Kazushi Tanimoto

Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m(2)/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56(bright) NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

Absence of Chlamydial Infection in Japanese Patients with Ocular Adnexal Lymphoma of Mucosa-Associated Lymphoid Tissue

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immuno-globulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, orChlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies againstChlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

Human herpesvirus 6 infection impairs Toll-like receptor signaling

Human herpesvirus 6 (HHV-6) has a tropism for immunocompetent cells, including T lymphocytes, monocytes/macrophages, and dendritic cells (DCs) suggesting that HHV-6 infection affects the immunosurveillance system. Toll-like receptor (TLR) system plays an important role in innate immunity against various pathogens. In the present study, we investigated the effect of HHV-6 infection on the expression and intracellular signaling of TLRs in DCs. Although expression levels of TLRs were not decreased or slightly elevated following HHV-6 infection, the amounts of cytokines produced following stimulation with ligands for TLRs appeared to be dramatically decreased in HHV-6-infected DCs as compared to mock-infected DCs. Similarly, phosphorylation levels of TAK-1, IκB kinase, and IκB-α following stimulation of HHV-6-infected DCs with lipopolysaccharide, which is the ligand for TLR4, appeared to be decreased. These data show that HHV-6 impairs intracellular signaling through TLRs indicating the novel mechanism of HHV-6-mediated immunomodulation.

Single-dose daily infusion of cyclosporine for prevention of Graft-versus-host disease after allogeneic bone marrow transplantation from HLA allele-matched, unrelated donors.

Peak blood concentration of cyclosporine (CsA) in renal transplantation patients was recently reported to be associated with clinical efficacy.We therefore evaluated the toxicity and efficacy of a regimen of once-daily infusion of CsA plus a short course of methotrexate as prophylaxis of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor. Nineteen patients with hematologic malignancies received CsA, 3 mg/kg per day, as a 4-hour intravenous (IV) infusion from day -1. After engraftment, patients received CsA orally at twice the IV dose. The CsA dose was adjusted to maintain the blood trough level between 150 and 200 ng/mL. Methotrexate was administered IV at doses of 10 mg/m2 on day 1 and 7 mg/m2 on days 3, 6, and 11. Bone marrow engraftment occurred in all patients. Grade 1 and grade 2 GVHD occurred in 6 (31.6%) and 7 (36.8%) of the 19 patients, respectively. No patient had grade 3 or 4 GVHD. Acute nephrotoxicity developed in 1 (5.3%) of the 19 patients, and hypertension developed in 3 (15.8%) of the 19 patients.We evaluated the pharmacokinetics of 4-hour CsA infusion in 10 patients.The mean trough concentration, mean peak concentration, mean time to peak concentration, and area under the curve (24 hours) were 161 ± 43 ng/mL, 1498 ± 387 ng/mL, 3.2 ± 1.0 hours, and 10,848 ± 1,991 ng ± h/mL, respectively. This regimen was well tolerated and did not enhance the risk of severe GVHD in patients undergoing allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor.

Gene-Modified Human α/β-T Cells Expressing a Chimeric CD16-CD3ζ Receptor as Adoptively Transferable Effector Cells for Anticancer Monoclonal Antibody Therapy

Ochi and colleagues engineered T cells to express chimeric CD16 V158-CD3ζ receptors that mediate antibody-dependent tumoricidal activity; upon stimulation these T cells proliferate and differentiate into effector memory T cells, which could combine with and enhance clinical responses by anticancer monoclonal antibodies. The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)–expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20+ lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2–treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs. Cancer Immunol Res; 2(3); 249–62. ©2014 AACR.

Activation of T-cell receptor signaling in peripheral T-cell lymphoma cells plays an important role in the development of lymphoma-associated hemophagocytosis.

Peripheral T-cell lymphoma (PTCL) is a biologically diverse lymphoid malignancy. The clinical aggressiveness associated with hemophagocytic syndrome (HS) is a characteristic of PTCL, being more distinctive in CD8+ PTCL. However, the underlying mechanism of PTCL-associated HS has not yet been fully investigated. We newly established a novel IL-2-dependent CD8+ PTCL lymphoma cell line (T8ML-1) from a patient with CD8+ PTCL who suffered recurrent HS accompanying disease flare-up. Focusing on the lymphoma cell T-cell receptor (TCR), we examined the lymphoma cell functions responsible for such clinical manifestations. First, T8ML-1.1 in which endogenous TCR-α/β chains were silenced by siRNAs, and T8ML-1.2 in which endogenous TCR-α/β chains were replaced with HLA-A*24:02-restricted and WT1235–243-specific TCR-α/β, were established. T8ML-1 exerted phytohemagglutinin (PHA)-dependent cytotoxicity via granular exocytosis. Additionally, soluble factors produced by PHA-stimulated T8ML-1, which included INF-γ and TNF-α, but not by simple-cultured T8ML-1, caused human monocytes to exhibit erythrophagocytosis and thrombophagocytosis in vitro. PHA binding induced phosphorylation of CD3ζ chain. Furthermore, both cytotoxicity and hemophagocytosis were completely inhibited by T8ML-1.1, but eventually restored by T8ML-1.2. These data suggest that exogenous activation of TCR signaling in PTCL cells might play an important role in the formation of PTCL-associated HS.

Development of Engineered T Cells Expressing a Chimeric CD16-CD3ζ Receptor to Improve the Clinical Efficacy of Mogamulizumab Therapy Against Adult T-Cell Leukemia.

Purpose: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. Experimental Design: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo. Results: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). Conclusions: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405–16. ©2016 AACR.

Genetically engineered fixed K562 cells: potent "off-the-shelf" antigen-presenting cells for generating virus-specific T cells

BACKGROUND AIMS The human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. METHODS In order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. RESULTS In preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8⁺ T cells in HLA-A∗0201⁺ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. CONCLUSIONS Fixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Clinical significance of co-expression of CD21 and LFA-1 in B-cell lymphoma

We previously reported that the prognosis of CD21-positive diffuse large B-cell lymphoma (DLBCL) is significantly favorable to that of CD21-negative DLBCL (Otsuka et al. in Br J Haematol 127:416–424, 2004). In this study, we attempted to clarify the biological significance of CD21 expression in B-cell lymphoma (BCL) by performing in vitro experiments using CD21 transfection into a CD21-negative lymphoma cell line and analyzing clinical data from lymphoma samples. Established clones of CD21 transfectants showed homotypic aggregation in suspension culture. Analysis of integrin expression revealed that LFA-1 appeared to be expressed on CD21 transfectants, and the cell aggregation was abrogated by anti-LFA-1 antibody. The CD21 transfectants could adhere to plastic plates coated with ICAM-1. Moreover, flow cytometry and/or immunohistochemical analyses of clinical BCL samples (n = 29) revealed positive for CD21 in all cases; LFA-1 was also expressed without exception. All BCL cells isolated from cavity fluids (n = 10) failed to express both CD21 and LFA-1. These data suggest that CD21 is tightly related to LFA-1 expression in BCL and the absence of CD21/LFA-1 expression is associated with pleural/peritoneal fluid involvement by BCL, a potential indicator of disease progression of BCL.

A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient’s own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this ‘siTCR’ vector. We then compared the activity of this vector against the original, ‘conventional’ vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.