Natural killer/T cell lymphoma (NKTCL) exhibits highly aggressive clinical behavior, and the outcomes for relapsed/refractory patients are still poor. Recently, the mechanism underlying the effect of Epstein-Barr virus (EBV) infection, which has not been fully defined in NKTCL, has attracted great attention. We explored how LMP1 promoted aerobic glycolysis via metabolic sequencing combined with mRNA sequencing and immunoprecipitation coupled to mass spectrometry. Experimental assays were used to determine the effects of LMP1 and its downstream pathway on the function and glucose metabolism of NKTCL cells. The correlations between LMP1 expression in patients and their clinical features, treatment response, and prognosis were analyzed. Results show that LMP1 enhances NKTCL cell proliferation in vitro and in vivo, inhibits apoptosis, and decreases gemcitabine sensitivity. In addition, LMP1 also enhances aerobic glycolysis in NKTCL cells, as indicated by increases in glucose uptake, lactate production, and extracellular acidification rate. Clinically, LMP1 expression is correlated with risk stratification, treatment response, and prognosis, and higher LMP1 expression indicates greater SUVmax for NKTCL patients. Mechanistically, LMP1 competitively binds to TRAF3 to promote cell proliferation and aerobic glycolysis by regulating the noncanonical NF-κB pathway. The application of an NF-κB pathway inhibitor or reactivation of the NF-κB pathway affects aerobic glycolysis and the biological function of NKTCL cells. In summary, this study is the first to describe and define in detail how LMP1 affects glucose metabolism in NKTCL and might provide a novel perspective for further treatment.

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Natural killer/T-cell lymphoma (NKTCL) is an aggressive lymphoma with poor prognosis. In the past decade, chimeric antigen receptor transduced T (CAR-T) cell therapy has been a promising strategy against hematologic malignancies. Researchers have evaluated the efficacy of CAR-T cell targeting CD38 in multiple myeloma and NKTCL. LMP1 may induce a malignant transformation in B cells and epithelial cells, making it a non-negligible molecule in NKTCL. These suggest the potential clinical administration of CD38 and LMP1 targeting therapy. Aims: We aimed to provide an immunotherapy for clinical practice and improve the treatment efficiency of NKTCL. Methods: Four second-generation CAR-T cells including two single-targeted CAR (CD38-CAR and LMP1-CAR) and two tandem CAR (CD38-LMP1 Tan CAR 1 and Tan CAR 2) were constructed. Then the effects of four CAR-T cells on NKTCL cells were evaluated both in vitro and in vivo. Results: Four CAR-T cells could effectively eliminate malignant target-positive NKTCL cells in vitro. They could be activated and produce inflammatory cytokines in a target-dependent manner. Stimulated by CD38+LMP1+ NKTCL cells, two Tan CAR-T cells produced more IL-13, Granzyme B, IFN-γ and TNF-α than LMP1-CAR-T cells. In vivo test showed that CAR-T cells also exhibit significant anti-tumor effects in a xenotransplant NKTCL mice models. Two Tan CAR-T cells showed higher cytolytic activity against CD38+LMP1+ NKTCL cells than single-targeted CAR-T cells both in vitro and in vivo. Summary/Conclusion: We explored the feasibility of CAR-T cell therapy targeting both CD38 and LMP1 molecule. Our results indicated a promising effective therapeutic strategy for NKTCL. Keywords: Non-Hodgkin’s lymphoma, CD38, EBV, CAR-T

Background: Multiple myeloma (MM) is an aggressive plasma cell malignancy, causing a number of deaths worldwide every year. Chimeric antigen receptor (CAR) transduced T-cell therapy has been a promising immunotherapy against hematological malignancies.Methods: In this study, we developed a second-generation CAR construct and generated CAR-T cells targeting CD38 molecule. Then effects of CAR-T cells against MM cell lines were evaluated.Results: CD38-CAR-T cells showed higher cytotoxicity to MM cell lines and primary MM cells than that of control T cells in vitro. Over 50% MM1.s and RPMI8226 cells were killed by CAR-T cells even at effector to target ratio of 1:100. CAR-T cells also showed an enhanced cytotoxicity against primary MM cells. CAR-T cells could be activated and produced a variety of cytokines in a target-dependent manner. In vivo test indicated that CAR-T cells also showed significant antitumor effect on xenograft mice models.Conclusion: These results indicated a promising therapeutic strategy of CD38-CAR-T cells against MM.

Background Natural killer/T cell lymphoma (NKTCL) is an aggressive lymphoma with a poor prognosis. Chimeric antigen receptor-transduced T (CAR-T) cell therapy has become a promising immunotherapeutic strategy against haematologic malignancies. Methods In this study, four CAR-T cell lines (CD38-CAR, LMP1-CAR, CD38-LMP1 tandem CAR 1 and CD38-LMP1 tandem CAR 2) were generated. The effect of CAR-T cells against NKTCL cells was evaluated both in vitro and in vivo. Expression of T cell activation markers and cytokines produced by CAR-T cells were detected by flow cytometry. Results The four CAR-T cell lines could effectively eliminate malignant NKTCL cells. They could be activated and produce inflammatory cytokines in a target-dependent manner. In vivo tests showed that the CAR-T cells exhibited significant antitumour effects in a xenotransplanted NKTCL mouse model. Conclusions In summary, four CAR-T cell lines exhibited significant cytotoxicity against NKTCL cells both in vitro and in vivo. These results indicated the effective therapeutic promise of CD38 and LMP1 CAR-T cells in NKTCL.

Gut microbiota represents a hidden treasure vault encompassing trillions of microorganisms that inhabit the intestinal epithelial barrier of the host. In the past decade, numerous in-vitro, animal and clinical studies have revealed the profound roles of gut microbiota in maintaining the homeostasis of various physiological functions, especially immune modulation, and remarkable differences in the configuration of microbial communities between cancers and healthy individuals. In addition, although considerable efforts have been devoted to cancer treatments, there remain many patients succumb to their disease with the incremental cancer burden worldwide. Nevertheless, compared with the stability of human genome, the plasticity of gut microbiota renders it a promising opportunity for individualized treatment. Meanwhile, burgeoning findings indicate that gut microbiota is involved in close interactions with the outcomes of diverse cancer immunotherapy protocols, including immune checkpoint blockade therapy, allogeneic hematopoietic stem cell transplantation, and chimeric antigen receptor T cell therapy. Here, we reviewed the evidence for the capacity of gut microflora to modulate cancer immunotherapies, and highlighted the opportunities of microbiota-based prognostic prediction, as well as microbiotherapy by targeting the microflora to potentiate anticancer efficacy while attenuating toxicity, which will be pivotal to the development of personalized cancer treatment strategies.

The treatment of natural killer/T-cell lymphoma (NKTCL) presents an onerous challenge, and a search for new therapeutic targets is urgently needed. Poly ADP-ribose polymerase inhibitors (PARPi) were initially used to treat breast and ovarian cancers with BRCA1/2 mutations. Their excellent antitumor efficacy led to a series of clinical trials conducted in other malignancies. However, the exploration of PARPi and their potential use in combination treatments for NKTCL remains unexplored. We treated NKTCL cell lines with fluzoparib (a novel inhibitor of PARP) and chidamide (a classical inhibitor of HDACs) to explore their cytotoxic effects in vitro. Then, their antitumor efficacy in vivo was confirmed in YT-luciferin xenograft mouse models. Fluzoparib or chidamide alone inhibited NKTCL cell proliferation in a dose-dependent manner. Cotreatment with both drugs synergistically induced excessive accumulation of DNA double-strand breaks and massive apoptotic cell death by inhibiting the DNA damage repair pathway, as shown by the decreased protein levels of p-ATM, p-BRCA1, p-ATR, and Rad51. Moreover, the combination treatment apparently increased the level of intracellular reactive oxygen species (ROS) to enhance apoptosis, and pretreatment with an ROS scavenger reduced the proapoptotic effect by 30-60% in NKTCL cell lines. In vivo, this combined regimen also showed synergistic antitumor effects in xenograft mouse models. The combination of fluzoparib and chidamide showed synergistic effects against NKTCL both in vitro and in vivo and deserves further exploration in clinical trials.

e19529 Background: Natural killer/T-cell lymphoma (NKTCL) is an aggressive lymphoma with poor prognosis. In the past decade, chimeric antigen receptor transduced T (CAR-T) cell therapy has been a promising strategy against hematologic malignancies. Researchers have evaluated the efficacy of CAR-T cell targeting CD38 in multiple myeloma and NKTCL. LMP1 may induce a malignant transformation in B cells and epithelial cells, making it a non-negligible molecule in NKTCL. These suggest the potential clinical administration of CD38 and LMP1 targeting therapy. Methods: Four second-generation CAR-T cells including two single-targeted CAR (CD38-CAR and LMP1-CAR) and two tandem CAR (CD38-LMP1 Tan CAR 1 and Tan CAR 2) were constructed. Then the effects of four CAR-T cells on NKTCL cells were evaluated both in vitro and in vivo. Results: Four CAR-T cells could effectively eliminate malignant target-positive NKTCL cells in vitro. They could be activated and produce inflammatory cytokines in a target-dependent manner. Stimulated by CD38+LMP1+ NKTCL cells, two Tan CAR-T cells produced more IL-13, Granzyme B, IFN-γ and TNF-α than LMP1-CAR-T cells. In vivo test showed that CAR-T cells also exhibit significant anti-tumor effects in a xenotransplant NKTCL mice models. Two Tan CAR-T cells showed higher cytolytic activity against CD38+LMP1+ NKTCL cells than single-targeted CAR-T cells both in vitro and in vivo. Conclusions: We explored the feasibility of CAR-T cell therapy targeting both CD38 and LMP1 molecule. Our results indicated a promising effective therapeutic strategy for NKTCL.

Additional file 1: Fig. S1. A: CD38 and LMP1 expression in NKTCL patients. Fig. S2. CAR expression was detected by FITC-labeled human CD38 protein or EGFP. Fig. S3. Activation markers (CD69, CD25 and HLA-DR) expression on CD4+ and CD8+ subgroups of CAR-T cells co-cultured with YT, KAI3, SNK6 or SNT16. Fig. S4. Statistical results of activation markers expression on CAR-T cells co-cultured with YT, KAI3, SNK6 or SNT16. Fig. S5. Statistical results of cytokines releasing of CAR-T cells co-cultured with YT, KAI3, SNK6 or SNT16. Fig. S6. Flow histogram showed fluorescence intensity of each cytokine. Fig. S7. Survival curve data for each experimental group.

Natural killer/T-cell lymphoma (NKTCL) is an incurable aggressive T-cell lymphoma closely correlated with Epstein‒Barr virus (EBV) infection. Chronic and consistent viral infection induces T-cell exhaustion. Herein, we describe T-cell dysfunction in NKTCL patients for the first time. Peripheral blood mononuclear cells (PBMCs) from age-matched healthy donors (HDs) and NKTCL patients were collected, and lymphocyte distributions, multiple surface inhibitory receptors (IRs), effector cytokine production and cell proliferation were determined by flow cytometry. PBMCs from HDs were cocultured with NKTCL cell lines to verify the clinical findings. IR expression was further assessed in NKTCL tumor biopsies using multiplex immunohistochemistry (mIHC). NKTCL patients have higher frequencies than HDs of inhibitory T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). T-cell distribution also varies between NKTCL patients and HDs. T cells from NKTCL patients demonstrated higher expression levels of multiple IRs than HDs. Meanwhile, T-cell proliferation and interferon-γ production was significantly reduced in NKTCL patients. More importantly, the number of EBV-specific cytotoxic cells was lower in NTKCL patients, and these cells demonstrated upregulation of multiple IRs and secreted fewer effector cytokines. Interestingly, NKTCL cells caused normal PBMCs to acquire T-cell exhaustion phenotypes and induced generation of Tregs and MDSCs. In line with ex vivo finding, mIHC results showed that CD8+ T cells from NKTCL tumor biopsies expressed much higher level of IRs compared with reactive lymphoid hyperplasia individuals. The immune microenvironment of NKTCL patients exhibited T-cell dysfunction and accumulation of inhibitory cell components, which may contribute to impaired antitumor immunity.

BACKGROUND:PARP inhibitor (PARPi), as a kind of DNA damage repair inhibitor, has been shown to be effective in various solid tumors and hematologic malignancies. Natural killer/T cell lymphoma (NKTCL) is a highly aggressive malignancy, the treatment of which has long been a major challenge in the clinic. Here, we investigated the efficacy and mechanism of PARPi, and the therapeutic value of PARPi combined with cisplatin in NKTCL.METHODS:The cell proliferation, cell apoptosis, and cell cycle of NKTCL cells were detected respectively by CCK-8 and flow cytometry. The changes of mRNA expression and protein level were measured respectively by mRNA-sequencing, quantitative real-time PCR, western blotting, and immunofluorescence. LMO2 expression was detected by immunohistochemistry and western blotting. Targeted knockdown of LMO2 was conducted by short hairpin RNA. The tumor xenograft models were established to evaluate the efficacy of drugs in vivo.RESULTS:PARPi inhibited cell proliferation, promoted cell apoptosis, and induced S-phase cell cycle arrest in NKTCL cells. PARPi led to the accumulation of DNA damage by blocking DNA repair and DNA replication. Additionally, LMO2 deficiency reduced the sensitivity of NKTCL cells to PARPi. Finally, the combination of PARPi and cisplatin exhibited significant synergistic effects both in vitro and in vivo.CONCLUSIONS:In summary, we found that PARPi exerted an anti-tumor effect via LMO2 and synergized with cisplatin in NKTCL, which provides the theoretical basis for the clinical application of PARPi.

The development of chimeric antigen receptor T (CAR-T) cell therapy, a specific type of immunotherapy, in recent decades was a fantastic breakthrough for the treatment of hematological malignancies. However, difficulties in collecting normal T cells from patients and the time cost of manufacturing CAR-T cells have limited the application of CAR-T-cell therapy. In addition, the termination of related clinical trials on universal CAR-T cell therapy has made further research more difficult. Natural killer (NK) cells have drawn great attention in recent years. Chimeric antigen receptor-NK (CAR-NK) cell therapy is a promising strategy in the treatment of malignant tumors because of its lack of potential for causing graft-versus-host disease (GVHD). In this review, we will address the advances in and achievements of CAR-NK cell therapy.

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive hematological malignancy. However, there is currently no consensus on therapies for refractory/relapsed patients. In this study, we investigated the synergistic anticancer effect and potential mechanism of combining chidamide, a histone deacetylases (HDACs) inhibitor, and etoposide, a DNA-damaging agent, in NKTCL. We demonstrated that chidamide or etoposide alone dose- and time-dependently inhibited the cell viability of NKTCL cell lines, YT, NKYS and KHYG-1. Functional experiments suggested that combined chidamide and etoposide treatment exerted synergistic antiproliferation effect and enhanced cell apoptotic death in vitro and in vivo. Furthermore, the expression of DNA damage related proteins was detected and we also examined the alternations in histone acetylation, cell cycle progression, and mitochondrial membrane potential (MMP). The results suggested that increased histone acetylation, cell cycle arrest at the G2/M phase and loss of MMP, converging to greater DNA damage, might account for the synergism of the combination of chidamide and etoposide in NKTCL. Taken together, our study provides an evident for possible application on combining HDACs inhibitors and DNA-damaging agents for the treatment of NKTCL.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and numerous oncogenes are associated with this disease. Oxysterol-binding protein-related protein 8 (ORP8) is essential for cell growth, migration and the modulation of mitochondrial respiration and morphology. However, the underlying role of ORP8 in NSCLC remains unclear. In the present study, it was reported that the expression of ORP8 was low in NSCLC cells and tissues. The ORP8 expression levels were analyzed by immunohistochemistry (IHC), quantitative real-time PCR (qPCR) and western blot analysis. ORP8 overexpression inhibited cell growth and induced apoptosis in NSCLC cells with MTS, anchorage-independent growth and Hoechst 33342 staining assay. Further experiments demonstrated that ORP8 overexpression induced the apoptosis of NSCLC cells via the release of cytochrome c from mitochondria into the cytoplasm with western blot analysis and confocal microscopy results. In addition, qPCR analysis showed that miR-421 was upregulated in NSCLC cell lines, with the bioinformatics analysis, western blot analysis and Dual-Luciferase reporter assay, it was determined that miR-421 could target ORP8. The inhibition of cell proliferation via ORP8 overexpression was rescued by a miR-421 mimic, which aided in maintaining the proliferative potential of the cells. Overall, the present study revealed that ORP8 may be a candidate target in the prevention and treatment of NSCLC.

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA.

Objective To investigate the roles of IP-10 and interferon-γin the pathogenesis of psoriasis and compare the effects of quercetin and dexamethasone on psoriasis like HaCaT cells.Methods Cultured HaCaT cells were divided into 5 groups:①nQ group (40、50、60 μmol/L),②Dex group (10-7、10-6、10-5 mol/L),③IFN-γgroup(50,100,150 U/ml),④ IP-10 group(20,30,40 ng/μl) and ⑤control group(C).The growth rate(GR) or growth suppression rate(GSR) of nQ,Dex,IFN-γ and IP-10 were determined by MTT assay.DNA and RNA of IP-10 and c-myc hybrid signals were detected by in situ hybridization.Results The GR/GSR of each group showed a dose-dependent manner,and the GSR of nQ was higher than Dex,the difference was significant(P ＜0.01).In situ hybridization showed that the hybrid signal was up-regulated in IFN-γ group and IP-10 group,while down-regulated in nQ group and Dex group,the differences were significant (P ＜ 0.01).Conclusions Certain concentrations of interferon-γ and IP-10 can promote proliferation of HaCaT cells,while quercetin and dexamathasone can inhibit proliferation of HaCaT cells.Besides,quercetin is more potent than dexamethasone.

Objective To explore the effects of natural avocado oil and olive oil on the proliferation and differentiation of HaCaT cells. Methods MTT assay was performed to determine the optimal work concentration of avocado oil and olive oil. Cultured HaCaT cells were divided into 3 groups, i.e., avocado oil group treated with avocado oil of 3% (v/v), olive oil group treated with olive oil of 3% (v/v), and control group without any treatment. Immunocytochemistry and immuno-dot-blot method were used to detect the expressions of c-myc, mitogen-activated protein kinase ( MAPK ), nuclear factor ( NF)-κB, filaggrin, involucrin and keratin10 in HaCaT cells. Results As immunocytochemistry showed, the mean grey values (staining intensity) of c-myc,MAPK, and NF-κB in HaCaT cells were 131.4 ± 6.6,136.3 ± 4.5 and 134.3 ± 5.2 respectively in the avocado oil group, 121.1 ± 4.5, 107.9 ± 7.3 and 106.4 ± 5.4 respectively in the olive oil group, significantly higher than that in the control group (101.9 ± 8.9,91.4 ± 5.1 and 94.3 ± 7.0, respectively, all P＜ 0.05), and the avocado oil group was higher than the olive oil group in all the above parameters (all P ＜ 0.05). Increased expressions of filaggrin, involucrin and keratin 10 were observed in the avocado oil group and olive oil group compared with the control group (all P＜ 0.05), and in the olive oil group than in the avocado oil group (all P＜ 0.05).The mean grey values of these proteins obtained by immunocytochemisty were significantly correlated with those obtained by immuno-dot-blot method in avocado oil group (r = 0.94, P ＜ 0.01 ) and olive oil group (r=0.97, P ＜ 0.01 ). Conclusions Certain concentrations of avocado oil and olive oil can promote the proliferation and differentiation of HaCaT cells; avocado oil is more capable to accelerate their growth and proliferation, and olive oil to enhance their differentiation.

Objective To explore the effects of vitamin C and niacinamide on the growth and differentiation of human primary cultured keratinocytes.Methods Normal human foreskin was used in this study.The epidermis was separated enzymatically from the dermis by thermolysin,and keratinocytes were isolated from the epidermis by digestion with trypsin plus EDTA.The single keratinocytes were cultured with undedying NIH-3T3 cells as feeder cells in a complete medium supplied with 50 mg/L (vitamin C group),niacinamide of 400 μmol/L(niacinamide group)or vehicle(control group).Immunocytochemistry and immunodot blot were performed using monoclonal antibodies directed against C-myc,cyclin D1,filaggrin and involucrin.Results The colony number was highest in vitamin C group,followed by the control group and niacinamide group,and the colony morphology in vitamin C group was similar to that in the control group,but distinct from that in the niacinamide group.A significant increase was noticed in the expression of C-myc,cyclin D1,filaggrin and involucrin in vitamin C-treated keratinocytes compared with the control keratinocytes(all P＜0.05);however,in niacinamide-treated keratinocytes,the expression of filaggrin was significantly enhanced(P＜0.01),that of involucrin remained unchanged(P＞0.05),while that of C-myc was depressed(P＜0.05).Conclusions These results demonstrate that vitamin C has a favorable effect on both the growth and differentiation of human keratinocytes,while niacinamide seems to only promote the differentiation but attenuate the growth of human keratinocytes.

Objective To observe the clinical efficacy of acitretin combined with NB-UVB phototherapy in treating patients with psoriasis vulgaris.Methods 30 patients treated with acitretin 10 per time and three times a day,and NB-UVB(three times per week),28 patients treated with acitretin only,total time was 8 weeks.Results The effective rate in treating group was 90.00%,64.28% in control group( P0.05).Conclusion Acitretin combined with NB-UVB in treating patients with psoriasis vulgaris is more effective and safe .