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PURPOSE Relapsed or refractory extranodal natural killer/T-cell lymphoma (R/R ENKTL) is a rare and aggressive type of non-Hodgkin lymphoma with limited treatment options. This phase II study evaluated the efficacy and safety of sugemalimab, an anti–PD-L1 monoclonal antibody, in R/R ENKTL. METHODS Eligible patients received sugemalimab 1,200 mg intravenously once every 3 weeks for up to 24 months or until progression, death, or study withdrawal. The primary end point was objective response rate (ORR) assessed by an independent radiologic review committee. Key secondary end points included ORR assessed by the investigators, complete response rate, duration of response, and safety. RESULTS At the data cutoff (February 23, 2022), 80 patients were enrolled and followed for a median of 18.7 months. At baseline, 54 (67.5%) had stage IV disease and 39 (48.8%) had received ≥2 lines of prior systemic therapy. Independent radiologic review committee–assessed ORR was 44.9% (95% CI, 33.6 to 56.6); 28 (35.9%) patients achieved a complete response and seven (9.0%) achieved a partial response, with a 12-month duration of response rate of 82.5% (95% CI, 62.0 to 92.6). Investigator-assessed ORR was 45.6% (95% CI, 34.3 to 57.2), and 24 (30.4%) patients achieved a complete response. Most treatment-emergent adverse events were grade 1-2 in severity, and grade ≥ 3 events were reported in 32 (40.0%) patients. CONCLUSION Sugemalimab showed robust and durable antitumor activity in R/R ENKTL. Treatment was well tolerated with expected safety profile for this drug class.

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.

Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only curative method in treating haematologic malignant diseases. Graft‐versus‐host disease (GVHD) is a common complication post–allo‐HSCT, which can be life‐threatening. Mesenchymal stem cells (MSCs) as an adult stem cell with immunoregulatory function have demonstrated efficacy in steroid resistant acute GVHD (aGVHD). However, the outcome of aGVHD treated with MSCs in clinical trials varied and its underlying mechanism is still unclear. TGF‐β1 is a potent cytokine, which plays a key role in immunoregulation. In the present study, we firstly transduced the lentivirus vector containing TGF‐β1 gene with mouse bone marrow‐derived MSCs. Then, we investigated the immunosuppressive effect of TGF‐β1 gene‐modified MSCs on lymphocytes in vitro and its preventive and therapeutical effects on murine aGVHD model in vivo. Murine MSC was successfully isolated and identified. TGF‐β1 was efficiently transduced into mouse MSCs, and high level TGF‐β1 was detected. MSC‐TGF‐β1 shared the same morphology and immunotypic features of normal MSC. In vitro, MSC‐TGF‐β1 showed enhanced immunosuppressive function on lymphocyte proliferation. In vivo, MSC‐TGF‐β1 showed enhanced amelioration on the severity of aGVHD both in prophylactic and therapeutic murine models. Finally, the macrophages (MØs) derived from MSC‐TGF‐β1–treated mice showed a remarkably increasing of anti‐inflammatory M2‐like phenotype. Furthermore, the differentiation of CD4+ CD25+ Foxp3+ Treg cells was significantly increased in MSC‐TGF‐β1–treated group. Taken together, we proved that MSC‐TGF‐β1 showed enhanced alleviation of aGVHD severity in mice by skewing macrophages into a M2 like phenotype or increasing the proportion of Treg cells, which opens a new frontier in the treatment of aGVHD.

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.

7552 Background: Effective treatment choices for patients (pts) with relapsed/refractory (R/R) mature T/NK-cell neoplasms after failure of standard therapies are limited. Tislelizumab (TIS), a humanized anti-PD-1 mAb, demonstrated outstanding efficacy and favorable safety in pts with R/R classical Hodgkin lymphoma or solid tumors. We present safety and efficacy of the phase 2 study of TIS in pts with R/R mature T/NK-cell neoplasms. Methods: Pts were enrolled into 3 cohorts stratified by the type of T/NK-cell neoplasm to receive TIS 200 mg intravenously every 3 weeks until disease progression or intolerable toxicity. Eligible pts had ≥1 prior systemic therapy, disease progression during/after most recent therapy completion or refractory disease, ECOG ≤2, and life expectancy ≥6 mo. Primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DoR), complete response (CR) rate, progression-free survival (PFS), overall survival (OS) in cohorts 1 and 2, and safety. Results: 77 pts were treated. Cohort 1: R/R extranodal NK/T-cell lymphoma (n = 22); cohort 2: R/R mature T-cell neoplasms (n = 44; 21 peripheral T-cell lymphoma not otherwise specified; 11 angioimmunoblastic T-cell lymphoma; 12 anaplastic large cell lymphoma); cohort 3: R/R cutaneous T-cell lymphomas (CTCL; stage ≥1B; n = 11; 8 mycosis fungoides and 3 Sézary syndrome). Of all pts, 76.6% had advanced-stage disease, 51.9% had refractory disease, and 49.4% had ≥3 prior systemic regimens. Median treatment cycles for cohorts 1, 2, and 3 were 5 (range, 1-37), 4.5 (range, 1-38), and 17 (range, 3-25), respectively. Cohort 3 had promising efficacy (median follow-up [FU] 16.6 mo): ORR 45.5%; CR 9.1%; median DoR 11.3 mo (95% CI: 2.76-11.30); median PFS 16.8 mo; median OS not reached (NR). Modest efficacy was reported in cohort 1 (median FU 8.4 mo): ORR 31.8%; CR 18.2%; median DoR NR (95% CI: 2.66-not estimable [NE]); median PFS 2.7 mo; median OS 8.8 mo and also in cohort 2 (median FU 9.3 mo): ORR 20.5%; CR 9.1%; median DoR 8.2 mo (95% CI: 2.50-NE); median PFS 2.7 mo; median OS 13.3 mo. Most frequent adverse events (AEs) were pyrexia (32.5%), anemia (18.2%), arthralgia (18.2%), and diarrhea (15.6%); most frequent grade ≥3 AEs were anemia (7.8%), pneumonia (6.5%), and neutropenia (5.2%). Any grade immune-mediated AEs occurred in 22 (28.6%) pts, most frequently hypothyroidism (10.4%), hyperglycemia (5.2%), and rash (5.2%); and grade ≥3 in 4 (5.2%) pts (blood creatine phosphokinase increased, hepatitis, hypothyroidism, rash, and urticaria [1 pt each]). No treatment-related AEs led to death. Conclusions: TIS was well tolerated, achieving modest efficacy in R/R mature T/NK-cell neoplasms, with some long-lasting remissions particularly in CTCL. Further studies are warranted to determine the biologic features associated with response and explore optimal combination therapies. Clinical trial information: NCT03493451.

7525 Background: Dysregulation of histone methyltransferase EZH2 plays critical roles in lymphomagenesis. Emerging evidence shows that EZH2 also contributes to tumor immune evasion. SHR2554 is an oral, small-molecule inhibitor exhibiting potent selectivity for EZH2. We initiated a first-in-human study to assess SHR2554 across a broad spectrum of lymphoid neoplasms that had relapsed or was refractory to standard systemic therapies, including B-cell lymphomas, T-cell lymphomas, and classical Hodgkin lymphoma (cHL). Methods: This multicenter, phase 1 study was composed of 3 parts: dose escalation phase according to mTPI-2 design, dose expansion phase, and clinical expansion phase in selected tumor subtypes. Dose was escalated at 50, 100, 200, and 400 mg BID, and then de-escalated at 300 and 350 mg based on the observed toxicities. Two dose levels (300 and 350 mg) were expanded. Subsequently, follicular lymphoma (FL), peripheral T-cell lymphoma (PTCL), and cHL were selected for clinical expansion at RP2D. Primary endpoints were to assess the safety and determine the MTD and RP2D. Key secondary endpoint included clinical activity at RP2D in different subtypes. Results: As of Sep 10, 2021, 113 heavily pretreated pts were enrolled, with 53 (46.9%) having received ≥3 lines of prior systemic therapies. In dose escalation phase, DLTs occurred in 2 of 3 pts at 400 mg and 1 of 6 pts at 350 mg; thus, 350 mg BID was the MTD. Combined with the safety, tolerability, PK/PD, and preliminary efficacy findings observed in dose escalation and expansion phases, RP2D was determined to be 350 mg BID. As of cutoff date, 41 FL, 22 PTCL, and 21 cHL pts who received SHR2445 at 350 mg BID completed at least one post-baseline efficacy assessment. ORR in FL was 58.5% (95% CI 42.1-73.7); majority of responses (66.7%) were still ongoing, and the estimated median DoR was 9.3 mos (95% CI 5.6-NR). EZH2mut FL pts showed a slightly higher ORR than EZH2WT FL pts (62.5% vs 55.2%). EZH2 mutations were not detected in pts with PTCL or cHL. ORR in PTCL was 63.6% (95% CI 40.7-82.8); majority of responses (71.4%) were still ongoing, and the estimated median DoR was 7.4 mos (95% CI 2.9-NR). All cHL pts had received prior chemotherapy and anti-PD-1/PD-L1 antibodies. Shrinkage in target lesions was shown in 76.2% of cHL pts; ORR was 19.0% (95% CI 5.4-41.9); majority of responses (75.0%) were still ongoing, and the median DoR had not been reached yet. Grade ≥3 treatment-related AEs occurred in 38 of the 113 pts (33.6%), with the most common being decreased platelet count (17.7%), decreased neutrophil count (8.8%), decreased white blood cell count (8.0%), and anemia (6.2%). Conclusions: SHR2554 showed a manageable safety profile and promising anti-tumor activity in pts with r/r lymphomas, supporting further explorations in FL, PTCL, and cHL. Clinical trial information: NCT03603951.

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.

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 comprises autologous T cells transduced with a second-generation chimeric antigen receptor (CAR) utilizing a fully human BCMA-specific single-chain fragment variant (25C2) with high binding affinity. CT053 was firstly studied in a single-arm, open-label Phase I, investigator-initiated program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Here we present the 24-month follow-up results of the study. Methods: The multicenter exploratory Phase 1 studies included adult subjects with relapsed/ refractory multiple myeloma (RRMM) who had received at least 2 prior lines of myeloma treatment. After preconditioning treatment with fludarabine and cyclophosphamide for 2-4 days, 21 subjects received one cycle of 1.5 × 108 T cells CT053 CAR-BCMA T cells. Three subjects respectively received 0.5 × 108, 1 × 108, or 1.8 × 108 cells. The primary objective was subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. Results: A total of 24 subjects with a median age of 60.1 years (range, 38.5-69.9) were enrolled from Sept. 10, 2017 to Sept. 22, 2018. The subjects had a median of 4.5 (range, 2-11) prior lines of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, 10 subjects (41.7%) had concomitant extramedullary involvement, 8 subjects (33.3%) had ECOG scores 2-3, and 9 subjects (37.5%) reported ISS Grade III. As of June 30, 2020, 9 subjects completed 24 months of follow-up with responses including 8 stringent complete response (sCR) and 1 complete response (CR). Also, 15 subjects discontinued prior to completing the 24-month follow-up, of whom 13 discontinued due to disease progression (PD), and 2 discontinued for other anticancer therapy. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete responses (3 CR, 16 sCR). The median duration of response (DOR) was 21.8 months (95%CI: 9.2, not evaluable [NE]). The median progression-free survival (PFS) was 18.8 months (95%CI: 10.1, NE), with 6-month and 12-month PFS rates of 87% and 60.9%, respectively. Thirteen subjects progressed with median PFS of 10.2 months (range, 0.9-23 months): 3 progressed within 6 months, 6 progressed within 6-12 months, and 4 within 12-24 months. Compared to 9 subjects with persistent CR/sCR, the 13 progressed subjects had a higher percentage of ECOG scores 2-3 (46.2% vs 22.2%), ISS Grade III (53.9% vs 11.1%) and high-risk cytogenetics profiles (53.8% vs 33.3%). Rates of concomitant extramedullary diseases were similar, 46.2% and 44.4%, respectively. Hematological toxicities were the most common treatment-related adverse events of grade (G) 3 or higher, including leukopenia (83.3%), neutropenia (85%), lymphocytopenia (79.2%) and thrombocytopenia (20.8%). In general, cytokine release syndrome (CRS) occurred at 1-4 days and resolved in a median 6 days (range, 3-9 days). Low-grade CRS was reported in 15 of 24 (62.5%) subjects. All CRS events (4 G1, 11 G2) resolved within 2-8 days; among them, 9 patients received a low dose of tocilizumab 4-6 mg/kg. One patient experienced G3 neurotoxicity, presenting as epilepsy and accompanied by simultaneous G2 CRS. This patient fully recovered within 3 days after treatment with methylprednisolone, diazepam and sodium valproate. Six patients (25%) experienced 10 cases of treatment-related serious adverse events (SAEs), including lung infection (3), gastroenteritis (1), neutropenic infection (1), fever (1) and hematological toxicities (4). By the cutoff date, one subject died of SAE a (bone morrow failure and neutropenic infection) and PD, and seven subjects died of PD. CAR-BCMA T cell expansion was detectable as early as day 1-7 after infusion and reached peak values on day 7-21 with the highest concentration at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. No immunogenicity was detected. Conclusion: These studies demonstrated that CT053 had excellent efficacy in RRMM, showing early, deep and durable response with 21.8 months DOR. CT053 was well tolerated among the subjects. Figure Disclosures Li: CARsgen Therapeutics Co. LtD: Current Employment, Current equity holder in private company. Wang:CARsgen Therapeutics Corp.: Current Employment. Xiao:CARsgen Therapeutics Corp.: Current Employment. Yuan:CARsgen Therapeutics Corp.: Current Employment. Ma:CARsgen Therapeutics Corp.: Current Employment.

Acute myeloid leukemia (AML) is one of the most common hematological malignancies. It is difficult to treat since it easily develops resistance to therapeutic drugs. Myeloid cell leukemia 1 (MCL-1), BCL-2 and BCL-XL, which belong to the anti-apoptotic group of proteins in the BCL-2 family, are overexpressed in AML. The effects of inhibitors that target anti-apoptotic proteins of the BCL-2 family in AML were evaluated in the present study. MCL-1 protein levels of HL60, MOLM13, OCI-AML3 and MV4-11 cell lines were investigated. Furthermore, following treatment with MCL-1-selective antagonist A-1210477 and/or BCL-2/BCL-XL antagonist ABT-737, cell viability was detected. The chimera rate of human CD45(+) cells of bone marrow from mouse models was analyzed via flow cytometry and immunohistochemistry using murine tissues (lung, spleen and liver). The data revealed that the HL-60 cell line, which exhibited a low MCL-1 protein level, and MOLM-13 and MV4-11 cell lines, whose MCL level was intermediate, were sensitive to ABT-737, whereas OCI-AML3 cells, which exhibited a high MCL-1 level, were insensitive to ABT-737. However, multiple AML mouse models and AML cell lines were sensitive to the MCL-1-selective antagonist A-1210477. The results of the present study indicated that the MCL-1-selective antagonist could overcome the resistance to the BCL-2/BCL-XL antagonist (ABT-737) in vitro and in vivo.

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.

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 CAR-BCMA, which is a BCMA-specific chimeric antigen receptor (CAR) T cell, consists of autologous T cells genetically modified with a second-generation CAR incorporating a fully human anti-BCMA single chain fragment variant, a 4-1BB co-stimulatory domain and a CD3-zeta signaling domain. CT053 was studied in a single-arm, open-label phase I program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Methods: This investigation is a 3-site study in adult subjects with relapsed/refractory multiple myeloma (rrMM) who had received at least 2 prior myeloma regimens. Subjects received one cycle of CT053 CAR-BCMA after fludarabine/cyclophosphamide infusion. The primary objective was the assessment of subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. The data cutoff date was June 30, 2019. Results: A total of 24 subjects with median age of 60.1 years (range, 38.5-69.9) were enrolled from Sep 10, 2017 to Sep 22, 2018 (Table 1). The subjects had a median of 4.5 (range, 2-11) prior regimens of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, eleven out of 24 subjects (45.8%) had concomitant extramedullary involvement. Eight subjects (33.3%) had ECOG score 2-3, and 9 subjects (37.5%) reported ISS grade III. All subjects received lymphodepletion preconditioning of fludarabine/cyclophosphamide for 2-4 days, followed by one cycle of CT053 at a dose of 1.5 x 108 T cells except 3 subjects who received 0.5 x 108, 1 x 108, and 1.8 x 108 cells, respectively. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete response (5 CR, 14 sCR). As shown in Figure 1, P1 who received the lowest dose of 0.5 x 108 experienced partial response (PR) at M1 and very good partial response (VGPR) at M2. P1 then converted to CR at M14 and sCR at M16. Among 20 subjects who underwent the evaluation of minimal residual disease (MRD) status, 17 achieved MRD-negative (≤10−4 nucleated cells) and reported a tumor response (17 CR/sCR). In 13 subjects with ongoing CR/sCR, the median follow-up after CT053 infusion was 383 days (range, 301-467). Nine subjects progressed with median progression-free period of 281 days (range, 57-573); among them, 5 progressed within 6-12 months, 1 at 13 months and 1 at 19 months. Compared to 13 subjects with persistent CR/sCR, the 9 progressed subjects were observed to have a higher percentage of ECOG score 2-3 (66.7% vs 15.4%), ISS Grade III (55.6% vs 15.4%), and concomitant extramedullary diseases (66.7% vs 38.5%) and a decreased hemoglobin (70g/L vs 92g/L) at baseline. Three subjects died of disease progression at the time of analysis. Hematologic toxic effects were the most common treatment-related adverse events of grade (G) 3 or higher, including white blood cell count decreased (87.5%), neutrophil count decreased (66.7%), lymphocyte count decreased (79.2%) and thrombocytopenia (25%). No dose limiting toxicities were observed. Low-grade cytokine release syndrome (CRS) was reported in 15 of 24 (62.5%) subjects. All CRS events (3 G1, 12 G2) recovered within 2-8 days; among them 8 received tocilizumab. Three subjects (12.5%) had neurotoxicity (2 G1, 1 reversible G3). One subject died of bone marrow failure and neutropenic infection. CAR-BCMA T cell expansion was detected as early as Day 1-7 after infusion and reached peak values on Day 7-21 with the highest at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. Conclusion: This study demonstrated that CT053 had an excellent efficacy and a good safety profile in subjects with rrMM. Disclosures Li: CARsgen Therapeutics Co. Ltd: Employment, Equity Ownership. Xiao:CARsgen Therapeutics Co. Ltd: Employment. WANG:CARsgen Therapeutics Co. Ltd: Employment. Yuan:CARsgen Therapeutics Co. Ltd: Employment. Ma:CARsgen Therapeutics Co. Ltd: Employment.

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...

Introduction: B Cell Mature Antigen (BCMA)-targeted chimeric antigen receptor T (CAR-T) cell therapy emerges as promising treatment for patients with relapse/refractory multiple myeloma (RRMM). Previous studies indicate patients who receive high-dose CAR-T cells may achieve better remission but have worse adverse events, like cytokine release syndrome (CRS). To solve this dilemma, we have developed novel autologous CAR-T therapeutics CT053 that are genetically modified T cells comprising an extracellular anti-BCMA human scFv and an intracellular 4-1BB costimulatory motif connected to a CD3-zeta T cell activation domain. Methods: A multi-center investigator-initiated clinical study is designed to evaluate CT053 in patients with RRMM who have failed in the prior treatment with ≥2 regimens, including a proteasome inhibitor, an immunomodulatory agent, and anti-CD38 monoclonal antibody. All patients have ≥50% BCMA expression on malignant cells. Patients are subjected to the lymphodepletion with 20-25 mg/m2 fludarabine and 300-500 mg/m2 cyclophosphamide daily for 2-4 days prior to receiving single-dose infusion of CT053 CAR-T cells. In case of progressive disease, patient may be dosed again on basis of investigators' evaluation of the disease status, BCMA expression and CAR-T persistence. Most enrolled patients received a single dose of 1.5 x 108 cells, except for 1 patient who received 0.5 x 108 cells and 1 patient who was infused with 1.8 x 108 cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs) and CAR T related AEs. Additional outcome measures include clinical response assessed according to the IMWG Uniform Response Criteria for Multiple Myeloma, overall and progression-free survival, pharmacokinetics and pharmacodynamic of CT053. Results: The study was performed in compliance with the declaration of Helsinki. As of the data cut-off date (July 10th, 2018), 16 patients (median 55 [39 to 67] years old) with a median of 3.9 (0.4 to 16.7) years since MM diagnosis, were infused with CT053. Patients had a median of 4 prior different regimens (range 2 to 10), and 56% (9/16) patients received prior autologous or allogeneic stem cell transplant. Among 16 patients, no neurotoxicity and no dose-limiting toxicities (DLT) were observed. The most common grade≥3 CAR-T related AEs were 3 thrombocytopenia (19%), 3 leukopenia (19%), 2 anemia (13%), 2 neutropenia (13%), 2 fever (13%) (Figure 1A). CRS was reported in 3 patients, including 1 Grade 3, 1 Grade 2 and 1 Grade 1, who had rapid recovery after Tocilizumab administration. 13/16 patients were eligible for initial evaluation of early clinical response with a median observation period of 8 (4 to 36) weeks. Overall response rate (ORR) in 13/13 patients was 100% post treatment. 12/13 patients (92%) quickly achieved partial response (4 PR), very good PR (6 VGPR), and complete response (2 CR) within 4 weeks post single-dose infusion (Figure 1B). 5/12 patients (42%) who were dosed at ≥1.5 x 108 CT053 CAR-T cells obtained CR at a median of 8 weeks post treatment. Durable responses from 4 weeks towards the data cut-off date were found in 12/13 patients (92%). One relapse from VGPR by the Week 12 was reported in a patient who had aggressive RRMM at enrollment and received the reduced dose of lymphodepletion regimen at 19 mg/m2 fludarabine and 192 mg/m2 cyclophosphamide for 2 days prior to CT053 infusion. Because positive BCMA expression on malignant cells was verified at relapse, the patient was re-dosed with CT053 at the Week 16 and subjected to the further evaluation. All patients had detectable CAR-T expansion from Day 3 post CT053 infusion. Expansion peaks were found on Day 7 (5/13), Day 14 (6/13) and Day 21 (2/13). 11/13 patients had notable persistence of CT053 CAR-T cells up to 4-6 months. The only relapsed patient had the lowest CAR-T expansion peak among 13 patients, indicating the potential correlation between CAR-T expansion and response outcome. Conclusions: Data from this early-stage clinical study showed the unparalleled safety and efficacy of CT053 CAR-T cells. Major AEs were transient, manageable, and reversible. 100% ORR in 13/13 evaluable patients were reported post single-dose infusion of 0.5~1.8 x 108 cells. 5/12 patients who were dosed at ≥1.5 x 108 CAR-T cells rapidly achieved durable CR at median of 8 weeks, suggesting CT053 could be developed as competitive therapeutics to treat patients with RRMM. Disclosures Ruan: CARsgen Therapeutics: Employment. Xiao:CARsgen Therapeutics: Employment, Equity Ownership. Wang:CARsgen Therapeutics: Employment, Equity Ownership. Li:CARsgen Therapeutics: Employment, Equity Ownership.

Intravenous busulfan (IV-Bu) or total body irradiation (TBI) based regimens are currently the most widely used myeloablative conditioning regimens for patients with hematologic malignancies undergoing allogeneic stem-cell transplantation(allo-SCT). Numerous trials have been undertaken on the clinical outcomes between IV Bu and TBI, but there are no comparative data for cord blood transplantation(CBT). We conducted a prospective registry-based study to analysis the outcomes of IV Bu and TBI in CBT patients with hematologic malignancies. From May 1, 2008 to Mar 31, 2016, a total of 331 consecutive patients with hematologic malignancies recieved singe unrelated CBT were involved in the study. Eligibility criteria for this analysis included:(1)Weigh ≧35 kilograms and age ≦ 60 years; (2)All patients recieved a single unit CBT but not a double units CBT; (3)Consensus criteria preparative regimens were based on full dose IV Bu(total 12.8 mg/kg, 0.8mg/kg every 6 h for 4 days) or TBI(total 12 Gy, 4 fractions) combined with Cy(60mg/kg × 2d); (4)GVHD prophylaxis regimens include cyclosporine(CSA) and mycophenolate mofetil(MMF) without Antithymocyte Globulin(ATG). Patients who has recieved a previous autologous or allogeneic transplantation was excluded in the study. The cumulative incidence of neutrophil engraftment were 91.6% in IV Bu/Cy cohort and 98.0% in Cy/TBI cohort(P < .001), respectively. The median follow-up time in IV Bu/Cy and Cy/TBI cohorts was 28.7(range, 12.2 to 91.3) months and 55.5(range, 13.1 to 117.1) months, respectively(P Our resluts demonstrates that, compared with TBI, IV Bu regimen was associated with a higher incidence of graft rejection in CBT. But there was no difference in survival with no increased risk for NRM or relapse between two regimens. For those centers lack of radiation facilities, IV Bu may be a valid and efficient alternative to TBI. With the restriction of a retrospective registry analysis and limited patient munbers, rigorously designed prospective randomized controlled trials are needed to further investigated the availability of IV Bu and TBI for CBT. Disclosures No relevant conflicts of interest to declare.

Interleukin-10 (IL-10) has been identified as a key immunomodulatory cytokine on T cells. However, both immunosuppressive and immunostimulatory effects of IL-10 on T cells also have been reported. The discrepancy between these in vitro effects of IL-10 may be due to the different T cells (naive vs. active or resting active T cells) used under various experimental conditions in these studies. Therefore, it is necessary to clearly define the IL-10 effect on T cell subsets in their different statuses. In this study, we used a molecularly defined T cell system, the ovalbumin (OVA)-specific CD4(+) and CD8(+) T cells from transgenic OT-I and OT-II mice expressing OVA-specific T cell receptor (TCR). We investigated the effect of IL-10 on these OVA-specific T cell subsets in their different statuses (i.e., naive and active T cells). Our data demonstrate that IL-10 has distinct immunoregulatory effects on naive and active T cell subsets. IL-10 inhibits active CD4(+) T cell proliferation, whereas it stimulates and suppresses active CD8(+) T cell proliferation and cytotoxicity, respectively. IL-10-treated dendritic cells (DCs) stimulate anergic cytotoxic T lymphocyte-associated molecule-4 (CTLA)-4-expressing CD4(+) T cell responses possibly through downregulation of major histocompetibility complex (MHC) class II and costimulatory molecule expression on DCs. The anergic CD4(+) T cells suppress T cell proliferation mainly through a CTLA-4-mediated pathway. The distinct role of IL-10 on T cell subsets may be useful in designing T cell-based immunotherapy of cancer and infectious diseases.

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.

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DCOVA), and act as Th-APCs in stimulation of CD8+CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DCOVA stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DCOVA and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).

The CXC chemokine SDF-1 has been characterized as a T-cell chemoattractant both in vitro and in vivo. To determine whether SDF-1 expression within tumors can influence tumor growth, we transfected an expression vector pCI-SDF-1 for SDF-1 into J558 myeloma cells and tested their ability to form tumors in BALB/c. Production of biologically active SDF-1 (1.2 ng/mL) was detected in the culture supernatants of cells transfected with the expression vector pCI-SDF-1. J558 cells gave rise to a 100% tumor incidence, whereas SDF-1-expressing J558/SDF-1 tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells. Regression of the J558/SDF-1 tumors was dependent on both CD4(+) and CD8(+) T-cells. Our data also indicate that TIT cells containing both CD4(+) and CD8(+) T-cells within J558/SDF-1 tumors express the SDF-1 receptor CXCR4, and that SDF-1 specifically chemoattracts these cells in vitro. Furthermore, immunization of mice with engineered J558/SDF-1 cells elicited the most potent protective immunity against 0.5 x 10(6) cells J558 tumor challenge in vivo, compared to immunization with the J558 alone, and this antitumor immunity mediated by J558/SDF-1 tumor cell vaccination in vivo appeared to be dependent on CD8(+) CTL. Thus, SDF-1 has natural adjuvant activities that may augment antitumor responses through their effects on T-cells and thereby could be important in gene transfer immunotherapies for some cancers.

The P815 and P198 cell lines are clonally related mouse mastocytoma cell lines. They differ in their biologic behavior in that P815 is a progressive tumor cell line, whereas P198 is a regressive one. These cell lines have been extensively used as models for the study of tumor-host relationships and tumor immunology. Although some of their biological properties have been well documented, the molecular mechanisms underlying tumor progression or regression have not been completely elucidated. In this study, we characterized the growth behavior and immunophenotype of these two cell lines, and analyzed their gene profiles using a complementary deoxynucleic acid (cDNA) microarray composed of 514 immunologically relevant genes. Our data showed that the two cell lines exhibited quite dissimilar and contrasting growth characteristics when inoculated into syngeneic mice. P815 tumors grew unremittingly, while P198 tumors gradually regressed. From a molecular viewpoint, P815 cells showed a higher expression of genes promoting tumor growth, such as IGF-1, IL-8R, FGFR1, VEGF-A, and VEGF-B. On the other hand, P198 tumor cells expressed CD11b and CD80, which favor the recruitment of lymphocytes and antigen-presenting cells (APCs), as well as the elicitation of antitumor immunity. P198 tumor cells also depicted a higher expression of genes inhibiting tumor growth, such as TNF-alpha, SOCS-1, CIS1, 4-1BB, and GDF-10. In conclusion, our results contribute further information in the understanding of the molecular mechanisms associated with the regression and progression of P815 and P198 tumor cells.

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

Tumor cell-derived exosomes (TEX) have been widely used to induce antitumor immune responses in animal models and clinical trials. However, the efficiency of the antitumor immunity that is induced by TEX is still relatively weak. In this study, we compared the antitumor immunities between EG7 tumor cell-derived exosomes (EXO(EG7)) and EXO(EG7)-targeted dendritic cells (DC(EXO)). We found that EXO(EG7) harbored OVA and peptide major histocompatibility complex I (pMHC-I), which were expressed on its parental EG7 tmor cells, and they could transfer OVA and pMHC-I to dendritic cells (DCs) in vitro. DC(EXO) could more efficiently induce antitumor immunity than EXO(EG7). In addition, we showed that the immune stimulatory effects of EXO(EG7) were dependent on the host DCs and, whereas those of DC(EXO) were not, indicating the important role of the host DCs in TEX vaccines. Taken together, TEX-targeted DCs may be more effective for EXO-based vaccines for the induction of antitumor immunity.

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.

CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cell's help is delivered to CD8+ T cells in vivo is still unclear. We previously demonstrated that CD4+ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC(OVA)) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4+ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC(OVA)-activated CD4+ Th cells (3 x 10(6) cells/mouse) can directly stimulate OVA-specific CD8+ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4+ T cells. A large amount of CD4+ Th cells (12 x 10(6) cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8+ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4+ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4+ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXOOVA) express exosomal peptide/MHC class I and costimulatory molecules. These EXOOVA-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXOOVA-uptaken (targeted)CD4+ aT cell’s stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.

The cooperative role of CD4+ helper T (Th) cells has been reported for CD8+ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4+ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naive CD8+ T cell proliferation and activation. In this study, we used CD4+ Th1 and CD8+ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4+ Th1 cell's help effects on active CD8+ Tc1 cells and the molecular mechanisms involved in CD8+ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4+ Th1 cells with acquired pMHC I by OVA-pulsed DC (DCOVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8+ Tc1 cells in vitro, and promoting CD8+ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8+ Tc1 with CD4+ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4+ Th1's help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8+ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.