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Dysregulation of EZH2 has a crucial role in lymphomagenesis. We did a first-in-human study to assess the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of SHR2554, an oral EZH2 inhibitor, in patients with relapsed or refractory mature lymphoid neoplasms, including B-cell lymphomas, T-cell lymphomas, and classical Hodgkin lymphoma.This was a multicentre, dose-escalation, dose-expansion, and clinical expansion phase 1 study done at 13 hospitals in China. Eligible patients had histologically or cytologically confirmed mature lymphoid neoplasms that had relapsed or were refractory to standard systemic therapies or had no standard-of-care. The study included a dose-escalation phase, at doses of SHR2554 from 50 mg to 800 mg twice daily; a dose-expansion phase, at two selected doses; and a subsequent clinical expansion phase at the recommended phase 2 dose in selected tumours. Primary endpoints were the safety, maximum tolerated dose, and recommended phase 2 dose. Objective response rate was a secondary endpoint. Safety and activity were assessed in all patients who received at least one dose of SHR2554 and had at least one post-baseline evaluation. This study is registered with ClinicalTrials.gov, NCT03603951, and follow-up is ongoing.Between Aug 14, 2018, and July 13, 2021, 113 patients received SHR2554. At data cutoff (Sept 10, 2021), the median follow-up duration was 7·0 months (IQR 3·7-12·0). 71 (63%) patients were men and 42 (37%) were women, 110 (97%) were of Han ethnicity and 3 (3%) of other ethnicities, and 53 (47%) had received three or more lines of previous anticancer therapies. Dose-limiting toxicities occurred in two (67%) of three patients who received 400 mg SHR2554 twice daily and one (17%) of six patients who received 350 mg SHR2554 twice daily. The maximum tolerated dose and recommended phase 2 dose was determined to be 350 mg twice daily. The most common grade 3 or 4 treatment-related adverse events in all 113 patients were decreased platelet count (20 [18%]), decreased neutrophil count (ten [9%]), decreased white blood cell count (nine [8%]), and anaemia (seven [6%]). 18 (16%) patients had serious treatment-related adverse events. Two patients (2%) died due to treatment-related adverse events: one (1%) due to skin infection and toxic epidermal necrolysis and one (1%) due to respiratory failure. 107 (95%) of the 113 enrolled patients had post-baseline assessments for tumour response and were included in the activity analysis. 46 (43%; 95% CI 33-53) of these 107 patients had an overall response.SHR2554 showed an acceptable safety profile and promising antitumour activity in patients with relapsed or refractory lymphomas, providing evidence for future investigations.Jiangsu Hengrui Pharmaceuticals.For the Chinese translation of the abstract see Supplementary Materials section.

Early-stage natural killer/T-cell lymphoma (NK/TCL) patients usually receive a combination of chemotherapy and radiotherapy, but the optimal treatment approach has not yet been established. This study aimed to investigate the efficacy and safety profile of a novel chemotherapy regimen and sandwiched radiotherapy in early-stage NK/TCL. Patients with newly diagnosed stage IE/IIE disease were eligible. Patients were initially treated with two courses of the GELAD regimen (gemcitabine 1·0 g/m2 day 1, etoposide 60 mg/m2 days 1-3, pegaspargase 2000 units/m2 day 4, and dexamethasone 40 mg days 1-4), followed by intensity-modulated radiotherapy (IMRT; 50-56 Gy in 25-28 fractions) and two additional courses of GELAD chemotherapy. A total of 52 patients were enrolled. The overall response rate and complete response rate per Lugano 2014 criteria were 94·2% and 92·3% respectively. With a median follow-up of 32 months, the estimated four-year overall survival rate and progression-free survival rate were 94·2% [95% confidence interval (CI), 83·2% to 93·1%] and 90·4% (95% CI, 78·4% to 95·9%) respectively. The most common adverse events were related to pegaspargase. Haematological toxicities were mild, with grade 3/4 neutropenia in 15·4% of patients. Our study provides a new approach with high activity and improved safety for the treatment of early-stage NK/TCL patients. This study was registered at www.clinicaltrials.gov as NCT02733458.

Background: Tumor cell-derived exosomes (TEXs) have been widely used to induce antitumor immune responses in animal models and clinical trials. Similarly, leukemia cell-derived exosomes (LEXs) can induce antileukemia immune responses in animal models. However, the antileukemia immunity induced by LEXs is less effective, which may be due to an inadequate costimulatory capacity.Methods: In this study, we transduced L1210 leukemia cells with a lentiviral vector encoding two B7 costimulatory molecules (CD80, CD86) and obtained LEXs that highly expressed CD80 and CD86. The antileukemia immune response derived from these LEXs was examined in vitro and in vivo in animal models.Results: We found that B7 gene-modified LEXs, including LEX-CD80, LEX-CD86, and LEX-8086, could significantly boost the expression of CD80 and CD86 in dendritic cells (DCs) and promote the secretion of functional cytokines such as TNF-α and IL-12. Moreover, these B7 gene-modified LEXs, particularly LEX-CD8086, could effectively induce CD4+ T cell proliferation, Th1 cytokine secretion, and an antigen-specific anti-leukemia cytotoxic T lymphocyte (CTL) response. Additional animal studies indicated that immunization with B7 gene-modified LEXs, in particular LEX-CD8086, could significantly retard tumor growth compared to the control LEXnull group.Conclusions: This study sheds light on the feasibility of obtaining LEXs that overexpress costimulatory molecules via genetically modified leukemia cells, thereby enhancing their anti-leukemia immunity and providing a potential therapeutic strategy that contributes to leukemia immunotherapy.

Limited to inadequate stem-cell doses, cord blood transplantation (UCBT) is accompanied by increased graft failure and delayed haematopoietic recovery. The conditioning regimen is critically important for engraftment, and numerous trials have been undertaken comparing the outcomes of IV Bu and TBI, but there are no comparative data for UCBT. We conducted a retrospective multicentre study to analyse the outcomes of IV Bu and TBI in UCBT patients with haematologic malignancies. Between 1 May, 2008 and 31 Mar, 31 2018, a total of 331 patients from the China Umbilical Cord Blood Transplantation Corporation (IV Bu, n = 131; TBI, n = 200) were evaluated. The cumulative incidence of neutrophil engraftment was 91.6% in the IV Bu/Cy cohort and 98.0% in the Cy/TBI cohort (P

Background: The preferred type of postremission therapy (PRT) for intermediate-risk acute myeloid leukemia (AML) in first complete remission (CR1) is a subject of continued debate. Although allogeneic stem cell transplantation (alloSCT) is regarded as a curative strategy for AML, the efficacy of autologous stem cell transplantation (autoSCT) for patients without a matched sibling donor (MSD) has remained controversial. Methods: To compare survival outcomes after alloSCT versus autoSCT for patients with intermediate-risk AML in CR1, we performed a meta-analysis of 11 clinical studies. The outcomes included relapse-free survival (RFS), overall survival (OS), relapse rate (RR), and treatment-related mortality (TRM). Results: Compared with autoSCT, alloSCT showed better RFS, OS, and RR benefits, but higher TRM. Subgroup analysis based on donor category (MSD and matched unrelated donor [MUD]) of alloSCT showed alloSCT from MSD rather than from MUD had better OS benefits compared to autoSCT. For fms-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) wild-type patients, alloSCT and autoSCT had comparable RFS and OS outcomes. Conclusion: Our results suggest that, in the absence of an available MSD, autoSCT remains a viable PRT alternative for intermediate-risk AML in CR1, especially for FLT3-ITD wild-type patients.

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

Hemophagocytic lymphohistiocytosis (HLH) is a rare fatal clinical syndrome characterized by a hyperinflammatory condition caused by aberrantly activated macrophages and cytotoxic T cells, resulting in a cytokine storm and organ impairment. Lymphoma, especially B-cell lymphoma in Japan, is a common trigger of secondary HLH. In China, however, most cases of HLH secondary to lymphoma occur in patients with T-cell/natural killer-cell lymphoma or Hodgkin`s lymphoma; HLH is relatively uncommon in patients with B-cell non-Hodgkin’s lymphoma. We herein describe a man with diffuse large B-cell lymphoma (DLBCL) and secondary HLH who was successfully treated by R-CHOP-E chemotherapy. All symptoms resolved and laboratory indications of HLH normalized, and complete remission of the lymphoma was achieved. This rare case highlights not only the possibility of HLH secondary to DLBCL but also the importance of early initiation of R-CHOP-E chemotherapy.

Background Consolidative allogeneic hematopoietic stem cell transplantation is a controversial option for patients with relapsed/refractory acute lymphoblastic leukemia after chimeric antigen receptor T cell (CAR-T) therapy. We performed a multicenter retrospective study to assess whether patients can benefit from haploidentical hematopoietic stem cell transplantation after CAR-T therapy. Methods A total of 122 patients after CAR-T therapy were enrolled, including 67 patients without subsequent transplantation (non-transplant group) and 55 patients with subsequent haploidentical hematopoietic stem cell transplantation (transplant group). Long-term outcome was assessed, as was its association with baseline patient characteristics. Results Compared with the non-transplant group, transplantation recipients had a higher 2-year overall survival (OS; 77.0% versus 36.4%; P < 0.001) and leukemia-free survival (LFS; 65.6% versus 32.8%; P < 0.001). Multivariate analysis showed that minimal residual disease (MRD) positivity at transplantation is an independent factor associated with poor LFS (P = 0.005), OS (P = 0.035), and high cumulative incidence rate of relapse (P = 0.045). Pre-transplant MRD-negative recipients (MRD− group) had a lower cumulative incidence of relapse (17.3%) than those in the non-transplant group (67.2%; P < 0.001) and pre-transplant MRD-positive recipients (MRD+ group) (65.8%; P = 0.006). The cumulative incidence of relapse in MRD+ and non-transplant groups did not differ significantly (P = 0.139). The 2-year LFS in the non-transplant, MRD+, and MRD− groups was 32.8%, 27.6%, and 76.1%, respectively. The MRD− group had a higher LFS than the non-transplantation group (P < 0.001) and MRD+ group (P = 0.007), whereas the LFS in the MRD+ and non-transplant groups did not differ significantly (P = 0.305). The 2-year OS of the MRD− group was higher than that of the non-transplant group (83.3% versus 36.4%; P < 0.001) but did not differ from that of the MRD+ group (83.3% versus 62.7%; P = 0.069). The OS in the non-transplant and MRD+ groups did not differ significantly (P = 0.231). Conclusion Haploidentical hematopoietic stem cell transplantation with pre-transplant MRD negativity after CAR-T therapy could greatly improve LFS and OS in patients with relapsed/refractory acute lymphoblastic leukemia. Trial registration The study was registered in the Chinese clinical trial registry (ChiCTR1900023957).

Leukemia and lymphoma are common hematological malignancies in children and young adults, which pose a tremendous threat to the survival of these young patients worldwide, despite availability of various effective treatments. The Hippo pathway is a novel‑signaling pathway that regulates organ size, cell proliferation, apoptosis and tumorigenesis. The chief component of this pathway is the transducer yes‑associated protein (YAP) which is over‑expressed in numerous categories of tumors. However, little is known about the effect of YAP in hematological malignancies. In the present study, YAP expression was screened in several leukemia and lymphoma cell lines, and high YAP expression was demonstrated in Jurkat cells. To further unravel its effect on the biological behavior of Jurkat cells, lentivirus transduced short hairpin RNA (shRNA) technique was used to silence YAP. As expected, the YAP‑specific shRNA dramatically inhibited YAP expression at the mRNA and protein levels. Reduced leukemia cell proliferation and increased cell apoptosis were demonstrated in YAP knockdown Jurkat cells. It was also demonstrated that YAP knockdown resulted in deregulated expression of a cluster of downstream genes crucial to cell proliferation or apoptosis, including protein kinase B, B‑cell lymphoma 2 (BCL2) and BCL2 like protein 1. Consequently, the results of the present study established that suppression of YAP expression serves an important role in Jurkat cell proliferation and apoptosis, which may serve as a potential therapeutic target.

The standard treatment in elderly patients with diffuse large B cell lymphoma (DLBCL) has not yet been finely established. We investigated the efficacy and safety of rituximab with a reduced-dose of EPOCH chemotherapy in elderly patients who had advanced DLBCL with high IPI scores. The dose of 70% EPOCH was given to patients aged 75 to 79 years, and dose of 50% to patients aged over 80 years. Thirty-one patients with a median age of 79 years (range 75–86 years) were enrolled. Patients received a median of 6 cycle’s chemotherapy. The complete response rate was 71.0%. The 3-year overall survival (OS) and progression-free survival rates were 62.8 and 60.3%, respectively. The most frequent grade 3/4 adverse effects were neutropenia (3 patients, 7 events), febrile neutropenia (3 patients, 5 events), and pulmonary infection (3 patients, 3 events). Our study showed that ECOG score 3–4, bulky disease, β2-MG > 5.0 mg/L, and loss of any IADL are prognostic factors for OS in univariate analysis. In summary, reduced-dose EPOCH-R chemotherapy for very elderly patients is very effective with acceptable toxicities. Our preliminary study may provide an alternative approach to manage very elderly fit patients with advanced and poor risk DLBCL by first-line treatment with reduced-dose EPOCH-R.

Background/Aims: Minimal residual leukemia cells (MRLs) are difficult to eradicate through traditional treatment and therefore remain to be a major threat to the long-term survival of leukemia patients. Tumor-derived exosomes (TEXs), which carry tumor associated antigens (TAA), may be a potential cell-free tumor vaccine for the specific eradication of MRLs. However, TEXs are intended to be less immunogenic due to exosomal TGF-β1. To further optimize the efficacy of TEX-based vaccines, we investigated whether exosomes from TGF-β1 silenced leukemia cells (LEXTGF-β1si) had an increased potential to induce a specific antitumor effect compared with non-modified exosomes. Methods: Exosomal TGF-β1 was downregulated via lentiviral shRNA silencing of TGF-β1 in leukemia cells. The characteristics of LEXTGF-β1si were determined via electron microscopy, western blot analysis, and flow cytometry. The antitumor effect of LEXTGF-β1si was evaluated by detecting the properties of LEXTGF-β1si-pulsed dendritic cells (DCs), CD4+ T-cell proliferation, Th1 cytokine secretion, specific CTL activity, and NK cell function. Moreover, to verify the superiority of LEXTGF-β1si immunization, LEXTGF-β1si was subcutaneously injected into DBA/2 mice: either followed by tumor challenge or tumor bearing. Results: The lentiviral shRNA silencing of TGF-β1 in parental leukemia cells successfully downregulated the TGF-β1 level in leukemia cell derived exosomes (LEX). LEXTGF-β1si was uptaken by DCs and was more potent in promoting DC function by upregulating the surface expression of costimulatory factors and MHC class II molecules, while inducing the secretion of IL-12p70 and TNF-α. Furthermore, immunization with LEXTGF-β1si facilitated CD4+ T-cell proliferation and Th1 cytokine secretion, and stimulated stronger specific cytotoxic lymphocyte (CTL) response and nature killer (NK) cell cytotoxicity more efficiently compared to non-modified LEX. In mice models, immunization with LEXTGF-β1si resulted in a more potent capability to inhibit tumor growth and to prolong survival, suggesting that LEXTGF-β1si was more effective in both protective and therapeutic antitumor tests than non-modified LEX. Conclusions: These data suggested that down-regulation of exosomal TGF-β1 effectively induced potent anti-tumor immunity. Our strategy of optimizing exosome vaccine may have promising potential for leukemia immunotherapy.

Aberrant over-expression of BCL-2 family proteins (BCL-2, BCL-xL, MCL-1) are associated with hematological malignancies. Antagonists of BCL-2 family proteins include BCL-2-selective inhibitor ABT-199, MCL-1-selective inhibitor A-1210477, BCL-xL-selective inhibitor A-1155463. In this study, we evaluated their potential inhibitory effectiveness. Our data showed that OCI-AML3 cells and U937 cells were resistant to BCL-2-selective inhibitor ABT-199 in vitro and in vivo, however, while OCI-AML3 cells were sensitive to MCL-1-selective inhibitor A-1210477 in vitro and in vivo, indicating that A-1210477 could counteract the resistance of AML cells to ABT-199 as a single agent in MCL-1-dependent AML cells. U-937 cell line and mouse model were resistant to A-1210477 or ABT-199, and expressed high level of BCL-xL, indicating that BCL-xL might play an important role in the resistance of A-1210477 or ABT-199. Besides, this study also showed that ABT-199 could synergize with A-1210477 in vitro or in vivo.

Although 1,25‑dihydroxyvitamin D3 (VD3) is regarded as a promising inducing agent for leukemia cell differentiation, it is not as effective an agent as all‑trans‑retinoic acid, and its usefulness is also limited by the adverse effects of hypercalcemia. The aim of the present study was to determine whether combining VD3 with adenanthin, a peroxiresoxin I (Prx I)‑targeting natural compound, improves the efficacy of VD3. Cell viability was assessed using a trypan blue exclusion assay and flow cytometry was used to evaluate the expression of cell surface markers, CD11b/CD14, and the level of reactive oxygen species (ROS). Wright's staining was used to examine morphological changes and RNA‑interference was used to knockdown Prx I and p65 gene expression. Protein expression was determined by western blot analysis. The results demonstrated that adenanthin markedly enhanced VD3‑induced cell differentiation of leukemia NB4 cells, as evidenced by the increased percentage of CD11b‑ and CD14‑positive cells, the mature morphology of the monocytes and the increased phagocytic ability. Consistent with these results, knockdown of Prx I, but not nuclear factor‑κB (p65), enhanced VD3‑induced cell differentiation. The combinatorial effects of adenanthin and VD3 were shown to be associated with the ROS‑CCAAT‑enhancer‑binding protein (C/EBP)β axis, since N‑acetylcysteine, a ROS scavenger, was able to abrogate the differentiation‑enhancing effects of adenanthin, and the knockdown of C/EBPβ also inhibited the combinatorial effects of adenanthin and VD3. In addition, co‑treatment with adenanthin and VD3 was able to induce differentiation in other non‑acute promyelocytic leukemia cells and primary leukemia cells. In conclusion, the results of the present study revealed a novel role for Prx I in VD3‑induced cell differentiation, and suggested that targeting Prx I may represent a novel strategy to enhance VD3‑induced leukemia cell differentiation.

To better understand predictive factors and improve the clinical outcome of allogeneic transplant for patients with Philadelphia positive acute lymphoblastic leukemia, we analyzed 67 Southeast Asian patients transplanted in our institutions. Multivariate analysis showed that disease status before transplant, year of transplant and, interestingly, French-American-British (FAB) subtype had a significant impact on overall survival (OS) and non-relapse mortality. Patients who were minimal residual disease (MRD) negative at transplant had a 3-year OS of 73% compared to those who were MRD positive (45%) and refractory (0%). The 3-year cumulative incidence of relapse was 18% and 36% for the MRD negative and positive groups, respectively. FAB L1 subtype had a significantly superior 3-year OS of 63% vs. 29% for L2 subtype. Pre-transplant use of a tyrosine kinase inhibitor significantly improved outcomes in univariate but not multivariate analysis, as it served to induce more patients into MRD negativity, which was the factor that directly improved transplant outcome.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland ‘bioreactor’ is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P

Tumor-derived exosomes (TEX) can induce a specific antitumor immune response and have been developed as a promising tumor vaccine. Despite promising preclinical data, TEX exhibit relatively low efficacy and limited clinical benefit in clinical trials. In the present study, we investigated whether exosomes from the TGF-β1 silenced L1210 cells (LEXTGF-β1si) can enhance the efficacy of DC-based vaccines. We silenced TGF-β1 in L1210 cells with a lentiviral shRNA vector and prepared the LEXTGF-β1si. It was shown that LEXTGF-β1si can significantly decrease TGF-β1 expression of dendritic cells (DC) and effectively promote their maturation and immune function. In addition, DC pulsed with LEXTGF-β1si (DCLEX-TGF-β1si) more effectively promoted CD4+ T cell proliferation in vitro and Th1 cytokine secretion and induced tumor-specific CTL response. This response was higher in potency compared to that noted by the other two formulations. Moreover, DCLEX-TGF-β1si inhibited tumor growth more efficiently than other formulations did as the preventive or therapeutic tumor vaccine. Accordingly, these findings revealed that DCLEX-TGF-β1si induced a more potent antigen-specific anti-leukemic immunity than DC pulsed with exosomes from non-manipulated L1210 cells. This indicated that the targeting of DC by LEXTGF-β1si may be used as a promising approach for leukemia immunotherapy.

Hemophagocytic lymphohistiocytosis (HLH) in adult patients is a rare and life-threatening disorder complicated by a variety of underlying diseases including malignant, infectious, and autoimmune di...

Exosomes (EXOs) are nanometer-sized membrane vesicles secreted from epithelial and hematopoietic cells. They display a spectrum of molecules involved in immune responses and signal transductions. Previous studies showed that tumor antigen-loaded dendritic cell (DC)- and tumor cell-derived EXOs (Dexo and Texo) induce tumor antigen-specific CD8(+) cytotoxic T-lymphocyte responses and antitumor immunity in experimental animal models and human clinical trials. This review will present the main biologic features of Dexo and Texo as cell-free cancer vaccines with emphasis on their immunostimulatory properties and their potential efficacy in cancer immunotherapy.

Dendritic cell (DC) and DC-derived exosomes (EXO) have been used extensively for tumor vaccination. However, its therapeutic efficiency is limited to only production of prophylactic immunity against tumors. T cells can uptake DC-released EXO. However, the functional effect of transferred exosomal molecules on T cells is unclear. In this study, we demonstrated that OVA protein-pulsed DC-derived EXO (EXOOVA) can be taken up by Con A-stimulated, nonspecific CD4+ T cells derived from wild-type C57BL/6 mice. The active EXO-uptaken CD4+ T cells (aTEXO), expressing acquired exosomal MHC I/OVA I peptide (pMHC I) complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of stimulating OVA-specific CD8+ T cell proliferation in vitro and in vivo and inducing efficient CD4+ Th cell-independent CD8+ CTL responses in vivo. The EXOOVA-uptaken CD4+ aTEXO cell vaccine induces much more efficient CD8+ T cell responses and immunity against challenge of OVA-transfected BL6-10 melanoma cells expressing OVA in wild-type C57BL/6 mice than EXOOVA. The in vivo stimulatory effect of the CD4+ aTEXO cell to CD8+ T cell responses is mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and pMHC I complexes, respectively. In addition, CD4+ aTEXO vaccine stimulates a long-term, OVA-specific CD8+ T cell memory. Therefore, the EXOOVA-uptaken CD4+ T cells may represent a new, effective, EXO-based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEXK562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (e.g., intercellular adhesion molecule-1) and immunologically associated molecules (e.g., heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (P

A 3-year-old girl presented with petechial hemorrhages and repeated nosebleeds. Two weeks earlier she had been admitted to a local hospital with nosebleeds accompanied by 2 episodes of vomiting dark red blood. Results of the laboratory evaluation included: white blood cell count, 8.2 × 109/L [reference interval (RI),3 4 × 109/L to 10 × 109/L]; hemoglobin, 10.7 g/dL (RI, 11–15 g/dL); platelet count, 142 × 109/L (RI, 100 × 109/L to 300 × 109/L), prothrombin time, 11.5 s (RI, 9–13 s); activated partial thromboplastin time, 31.2 s (RI, 26–39 s); and fibrinogen, 2.5 g/L (RI, 2.0–4.0 g/L). The patient was discharged in good condition after insertion of nasal packs. One day before the current admission, the patient had nose bleeds once again, this time accompanied by 4 episodes of hematemesis and tarry stool. At presentation, she had no fever and no diarrhea. She was not on any medications. The patient had a history of easy bruising, repeated gum bleeding, but not hemarthrosis. There was no family history of abnormal bleeding. On examination, she appeared pale, with normal vital signs. Her skin had scattered petechiae. The physical examination was otherwise unremarkable. At presentation, laboratory findings included the following: hemoglobin, 6.2 g/dL (RI, 11–15 g/dL); reticulocyte count, 6.8% (RI, 0.5%–1.5%). Other laboratory results are shown in Table 1. View this table: Table 1. Patient's laboratory results. In vitro testing showed that the patient's platelets did not aggregate in response to ADP, epinephrine, arachidonic acid, or collagen, but platelets had relatively normal ristocetin-induced aggregation. These findings were confirmed on repeat testing. A smear of peripheral blood showed no clusters of normal platelets. A flow cytometry evaluation revealed marked reduction in glycoprotein IIb/IIIa (GPIIb/IIIa). ### QUESTIONS TO CONSIDER 1. What disorders should be considered in the workup of children with repeated nose bleeds? …

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

Oncolytic adenovirus (rAd)-mediated E1A gene therapy of cancer has become a novel therapeutic modality. In this study, we constructed a recombinant oncolytic adenovirus (rAd-E1A) expressing the tumor suppressor E1A gene. We demonstrated that the rAd-E1A replicated in HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cells but attenuated in the normal liver cell line HL-7702. It induced HCC cell apoptosis through upregulation of apoptosis-associated Bax, caspase-3, and Fas and downregulation of survivin and Bcl-2 in a p53-dependent pathway. It also downregulated the expression of angiogenesis- associated vascular endothelial growth factor (VEGF) and CD34 genes and reduced tumor vessel formation and angiogenesis. In mice bearing SMMC-7721 tumors, intratumoral injections of rAd- E1A significantly inhibited HCC growth. Therefore, the oncolytic adenovirus-mediated E1A gene therapy may be a useful therapeutic approach for HCC treatment.

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce antitumor immunity and to study the antitumor immune mechanisms derived from immunogene expression. In this chapter, we describe the design and methods for cloning a cDNA gene coding for the mouse CD40L molecule and for construction of the expression vector pcDNA-CD40L, as well as the methods for generation of engineered myeloma cells J558/CD40L expressing CD40 ligand. We also demonstrate that the engineered J558/CD40L tumor cells lose their tumorigenicity in syngeneic mice, and that the inoculation of J558/CD40L tumor cells further leads to protective immunity against wild-type J558 tumors.

Immunoisolation technology using microencapsulated nonautologous cells is a novel alternative approach to the treatment of cancer. The work was aimed on investigation of the effect of implantation of microencapsulates on tumor growth in vivo.In this study, we constructed an engineered tumor cell line J558/TNF-alpha that secreted functional tumor necrosis factor-alpha (TNF-alpha) (2 ng/ml), and went on to encapsulate the engineered cells into microencapsules.Our data showed that the microencapsulates thus produced could release functional TNF-alpha (1.2 ng/ml), which then diffused through the microencapsule membrane into the supernatant, and produced a cytotoxic effect on L929 cells in vitro. Microencapsulated cells were intratumorally (i.t.) implanted into athymic nude mice bearing the human breast cancer MCF-7. The results showed that the i.t. implantation induced extensive tumor cell apoptosis and necrosis leading to significant tumor regression and slower tumor growth than in the control groups that were i.t. injected with microencapsulated J558 or PBS respectively (p0.05).This study provides further evidence that the microencapsulation of recombinant tumor cells secreting cytokines may be an alternative approach in treatment of cancer.

Dendritic cell-tumor cell fusion hybrid vaccines which facilitate antigen presentation represent a new powerful strategy in cancer immunotherapy. The clinical frequency of objective responses to the conventional fusion hybrid vaccines is still quite low, indicating that the current conventional protocol of simply fusing dendritic cells (DCs) and tumor cells needs further improvement to enhance its antitumor efficiency.In the present study, we generated a novel fusion hybrid DC/J558(CD40L) by fusing DCs and an engineered J558(CD40L) myeloma cells expressing CD40 ligand (CD40L) molecule using polyethylene glycol (PEG). The fusion efficiency was approximately 20%. We investigated the antitumor immunity derived from vaccination of the fusion hybrid DC/J558(CD40L).Our results showed that vaccination of mice with DC/J558(CD40L) hybrids induced more efficient cytotoxic T lymphocyte (CTL) responses and protective immunity against J558 tumor cells, than that of the conventional fusion hybrid DC/J558 from the fusion of DCs and J558 tumor cells. The antitumor immunity derived from vaccination of DC/J558(CD40L) was mainly mediated by CD4(+) and CD(8+)cT cells, but not natural killer (NK) cells.Therefore, this novel fusion hybrid vaccine which combines gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Background Exosomes secreted by tumor cells contain specific antigens that may have immunotherapeutic purposes. The aim of this study was to characterize the proteomic content of lymphoma cell-derived exosomes (LCEXs). Methods In this study, exosomes derived from Raji cells (EXORaji) were purified and proteins of EXORaji were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein bands were identified by mass spectrometry. The protein components of EXORaji were analyzed using shotgun technology, and the function proteins of EXORaji were defined and described using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results A total of 197 proteins were identified in EXORaji; 139 proteins were also identified in Raji cells, showing an overlap of 70.56% of the total proteins in EXORaji. Interestingly, the remaining 58 proteins were unique to EXORaji. The GO database and KEGG were used to define and describe the function of proteins. The data showed that some important proteins involved in antigen procession and presentation as well as cell migration and adhesion were also identified in EXORaji, such as MHC-I and II, HSC70, HSP90, and ICMA-1. Conclusions LCEXs express a discrete set of proteins involved in antigen presentation and cell migration and adhesion, suggesting that LCEXs play an important role in the regulation of immunity and interaction between lymphoma cells and their microenvironment. LCEXs harbor most of the proteins of lymphoma cells and could be one of the sources of lymphoma-associated antigens for immunotherapeutic purposes.