Chimeric antigen receptor (CAR) T-cell therapy is highly effective in hematologic malignancies, yet its durability is limited by insufficient expansion, persistence, and T-cell exhaustion. Basic leucine zipper ATF-like transcription factor (BATF) promotes CD8+ T-cell effector differentiation but can drive exhaustion under chronic stimulation. Here, we developed a transient, non-viral strategy to modulate BATF expression in therapeutic T cells using clinically approved lipid nanoparticles (LNPs). Among 3 Food and Drug Administration (FDA)-approved ionizable lipids, SM-102-based LNPs achieved the highest mRNA delivery efficiency in primary T cells. Transient BATF overexpression enhanced T-cell cytotoxicity in vitro without inducing exhaustion. In vivo, BATF mRNA transfection enhanced T-cell expansion, reduced exhaustion, and improved anti-tumor activity for both OT-1 TCR-T cells in melanoma and CD19 CAR-T cells in acute lymphoblastic leukemia. These findings establish a safe and reversible platform for transient transcription factor modulation to optimize T-cell differentiation and function, thereby enhancing the efficacy of adoptive T-cell therapies and supporting clinical translation.

Age-related macular degeneration (AMD) is a prevalent neuroinflammation condition and the leading cause of irreversible blindness among the elderly population. Smoking significantly increases AMD risk, yet the mechanisms remain unclear. Here, we investigate the role of Sema4D-PlexinB1 axis in the progression of AMD, in which Sema4D-PlexinB1 is highly activated by smoking. Using patient-derived samples and mouse models, we discover that smoking increases the presence of Sema4D on the surface of CD8+ T cells that migrate into the choroidal neovascularization (CNV) lesion via CXCL12-CXCR4 axis and interact with its receptor PlexinB1 on choroidal pericytes. This leads to ROR2-mediated PlexinB1 phosphorylation and pericyte activation, thereby disrupting vascular homeostasis and promoting neovascularization. Inhibition of Sema4D reduces CNV and improves the benefit of anti-VEGF treatment. In conclusion, this study unveils the molecular mechanisms through which smoking exacerbates AMD pathology, and presents a potential therapeutic strategy by targeting Sema4D to augment current AMD treatments.

Background Approximately two-thirds of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) do not respond to or relapse after anti-CD19 chimeric antigen receptor T (CAR T)-cell therapy, leading to poor outcomes. Previous studies have suggested that intensified lymphodepletion and hematological stem cell infusion can promote adoptively transferred T-cell expansion, enhancing antitumor effects. Therefore, we conducted a phase I/II clinical trial in which CNCT19 (an anti-CD19 CAR T-cell) was administered after myeloablative high-dose chemotherapy and autologous stem cell transplantation (HDT/ASCT) in patients with R/R LBCL.Methods Transplant-eligible patients with LBCL who were refractory to first-line immunochemotherapy or experiencing R/R status after salvage chemotherapy were enrolled. The study aimed to evaluate the safety and efficacy of this combinational therapy. Additionally, frozen peripheral blood mononuclear cell samples from this trial and CNCT19 monotherapy studies for R/R LBCL were used to evaluate the impact of the combination therapy on the in vivo behavior of CNCT19 cells.Results A total of 25 patients with R/R LBCL were enrolled in this study. The overall response and complete response rates were 92.0% and 72.0%, respectively. The 2-year progression-free survival rate was 62.3%, and the overall survival was 68.5% after a median follow-up of 27.0 months. No unexpected toxicities were observed. All cases of cytokine release syndrome were of low grade. Two cases (8%) experienced grade 3 or higher CAR T-cell-related encephalopathy syndrome. The comparison of CNCT19 in vivo behavior showed that patients in the combinational therapy group exhibited enhanced in vivo expansion of CNCT19 cells and reduced long-term exhaustion formation, as opposed to those receiving CNCT19 monotherapy.Conclusions The combinational therapy of HDT/ASCT and CNCT19 demonstrates impressive efficacy, improved CNCT19 behavior, and a favorable safety profile.Trial registration numbers ChiCTR1900025419 and NCT04690192.

Relapse and treatment resistance pose significant challenges in the management of pediatric B cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The efficacy of immunotherapy in leukemia remains limited due to factors such as the immunosuppressive tumor microenvironment (TME) and lack of suitable immunotherapeutic targets. Thus, an in-depth characterization of the TME in pediatric leukemia is warranted to improve the efficacy of immunotherapy. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the TME of pediatric B-ALL and AML, focusing specifically on bone-marrow-derived T cells. Moreover, we investigated the transcriptome changes during the initiation, remission, and relapse stages of pediatric AML. Our findings revealed that specific functional expression programs correlated with fluctuations in various T cell subsets, which may be associated with AML progression and relapse. Furthermore, our analysis of cellular communication networks led to the identification of VISTA, CD244, and TIM3 as potential immunotherapeutic targets in pediatric AML. Finally, we detected elevated proportions of γδ T cells and associated functional genes in samples from pediatric patients diagnosed with B-ALL and AML, which could inform the development of novel therapeutic approaches, potentially focusing on γδ T cells.

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.

Cancer nanovaccine is a promising therapeutic strategy; however, the complicated composition and ambiguous structure of cancer nanovaccines hinder their clinical application. Thus, developing minimalist nanovaccine with simple structural molecule and potent immunostimulatory function is with great value. Herein, an organic small molecule with well-defined molecular structure and strong aggregation-induced emission (AIE) activity is synthesized. This molecule can self-assemble into nanoparticle (NM-7) acting as not only adjuvant but also carrier to capture model antigen ovalbumin to form nanovaccine, which can effectively deliver and chronically release antigens and induce effective antigen cross-presentation in draining lymph nodes. Additionally, the photophysical property of AIE molecule facilitates the determination of in vivo tissue distribution of NM-7. Notably, NM-7/ovalbumin (OVA) nanovaccine induces robust tumor-specific CD8(+) T-cell responses with greatly reduced cell exhaustion. Accordingly, NM-7/OVA nanovaccine shows potent efficacy in both prevention and therapeutic tumor models, and produces an impressive synergistic therapeutic effect when combined with immune checkpoint blockade (ICB) therapy. Therefore, AIE nanomaterial NM-7 with simple synthesis, well-defined molecular structure and self-adjuvanted function provides a new reference and general strategy for cancer vaccine development.

Background A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for haemophilia B gene therapy. We aimed to explore its safety and activity in increasing FIX concentrations and reducing bleeding frequency. Methods We did a single-centre, single-arm, phase 1, pilot trial evaluating the safety and activity of a single intravenous infusion of BBM-H901 at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Tianjin, China). We enrolled adult patients with haemophilia B (aged >18 years) with baseline FIX coagulation activity (FIX:C) of less than 2 IU/dL, no FIX inhibitor, and low titre of neutralising antibodies (<= 1:4) against vector capsid. Eligible participants were intravenously infused with a single dose of 5 x10(12) vector genomes (vg)/kg of BBM-H901 after 1 week of prophylactic prednisone treatment (1 mg/kg per day). Primary endpoints were the incidence of treatment-related adverse events, change in alanine aminotransferase (ALT) and aspartate amino transferase (AST), and development of antibodies against vector capsid within 1 year of infusion. We report the results of the prespecified 1-year analysis following complete enrolment. The trial is registered with ClinicalTrials.gov , NCT04135300, and is complete. Findings Between Oct 16, 2019, and Jan 13, 2021, 12 male participants were assessed, and ten Chinese participants were enrolled and infused with BBM-H901. After a median follow-up of 58 weeks (IQR 51.5-99.5), mean FIX:C reached mean 36.9 IU/dL (SD 20.5). No serious adverse events, no grade 3-4 adverse events were observed. Grade 1-2 adverse events related to BBM-H901 include pyrexia (1 [10%]) and elevation of aminotransferase (1 [10%]). No FIX inhibitors were observed. All participants developed antibodies against vector capsid after infusion. Eight (80%) participants had All and AST concentrations below the upper limit of normal throughout the follow-up period. Two (20%) participants had elevation of ALT and AST accompanied with decrease of FIX:C, which remained at 7 IU/dL and 11.8 IU/dL, respectively. Interpretation This pilot study suggests that liver-tropic BBM-H901 is safe 1 year after infusion. Vector derived FIX:C concentration is sufficiently high to prevent bleeding events and minimise the need for replacement therapy in small populations with haemophilia B. These findings support further study. Copyright (C) 2022 Published by Elsevier Ltd. All rights reserved.

Mesenchymal stem cells (MSCs) are a heterogeneous population with the capacities of self-renewal, tissue regeneration and immunoregulation, which have attracted intensive attention for their potential application in the treatment of various immune disorders. However, the immunosuppressive efficacy of these therapies based on MSC products has not been thoroughly characterized, which may lead to inconsistent outcomes of MSC therapy in clinical trials. Cellular senescence is a state that imposes permanent proliferative arrest on cells in response to various types of stress and damage. Evidence suggests that aging MSCs tend to have a compromised immunosuppressive capability for inhibiting T cells. However, the underlying cellular and molecular mechanisms are poorly understood. It is also unknown whether the proportions and states of senescent cells in MSC products derived from various donors and different culture passages is associated with dissonant outcomes across clinical trials of MSC therapies. Immune thrombocytopenia (ITP) is an autoimmune disease characterized by immune-mediated destruction of circulating platelets (PLTs) and suppression of PLT production. It has been reported that autoantibody-coated PLTs undergo accelerated clearance through the spleen macrophage. However, anti-PLT antibodies are detected in less than 50% of patients, which raises the possibility of alternative mechanisms of PLT destruction. In line with this, splenectomy, which removes the primary site of PLT destruction and inhibits B-cell-mediated autoantibody production, has been shown to be the most effective therapy for steroid-refractory or steroid-dependent ITP and offers the highest rate of durable response (60% to 70%) compared with other therapies. Nevertheless, 30% to 40% of refractory ITP patients still show no response to splenectomy, indicating a B-cell-independent pathogenic mechanism. Abnormal function of T cells has been reported in ITP patients, which could collectively drive the autoimmune process. Therefore, the immune mechanism underpinning ITP should be further investigated. Additionally, it is of great value to explore new therapeutic options, such as MSC-based cellular therapy, especially for splenectomy-nonresponsive refractory ITP. Here, we performed a single-cell transcriptomic analysis of clinically used MSCs derived from adipose tissue (AD), the bone marrow (BM), the placental chorionic membrane (PM) and the umbilical cord (UC). The transcriptomic profiles of a total of 45,955 cells revealed an unprecedented level of heterogeneity in cellular senescence. We found that cultured MSCs underwent progressive cell aging, accompanied by the gradual loss of immunosuppressive function. Moreover, compared to adult MSCs (derived from AD and the BM), perinatal MSCs (derived from the PM and UC) had fewer aging cells and exhibited more potent suppressive activity. Mechanistically, we found that the impaired immunomodulatory function of senescent MSCs was associated with reduced expression of the immunosuppressive surface molecule PD-L1, which was demonstrated to be crucial for mediating the immunosuppressive activity of MSCs. Next, we performed single-cell transcriptome analysis and revealed that T-cell hyperactivation was evident in a cohort of refractory ITP patients who had no long-lasting remission after splenectomy. We therefore conducted the registered clinical trial (NCT04014166) of the use of UC-derived MSCs for refractory ITP. In this single-center, phase I trial, six refractory ITP patients received four rounds of three different doses of UC-derived MSC transfusions (0.5 × 106 MSCs/kg, n = 3; 1.0 × 106 MSCs/kg, n = 2; 2.0 × 106 MSCs/kg, n = 1). Notably, all three participants who received 1.0 × 106 or 2.0 × 106 MSCs/kg responded to the transfusions, with a manageable safety profile, while all three participants who received 0.5 × 106 MSCs/kg had no response to the transfusions. Our findings reveal the pivotal roles of cell aging and PD-L1 expression in determining the immunosuppressive efficacy of MSCs and demonstrate that PD-L1 abundant perinatal MSCs are effective in treating refractory ITP with T cell hyperactivation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

既往研究表明,造血细胞的细胞遗传学、分子遗传学或表观遗传学异常导致血液系统恶性肿瘤发生.然而,近期的研究表明骨髓微环境在血液系统肿瘤性疾病的发生、疾病进展和对化疗药物耐受中发挥着重要的作用.该文总结了稳态条件下骨髓微环境的组成以及对正常造血的调控,异常的骨髓微环境如何驱动血液系统肿瘤性疾病发生,以及血液系统肿瘤性疾病如何重塑骨髓微环境;最后,对靶向免疫抑制性的骨髓微环境治疗血液系统恶性肿瘤的研究进展进行了讨论和展望.

Chimeric antigen receptor (CAR)-T-cell therapeutic efficacy is associated with long-term T-cell persistence and acquisition of memory. Memory-subset formation requires T-cell factor 1 (TCF-1), a master transcription factor for which few regulators have been identified. Here, we demonstrate using an immune-competent mouse model of B-cell acute lymphoblastic leukemia (ALL; B-ALL) that Regnase-1 deficiency promotes TCF-1 expression to enhance CAR-T-cell expansion and memory-like cell formation. This leads to improved CAR-T-mediated tumor clearance, sustained remissions, and protection against secondary tumor challenge. Phenotypic, transcriptional, and epigenetic profiling identified increased tumor-dependent programming of Regnase-1-deficient CAR-T cells into TCF-1+ precursor exhausted T cells (TPEX) characterized by upregulation of both memory and exhaustion markers. Regnase-1 directly targets Tcf7 messenger RNA (mRNA); its deficiency augments TCF-1 expression leading to the formation of TPEX that support long-term CAR-T-cell persistence and function. Regnase-1 deficiency also reduces exhaustion and enhances the activity of TCF-1- CAR-T cells. We further validate these findings in human CAR-T cells, where Regnase-1 deficiency mediates enhanced tumor clearance in a xenograft B-ALL model. This is associated with increased persistence and expansion of a TCF-1+ CAR-T-cell population. Our findings demonstrate the pivotal roles of TPEX, Regnase-1, and TCF-1 in mediating CAR-T-cell persistence and recall responses, and identify Regnase-1 as a modulator of human CAR-T-cell longevity and potency that may be manipulated for improved therapeutic efficacy.

Chimeric Antigen Receptor (CAR) modified T cells have been successful in treatment of acute lymphoblastic leukemia (ALL), however the efficacy of CAR T cell therapy is often limited by the poor persistence and function of adoptively transferred cells. We identified REGNASE-1 as a negative regulator of antitumor responses that modulates the persistence and effector function of adoptively transferred CD8+ T cells. [1] We developed a novel model to study the efficacy of Regnase-1-deficient CAR T cells in an immune replete environment. T cells from transgenic mice expressing a clinically relevant anti-human CD19 CAR were used to treat primary mouse ALL cells that express human CD19. We find that REGNASE-1-deficient CD8+ CAR T cells have improved in vivo persistence and exhibit markedly improved therapeutic efficacy against ALL. REGNASE-1-deficient CD8+ CAR T cells undergo antigen-dependent reprogramming to a naïve/memory-like phenotype, with simultaneous antigen-independent reduction in exhaustion marker expression. Regnase-1-deficiency also leads to enhanced memory responses of CAR T cells after primary tumor clearance. Our findings indicate that Regnase-1 modulates long term persistence and memory in CAR T cells. Depletion of Regnase-1 provides a new approach to improve the therapeutic efficacy of CAR T cell therapy. Supported by ALSAC/St. Jude Children’s Research Hospital and The Assisi Foundation of Memphis

Cross-species regulation of gene expression by microRNA is a possible untapped opportunity for miRNA-based therapy. In this study, we report a novel approach to ablate melanoma stem-like cells by targeting the transcription factor YB-1, which is significantly and selectively upregulated in these cells in melanoma. Silencing YB-1 expression was sufficient to significantly inhibit the stemness of melanoma stem-like cells. In exploring YB-1 targeting, we discovered that the shrimp microRNA miR-S8 could suppress human YB-1 expression in melanoma stem-like cells. Mechanistic investigations revealed that miR-S8 recognized the 3'UTR of YB-1 mRNA and mediated its degradation. In tumor cell and xenograft experiments, miR-S8 suppressed the tumorigenic capacity of melanoma stem-like cells by targeting human YB-1. Overall, our results illuminated a novel aspect of miRNA-mediated cross-species gene expression and its use in regulating cancer stem-like cells. Cancer Res; 77(20); 5543-53. ©2017 AACR.

Tregs (Foxp3+CD4+) are enriched in tumors to foster a tolerant microenvironment that inhibits antitumor immune response. IL-27 is reported to regulate the development and function of Tregs in vitro and in vivo; however, the effects of endogenous IL-27 on Tregs in the tumor microenvironment remain elusive. We demonstrated that in the absence of DC-derived IL-27, Tregs were decreased significantly in transplanted B16 melanoma, transplanted EL-4 lymphoma, and MCA-induced fibrosarcoma by using IL-27p28 conditional KO mice. Further studies revealed that IL-27 promoted the expression of CCL22, which is established to mediate the recruitment of peripheral Tregs into tumors. Tumor-associated DCs were identified as the major source of CCL22 in tumor sites, and IL-27 could induce CCL22 expression in an IL-27R-dependent manner. Intratumoral reconstitution of rmCCL22 or rmIL-27, but not rmIL-27p28, significantly restored the tumor infiltration of Tregs in IL-27p28 KO mice. Correlated with a decreased number of Tregs, tumor-infiltrating CD4 T cells were found to produce much more IFN-γ in IL-27p28 KO mice, which highlighted the physiological importance of Tregs in suppressing an antitumor immune response. Overall, our results identified a novel mechanism of action of IL-27 on Tregs in the context of cancers.

Meeting abstracts Roles of dendritic cells (DCs) in tumor immune responses have been reported to be very complex. It has been shown that in some tumor types, dendritic cells play an protective role by activating anti-tumor cells, while in other tumor types, they accelated tumor growth by inducing

The current study evaluated the glycoproteomic profile of tissues from colon cancer patients. The lectin microarray was first performed to compare the glycoprotein profiles between colon cancer and matched normal tissues. Level of N-acetylglucosamine (GlcNAc) that Solanum tuberosum lectin (STL) bound was found to be elevated in colon cancer, which was verified through lectin histochemistry. The subsequent glycoproteomic analysis based on STL enrichment of glycoproteins followed by label-free quantitative nano liquid chromatography-mass spectrometry/mass spectrometry (nanoLC-MS/MS) analysis identified 72 proteins in high confidence. Among these proteins, 17 were exclusively detected in cancer tissues, and 14 were significantly upregulated in tumor tissues. Annexin A1 and HSP90β were chosen for further investigation by immunoprecipitation coupled with lectin blots, western blots and tissue microarrays. Both Annexin A1 and HSP90β were GlcNAcylated, and their protein expressions were elevated in colon cancer, compared to normal tissues. Moreover, specific changes of GlcNAc abundances in Annexin A1 and HSP90β suggested that tumor-specific glycan patterns could serve as candidate biomarkers of colon cancer for distinguishing cancer patients from healthy individuals.

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene-induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell-dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Nanogels are promising carriers for the delivery of anti-cancer drugs for cancer therapy. We report in this study on a Janus nanogel system formed by mixing a prodrug of Taxol (PEGylated Taxol) and a copolymer of PLGA-PEG-PLGA. The Janus nanogels have good stability over months in aqueous solutions and the freeze-dried powder of nanogels can be re-dispersed instantly in aqueous solutions. The Janus nanogels show an enhanced inhibition effect on tumor growth in a mice breast cancer model probably due to the enhanced uptake of the nano-sized materials by the EPR effect. What is more, the nanogels can also serve as physical carriers to co-deliver other anti-cancer drugs such as doxorubicin to further improve the anti-cancer efficacy. The results obtained from H&E staining and TUNEL assay also support the observation of tumor growth inhibition. These results suggest the potential of this novel delivery system for cancer therapy.

Background. gamma delta T cells comprise a small subset of T cells and play a protective role against cancer and viral infections; however, their precise role in patients with chronic hepatitis B remains unclear.Methods. Flow cytometry and immunofunctional assays were performed to analyze the impact of V delta 2 gamma delta (V delta 2) T cells in 64 immune-activated patients, 22 immune-tolerant carriers, and 30 healthy controls.Results. The frequencies of peripheral and hepatic V delta 2 T cells decreased with disease progression from immune tolerant to immune activated. In the latter group of patients, the decreases in peripheral and intrahepatic frequencies of V delta 2 T cells reversely correlated with alanine aminotransferase levels and histological activity index. These activated terminally differentiated memory phenotypic V delta 2 T cells exhibited impaired abilities in proliferation and chemotaxis, while maintained a relative intact interferon (IFN) gamma production. Importantly, V delta 2 T cells, in vitro, significantly suppressed the production of cytokines associated with interleukin 17-producing CD4(+) T (Th17) cells through both cell contact-dependent and IFN-gamma-dependent mechanisms.Conclusions. Inflammatory microenvironment in IA patients result in decreased numbers of V delta 2 T cells, which play a novel role by regulating the pathogenic Th17 response to protect the liver in patients with chronic hepatitis B.