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The clinical efficacy of T-cell therapies based on T cells transduced with genes encoding tumor-specific T-cell receptors (TCR-T) is related to the in vivo persistence of the T cells. To improve persistence without modifying TCR affinity, we instead modified intracellular signaling, using artificial T cell–activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3ζ. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3ζ, and enhanced downstream signaling from the supramolecular activation cluster. ATAMs were retrovirally introduced into human CMV-specific or NY-ESO-1–specific TCR-transduced CD8+ T lymphocytes, and downstream functionality was then examined. ATAM-transduced NY-ESO-1 TCR-T cells were also investigated using the U266-xenograft mouse model. ATAMs were successfully transduced and localized to the cell membrane. ATAM-transduced CMV-specific T cells retained their cytotoxic activity and cytokine production against peptide-pulsed target cells without altering antigen-specificity and showed resistance to activation-induced cell death. Upon both single and repeated stimulation, CD3ζ/4-1BB–transduced T cells had superior proliferation to the CD3ζ-transduced T cells in both the CMV-specific and the NY-ESO-1 TCR-T models and significantly improved antitumor activity compared with untransduced T cells both in vitro and in a mouse xenograft model. ATAM-transduced TCR-T cells demonstrated improved proliferation and persistence in vitro and in vivo. This strategy to control the intracellular signaling of TCR-T cells by ATAM transduction in combination with various tumor-specific TCRs may improve the efficacy of TCR-T therapy. Cancer Immunol Res; 6(6); 733–44. ©2018 AACR.

pRetroX-TetOne vector sequence with CD19CAR and tEGFR

T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8+ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19+ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(+) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the “On” and “Off” status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy–related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene–modified T cells. Cancer Immunol Res; 4(8); 658–68. ©2016 AACR.See related Spotlight by June, p. 643.

Sequence data of CD3zeta and ATAM

Sequence data of CD3 and ATAM

A left ventricular assist device (LVAD) is a treatment option for patients with end-stage heart failure; however, a certain number of patients on durable LVADs are diagnosed with malignancy. Radiation therapy (RT) for patients with durable LVADs has safety concerns, because RT may interfere with the device. Herein, we report a case of RT during durable LVAD management. A 48-year-old man with a durable LVAD was diagnosed with sinusitis. As his symptoms were resistant to drug therapy, endoscopic sinus surgery was performed, and extranodal NK/T-cell lymphoma, nasal type (ENKL) was pathologically detected. Since RT was the first-line treatment for ENKL, we conducted two types of irradiation experiments to determine whether RT can be safely performed in patients with durable LVADs as follows: (1) assessing the extent of the radiation levels at each site and evaluating device malfunction by irradiating the lesion sites in the patient model with the same protocol as planned, and (2) evaluating device malfunction by directly irradiating the durable LVAD equipment once at the scheduled total dose. The radiation doses at the pump, driveline, system controller, power cable, and power module of the durable LVAD reached 7.86 cGy, 6.34 cGy, 0.66 cGy, 0.38 cGy, and 0.14 cGy, respectively. In both experiments, durable LVAD malfunction or any type of alarm was not observed. We concluded that RT could be safely performed with chemotherapy in this patient and our irradiation experiments can be applied to RT for other malignancies.

CCR Translation for the Article from Development of Tumor-Reactive T Cells After Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplant for Chronic Lymphocytic Leukemia

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19(+) target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19(+) target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

Despite the remarkable initial efficacy of CD19 chimeric Ag receptor T (CAR-T) cell therapy, a high incidence of relapse has been observed. To further increase treatment efficacy and reduce the rate of escape of Ag-negative cells, we need to develop CAR-T cells that target other Ags. Given its restricted expression pattern, CD37 was considered a preferred novel target for immunotherapy in hematopoietic malignancies. Therefore, we designed a CD37-targeting CAR-T (CD37CAR-T) using the single-chain variable fragment of a humanized anti-CD37 Ab, transmembrane and intracellular domains of CD28, and CD3ζ signaling domains. High levels of CD37 expression were confirmed in B cells from human peripheral blood and bone marrow B cell precursors at late developmental stages; by contrast, more limited expression of CD37 was observed in early precursor B cells. Furthermore, we found that human CD37CAR-T cells with longer spacer lengths exhibited high gene transduction efficacy but reduced capacity to proliferate; this may be due to overactivation and fratricide. Spacer length optimization resulted in a modest transduction efficiency together with robust capacity to proliferate. CD37CAR-T cells with optimized spacer length efficiently targeted various CD37+ human tumor cell lines but had no impact on normal leukocytes both in vitro and in vivo. CD37CAR-T cells effectively eradicated Raji cells in xenograft model. Collectively, these results suggested that spacer-optimized CD37CAR-T cells could target CD37-high neoplastic B cells both in vitro and in vivo, with only limited interactions with their normal leukocyte lineages, thereby providing an additional promising therapeutic intervention for patients with B cell malignancies.

To overcome the delayed or failed engraftment after unrelated cord blood transplantation (CBT), we conducted a multicenter phase II study of intrabone single-unit CBT without antithymocyte globulin (ATG) for adult patients with hematological malignancies (UMIN-CTR, UMIN000020997). Sixty-four patients received an intrabone injection of unwashed (n = 61) or washed (n = 3) cord blood after local anesthesia. All injection-related adverse events were mild and resolved spontaneously. Sixty-two patients were evaluable for the efficacy of intrabone CBT of serological HLA-A, -B, and -DR ≥ 4/6 matched cord blood with a median number of 2.57 × 10^7/kg cryopreserved total nucleated cells. The probability of survival with neutrophil engraftment on day 28 was 77.4% (95% confidence interval, 67.0–85.8%), which exceeded the threshold value. The cumulative incidences of neutrophils ≥ 0.5 × 10^9/L on day 60 was 80.6% (68.2–88.6%), with a median time to recovery of 21 days after transplantation. The cumulative incidences of platelets ≥ 20 × 10^9/L and platelets ≥ 50 × 10^9/L on day 100 were 75.8% (62.6–84.9%) and 72.6% (59.4–82.1%), respectively, with median time to platelets ≥ 20 × 10^9/L and platelets ≥ 50 × 10^9/L of 38 and 45 days after transplantation, respectively. The cumulative incidences of grade II–IV and III–IV acute graft-versus-host disease were 29.0% and 6.5%, respectively. All responded to steroid therapy, and secondary treatments were not required. The present study suggests the efficacy of intrabone single-unit CBT without ATG in terms of early engraftment and controllable acute graft-versus-host disease.

Mesenchymal stem cells (MSCs) have immunomodulatory properties and support hematopoiesis in the bone marrow (BM). To develop a new strategy to not only prevent graft‐vs‐host disease (GVHD) but also to enhance engraftment, a phase I trial of cord blood transplantation (CBT) combined with intra‐BM injection of MSCs (MSC‐CBT) was designed. Third‐party BM‐derived MSCs were injected intra‐BM on the day of CBT. The conditioning regimen varied according to patient characteristics. GVHD prophylaxis was tacrolimus and methotrexate. The primary endpoint was toxicity related to intra‐BM injection of MSCs. Clinical outcomes were compared with those of six controls who received CBT alone. Five adult patients received MSC‐CBT, and no adverse events related to intra‐BM injection of MSCs were observed. All patients achieved neutrophil, reticulocyte, and platelet recoveries, with median times to recoveries of 21, 35, and 38 days, respectively, comparable with controls. Grade II‐IV acute GVHD developed in three controls but not in MSC‐CBT patients. No patients developed chronic GVHD in both groups. At 1 year after transplantation, all MSC‐CBT patients survived without relapse. This study shows the safety of MSC‐CBT, and the findings also suggest that cotransplantation of MSCs may prevent GVHD with no inhibition of engraftment. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as number 000024291., Mesenchymal stem cells (MSCs), which were derived from bone marrow (BM) of third‐party donor, were injected intra‐BM of recipient 4 hours before cord blood transplantation (CBT). This study shows the safety of CBT combined with intra‐BM injection of MSCs, and also suggests that co‐transplantation of MSCs may prevent graft‐vs‐host disease without inhibition of engraftment.

An artificial T cell adaptor molecule (ATAM) was generated to improve persistence of T cell receptor (TCR) gene-transduced T (TCR-T) cells compared to such persistence in a preceding study. ATAMs are gene-modified CD3ζ with the intracellular domain of 4-1BB inserted in the middle of CD3ζ. NY-ESO-1 TCR-T cells transduced with an ATAM with two separated virus vectors demonstrated superior proliferation upon antigen stimulation. To further develop clinically applicable ATAM-transduced TCR-T cells, we attempted to make a single virus vector to transduce the TCR and ATAM simultaneously. Because we failed to observe improved proliferation capacity upon stimulation after one virus vector (1vv) transduction, we compared TCR-T cells transduced with 1vv and two virus vector (2vv) methods to elucidate the reason. In Jurkat reporter cells, an ATAM transduced by the 2vv method demonstrated a higher intensity than by the 1vv method, and the ATAM intensity was associated with increased nuclear factor κB (NF-κB) signals upon stimulation. In ATAM-transduced primary T cells, a transduced ATAM by the 2vv method showed higher intensity and better proliferation. ATAM-transduced TCR-T cells demonstrated improved proliferation only when the ATAM was transduced at a higher intensity. To create a simpler transduction method, we need to develop a strategy to make a higher ATAM expression to prove the efficacy of ATAM transduction in TCR-T therapy., Graphical Abstract, An artificial T cell adaptor molecule (ATAM) with a 4-1BB intracellular domain in the middle of CD3ζ was developed and transduced into T cells simultaneously with the NY-ESO-1 TCR gene. We demonstrated that gene transfer of an ATAM above a certain threshold would be necessary to boost cell proliferation after stimulation.

Hydroxyapatite/collagen (HAP/Col) composite has a nanostructure and composition similar to that of natural bone. Herein, we have evaluated the beneficial effects of acid-electrolyzed functional water (FW) in combination with HAP/Col composite as an irrigation material in a rat calvarium defect model. The rats were divided into four groups: control, PBS irrigation; FW, FW irrigation; HAP/Col, filled with HAP/Col; FW + HAP/Col, FW irrigation prior to HAP/Col filling. Bone volume (BV) and bone mineral density (BMD) of the newly formed bone were analyzed by microcomputed tomography. The results indicated that the combined use of FW and HAP/Col significantly augmented both BV (12.25 ± 1.93 mm3 , control: 3.22 ± 0.55 mm3 , 6 weeks) and BMD (120.09 ± 14.76 cm3 /mg vs. control: 54.67 ± 7.20 cm3 /mg, 6 weeks) in a time-dependent manner, which might be attributed to the soluble factor-inducing ability of FW. Based on this assumption, bFGF concentration in peripheral blood was measured. bFGF concentration was significantly increased in the FW + HAP/Col group (68.25 ± 9.2 pg/ml vs. control: 21.70 ± 8.18 pg/ml, 6 hr). Real-time PCR demonstrated significant augmentation of MCSF (2.82 ± 0.59-fold), RANKL (2.51 ± 0.33-fold) and BMP7 (1.66 ± 0.25-fold) (bone regeneration-related genes) and PDGF (1.31 ± 0.15-fold), VEGF (3.27 ± 0.42-ld) and IL-8 (6.77 ± 2.02-fold) (angiogenic genes) mRNAs in the FW + HAP/Col group. Taken together, these results suggest that the combined use of FW and HAP/Col induces bone regeneration, presumably by inducing the factors contributing to bone regeneration and angiogenesis.

Genomic deletion of donor-patient-mismatched HLA alleles in leukemic cells is a major cause of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Mismatched HLA is frequently lost as an individual allele or a whole region in HLA-class I, however, it is downregulated in HLA-class II. We hypothesized that there might be a difference in T cell recognition capacity against epitopes associated with HLA-class I and HLA-class II and consequently such allogeneic immune pressure induced HLA alterations in leukemic cells. To investigate this, we conducted in vitro experiments with T cell receptor-transduced T (TCR-T) cells. The cytotoxic activity of NY-ESO-1-specific TCR-T cells exhibited similarly against K562 cells with low HLA-A*02:01 expression. However, we demonstrated that the cytokine production against low HLA-DPB1*05:01 expression line decreased gradually from the HLA expression level approximately 2-log lower than normal expressors. Using sort-purified leukemia cells before and after HSCT, we applied the next-generation sequencing, and revealed that there were several marked downregulations of HLA-class II alleles which demonstrated consistently low expression from pre-transplantation. The marked downregulation of HLA-class II may lead to decreased antigen recognition ability of antigen-specific T cells and may be one of immune evasion mechanism associated with HLA-class II downregulation.

Cytomegalovirus (CMV) infection is a major complication in allogeneic stem cell transplantation. The utility of CMV prophylaxis with letermovir has been reported; however, the specific applications remain unclear. In this study, we retrospectively analyzed large-scale registry data (N = 10 480) to clarify the risk factors for nonrelapse mortality (NRM) in connection with CMV reactivation. First, we identified risk factors for CMV reactivation using multivariate analysis and developed a scoring model. Although the model effectively stratified reactivation risk into 3 groups (43.7% vs 60.9% vs 71.5%; P < .001), the 3-year NRM was significantly higher in patients with CMV reactivation, even in the low (20.9% vs 13.0%, P < .001), intermediate (21.4% vs 15.6%; P < .001), and high (29.3% vs 18.0%; P < .001) reactivation risk groups. Next, survival analysis considering competing risks, time-dependent covariates, and interaction terms for exploring the heterogeneous impact of CMV reactivation on NRM in the training cohort revealed that chronic myeloid leukemia (CML) (hazard ratio [HR], 1.76; 95% confidence interval [CI], 1.05-2.96; P = .033), good performance status (PS) (HR, 1.42; 95% CI, 1.04-1.94; P = .028), HLA-matched donor (HR, 1.34; 95% CI, 1.06-1.70; P = .013), and standard-risk disease (HR, 1.28; 95% CI, 1.04-1.58; P = .022) were associated with increased NRM. In the test cohort, CMV reactivation was significantly associated with increased 3-year NRM among patients with 2 to 4 factors (22.1% vs 13.1%; P < .001) but was comparable among patients with 0 or 1 factor (23.2% vs 20.4%; P = .62). We propose that CMV prophylaxis should be determined based on reactivation risk, as well as these other factors.

A prospectively registered observational study was conducted to assess the significance of allogeneic hematopoietic stem cell transplantation from highly HLA-matched unrelated donors (UD) and cord blood (CB) on outcomes in adult acute leukemia (AL) and myelodysplastic syndrome (MDS). Between 2007 and 2015, 231 transplant-eligible patients were registered for a phase 2 study of alternative donor transplantation. After registration, a sufficient time period was given to find appropriate UD. Patients received CB transplantation (CBT) if an appropriate UD was unavailable. In total, 119 patients received CBT (106 AL and 13 MDS) and 91 patients received UD transplantation (UDT) (86 AL and 5 MDS). The median age was 39 years in both groups. The primary objective was overall survival (OS); secondary objectives included cumulative incidences of non-relapse mortality (NRM) and relapse, and disease-free survival. Diagnosis, disease status at transplantation, refined disease risk index, and hematopoietic cell transplant-specific comorbidity index did not differ between UDT and CBT. In multivariate analyses, graft source was not a significant risk factor for all objectives. In adjusted analyses, UDT and CBT showed similar OS, NRM, and relapse in this prospective study. CB can be a comparable alternative stem cell source to UD by achieving a timely transplant.

Objective Little information is available about the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with secondary myelofibrosis from essential thrombocythemia (ET) and polycythemia vera (PV). A nationwide retrospective study of the outcome of HSCT for post-ET and post-PV myelofibrosis was conducted in Japan. Patients and Methods Clinical data for patients with post-ET (n=29) and post-PV (n=9) myelofibrosis who had received first allogeneic HSCT were extracted from the Transplant Registry Unified Management Program, which is a registry of the outcomes of HSCT in Japan. Results Five patients died without neutrophil recovery within 60 days after transplantation. The incidence of neutrophil recovery was significantly lower in umbilical cord blood (UCB) transplantation than in related donor transplantation (40% vs. 92%, p=0.010). The 1-year non-relapse mortality for post-ET and post-PV myelofibrosis was 35% and 27%, respectively (p=0.972). No patient or transplantation characteristics were associated with non-relapse mortality. The 4-year overall survival for post-ET and post-PV myelofibrosis was 46% and 65%, respectively (p=0.362). A univariate analysis identified UCB transplantation (vs. related donor, p=0.017) and ≥10 times red blood cell transfusions before transplantation (vs.

Almost comparable transplantation outcomes have been reported with HLA-matched unrelated donor transplantation (UDT) and cord blood transplantation (CBT). We conducted a prospective phase 2 study to assess the efficacy and safety of single-unit myeloablative CBT in adult leukemia and myelodysplastic syndrome. Because the day 180 survival of UDT was approximately 80%, we determined the alternative hypothesis of expected day 180 survival with a successful engraftment rate of 80% and set the null hypothesis of threshold rate at 65%. Sixty-two patients (median age, 37 years) were registered, including 28 with acute myelogenous leukemia, 25 with acute lymphoblastic leukemia, and 9 with myelodysplastic syndrome. Of 61 eligible patients, 52 were successfully engrafted and survived at day 180 (85%; 95% confidence interval, 74% to 93%). Single-unit CBT was judged to be effective because the null hypothesis was rejected (P.001). Furthermore, neutrophil engraftment was observed in 57 patients (92%); the incidences of grade II-IV acute and chronic graft-versus-host disease were 30% and 32%, respectively; and the cumulative incidences of nonrelapse mortality and relapse at 2 years were 18% and 13%, respectively. The present study showed favorable survival outcomes with single-unit CBT. Therefore, this method may be considered if a well-HLA-matched UDT cannot be obtained.

Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) is an enzyme that converts cholesterol to cholesteryl esters. A recent in vivo study reported that inhibiting ACAT1 enzyme activity upregulates the membrane cholesterol levels of T cells, enhancing their cytotoxic function. In the present study, we investigated whether the presence of the ACAT1 single nucleotide polymorphism rs11545566 in transplant donors affected the risk of graft-versus-host disease (GVHD) in 116 adult patients who underwent bone marrow transplantation from human leukocyte antigen-identical sibling donors, and who received GVHD prophylaxis with short-term methotrexate and cyclosporine. The frequencies of the AA, AG, and GG genotypes in the donors were 31%, 45%, and 24%, respectively. The cumulative incidences of grade II–IV acute GVHD on day 100 in patients whose donors had AA vs. non-AA genotypes were 6% and 18%, respectively, and those of extensive chronic GVHD at 2 years were 7% and 32%, respectively. Multivariate analyses demonstrated that donor rs11545566 non-AA genotypes showed a trend toward a higher incidence of grade II–IV acute GVHD (P = 0.079), and were significantly associated with a higher incidence of extensive chronic GVHD (P = 0.021). These results suggest that donor ACAT1 rs11545566 genotype may be predictive of GVHD., ファイル公開：2021/01/01

IntroductionA better long-term quality of life after umbilical cord blood transplantation (CBT) is observed compared with transplants from other alternative donors, whereas graft failure and relapses after CBT are still major issues. To minimise graft failure and relapse after CBT, intensification of conditioning by the addition of high-dose cytosine arabinoside (CA) and concomitant continuous use of granulocyte-colony stimulating factor (G-CSF) are reported to convey a significantly better survival after CBT in some retrospective studies. To confirm the effect of G-CSF plus CA combination, in addition to the standard conditioning regimen, cyclophosphamide (CY)/total body irradiation (TBI), we design a randomised controlled study comparing CA/CY/TBI with versus without G-CSF priming (G-CSF combined conditioned cord blood transplantation [G-CONCORD] study).Methods and analysisThis is a multicentre, open-label, randomised phase III study that aimed to compare G-CSF+CA/CY/TBI as a conditioning regimen for CBT with CA/CY/TBI. Patients with acute myeloid leukaemia or myelodysplastic syndrome, aged 16–55 years, are eligible. The target sample size is 160 and the registration period is 4 years. The primary endpoint is the 2-year disease-free survival rate after CBT. The secondary endpoints are overall survival, relapse, non-relapse mortality, acute and chronic graft-versus-host disease, engraftment rate, time to neutrophil recovery, short-term adverse events, incidence of infections and causes of death.This study employs a single one-to-one web-based randomisation between the with-G-CSF versus without-G-CSF groups after patient registration. Combination of high-dose CA and CY/TBI in both groups is used for conditioning.Ethics and disseminationThe study protocol was approved by the central review board, Nagoya University Certified Review Board, after the enforcement of the Clinical Trials Act in Japan. The manuscripts presenting data from this study will be submitted for publication in quality peer-reviewed medical journals. Study findings will be disseminated via presentations at national/international conferences and peer-reviewed journals.Trial registration numbersUMIN000029947 and jRCTs041180059.

In a highly immunodeficient state following allogeneic hematopoietic stem cell transplantation (HSCT), the reactivation of cytomegalovirus (CMV) is occasionally uncontrollable, which leads to various clinical symptoms, such as pneumonia, enteritis, and hepatitis and finally to lethal outcomes. Although preemptive anti-CMV therapy based on monitoring of CMV infection by quantitative polymerase chain reaction and CMV antigenemia assays has reduced lethal CMV disease, CMV infection is associated with increased non-relapse mortality and reduced survival. A new antiviral drug, letermovir, which exhibited high tolerability and preventive effect on CMV infection, was approved as a prophylactic agent for CMV infection. In the future, it is expected that survival after allogeneic HSCT will be improved by preventing CMV infection. However, further investigations are warranted to solve problems, such as the establishment of the appropriate prophylaxis for CMV infection and the management of late-onset and drug-resistant CMV infection.

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.

Epstein-Barr virus (EBV) infection is highly prevalent in humans and is implicated in various diseases, including cancer1,2. Chronic active EBV infection (CAEBV) is an intractable disease classified as a lymphoproliferative disorder in the 2016 World Health Organization lymphoma classification1,2. CAEBV is characterized by EBV-infected T/natural killer (NK) cells and recurrent/persistent infectious mononucleosis-like symptoms3. Here, we show that CAEBV originates from an EBV-infected lymphoid progenitor that acquires DDX3X and other mutations, causing clonal evolution comprising multiple cell lineages. Conspicuously, the EBV genome in CAEBV patients harboured frequent intragenic deletions (27/77) that were also common in various EBV-associated neoplastic disorders (28/61), including extranodal NK/T-cell lymphoma and EBV-positive diffuse large B-cell lymphoma, but were not detected in infectious mononucleosis or post-transplant lymphoproliferative disorders (0/47), which suggests a unique role of these mutations in neoplastic proliferation of EBV-infected cells. These deletions frequently affected BamHI A rightward transcript microRNA clusters (31 cases) and several genes that are essential for producing viral particles (20 cases). The deletions observed in our study are thought to reactivate the lytic cycle by upregulating the expression of two immediate early genes, BZLF1 and BRLF14-7, while averting viral production and subsequent cell lysis. In fact, the deletion of one of the essential genes, BALF5, resulted in upregulation of the lytic cycle and the promotion of lymphomagenesis in a xenograft model. Our findings highlight a pathogenic link between intragenic EBV deletions and EBV-associated neoplastic proliferations.

Extracellular matrix metalloproteinase inducer (EMMPRIN) secretion was induced in the oral squamous cell carcinoma cell line HSC3 cell by acid-electrolyzed functional water (FW) stimulation. Augmented EMMPRIN secretion was not under transcriptional control; rather, it was derived from the intracellular storages. EMMPRIN secretion was also induced under oxidative stress and accompanied by the release of lactate dehydrogenase (LDH). The molecules released from cells undergoing necrosis are called as alarmins, and the secretion of IL-1α, a typical alarmin, was induced by FW stimulation and oxidative stress. Intracellular localization was examined by cell fractionation. A significant amount of EMMPRIN was localized in the triton X-100 and DNase sensitive fractions; the levels were drastically reduced following FW treatment. The function of the released EMMPRIN was examined using the monocytic cell line THP1. Culture supernatant derived from FW-treated HSC3 cells induced the expression of matrix metalloproteinases (MMPs) 1, 2, 8, 9, 13, and 14, platelet-derived growth factor, and interleukin-8. In contrast, vascular endothelial growth factor expression was reduced. Induction of these factors was abolished following eliminating of EMMPRIN by immunoprecipitation. These results indicate that EMMPRIN might be considered as a type of alarmin that transduces danger signals to the surrounding cells.

A 55-year-old Japanese man was diagnosed with VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome, which is a newly documented adult-onset autoinflammatory disease caused by somatic UBA1 mutations [1], after four years of symptoms. He had suffered from recurrent fever after the onset of systemic arthralgia, scleritis, periorbital/orbital inflammation, optic perineuritis (A, B, T1-weighted contrast-enhanced magnetic resonance imaging in C and D, arrow in D), and myelodysplastic syndrome.

The present study was designed to compare the bone augmentation ability of absorbable collagen sponge (ACS) with that of hydroxyapatite/collagen composite (HAP/Col) using a rat calvaria defect model. Bone defects were created artificially on the surface of the calvariae of 10-week-old male Fisher rats, and then cylindral plastic caps filled with ACS or HAP/Col were placed on the defects. This area was designated as the region of interest (ROI) and new bone formation was observed at 0, 4, 8, and 12 weeks after surgery using micro-CT. Histological examinations were performed using sections obtained from 12-week-old rats. Prominent new bone formation was observed in the HAP/Col group relative to the ACS group; onset of new bone augmentation was evident from 4 weeks after surgery in the HAP/Col group and from 8 weeks in the ACS group. Histological examination revealed that the entire area of the cap was filled with newly formed bone intermingled with the HAP/Col composite. Bone mineral density in the HAP/Col group was double that in the ACS group. These results indicate that the use of HAP/Col contributes significantly to new bone augmentation.

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.

The outcomes of cord blood transplantation with non-irradiated reduced-intensity conditioning for hematological malignancies need to be improved because of graft failure and delayed engraftment. Intrabone infusion of cord blood cells has the potential to resolve the problems. In this phase 2 study, twenty-one adult patients with hematological malignancy received intrabone transplantation of serological HLA-A, B, and DR ≥4/6 matched single cord blood with a median number of cryopreserved total nucleated cells of 2.7 × 107/kg (range, 2.0-4.9 × 107/kg) following non-irradiated fludarabine-based reduced-intensity conditioning. Short-term methotrexate and tacrolimus were given as graft-versus-host disease prophylaxis, and granulocyte colony-stimulating factor was administered after transplantation. No severe adverse events related to intrabone injection were observed. The cumulative incidences of neutrophils ≥0.5 × 109/L, reticulocytes ≥1%, and platelets ≥20 × 109/L recoveries were 76.2%, 71.4%, and 76.2%, respectively, with median time to recoveries of 17, 28, and 32 days after transplantation, respectively. The probability of survival with neutrophil engraftment on day 60 was 71.4%, and overall survival at 1 year after transplantation was 52.4%. The incidences of grade II-IV and III-IV acute graft-versus-host disease were 44% and 19%, respectively, with no cases of chronic graft-versus-host disease. The present study demonstrated the safety of direct intrabone infusion of cord blood. Further analysis is required to confirm the efficacy of intrabone single cord blood transplantation with non-irradiated reduced-intensity conditioning for adult patients with hematological malignancy. This article is protected by copyright. All rights reserved.

Background Diabetes and periodontitis are interrelated, and patient education and guidance are important. Therefore, we conducted a periodontitis care program for patients with type 2 diabetes to provide education about diabetes and periodontitis and to promote self-care skills, and we evaluated the effectiveness of this program. Methods This was a randomized controlled trial. Thirty-eight and 39 adult patients diagnosed with type 2 diabetes were allocated to the intervention and control groups, respectively. The program comprised content that promoted optimal behavior for the improvement of diabetes and periodontitis. Periodontitis status, diabetes status, tumor necrosis factor-α levels, self-efficacy in relation to periodontitis, and teeth-brushing behaviors were evaluated before and after the intervention program. Results After the intervention program, the intervention group demonstrated significant improvements in bleeding on probing, which was used to evaluate periodontitis status (F=7.919; P