Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

The H3K9 demethylase plant homeodomain finger protein 2 regulates interleukin 4 production in CD4+ T cells.

CD4+ T cells play a key role in the immune response via their differentiation into various helper T cell subsets that produce characteristic cytokines. Epigenetic changes in CD4+ T cells are responsible for cytokine production in these subsets, although the exact molecular mechanisms remain unclear. Therefore, we investigated the effects of plant homeodomain finger protein 2 (PHF2), a histone H3K9 demethylase, on cytokine production in CD4+ T cells using T cell-specific Phf2-conditional knockout (cKO) mice in this study. we showed that interleukin 4 (Il4) expression was significantly decreased in Phf2-cKO CD4+ T cells compared to that in wild-type cells. To further elucidate the role of PHF2 in vivo, we assessed immune responses in a mouse model of ovalbumin (OVA)-induced atopic dermatitis. Phf2-cKO mice exhibited lower serum levels of OVA-specific IgE than those in wild-type mice. These findings suggest that PHF2 plays a role in promoting T helper 2 cell (Th2) function and may contribute to the pathogenesis of Th2-related allergies such as atopic dermatitis. This study demonstrated the impact of PHF2 on cytokine production in CD4+ T cells for the first time. Further studies on the PHF2-mediated epigenetic mechanisms may lead to the development of treatments for a variety of immune diseases.

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8+ T Cells.

Although the methylation status of histone H3K27 plays a critical role in CD4+ T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8+ T cell-dependent immune response remains unclear. We therefore generated T cell-specific Utx flox/flox Cd4-Cre Tg (Utx KO) mice to determine the role of Utx in CD8+ T cells. Wild-type (WT) and Utx KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. There was no significant difference in the number of Ag-specific CD8+ T cells upon primary infection between WT and Utx KO mice. However, Utx deficiency resulted in more Ag-specific CD8+ T cells upon secondary infection. Adoptive transfer of Utx KO CD8+ T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including Prdm1 encoding Blimp1, in Utx KO CD8+ T cells. We confirmed that the trimethylation level of histone H3K27 in the Prdm1 gene loci in the Utx KO cells was higher than in the WT cells. The treatment of CD8+ T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8+ T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8+ T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8+ T cells.

[Glutamine Regulates the Antitumor Activity of CD8 T Cells].

The cancer immunotherapies based on adoptive T cell therapy(ACT)has been receiving increased attention by improvement of the curative effect. T cells for ACT are harvested from the patient, then activated and expanded in vitro. However, in vitro activated T cells frequently show dysfunction after adoptive transfer, such as the exhaustion and the senescence. The exhausted/senescent T cells reduces the effector functions and fails to eliminate tumor cells. Therefore, the development of the culture method avoiding a T cellexhaustion and senescence. Recent findings revealthe dramatic changes of the metabolic status in T cells during T-cell receptor(TCR)-mediated activation. We recently reported that the activation status of glutaminolysis during TCR-stimulation determines the activated CD8 T cell fate. We considered that the therapeutic effect of ACT will be improved by the modulation of glutaminolysis. We demonstrated that the CD8 T cell exhaustion and/or senescence is prevented and the antitumor activity of adoptively transferred CD8 T cells is reinforced by the glutamine restriction during in vitro culture. The adoptively transferred CD8 T cells cultured under glutamine-restricted conditions shows higher infiltration in the tumor sites than that of CD8 T cells cultured under normal conditions. The expression of inhibitory receptors, such as PD-1 is decreased in tumor-infiltrating CD8 T cells cultured under glutamine-restricted conditions. Furthermore, the restriction of glutamine during CD8 T cell activation in vitro drives memory T cell development after adoptive transfer. The effect of glutamine restriction is antagonized by a-ketoglutarate, a metabolite of glutaminolysis. Thus, our recent findings suggest that the glutamine-restricted culture of CD8 T cells in vitro will improve the efficacy of CD8 T cell-based ACT.

Safety and persistence of WT1-specific T-cell receptor gene-transduced lymphocytes in patients with AML and MDS.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.

Reinforce the antitumor activity of CD8+ T cells via glutamine restriction.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T-cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8+ T cells using a tumor-inoculated mouse model. The adoptive transfer of tumor-specific CD8+ T cells cultured under glutamine-restricted (dGln) conditions or CD8+ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor-inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD-1 and increased Ki67 positivity among tumor-infiltrating CD8+ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8+ T-cell exhaustion in vivo. Furthermore, the transferred CD8+ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8+ T cells under Ctrl conditions. We found that the expression of a pro-survival factor and memory T cell-related transcription factors was significantly higher in CD8+ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8+ T cells. The novel adoptive transfer of tumor-specific CD8+ T cells cultured in glutamine-restricted conditions may be a promising approach to improve the efficacy of cell-based adoptive immunotherapy.

Induction of WT1-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD/SCID/IL2rgKO mice.

Induction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8(+) T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide-loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene-transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8(+) T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies.

Menin Plays a Critical Role in the Regulation of the Antigen-Specific CD8+ T Cell Response upon Listeria Infection.

Menin, a tumor suppressor protein, is encoded by the MEN1 gene in humans. Certain germinal mutations of MEN1 induce an autosomal-dominant syndrome that is characterized by concurrent parathyroid adenomas and several other tumor types. Although menin is also expressed in hematopoietic lineages, its role in CD8+ T cells remains unclear. We generated Meninflox/flox CD4-Cre (Menin-KO) mice by crossing Meninflox/flox mice with CD4-Cre transgenic (Tg) mice to determine the role of menin in CD8+ T cells. Wild-type (WT) and Menin-KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. Menin deficiency resulted in an impaired primary immune response by CD8+ T cells. On day 7, there were fewer Menin-KO OVA-specific CD8+ T cells compared with WT cells. Next, we adoptively transferred WT and Menin-KO OT-1 Tg CD8+ T cells into congenic recipient mice and infected them with L. monocytogenes expressing OVA to determine the CD8+ T cell-intrinsic effect. Menin-KO OT-1 Tg CD8+ T cells were outcompeted by the WT cells upon infection. Increased expression of Blimp-1 and T-bet, cell cycle inhibitors, and proapoptotic genes was observed in the Menin-KO OT-1 Tg CD8+ T cells upon infection. These data suggest that menin inhibits differentiation into terminal effectors and positively controls proliferation and survival of Ag-specific CD8+ T cells that are activated upon infection. Collectively, our study uncovered an important role for menin in the immune response of CD8+ T cells to infection.

Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells.

A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T-cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1-type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1-deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin-12- and interferon-γ-independent manner. The increase of Th1-type immune responses was confirmed in vivo in Gfi1-deficient mice using a murine model of nickel allergy and delayed-type hypersensitivity (DTH). The expression levels of Th1-related transcription factors were found to increase in Gfi1-deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1-type immune response.

Human tolerogenic dendritic cells generated with protein kinase C inhibitor are optimal for functional regulatory T cell induction - A comparative study.

Tolerogenic dendritic cells (tDCs) are a promising therapeutic tool for specific induction of immunological tolerance. Human tDCs can be generated ex vivo using various compounds. However, the compound(s) most suitable for clinical application remain undefined. We compared the tolerogenic properties of tDCs treated with protein kinase C inhibitor (PKCI), dexamethasone, vitamin D3 (Vit D3), rapamycin (Rapa), interleukin (IL)-10, transforming growth factor (TGF)-ß, and a combination of peroxisome proliferator-activated receptor γ agonist and retinoic acid. All tDCs had a semi-mature DC phenotype. PKCI-, TGF-ß-, and Rapa-tDCs showed CCR7 expression and migration to CCL19, but other tDCs showed little or none. PKCI- and IL-10-tDCs induced functional regulatory T cells more strongly than other tDCs. The tolerogenic properties of all tDCs were stable against proinflammatory stimuli. Furthermore, PKCI-tDCs were generated from patients with rheumatoid arthritis and primary Sjögren's syndrome. Therefore, PKCI-tDCs showed the characteristics best suited for tolerance-inducing therapy.

Successful rituximab treatment in an elderly patient with recurrent thrombotic thrombocytopenic purpura.

An 81-year-old man presenting with fever, neurological symptoms, thrombocytopenia, and hemolytic anemia was diagnosed with acquired idiopathic thrombotic thrombocytopenic purpura (TTP). His disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) activity was <1% and the ADAMTS13 inhibitor titer was 3.2 BU/ml. He received plasma exchange and steroid administration until remission was achieved. Seven months later, he suffered from paralysis of the right hand, hemolytic anemia, and thrombocytopenia. We confirmed TTP recurrence based on ADAMTS13 activity <1% and an ADAMTS13 inhibitor titer of 19.4 BU/ml. Four infusions of rituximab were administered in addition to plasma exchange and steroid pulse therapy. Platelet count recovery was observed within 5 days. No severe side effects related to rituximab occurred. Although rituximab has not been approved for TTP in Japan, we report the efficacy and safety of rituximab in an elderly patient with recurrent TTP. We suggest that rituximab therapy should be started as soon as possible for recurrent TTP in patients with high titers of ADAMTS13 inhibitor.

Development of a novel redirected T-cell-based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/ß genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Protein kinase C inhibitor generates stable human tolerogenic dendritic cells.

Tolerogenic dendritic cells (DCs) are a promising tool for a specific form of cellular therapy whereby immunological tolerance can be induced in the context of transplantation and autoimmunity. From libraries of bioactive lipids, nuclear receptor ligands, and kinase inhibitors, we screened conventional protein kinase C inhibitors (PKCIs) bisindolylmaleimide I, Gö6983, and Ro32-0432 with strong tolerogenic potential. PKCI-treated human DCs were generated by subjecting them to a maturation process after differentiation of immature DCs. The PKCI-treated DCs had a semimature phenotype, showing high production of IL-10, and efficiently induced IL-10-producing T cells and functional Foxp3(+) regulatory T cells from naive CD4(+) T cells, thus eliciting a strong immunosuppressive function. They also showed CCR7 expression and sufficient capacity for migration toward CCR7 ligands. Additionally, PKCI-treated DCs were highly stable when exposed to inflammatory stimuli such as proinflammatory cytokines or LPS. Conventional PKCIs inhibited NF-κB activation of both the canonical and noncanonical pathways of DC maturation, thus suppressing the expression of costimulatory molecules and IL-12 production. High production of IL-10 in PKCI-treated DCs was due to not only an increase of intracellular cAMP, but also a synergistic effect of increased cAMP and NF-κB inhibition. Moreover, PKCI-treated mouse DCs that had properties similar to PKCI-treated human DCs prevented graft-versus-host disease in a murine model of acute graft-versus-host disease. Conventional PKCI-treated DCs may be useful for tolerance-inducing therapy, as they satisfy the required functional characteristics for clinical-grade tolerogenic DCs.

[A Fatal Case of Severe Fever with Thrombocytopenia Syndrome Complicated by Hemophagocytic Lymphohistiocytosis].

Severe fever with thrombocytopenia syndrome (SFTS) is a recently identified emerging viral infectious disease in China that is caused by a novel phlebovirus in the family Bunyaviridae, SFTS virus, with an average case fatality rate of 12-30%. A cytokine storm with abnormally expressed cytokine profiles is associated with the disease severity. Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and lifethreatening syndrome associated with excessive immune activation. We report herein on a fatal case of SFTS complicated by HLH. Consecutive plasma exchange and immunomodulatory therapy was ineffective in our case. The pathognomonic histological feature was necrotizing lymphadenitis with massive hemophagocytosis of systemic lymphoid tissues with SFTS viruses and SFTS-RNA copies. No specific treatment of SFTS is available, and an effective treatment strategy for patients with rapidly progressing SFTS has not been established. Appropriate immunomodulatory therapy is necessary for SFTS patients complicated by HLH.

[Pulmonary Nocardiosis due to Nocardia asiatica in a Patient with ANCA-associated Vasculitis].

Nocardia asiatica is a rare causative organism responsible for opportunistic infection, and was first reported by Kageyama et al. in 2004. We report herein on a very rare case of N. asiatica infection in a 76-year old male patient with ANCA-associated vasculitis and a history of pulmonary tuberculosis. The patient developed pulmonary nocardiosis due to N. asiatica while receiving glucocorticoid therapy. Chest computed tomography demonstrated multiple granules and cavity formation mainly in the left lower lobe. From the images, we suspected opportunistic infection, possibly pulmonary tuberculosis or pulmonary damage due to ANCA-associated vasculitis. Nocardia sp. was detected from a bronchoalveolar lavage culture and N. asiatica was identified by 16S ribosomal DNA gene sequencing. Cranial magnetic resonance imaging revealed no abnormality. Administration of Doripenem (1.5g/day) and sulfamethoxazole-trimethoprim (4g/day) was started, and the patient's clinical and imaging findings promptly improved. Thereafter, he received sulfamethoxazole-trimethoprim (2g/day) and prednisolone (10 mg/day) as maintenance therapy for ANCA-associated vasculitis for more than one year, and there has since been no recurrence of the Nocardia infection.

[Severe fever with thrombocytopenia syndrome, an emerging infectious disease for hematologists].

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by tick-borne SFTS virus infection that was first described in rural areas of China in 2011. Since then, SFTS has also been found in the western part of Japan in 2013. The clinical pictures of SFTS are characterized by abrupt onset of fever and gastrointestinal symptoms, followed by a progressive decline in platelet and white blood cell counts. Disseminated intravascular coagulation and hemophagocytic lymphohistiocytosis are also frequently observed in the patients with advanced phase of SFTS. No specific treatment of SFTS is available, and avoiding tick bites is an important way to prevent the infection of SFTS virus. Standard precautions should be applied to SFTS patients to prevent human-to-human transmission of SFTS virus. Since ticks bearing SFTS virus are found in all area of Japan including Hokkaido, this disease has become a substantial risk to public health in all parts of Japan.

Severe fever with thrombocytopenia syndrome mimicking intravascular lymphoma.

A 63-year-old woman was admitted to our hospital, because of a high fever and general malaise which had persisted for several days. Laboratory findings showed leucopenia, thrombocytopenia, and abnormal liver functions. A bone marrow smear revealed hemophagocytosis. Since a diagnosis of intravascular large B cell lymphoma was strongly suspected, a random skin biopsy was performed but revealed no evidence of malignant lymphoma. She was treated with steroids, blood product transfusions, and antibiotics, and then gradually recovered. The severe fever with thrombocytopenia syndrome (SFTS) viral genome was detected in her serum obtained in the acute phase. Therefore, the final diagnosis was SFTS, which is among the major causes of hemophagocytic syndrome.

The first identification and retrospective study of Severe Fever with Thrombocytopenia Syndrome in Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression, but non-specifically enhanced on induction of Tax expression.

BACKGROUND: Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4(+) T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis. RESULTS: Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26-34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02. CONCLUSIONS: HTLV-1 gene expression in primary CD4(+) T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.