Cancer-associated fibroblasts contribute to the immunosuppressive landscape and influence the efficacy of the combination therapy of PD-1 inhibitors and antiangiogenic agents in hepatocellular carcinoma.

BACKGROUND: Chronic inflammation is considered the most critical predisposing factor of hepatocellular carcinoma (HCC), with inflammatory cell heterogeneity, hepatic fibrosis accumulation, and abnormal vascular proliferation as prominent features of the HCC tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) play a key role in HCC TME remodeling. Therefore, the level of abundance of CAFs may significantly affect the prognosis and outcome in HCC patients. METHODS: Unsupervised clustering was performed based on 39 genes related to CAFs in HCC identified by single-cell RNA sequencing data. Patients of bulk RNA were grouped into CAF low abundance cluster and high abundance clusters. Subsequently, prognosis, immune infiltration landscape, metabolism, and treatment response between the two clusters were investigated and validated by immunohistochemistry. RESULTS: Patients in the CAF high cluster had a higher level of inflammatory cell infiltration, a more significant immunosuppressive microenvironment, and a significantly worse prognosis than those in the low cluster. At the metabolic level, the CAF high cluster had lower levels of aerobic oxidation and higher angiogenic scores. Drug treatment response prediction indicated that the CAF high cluster could have a better response to PD-1 inhibitors and conventional chemotherapeutic agents for HCC such as anti-angiogenic drugs, whereas CAF low cluster may be more sensitive to transarterial chemoembolization treatment. CONCLUSIONS: This study not only revealed the TME characteristics of HCC with the difference in CAF abundance but also further confirmed that the combination therapy of PD-1 inhibitors and anti-angiogenic drugs may be more valuable for patients with high CAF abundance.

Tissue adaptation of regulatory T cells in adipose tissue.

Foxp3+ regulatory T (Treg) cells critically suppress over-activated immune responses and therefore maintain immune homeostasis. Adipose tissue-resident Treg (AT Treg) cells are known for modulating immunity and metabolism in adipose tissue microenvironment through various physiological signals, as well as their heterogeneous subsets, which potentially play disparate roles in aging and obesity. Recent single-cell studies of Treg cells have revealed specialized trajectories of their tissue adaptation and development in lymphoid tissues and at barrier sites. Here, we reviewed a T Cell Receptor (TCR)-primed environmental cue-boosted model of adipose Treg cells' tissue adaptation, especially in response to IL-33, IFN-α, insulin, and androgen signals, which trigger sophisticated transcriptional cascades and ultimately establish unique transcriptional modules in adipose Treg cell subsets. In addition, we further discuss potential therapeutic strategies against aging and obesity by blocking detrimental environmental cues, strengthening the functions of specific AT Treg subsets and modifying the communications between AT Treg subsets and adipocytes.

Combined overexpression of four transcription factors promotes effector T cell dedifferentiation toward early phenotypes.

Effector T cells, which are abundant but are short-lived after reinfusion into the body, are generally used for T-cell therapy, and antitumor immunity is typically not maintained over the long term. Genetic modification by early differentiated T cells and reinfusion has been shown to enhance antitumor immunity in vivo. This study overexpressed the characteristic transcription factors of differentiated early T cells by transfecting effector T cells with transcription factor recombinant lentivirus (S6 group: BCL6, EOMES, FOXP1, LEF1, TCF7, KLF7; S1 group: BCL6, EOMES, FOXP1, KLF7; S3 group: BCL6, EOMES, FOXP1, LEF1) to induce a sufficient number of effector T cells to dedifferentiate and optimize the transcription factor system. The results revealed that overexpression of early characteristic transcription factors in effector T cells upregulated the expression of early T cell differentiation markers (CCR7 and CD62L), with the S1 group having the highest expression level, while the rising trend of late differentiation marker (CD45RO) expression was suppressed. Moreover, the expression of early differentiation-related genes (ACTN1, CERS6, BCL2) was significantly increased, while the expression of late differentiation-related genes (KLRG-1) and effector function-related genes (GNLY, GZMB, PRF1) was significantly decreased; this difference in expression was more significant in the S1 group than in the other two experimental groups. The antiapoptotic ability of each experimental group was significantly enhanced, while the secretion ability of TNF-α and IFN-γ was weakened, with the effector cytokine secretion ability of the S1 group being the weakest. Transcriptomic analysis showed that the gene expression profile of each experimental group was significantly different from that of the control group, with differences in the gene expression pattern and number of differentially expressed genes in the S1 group compared with the other two experimental groups. The differentially expressed gene enrichment pathways were basically related to the cell cycle, cell division, and immune function. In conclusion, overexpression of early characteristic transcription factors in effector T cells induces their dedifferentiation, and induction of dedifferentiation by the S1 group may be more effective.

The effect of inulin-type fructans on the intestinal immune function of antibiotic-treated mice.

This study aimed to evaluate the effect of supplementation with inulin-type fructans (ITFs) on the intestinal immune function in the context of dysbiosis resulting from antibiotic cocktail (ABx) treatment. BALB/c mice (8-9 weeks of age) were treated with an ABx for 3 weeks and then allowed to recover spontaneously or with ITF supplementation (5%) for 4 weeks. Our results showed that ABx treatment can induce gut microbiota dysbiosis and intestinal inflammation in mice. After 4 weeks of recovery, ITF supplementation restored the composition of the intestinal microbial community. However, compared with spontaneous recovery, ITF supplementation delayed inflammation recovery in the intestine and upregulated diamine oxidase (DAO) activity and increased lipopolysaccharide (LPS) content in serum. In addition, ITF supplementation delayed the regulatory T (Treg) cell and B cell recovery in the lamina propria (LP). Furthermore, compared with spontaneous recovery, ITF supplementation inhibited the relative expression of certain proinflammatory genes, such as for inducible nitric oxide synthase (iNOS) and tumour necrosis factor α (Tnf-α), in the colon, but it reduced the secretion of the anti-inflammatory mediator transforming growth factor ß1 (TGF-ß1) in serum, reduced the secretion of secretory immunoglobulin A (SIgA) in the colon and promoted the secretion of the proinflammatory cytokine interleukin (IL)-17A. In conclusion, these data supported the hypothesis that the influence of ITFs on the host's intestinal status is not always beneficial in the context of ABx-induced biological disorder. However, the significance of these findings needs to be determined by advanced studies KEY POINTS: • ITFs did not promote the recovery of microbial community composition. • ITFs delayed the recovery of ABx-induced colonic inflammation. • ITFs reduced the secretion of TGF-ß1 and SIgA. • ITFs delayed the recovery of Treg and B cells in the LP.

Adaptive Responses of Citrus grandis Leaves to Copper Toxicity Revealed by RNA-Seq and Physiology.

Copper (Cu)-toxic effects on Citrus grandis growth and Cu uptake, as well as gene expression and physiological parameters in leaves were investigated. Using RNA-Seq, 715 upregulated and 573 downregulated genes were identified in leaves of C. grandis seedlings exposed to Cu-toxicity (LCGSEC). Cu-toxicity altered the expression of 52 genes related to cell wall metabolism, thus impairing cell wall metabolism and lowering leaf growth. Cu-toxicity downregulated the expression of photosynthetic electron transport-related genes, thus reducing CO2 assimilation. Some genes involved in thermal energy dissipation, photorespiration, reactive oxygen species scavenging and cell redox homeostasis and some antioxidants (reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics) were upregulated in LCGSEC, but they could not protect LCGSEC from oxidative damage. Several adaptive responses might occur in LCGSEC. LCGSEC displayed both enhanced capacities to maintain homeostasis of Cu via reducing Cu uptake by leaves and preventing release of vacuolar Cu into the cytoplasm, and to improve internal detoxification of Cu by accumulating Cu chelators (lignin, reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics). The capacities to maintain both energy homeostasis and Ca homeostasis might be upregulated in LCGSEC. Cu-toxicity increased abscisates (auxins) level, thus stimulating stomatal closure and lowering water loss (enhancing water use efficiency and photosynthesis).

Overexpression of early T cell differentiation-specific transcription factors transforms the terminally differentiated effector T cells into less differentiated state.

The in vivo proliferation and viability of transfused engineered T cells markedly limits the long-term effect of adoptive cell therapy on tumors. The therapeutic efficacy and proliferative potential of T cells are reported to be dependent on the differentiation status of T cells. The T cells at the early stage of progressive differentiation have a long lifespan, strong proliferative potential, and the ability to reconstruct intact T cell subsets. Thus, they are more suitable for adoptive immunotherapy. Previously, it was difficult to obtain a sufficient number of early differentiated T cells by inhibiting the progressive differentiation of T cells or by two-step programming. A more effective strategy is to directly reprogram and dedifferentiate the easily available terminal effector T (TEFF) cells, which are generated in large numbers, into early T cells. This study attempted to overexpress eight (candidate) early differentiation-specific transcription factors (TFs) (LEF1, KLF7, ID3, EOMES, BCL6, TCF7, FOXP1, and FOXO1) in the TEFF cells, which were activated by in vitro stimulation, to promote dedifferentiation into early T cells. In the mature TEFF cells simultaneously overexpressing these specific TFs, the expression pattern of T cell differentiation markers (CCR7 and CD45RO) exhibited a tendency to change to the pattern observed during early differentiation. The transcriptome analysis revealed that the function of differentially expressed genes was mainly concentrated in the cell cycle, growth and development, and effector function. Moreover, many genes related to early differentiated T cells (such as BCL2 and PIM1) were significantly upregulated, while those related to the effector function of TEFF cells were significantly downregulated (such as GZMB, PRF1, and GNLY). Additionally, the TEFF cells overexpressing characteristic TFs exhibited enhanced anti-apoptotic capabilities and decreased secretion of cytokines (IFN-γ and TNF-α). Based on these results, we believe that the TEFF cells were reprogrammed into a less differentiated state after overexpression of the eight specific TFs.

Distribution and roles of Ligilactobacillus murinus in hosts.

Ligilactobacillus murinus, a member of the Ligilactobacillus genus, holds significant potential as a probiotic. While research on Ligilactobacillus murinus has been relatively limited compared to well-studied probiotic lactic acid bacteria such as Limosilactobacillus reuteri and Lactobacillus gasseri, a mounting body of evidence highlights its extensive involvement in host intestinal metabolism and immune activities. Moreover, its abundance exhibits a close correlation with intestinal health. Notably, beyond the intestinal context, Ligilactobacillus murinus is gaining recognition for its contributions to metabolism and regulation in the oral cavity, lungs, and vagina. As such, Ligilactobacillus murinus emerges as a potential probiotic candidate with a pivotal role in supporting host well-being. This review delves into studies elucidating the multifaceted roles of Ligilactobacillus murinus. It also examines its medicinal potential and associated challenges, underscoring the imperative to delve deeper into unraveling the mechanisms of its actions and exploring its health applications.

miR-744-5p promotes T-cell differentiation via inhibiting STK11.

T cells utilized in adoptive T cell immunotherapy are typically activated in vitro. Although these cells demonstrate proliferation and anti-tumor activity following activation, they often face difficulties in sustaining long-term survival post-reinfusion. This issue is attributed to the induction of T cells into a terminal differentiation state upon activation, whereas early-stage differentiated T cells exhibit enhanced proliferation potential and survival capabilities. In previous study, we delineated four T cell subsets at varying stages of differentiation: TN, TSCM, TCM, and TEM, and acquired their miRNA expression profiles via high-throughput sequencing. In the current study, we performed a differential analysis of miRNA across these subsets, identifying a distinct miRNA, hsa-miR-744-5p, characterized by progressively increasing expression levels upon T cell activation. This miRNA is not expressed in TSCM but is notably present in TEM. Target genes of miR-744-5p were predicted, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, revealing that these genes predominantly associate with pathways related to the 'Wnt signaling pathway'. We established that miR-744-5p directly targets STK11, influencing its expression. Further, we investigated the implications of miR-744-5p on T cell differentiation and functionality. Overexpression of miR-744-5p in T cells resulted in heightened apoptosis, reduced proliferation, an increased proportion of late-stage differentiated T cells, and elevated secretion of the cytokine TNF-α. Moreover, post-overexpression of miR-744-5p led to a marked decline in the expression of early-stage differentiation-associated genes in T cells (CCR7, CD62L, LEF1, BCL2) and a significant rise in late-stage differentiation-associated genes (KLRG1, PDCD1, GZMB). In conclusion, our findings affirm that miR-744-5p contributes to the progressive differentiation of T cells by downregulating the STK11 gene expression.

Identification of a novel HLA-A2-restricted mutated Survivin epitope and induction of specific anti-HCC CTLs that could effectively cross-recognize wild-type Survivin antigen.

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

A microbubble contrast agent improves prediction of ablated areas during radiofrequency ablation: a rabbit liver study.

OBJECTIVES: The purpose of this study was to explore the effects of a microbubble contrast agent (SonoVue; Bracco SpA, Milan, Italy) on the spread of gas generated by heating during radiofrequency ablation in vivo. METHODS: Radiofrequency ablation was performed with or without SonoVue on 2 areas per rabbit liver in 20 rabbits under sonographic guidance. They were divided into groups 1 and 2 on the basis of ablation with and without SonoVue. In group 1, SonoVue (0.05 mL/kg) was injected as an intravenous bolus. After radiofrequency ablation, 3 orthogonal diameters of hyperechogenicity and defects of enhancement on contrast-enhanced sonography were measured sonographically. Coagulated zones and ablation times were also evaluated. RESULTS: There were no significant differences in the sizes of the hyperechoic areas, defects, or coagulated zones between the groups (P > .05). However, the total number of ablations with clearly visualized hyperechogenicity was 15 in group 1 versus 7 in group 2, as opposed to ablations with partial or not clearly visualized hyperechogenicity (P< .05). A comparison between hyperechoic and pathologic volumes provided correlation values of r(2) = 0.803 and r(2) = 0.624 for groups 1 and 2, respectively. In addition, the ablation time for group 1 was significantly shorter (P < .05). CONCLUSIONS: A microbubble agent would not change the size of the ablated area, but it can improve prediction of ablated areas and reduce the ablation time for achieving the same effects. Therefore, it might be useful to prevent unfavorable ablation of adjacent organs and vessels.

Using a single hydrophobic-interaction chromatography to purify pharmaceutical-grade supercoiled plasmid DNA from other isoforms.

CONTEXT: The recent developments in non-viral gene therapy and DNA vaccine have fostered the development of efficient plasmid DNA (pDNA) purification processes. OBJECTIVES: This work aimed to establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccine. MATERIALS AND METHODS: E. coli DH5α harboring pCDNA3.1-GFP (7200 base pairs) was used as a model plasmid. Hydrophobic-interaction chromatography (HIC) was employed to purify supercoiled plasmid DNA (sc pDNA). RESULTS: With this method, not only host contaminants, but also open circular plasmid DNA (oc pDNA) could be removed from sc pDNA. Anion-exchange HPLC analysis proved that the recovery of HIC could reach 75%. The plasmid DNA exhibited high purity with supercoiled percentage of 98 ± 1.2% and undetectable residual endotoxins, genomic DNA, RNA and protein. The purity of pDNA had nothing to do with the flow rate in the range at least up to 400 cm/h. Liposomes transfection experiment prove that the purified pDNA in this article had higher transfection efficiency than the control pDNA. DISCUSSION AND CONCLUSION: In the present work, we confirmed the possibility of separation of sc pDNA from oc pDNA and other host contaminants using a single HIC chromatography.

Advances in Off-Target Detection for CRISPR-Based Genome Editing.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing system exhibits marked potential for both gene editing and gene therapy, and its continuous improvement contributes to its great clinical potential. However, the largest hindrance to its application in clinical practice is the presence of off-target effects (OTEs). Thus, in addition to continuous optimization of the CRISPR system to reduce and eventually eliminate OTEs, further development of unbiased genome-wide detection of OTEs is key for its successful clinical application. This article summarizes detection strategies for OTEs of different CRISPR systems, to provide detailed guidance for the detection of OTEs in CRISPR-based genome editing.

Prognostic modeling of patients with metastatic melanoma based on tumor immune microenvironment characteristics.

Most of the malignant melanomas are already in the middle and advanced stages when they are diagnosed, which is often accompanied by the metastasis and spread of other organs. Besides, the prognosis of patients is bleak. The characteristics of the local immune microenvironment in metastatic melanoma have important implications for both tumor progression and tumor treatment. In this study, data on patients with metastatic melanoma from the TCGA and GEO datasets were selected for immune, stromal, and estimate scores, and overlapping differentially expressed genes were screened. A nine-IRGs prognostic model (ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22) was established by univariate COX regression, LASSO and multivariate COX regression. Receiver operating characteristic curves were used to test the predictive accuracy of the model. Immune infiltration was analyzed by using CIBERSORT and Xcell in high-risk and low-risk groups. The immune infiltration of the high-risk group was significantly lower than that of the low-risk group. Immune checkpoint analysis revealed that the expression of PDCD1, CTLA4, TIGIT, CD274, HAVR2 and LAG3 demonstrated the visible difference in groups with different levels of risk scores. WGCNA analysis found that the yellow-green module contained seven genes from the nine-IRG prognostic model, and the yellow-green module had the highest correlation with risk scores. The results of GO and KEGG suggested that the genes in the yellow-green module were mainly enriched in immune-related biological processes. Finally, the expression characteristics of ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22 were analyzed between metastatic melanoma and normal samples. Overall, a prognostic model for metastatic melanoma based on the tumor immune microenvironment characteristics was established, which left plenty of space for further studies. It could function well in helping people to understand characteristics of the immune microenvironment in metastatic melanoma.

Chemokine CCL17 Affects Local Immune Infiltration Characteristics and Early Prognosis Value of Lung Adenocarcinoma.

CCL17 is an important chemokine that plays a vital immunomodulatory role in the tumor microenvironment (TME). Analysis of lung adenocarcinoma (LUAD) data in Kaplan-Meier plotter databases found that the overall survival of patients in the CCL17 high-expression group was higher than that of the low-expression group, especially for patients with early (stages I and II) LUAD, which has a more positive prognostic value. Expression of CCL17 in LUAD was positively correlated with the proportion of tumor-infiltrating lymphocytes, immunostimulators, and major histocompatibility complexes using the TISIDB databases. Based on the RNA-seq and clinical data of 491 LUAD patients obtained from the TCGA database, 1,455 differential genes were found between the CCL17 high- and low-expression groups. Using WGCNA analysis confirmed that the expression of differential genes in the blue module is negatively correlated with poor survival and clinical stages of LUAD patients, and CCL17 and CCR4 genes belong to the hub genes in the blue module. Further analysis by the ESTIMATE and CIBERSORT algorithm found that the naive B cells and CD8+ T cells in the CCL17 high-expression group have a higher distribution ratio in the early LUAD patients, and the high immune score has a positive relationship with the overall survival rate. Using somatic mutation data of TCGA-LUAD, we found that 1) the tumor mutation burden values of the CCL17 high-expression group were significantly lower than those of the CCL17 low-expression group and 2) the expression levels of CCL17 and the tumor mutation burden values were negatively correlated. Transwell chemotaxis and cytotoxicity assays confirmed that CCL17 contributes to the migration of CCR4-positive lymphocytes into the H1993 LUAD TME and enhances the specific lysis of LUAD cells. In summary, high expression of CCL17 in the LUAD TME promotes local immune cell infiltration and antitumor immune response, which may contribute to the better survival and prognosis of patients with early LUAD.

Involvement of MicroRNA-27a-3p in the Licorice-Induced Alteration of Cd28 Expression in Mice.

Licorice has previously been shown to affect gene expression in cells; however, the underlying mechanisms remain to be clarified. We analyzed the microRNA expression profile of serum from mice treated by gavage with licorice decoction, and obtained 11 differentially expressed microRNAs (DEmiRNAs). We also screened differentially expressed genes (DEgenes) based on RNA-Seq data, and 271 common genes were identified by intersection analysis of the predicted target genes of 11 DEmiRNAs and the DEgenes. The miRNA-gene network showed that most of the hub genes were immune-related. KEGG enrichment analysis of the 271 genes identified three significant pathways, and the 21 genes involved in these three pathways, and the 11 DEmiRNAs, were constructed into a miRNA pathway-target gene network, in which mmu-miR-27a-3p stood out. Compared to ImmPort, there were 13 immune genes within the above group of 21 genes, and three intersected with the mmu-miR-27a-3p predicted target genes, Cd28, Grap2 and Cxcl12, of which the expression of Cd28 changed most significantly. We confirmed the regulation of Cd28 by mmu-miR-27a-3p using a dual-luciferase assay, and further confirmed that overexpression of mmu-miR-27a-3p could significantly downregulate the expression of Cd28 in lymphocytes. These results indicate that mmu-miR-27a-3p could be involved in the licorice-mediated regulation of the expression of Cd28 in mice.

miR-150 promotes progressive T cell differentiation via inhibiting FOXP1 and RC3H1.

T cells used in immune cell therapy, represented by T cell receptor therapy (TCR-T), are usually activated and proliferated in vitro and are induced to a terminally differentiated phenotype, with limited viability after transfusion back into the body. T cells exhibited a robust proliferative potential and in vivo viability in the early stages of progressive differentiation. In this study, we identified microRNAs that regulate T cell differentiation. After microRNA sequencing of the four subsets: Naïve T cells (TN), stem cell-like memory T cells (TSCM), central memory T cells (TCM）, and effector memory T cells (TEM), miR-150 was identified as the most highly expressed miRNA among the four subsets and was lowly expressed in the TSCM cells. We predicted the target genes of miR-150 miRNA and performed Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses. We observed that the target genes of miR-150 were enriched in pathways associated with T-cell differentiation. FOXP1 and RC3H1 were identified as key target genes of miR-150 in the regulation of T-cell function. We examined the effects of miR-150 on the differentiation and function of healthy donor T-cells. We observed that miR-150 overexpression promoted T-cell differentiation to effector T-cells and effector memory T-cells, enhanced apoptosis, inhibited cell proliferation and increased secretion of pro-inflammatory cytokines such as IFN-γ and TNF-α. In addition, the expressions of early differentiation-related genes (ACTN1, CERS6, BCL2, and EOMES), advanced differentiation-related genes (KLRG1), and effector-function-related genes (PRF1 and GZMB) were significantly decreased after overexpression of miR-150. Collectively, our results suggested that miR-150 can promote progressive differentiation of T cells and the downmodulation of miR-150 expression while performing adoptive immunotherapy may inhibit T-cell differentiation and increase the proliferative potential of T cells.

Changes in the TCR repertoire of T-cell subsets during culture of cytokine-induced killer cells.

Cytokine-induced killer (CIK) cells are a group of highly heterogeneous T cells expressing diverse T-cell antigen receptors (TCRs), but possessing wide major histocompatibility complex (MHC)-unrestricted antitumour activity, which have been used in the treatment of many tumours. However, the clonal diversity of different T-cell subsets and whether there is a certain clonal preference during CIK culture remains to be clarified. We found that the proportion of oligoclones seen in some subfamilies of freshly isolated T cells decreased in varying degrees after culturing. This indicates that the diversity of T-cell clones had been restored to a certain extent. The diversity differed among different T-cell subsets, as some TCR subfamily oligoclones mainly appeared in CD3+ CD56+ cells, which also indicates the heterogeneity of the CIK cell composition.

Molecular mechanisms for magnesium-deficiency-induced leaf vein lignification, enlargement and cracking in Citrus sinensis revealed by RNA-Seq.

Citrus sinensis (L.) Osbeck seedlings were fertigated with nutrient solution containing 2 [magnesium (Mg)-sufficiency] or 0 mM (Mg-deficiency) Mg(NO3)2 for 16 weeks. Thereafter, RNA-Seq was used to investigate Mg-deficiency-responsive genes in the veins of upper and lower leaves in order to understand the molecular mechanisms for Mg-deficiency-induced vein lignification, enlargement and cracking, which appeared only in the lower leaves. In this study, 3065 upregulated and 1220 downregulated, and 1390 upregulated and 375 downregulated genes were identified in Mg-deficiency veins of lower leaves (MDVLL) vs Mg-sufficiency veins of lower leaves (MSVLL) and Mg-deficiency veins of upper leaves (MDVUL) vs Mg-sufficiency veins of upper leaves (MSVUL), respectively. There were 1473 common differentially expressed genes (DEGs) between MDVLL vs MSVLL and MDVUL vs MSVUL, 1463 of which displayed the same expression trend. Magnesium-deficiency-induced lignification, enlargement and cracking in veins of lower leaves might be related to the following factors: (i) numerous transciption factors and genes involved in lignin biosynthesis pathways, regulation of cell cycle and cell wall metabolism were upregulated; and (ii) reactive oxygen species, phytohormone and cell wall integrity signalings were activated. Conjoint analysis of proteome and transcriptome indicated that there were 287 and 56 common elements between DEGs and differentially abundant proteins (DAPs) identified in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively, and that among these common elements, the abundances of 198 and 55 DAPs matched well with the transcript levels of the corresponding DEGs in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively, indicating the existence of concordances between protein and transcript levels.

TCR mispairing in genetically modified T cells was detected by fluorescence resonance energy transfer.

Adoptive transfer of T lymphocytes genetically modified with antigen-specific T cell receptor (TCR) constitutes a promising approach for the treatment of malignant tumors and virus infections. One of the challenges in this field of TCR gene therapy is TCR mispairing defining the incorrect pairing between an introduced TCR α or ß chain and an endogenous TCR ß or α chain, which results in diluted surface expression of the therapeutic TCR αß. Although there is currently no clinical evidence for TCR mispairing-induced autoreactivity, the generation of autoreactive TCRs upon TCR mispairing cannot be excluded. So it is important to detect TCR mispairing to evaluate the efficiency of TCR gene therapy. Currently there is no available quantitative assay for the measurement of TCR mispairing. Fluorescence resonance energy transfer (FRET) is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. In this study, we described the method of FRET for the measurement of TCR mispairing. It was found that the average FRET efficiency was 12.2 ± 7.5% in HeLa cells and 8.4 ± 3.3% in Jurkat cells (P = 0.026605). The reduction of FRET efficiency in lymphocytes reflected the presence of mispaired TCRs, indicating there were ~30% TCR mispairing in lymphocytes. This study provides a quantitative intracellular assay that can be used to detect TCR mispairing in genetically modified T lymphocytes.

Metabolomic analysis of serum reveals the potential effective ingredients and pathways of Danggui Buxue Tang in promoting erythropoiesis.

Danggui Buxue Tang has been used for menopausal women in China for more than 800 years. However, the potential effective ingredients and pathways require further investigation. The main objective of this work was to explore the potential effective ingredients and pathways. The optimal administration time was optimized by detecting the changes of reticulocytes in peripheral blood. Drug-containing serum (DCS) was taken every 30 min after last administration. Because of the different concentration of effective ingredients absorbed into blood at different time, the pharmacodynamic effect is different. Therefore, bone marrow stromal cells as a member of hematopoietic microenvironment were used to evaluate the pharmacodynamics of DCS. Metabolomics was used to detect changes of metabolites (DBT and endogenous metabolites). The correlation of the metabolites and pharmacodynamics was used to identify the metabolites associated with erythropoiesis. After 14 days, the number of reticulocytes in peripheral blood, erythroid-related cells and erythroid progenitor cells in bone marrow in the DBT group were significantly increased. In vitro experiments showed that DCS at different time had different proliferation effects on BMSCs. Metabolomic analysis showed that the concentration of metabolites in DCS at different time was significantly different. The correlation analysis identified 7 DBT metabolites and 15 endogenous metabolites related to erythropoiesis. 15 endogenous metabolites were finally connected to different pathways. Glutamate is a node molecule. 7 potential effective ingredients of DBT were found. DBT promoted erythropoiesis via promoting the metabolism of glutamate and further affect other pathways.