-1) can all stimulate human peripheral blood mononuclear cells (PBMC) at very minute doses of antigens (≥10^ ng/ml). By contrast, when murine peripheral lymphocytes are used, SEs and TSST-1 have been segregated into two groups according to potency. SEA and TSST-1 are equally strong stimulators of murine peripheral lymphocytes (≥10^ ng/ml), whereas the responses of murine peripheral lymphocytes to SEB and SEC require 10^3～10^4-fold greater doses. However, it is still unclear whether this difference between the murine and human responses to SEB and SEC is due to a difference in T cell responsiveness or a difference in the activity of AC. In this study, the response of identical murine T cell preparations to SEB showed a preference for human AC over murine AC. Thus, the results indicated that the difference in response to SEB is primarily due to a difference in the activity of AC. Futhermore, the Ca^ concentration of murine T cells responding to SEB presented by human AC was higher than that of those responding to SEB presented by murine AC, indicating that different accessory activity influences T cell activation from the early phase of signal transduction.

The etiopathogenesis of many autoimmune disorders has not been identified. The aim of this paper is to focus on the involvement of bacterial exposure in the pathogenesis of primary biliary cirrhosis (PBC) and autoimmune pancreatitis (AIP), both of which are broadly categorized as autoimmune disorders involving hepatobiliary-pancreatic lesions. Avirulent and/or commensal bacteria, which may have important role(s) as initiating factors in the pathogenesis of autoimmune disorders such as PBC and AIP, will be emphasized.

10 páginas, 10 figuras, 1 tabla -- PAGS nros. 3970-3979, Vascular endothelial cells (ECs) are key players in leukocyte recruitment into tissues and metastatic dissemination of tumor cells. ECs express B7h, which is the ligand of the ICOS T cell costimulatory molecule. The aim of this work was to assess the effect of B7h triggering by a soluble form of ICOS (ICOS-Fc) on the adhesion of colon carcinoma cell lines to HUVECs. We found that B7h triggering inhibited HUVEC adhesiveness to HT29 and DLD1 cells (by 50 and 35%, respectively) but not to HCT116 cells. The effect was dependent on the ICOS-Fc dose and was detectable as early as 30 min after treatment and was still present after 24 h. It was inhibited by soluble anti-ICOS reagents (mAb and B7h-Fc) and silencing of B7h on HUVECs, and it was not displayed by an F119S mutated form of ICOS-Fc that does not bind B7h. HUVEC treatment with ICOS-Fc did not modulate expression of adhesion molecules and cytokines, but it substantially downmodulated ERK phosphorylation induced by E-selectin triggering or osteopontin, which may influence HUVEC adhesiveness. Moreover, HUVEC treatment with ICOS-Fc also inhibited adhesion of polymorphonuclear cells and several tumor cell lines from different origins. Therefore, the B7h–ICOS interaction may modulate spreading of cancer metastases and recruitment of polymorphonuclear cells in inflammatory sites, which opens a view on the use of ICOS-Fc as an immunomodulatory drug, This work was supported by Associazione Italiana Ricerca sul Cancro (Milan), Compagnia di San Paolo (Torino), Fondazione Cassa di Risparmio di Torino, Alfieri Project (Torino), Regione Piemonte (Ricerca Sanitaria Finalizzata and Piattaforme Innovative), Fondazione Italiana Sclerosi Multipla 2008/R/711 (Genoa), Fondazione Amici di Jean (Torino), and funds from the University of Torino

18 p.-8 fig.-1 tab., The interaction between the T‐lymphocyte costimulatory molecule ICOS and its ligand (ICOS‐L) is needed for efficient immune responses, but expression levels are tightly controlled, as altered expression of ICOS or ICOS‐L may lead to immunodeficiency, or favor autoimmune diseases and tumor growth. Using cells of mouse B cell lymphoma (M12.C3) and melanoma (B16), or hamster CHO cells transfected with various forms of mouse ICOS‐L, and ICOS+ T cell lines, we show that, within minutes, ICOS induces significant downmodulation of surface ICOS‐L that is largely mediated by endocytosis and trans‐endocytosis. So, after interaction with ICOS+ cells, ICOS‐L was found inside permeabilized cells, or in cell lysates, with significant transfer of ICOS from ICOS+ T cells to ICOS‐L‐expressing cells, and simultaneous loss of surface ICOS by the T cells. Data from cells expressing ICOS‐L mutants show that conserved, functionally important residues in the cytoplasmic domain of mouse ICOS‐L (Arg300, Ser307 and Tyr308), or removal of ICOS‐L cytoplasmic tail have minor effect on its internalization. Internalization was dependent on temperature, and was partially dependent on actin polymerization, the GTPase dynamin, protein kinase C, or the integrity of lipid rafts. In fact, a fraction of ICOS‐L was detected in lipid rafts. On the other hand, proteinase inhibitors had negligible effects on early modulation of ICOS‐L from the cell surface. Our data add a new mechanism of control of ICOS‐L expression to the regulation of ICOS‐dependent responses., Supported by Grants PI13/01809 (to J.M.R.), PI13/02153 and PI16CIII/00012 (to P.P.) from “Acción Estratégica en Salud, Plan Estatal I+D+i”, Ministerio de Economía, Industria y Competitividad(MINECO, Spain) and by the Associazione Italiana Ricerca sul Cancro(Grant IG20714, AIRC, Milan), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017-0535) (to U.D.).

13 p.-7 fig.-1 tab., Inducible T-cell costimulator (ICOS) upon binding to its ligand (ICOSL) mediates adaptive immunity and antitumor response. Thus, antitumor therapies targeting the ICOS/ICOSL pathway hold great promise for cancer treatment. In this regard, ICOSL triggering by a soluble recombinant form of ICOS (ICOS-Fc) hampered adhesiveness and migration of dendritic, endothelial, and tumor cells in vitro. Furthermore, in vivo treatment with ICOS-Fc previously showed the capability to inhibit lung metastatization of ICOSL+ B16-F10 melanoma cells when injected intravenously in mice, but it failed to block the growth of established subcutaneous B16-F10 murine tumors. Thus, we asked whether passive targeting of solid tumors with ICOS-Fc-loaded biocompatible and biodegradable nanoparticles (NPs) could instead prove effectiveness in reducing tumor growth. Here, ICOS-Fc was loaded in two types of polymer nanoparticles, i.e. cross-linked β-cyclodextrin nanosponges (CDNS) and poly(lactic-co-glycolic acid) (PLGA) NPs and in vitro characterized. In vivo experiments showed that treatment of C57BL6/J mice with ICOS-Fc loaded into the two nanoformulations inhibits the growth of established subcutaneous B16-F10 tumors. This anticancer activity appears to involve both anti-angiogenic and immunoregulatory effects, as shown by decreased tumor vascularization and downmodulation of IL-10 and Foxp3, two markers of regulatory T cells (Tregs). Overall, the substantial in vivo anticancer activity of ICOS-Fc-loaded CDNS and PLGA NPs against different components of the tumor microenvironment makes these nanoformulations attractive candidates for future combination cancer therapy., This work was supported by the Associazione Italiana Ricerca sul Cancro (IG 20714, AIRC, Milano), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017–0535).

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14− cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS–B7h system.

ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by activated antigen presenting cells. This work investigated the role of B7h and B7.1 in TH17 and TH9 cell differentiation by assessing activity of recombinant B7h-Fc and B7.1-Fc on human naive TH cells activated in the presence of different combinations of exogenous cytokines. In the presence of TGF-β1 and IL-1β (TH17 promoting condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10 secretion, whereas B7.1-Fc was more effective in inducing IL-17F. Dual costimulation with B7h-Fc and B7.1-Fc displayed an intermediate pattern with predominance of IL-17F over IL-17A, secretion of high levels of IL-10, and secretion of IL-9 levels lower than those induced by B7.1-Fc alone. In the presence of TGF-β1 and IL-4 (TH9 promoting condition), B7h-Fc induced IL-17A only, whereas B7.1-Fc induced also IL-17F, IL-10, and high levels of IL-9. Experiments on memory TH cells showed that B7h-Fc mainly supported secretion of IL-17A and IL-10, whereas B7.1-Fc supported secretion of IL-17A, IL-17F, IL-10, and IL-9. These data indicate that B7h and B7.1 play different roles in modulation of TH17 and TH9 differentiation. This plasticity might be important in the immune response to pathogens and tumors, and in the development of autoimmune diseases, and should be taken in consideration in designing of immunotherapeutic protocols triggering ICOS or CD28.