Eisei Kondo

Efficient Generation of Antigen-Specific Cytotoxic T Cells Using Retrovirally Transduced CD40-Activated B Cells

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8+ T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.

Nodal cytotoxic lymphoma spectrum: a clinicopathologic study of 66 patients.

The expression of cytotoxic granule-associated proteins has been reported in some T-cell or natural killer (NK)-cell lymphomas of mostly extranodal origin, but rarely of nodal origin except for anaplastic large cell lymphoma (ALCL) and Hodgkins disease (HD). This study analyzed 66 nodal lymphomas expressing T-cell intracellular antigen-1 (TIA-1) and/or granzyme B to characterize the clinicopathologic spectrum of these neoplasms. Four main groups could be delineated. The first group consisted of p80/anaplastic lymphoma kinase (ALK)-positive ALCL (n = 35). The patients were 2 to 62 years of age (median age, 16 years), and the lymphomas pursued a relatively indolent clinical course. The tumors were phenotypically of either T- or null-cell type with constant expression of CD30, epithelial membrane antigen (EMA), and p80/ALK, but not CD15 or BCL2. None harbored Epstein-Barr virus (EBV). The second group consisted of peripheral T/NK-cell lymphoma, the nodal high-grade cytotoxic type (n = 13). The patients were 29 to 72 years in age (median age, 55 years), and the tumors pursued an aggressive clinical course. The tumors often showed pleomorphic, anaplastic, or centroblastoid morphology, and were featured by either EBV association or CD56 expression. The third group consisted of peripheral T-cell lymphoma, of the nodal low-grade cytotoxic type (n = 8). The patients, three men and five women, were 31 to 75 years old (median age, 61 years). Notably, six of them exhibited lymphoepithelioid (Lennerts) lymphoma. The fourth group consisted of cytotoxic Hodgkins-like ALCL/HD (n = 10), included seven cases of Hodgkins-like ALCL and three cases of HD, and was characterized by the presence of Reed-Sternberg cells and often the CD15+ phenotype. The patients were all men except for one woman, and they ranged in age from 24 to 84 years (median age, 62 years). The link among these four groups was reinforced by the presence of a highly characteristic large cell with horseshoelike or reniform nuclei-the frequent expression of CD30 and EMA-and the often lack of T-cell receptor-alphabeta. In this series, the expression of p80/ALK and CD56 was also associated with favorable and poor prognoses respectively (p<0.001, log-rank test).

Clinicopathologic analysis of TAFRO syndrome demonstrates a distinct subtype of HHV‐8‐negative multicentric Castleman disease

Multicentric Castleman disease (MCD) describes a heterogeneous group of disorders involving systemic inflammation, characteristic lymph node histopathology, and multi‐organ dysfunction because of pathologic hypercytokinemia. Whereas Human Herpes Virus‐8 (HHV‐8) drives the hypercytokinemia in a cohort of immunocompromised patients, the etiology of HHV‐8‐negative MCD is idiopathic (iMCD). Recently, a limited series of iMCD cases in Japan sharing a constellation of clinical features, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O) has been described as TAFRO syndrome. Herein, we report clinicopathological findings on 25 patients (14 males and 11 females; 23 Japanese‐born and two US‐born), the largest TAFRO syndrome case series, including the first report of cases from the USA. The median age of onset was 50 years old (range: 23–72). The frequency of each feature was as follows: thrombocytopenia (21/25), anasarca (24/25), fever (21/25), organomegaly (25/25), and reticulin fibrosis (13/16). These patients frequently demonstrated abdominal pain, elevated serum alkaline phosphatase levels, and acute kidney failure. Surprisingly, none of the cases demonstrated marked hypergammoglobulinemia, which is frequently reported in iMCD. Lymph node biopsies revealed atrophic germinal centers with enlarged nuclei of endothelial cells and proliferation of endothelial venules in interfollicular zone. 23 of 25 cases were treated initially with corticosteroids; 12 patients responded poorly and required further therapy. Three patients died during the observation period (median: 9 months) because of disease progression or infections. TAFRO syndrome is a unique subtype of iMCD that demonstrates characteristic clinicopathological findings. Further study to clarify prognosis, pathophysiology, and appropriate treatment is needed. Am. J. Hematol. 91:220–226, 2016.

Recurrent DUX4 fusions in B cell acute lymphoblastic leukemia of adolescents and young adults

The oncogenic mechanisms underlying acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA; 15–39 years old) remain largely elusive. Here we have searched for new oncogenes in AYA-ALL by performing RNA-seq analysis of Philadelphia chromosome (Ph)-negative AYA-ALL specimens (n = 73) with the use of a next-generation sequencer. Interestingly, insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. A transplantation assay in mice demonstrated that expression of DUX4-IGH in pro-B cells was capable of generating B cell leukemia in vivo. DUX4 fusions were preferentially detected in the AYA generation. Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

Activated human umbilical cord blood dendritic cells kill tumor cells without damaging normal hematological progenitor cells.

Apart from their role as antigen presenting cells, human peripheral blood monocyte and CD34+ cell‐derived dendritic cells (DC), have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumoricidal activity of human cord blood dendritic cells (CBDC). In this article, we report that human cord blood monocyte‐derived DC acquire the ability to kill hematological tumor cells, after activation with lipopolysaccharide (LPS) or γ‐interferon (IFN‐γ), associated with the enhanced TNF‐α‐related apoptosis‐inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14‐positive cells collected from cord blood were induced to CBDC in vitro. After activation with IFN‐γ for 12 h, CBDC exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS induced cytotoxicity against Daudi and Jurkat cells. However, both LPS‐ and IFN‐γ‐stimulated CBDC showed no cytotoxic activity against normal CD14‐negative cord blood mononuclear cells. The formation of umbilical cord hematopoietic progenitor colonies, identified as burst‐forming unit‐erythroid and colony‐forming unit granulocyte‐macrophage, was not inhibited by stimulated or unstimulated CBDC. IFN‐γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface tumor necrosing factor‐α and Fas Ligand as analyzed by flow cytometry. Our results show that activated CBDC can serve as cytotoxic cells against hematological tumor cells without damaging the normal hematopoietic progenitor cells. (Cancer Sci 2005; 96: 127–133)

Allogeneic hematopoietic stem cell transplantation for advanced extranodal natural killer/T-cell lymphoma, nasal type.

The prognosis for patients with advanced or refractory extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) is extremely poor. Thus, allogeneic stem cell transplantation (allo-HSCT) should be considered for this disease. However, reports of allo-HSCT for ENKL are limited because of the rarity of the disease. Here, we describe the clinical course of 12 cases of advanced and refractory ENKL treated with allo-HSCT, including five cases with cord blood transplant. With a median follow-up of 13 months (range, 1–168 months), seven patients are alive in remission, five have died, and one treatment-related death occurred. All patients with disease progression at transplant died of disease progression, whereas seven of eight patients with a complete or partial response are long-term survivors. Allo-HSCT is a feasible and promising consolidation therapy for advanced and relapsed ENKL. The disease status before allo-HSCT is well associated with general outcome, and thus induction treatment is very important for this disease.

Programmed Death-1 Pathway in Host Tissues Ameliorates Th17/Th1-Mediated Experimental Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation, but its pathogenesis remains unclear. We investigated the role of the programmed death-1 (PD-1) pathway in chronic GVHD using a well-defined mouse model of B10.D2 (H-2d) donor to BALB/c (H-2d) recipients. PD-1 expression on allogeneic donor T cells was upregulated continuously in chronic GVHD development, whereas PD-L1 expression in host tissues was transiently upregulated and declined to basal levels in the late posttransplant period. Blockade of the PD-1 pathway by anti–PD-1, anti–PD-L1, or anti–PD-L2 mAbs exacerbated clinical and pathologic chronic GVHD. Chimeric mice revealed that PD-L1 expression in host tissues suppressed expansion of IL-17+IFN-γ+ T cells, and that PD-L1 expression on hematopoietic cells plays a role in the development of regulatory T cells only during the early transplantation period but does not affect the severity of chronic GVHD. Administration of the synthetic retinoid Am80 overcame the IL-17+IFN-γ+ T cell expansion caused by PD-L1 deficiency, resulting in reduced chronic GVHD damage in PD-L1−/− recipients. Stimulation of the PD-1 pathway also alleviated chronic GVHD. These results suggest that the PD-1 pathway contributes to the suppression of Th17/Th1-mediated chronic GVHD and may represent a new target for the prevention or treatment of chronic GVHD.

Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group

The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR–ABL-negative ALL. Patients aged 15–24 years (n=139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58–75%) and 73% (95% CI 64–80%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, P<0.001). These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR–ABL-negative ALL.

Identification of an HLA-A24-restricted cytotoxic T lymphocyte epitope from human papillomavirus type-16 E6: The combined effects of bortezomib and interferon-γ on the presentation of a cryptic epitope

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV‐16), and since the HPV‐16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)‐mediated immunotherapy. Nevertheless, only a limited number of HPV‐16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV‐16 E649–57 epitope restricted by HLA‐A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA‐A*2402 and HPV‐16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV‐16 and HLA‐A*2402, the cells became susceptible to lysis when transduced with E6‐E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)‐γ alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN‐γ fully restored CTL‐mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV‐16 E6 and HLA‐A*2402 was found to induce IFN‐γ production by specific CTLs. Tetramer analysis further revealed that induction of E649–57‐specific T cells was possible in 5 of 7 patients with HPV‐16‐positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E649–57 peptide. Thus, these findings together indicate that E649–57 is a candidate epitope for immunotherapy and immunological monitoring of such patients.

CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC‐vaccine trials, induction of tumour antigen‐specific immunity is observed frequently and well‐documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40‐activated B cells (CD40‐B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell‐based vaccines we performed a single‐patient analysis of 502 patients from DC‐based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re‐evaluated the CD40‐B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen‐presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40‐B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen‐presentation and migratory properties.