Masashi Iwasaki

Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer

The ligands for programmed cell death 1 (PD-1), an immunoinhibitory receptor belonging to CD28/cytotoxic T lymphocyte antigen 4 family, are PD-1 ligand 1 and 2 (PD-Ls). Recent reports suggest that the aberrant expression of PD-Ls on tumor cells impairs antitumor immunity, resulting in the immune evasion of the tumor cells. Although an inverse correlation between the expression level of PD-Ls and patients prognosis has been reported for several malignant tumors, the follow-up period was limited because of the lack of the antibody (Ab) applicable to paraffin-embedded specimens. Here we generated a new Ab against PD-1 ligand 1 (PD-L1) and analyzed the expression level of PD-Ls in human ovarian cancer using paraffin-embedded specimens. Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-1 ligand 2 also had a poorer prognosis, the difference was not statistically significant. A significant inverse correlation was observed between PD-L1 expression and the intraepithelial CD8+ T lymphocyte count, suggesting that PD-L1 on tumor cells directly suppresses antitumor CD8+ T cells. Multivariate analysis showed the expression of PD-L1 on tumor cells and intraepithelial CD8+ T lymphocyte count are independent prognostic factors. The PD-1/PD-L pathway can be a good target for restoring antitumor immunity in ovarian cancer.

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives “exhausted” virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

Expression and function of PD-1 in human γδ T cells that recognize phosphoantigens

Programmed cell death‐1 (PD‐1) is an inhibitory receptor and plays an important role in the regulation of αβ T cells. Little is known, however, about the role of PD‐1 in γδ T cells. In this study, we investigated the expression and function of PD‐1 in human γδ T cells. Expression of PD‐1 was rapidly induced in primary γδ T cells following antigenic stimulation, and the PD‐1+ γδ T cells produced IL‐2. When PD‐1+ γδ T cells were stimulated with Daudi cells with and without programmed cell death ligand‐1 (PD‐L1) expression, the levels of IFN‐γ production and cytotoxicity in response to PD‐L1+ Daudi cells were diminished compared to the levels seen in response to PD‐L1− Daudi cells. The attenuated effector functions were reversed by anti‐PD‐L1 mAb. When PD‐1+ γδ T cells were challenged by PD‐L1+ tumors pretreated with zoledronate (Zol), which induced γδ TCR‐mediated signaling, the resulting reduction in cytokine production was only slight to moderate compared to the reduction seen when PD‐1+ γδ T cells were challenged by PD‐L1− tumors. In addition, cytotoxic activity of PD‐1+ γδ T cells against Zol‐treated PD‐L1+ tumors was comparable to that against Zol‐treated PD‐L1− tumors. These results suggest that TCR triggering may partially overcome the inhibitory effect of PD‐1 in γδ T cells.

On the convergence of a solution of the discrete Lotka–Volterra system

For arbitrary positive discrete step size ? a determinantal solution of the discrete-time Lotka?Volterra (dLV) system is shown to asymptotically converge to the square of some singular value of a given bidiagonal matrix, where the initial value of the dLV system is uniquely determined by the entries of the matrix. Some basic properties of the solution are proved which are important for designing a new numerical algorithm for computing all of the singular values. They are a positivity of solution, a dependence of the correct initial value on ?, a sorting property and an acceleration of convergence speed by enlarging ?.

An Application of the Discrete Lotka-Volterra System with Variable Step-size to Singular Value Computation

The discrete Lotka–Volterra system with variable step-size is applied to a numerical algorithm for computing singular values. A new version of integrable singular value decomposition algorithm is designed, where the step-size δ is then a stepwise parameter δ(n). Some examples demonstrate that a better choice of the step-size gives a benefit in both convergence speed and numerical accuracy.

Orally administered soymorphins, soy-derived opioid peptides, suppress feeding and intestinal transit via gut μ1-receptor coupled to 5-HT1A, D2, and GABAB systems

We previously reported that soymorphins, mu-opioid agonist peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic-like activity. The aim of this study was to investigate the effects of soymorphins on food intake and gut motility, along with their mechanism. We found that soymorphins decreases food intake after oral administration in fasted mice. Orally administered soymorphins suppressed small intestinal transit at lower dose than that of anorexigenic activity. Suppression of food intake and small intestinal transit after oral administration of soymorphins was inhibited by naloxone or naloxonazine, antagonists of mu- or mu(1)-opioid receptor, respectively, after oral but not intraperitoneal administration. The inhibitory activities of small intestinal transit by soymorphins were also inhibited by WAY100135, raclopride, or saclofen, antagonists for serotonin 5-HT(1A), dopamine D(2), or GABA(B) receptor, respectively. We then examined the order of activation of 5-HT(1A), D(2), and GABA(B) receptors, using their agonists and antagonists. The inhibitory effect of 8-hydroxy-2-dipropylaminotetralin hydrobromide, a 5-HT(1A) agonist, after oral administration on small intestinal transit was blocked by raclopride or saclofen. Bromocriptine, a D(2) agonist-induced small intestinal transit suppression, was inhibited by saclofen, but not by WAY100135. Baclofen, a GABA(B) agonist-induced small intestinal transit suppression, was not blocked by WAY100135 or raclopride. These results suggest that 5-HT(1A) activation elicits D(2) followed by GABA(B) activations in small intestinal motility. We conclude that orally administered soymorphins suppress food intake and small intestinal transit via mu(1)-opioid receptor coupled to 5-HT(1A), D(2), and GABA(B) systems.

Accurate computation of singular values in terms of shifted integrable schemes

A new scheme with a shift of origin for computing singular values σk is presented. A shift θ is introduced into the recurrence relation defined by the discrete integrable Lotka-Volterra system with variable step-size. A suitable shift strategy is given so that the singular value computation becomes numerically stable. It is proved that variables in the new scheme converge to σk2 - Σ θ2. A comparison of the zero-shift and the nonzero-shift routines is drawn. With respect to both the computational time and the numerical accuracy, it is shown that the nonzero-shift routine is more accurate and faster than a credible LAPACK routine for singular values at least in four different types of test matrices.

Zoledronic acid-induced expansion of γδ T cells from early-stage breast cancer patients: effect of IL-18 on helper NK cells

Human γδ T cells display potent cytotoxicity against various tumor cells pretreated with zoledronic acid (Zol). Zol has shown benefits when added to adjuvant endocrine therapy for patients with early-stage breast cancer or to standard chemotherapy for patients with multiple myeloma. Although γδ T cells may contribute to this additive effect, the responsiveness of γδ T cells from early-stage breast cancer patients has not been fully investigated. In this study, we determined the number, frequency, and responsiveness of Vγ2Vδ2 T cells from early- and late-stage breast cancer patients and examined the effect of IL-18 on their ex vivo expansion. The responsiveness of Vγ2Vδ2 T cells from patients with low frequencies of Vγ2Vδ2 T cells was significantly diminished. IL-18, however, enhanced ex vivo proliferative responses of Vγ2Vδ2 T cells and helper NK cells from patients with either low or high frequencies of Vγ2Vδ2 T cells. Treatment of breast cancer patients with Zol alone decreased the number of Vγ2Vδ2 T cells and reduced their ex vivo responsiveness. These results demonstrate that Zol can elicit immunological responses by γδ T cells from early-stage breast cancer patients, but that frequent in vivo treatment reduces Vγ2Vδ2 T cell numbers and their responsiveness to stimulation.

Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP receptor followed by CCK1 receptor

The anti-hypertensive peptide Arg-Ile-Tyr, which was isolated based on its inhibitory activity (IC(50)=28microM) for angiotensin I-converting enzyme (ACE) from the subtilisin digest of rapeseed protein, exhibited vasorelaxing activity (EC(50)=5.1microM) in an endothelium-dependent manner in the mesenteric artery of spontaneously hypertensive rats (SHRs). We named the peptide rapakinin. ACE inhibitors are reported to induce nitric oxide (NO)-dependent vasorelaxation by elevating the endogenous bradykinin level; however, the vasorelaxation induced by 10microM of rapakinin was blocked only insignificantly by HOE140 or N(G)-nitro-l-arginine methyl ester (l-NAME), antagonists of bradykinin B(2) receptor and an inhibitor of NO synthase, respectively. On the other hand, the vasorelaxation induced by 10microM rapakinin was significantly blocked by indomethacin and CAY10441, a cyclooxygenase (COX) inhibitor and an antagonist of the IP receptor, respectively. The vasorelaxing activity of rapakinin was also blocked by lorglumide, an antagonist of the cholecystokinin (CCK) CCK(1) receptor, although rapakinin has no affinity for the IP and CCK(1) receptors. The vasorelaxation induced by 10microM iloprost, an IP receptor agonist, was also blocked by lorglumide, suggesting that CCK-CCK(1) receptor system is activated downstream of the PGI(2)-IP receptor system. The anti-hypertensive activity of rapakinin after oral administration in SHRs was also blocked by CAY10441 and lorglumide. These results suggest that the anti-hypertensive activity of rapakinin might be mediated mainly by the PGI(2)-IP receptor, followed by CCK-CCK(1) receptor-dependent vasorelaxation.

Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug

Bisphosphonates have benefits in breast cancer and multiple myeloma patients and have been used with adoptive immunotherapy with γδ T cells expressing Vγ2u2009Vδ2 TCRs. Although treatment with γδ T cells is safe, it has shown limited efficacy. Present bisphosphonates stimulate γδ T cells but were designed to inhibit bone resorption rather than treating cancer and have limited oral absorption, tumor cell entry, and cause bone side effects. The development of phosphate and phosphonate nucleotide prodrugs has led to important drugs for hepatitis C and HIV. Using a similar approach, we synthesized bisphosphonate prodrugs and found that they efficiently limit tumor cell growth. Pivoxil bisphosphonate esters enter cells where esterases convert them to their active acids. The bisphosphonate esters stimulated γδ T cells to secrete TNF-α in response to a variety of tumor cells more efficiently than their corresponding acids. The most active compound, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1- bisphosphonate (7), specifically expanded γδ T cells and stimulated them to secrete interferon-γ and kill tumor cells. In preclinical studies, combination therapy with compound 7 and γδ T cells prolonged survival of mice inoculated with either human bladder cancer or fibrosarcoma cells. Therefore, bisphosphonate prodrugs could enhance the effectiveness of adoptive cancer immunotherapy with γδ T cells.