Yukimi Sakoda

B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance.

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti-B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were re-exposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance.

Redirecting Gene-Modified T Cells toward Various Cancer Types Using Tagged Antibodies

Purpose: To develop an adaptable gene-based vector that will confer immune cell specificity to various cancer types. Experimental Design: Human and mouse T cells were genetically engineered to express a chimeric antigen receptor (CAR) that binds a fluorescein isothiocyanate (FITC) molecule, termed anti-FITC CAR T cells. Various antibodies (Ab) currently in clinical use including cetuximab (Ctx), trastuzumab (Her2), and rituximab (Rtx) were conjugated with FITC and tested for their ability to bind tumor cells, activate T cells, and induce antitumor effects in vitro and in vivo. Results: Anti-FITC CAR T cells recognize various cancer types when bound with FITC-labeled Abs resulting in efficient target lysis, T-cell proliferation, and cytokine/chemokine production. The treatment of immunocompromised mice with human anti-FITC CAR T cells plus FITC-labeled cetuximab (FITC-Ctx) delayed the growth of colon cancer but unexpectedly led to the outgrowth of EGF receptor (EGFR)-negative tumor cells. On the other hand, in a human pancreatic cancer cell line with uniform EGFR expression, anti-FITC CAR T cells plus FITC-Ctx eradicated preestablished late-stage tumors. In immunocompetent mice, anti-FITC CAR T cells exhibited potent antitumor activity against syngeneic mouse breast cancer expressing Her2 and B-cell lymphoma expressing CD20 by combining with FITC-Her2 and FITC-Rtx, respectively. In addition, the activity of anti-FITC CAR T cells could be attenuated by subsequent injections of nonspecific FITC-IgG. Conclusion: These studies highlight an applicability of anti-tag CAR technology to treat patients with different types of cancers and a possibility to regulate CAR T-cell functions with competing FITC molecules. Clin Cancer Res; 18(23); 6436–45. ©2012 AACR.

Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway.

B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor that interacts with herpesvirus entry mediator (HVEM), and this interaction regulates pathogenesis in various immunologic diseases. In graft-versus-host disease (GVHD), BTLA unexpectedly mediates positive effects on donor T-cell survival, whereas immunologic mechanisms of this function have yet to be explored. In this study, we elucidated a role of BTLA in GVHD by applying the newly established agonistic anti-BTLA monoclonal antibody that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. Our results revealed that provision of BTLA signal inhibited donor antihost T-cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand that delivers HVEM prosurvival signal in donor T cells. Collectively, current study elucidated dichotomous functions of BTLA in GVHD to serve as a costimulatory ligand of HVEM and to transmit inhibitory signal as a receptor.

Leucocyte-associated immunoglobulin-like receptor-1 is an inhibitory regulator of contact hypersensitivity

Leucocyte‐associated immunoglobulin‐like receptor‐1 (LAIR‐1) is a membrane receptor of the immunoglobulin (Ig) superfamily that is expressed on most types of haematopoietic cells, and delivers inhibitory signals through interacting with collagens. In order to elucidate the immunological functions of LAIR‐1 in vivo, we established transgenic mice expressing a chimeric protein composed of the extracellular domain of LAIR‐1 fused with an Ig tag (LAIR‐1–Ig), which acts as a decoy by competing with endogenous LAIR‐1. The transgenic mice showed an increased susceptibility for development of contact hypersensitivity (CHS), an experimental model of allergic contact dermatitis, in association with enhanced hapten‐specific T‐cell responses. When T cells from the hapten‐sensitized donor mice were transferred into non‐sensitized recipients, treatment of either donor mice or recipient mice with LAIR‐1–Ig protein accelerated CHS, suggesting a potentially negative role of LAIR‐1 in both the sensitization and the elicitation of hapten‐reactive T cells. In vitro assays revealed that LAIR‐1 decreased the production of interleukin‐6 and interleukin‐12 in dendritic cells, and inhibited the proliferation and cytokine production of naïve and memory T cells along with G0/G1 cell cycle arrest. Collectively, our findings suggest that LAIR‐1 plays a crucial inhibitory role in CHS by regulating antigen‐presenting cell and T‐cell functions.

Herpesvirus entry mediator regulates hypoxia-inducible factor-1α and erythropoiesis in mice.

Erythropoiesis, the production of red blood cells, must be tightly controlled to ensure adequate oxygen delivery to tissues without causing thrombosis or stroke. Control of physiologic and pathologic erythropoiesis is dependent predominantly on erythropoietin (EPO), the expression of which is regulated by hypoxia-inducible factor (HIF) activity in response to low oxygen tension. Accumulating evidence indicates that oxygen-independent mediators, including inflammatory stimuli, cytokines, and growth factors, also upregulate HIF activity, but it is unclear whether these signals also result in EPO production and erythropoiesis in vivo. Here, we found that signaling through herpesvirus entry mediator (HVEM), a molecule of the TNF receptor superfamily, promoted HIF-1α activity in the kidney and subsequently facilitated renal Epo production and erythropoiesis in vivo under normoxic conditions. This Epo upregulation was mediated by increased production of NO by renal macrophages. Hvem-deficient mice displayed impaired Epo expression and aggravated anemia in response to erythropoietic stress. These data reveal that HVEM signaling functions to promote HIF-1α activity and Epo production, and thus to regulate erythropoiesis. Furthermore, our findings suggest that this molecular mechanism could represent a therapeutic target for Epo-responsive diseases, including anemia.

Preventive and therapeutic potential of placental extract in contact hypersensitivity.

Immunoregulatory effects of placental extract and placenta-derived factors have been demonstrated in various conditions. Accordingly, placental extract has been used as certain types of medical intervention in Asian countries, whereas experimental evidence supporting its therapeutic effects and mechanisms has yet to be fully demonstrated. In this study, we investigate preventive and therapeutic effects of placental extract in contact hypersensitivity (CHS), a mouse model of allergic contact dermatitis. Administration of placental extract prior to the sensitization of allergic antigen (Ag) significantly inhibited the severity of CHS induced by Ag challenge. This effect was associated with reduced numbers of CD4(+) T cells in peripheral blood, decrease of tissue-infiltrating lymphocytes, and preferential production of Th2-type cytokines in Ag-challenged sites. In addition, CHS caused by repetitive challenges of allergic Ag was also prevented and treated by administration of placental extract. Finally, administration of cyclo-trans-4-L-hydroxyprolyl-L-serine, a dipeptide derived from placental extract, also alleviated CHS, suggesting its potential role in the effects of placental extract in CHS. Taken together, our findings demonstrated experimental evidence supporting immunoregulatory effects of placental extract in allergic skin diseases and elucidated its potential mechanisms.

Combined adjuvants of poly(I:C) plus LAG-3-Ig improve antitumor effects of tumor-specific T cells, preventing their exhaustion.

Therapeutic cancer vaccines are designed to treat cancer by boosting the endogenous immune system to fight against the cancer. In the development of clinically effective cancer vaccines, one of the most practical objectives is to identify adjuvants that are capable of optimizing the vaccine effects. In this study, we explored the potential of polyinosinic–polycytidylic acid (poly(I:C)) and LAG‐3‐Ig (soluble recombinant protein of lymphocyte activation gene‐3 [LAG‐3] extracellular domain fused with human IgG Fc region) as adjuvants for P1A tumor antigen peptide vaccine in a pre‐established P815 mouse tumor model with a transfer of tumor‐specific T cells. Whereas the use of poly(I:C) or LAG‐3‐Ig as a signal adjuvant induced a slight enhancement of P1A vaccine effects compared to incomplete Freunds adjuvant, combined treatment with poly(I:C) plus LAG‐3‐Ig remarkably potentiated antitumor effects, leading to complete rejection of pre‐established tumor and long‐term survival of mice. The potent adjuvant effects of poly(I:C) plus LAG‐3‐Ig were associated with an enhanced infiltration of T cells in the tumor tissues, and an increased proliferation and Th1‐type cytokine production of tumor‐reactive T cells. Importantly, the combined adjuvant of poly(I:C) plus LAG‐3‐Ig downregulated expressions of PD‐1, LAG‐3, and TIGIT on P1A‐specific T cells, indicating prevention of T cell exhaustion. Taken together, the results of the current study show that the combined adjuvants of poly(I:C) plus LAG‐3‐Ig with tumor peptide vaccine induce profound antitumor effects by activating tumor‐specific T cells.

Vγ2Vδ2 T‐cell co‐stimulation increases natural killer cell killing of monocyte‐derived dendritic cells

Interactions between natural killer (NK) cells and dendritic cells (DC) affect maturation and function of both cell populations, including NK cell killing of DC (editing), which is important for controlling the quality of immune responses. We also know that antigen‐stimulated Vγ2Vδ2 T cells co‐stimulate NK cells via 4‐1BB to enhance the killing of tumour cell lines but we do not know what regulates 4‐1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK–γδ T‐cell cross‐talk. Here we show that antigen‐stimulated γδ T cells co‐stimulate NK cells through inducible T‐cell co‐stimulator (ICOS)– ICOS ligand (ICOSL) and this signal increases NK cell killing of autologous DC. Effects of NK–γδ T‐cell co‐culture, which could be reproduced with soluble ICOS‐Fc fusion protein, included increased CD69 and 4‐1BB expression, interferon‐γ, tumour necrosis factor‐α, macrophage inflammatory protein‐1β, I‐309, RANTES and sFas ligand production, as well as elevated mRNA levels for co‐stimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Hence, ICOS–ICOSL co‐stimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK–γδ T‐cell cross‐talk links innate and antigen‐specific lymphocyte responses in the control of cytotoxic effector function and DC killing.

IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor

Infiltration, accumulation, and survival of chimeric antigen receptor T (CAR-T) cells in solid tumors is crucial for tumor clearance. We engineered CAR-T cells to express interleukin (IL)-7 and CCL19 (7 × 19 CAR-T cells), as these factors are essential for the maintenance of T-cell zones in lymphoid organs. In mice, 7 × 19 CAR-T cells achieved complete regression of pre-established solid tumors and prolonged mouse survival, with superior anti-tumor activity compared to conventional CAR-T cells. Histopathological analyses showed increased infiltration of dendritic cells (DC) and T cells into tumor tissues following 7 × 19 CAR-T cell therapy. Depletion of recipient T cells before 7 × 19 CAR-T cell administration dampened the therapeutic effects of 7 × 19 CAR-T cell treatment, suggesting that CAR-T cells and recipient immune cells collaborated to exert anti-tumor activity. Following treatment of mice with 7 × 19 CAR-T cells, both recipient conventional T cells and administered CAR-T cells generated memory responses against tumors.

Pathogenic Function of Herpesvirus Entry Mediator in Experimental Autoimmune Uveitis by Induction of Th1- and Th17-Type T Cell Responses

Herpesvirus entry mediator (HVEM), a member of the TNFR superfamily, serves as a unique molecular switch to mediate both stimulatory and inhibitory cosignals, depending on its functions as a receptor or ligand interacting with multiple binding partners. In this study, we explored the cosignaling functions of HVEM in experimental autoimmune uveitis (EAU), a mouse model resembling human autoimmune uveitis conditions such as ocular sarcoidosis and Behcet disease. Our studies revealed that EAU severity significantly decreased in HVEM-knockout mice compared with wild-type mice, suggesting that stimulatory cosignals from the HVEM receptor are predominant in EAU. Further studies elucidated that the HVEM cosignal plays an important role in the induction of both Th1- and Th17-type pathogenic T cells in EAU, including differentiation of IL-17–producing αβ+γδ− conventional CD4+ T cells. Mice lacking lymphotoxin-like, inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT), B- and T-lymphocyte attenuator (BTLA) or both LIGHT and BTLA are also less susceptible to EAU, indicating that LIGHT–HVEM and BTLA–HVEM interactions, two major molecular pathways mediating HVEM functions, are both important in determining EAU pathogenesis. Finally, blocking HVEM cosignals by antagonistic anti-HVEM Abs ameliorated EAU. Taken together, our studies revealed a novel function of the HVEM cosignaling molecule and its ligands in EAU pathogenesis through the induction of Th1- and Th17-type T cell responses and suggested that HVEM-related molecular pathways can be therapeutic targets in autoimmune uveitis.