Factor Of T-cells Research Articles

Gαq Signaling Activates β-Catenin-Dependent Gene Transcription

The canonical Wnt signal transduction or the Wnt/β-catenin pathway plays a crucial role in both carcinogenesis and development of animals. Activation of the Gαq class of Gα proteins positively regulates Wnt/β-catenin pathway, and expression of Gαq in human embryonic kidney 293 (HEK293T) cells or Xenopus oocytes leads to the inhibition of glycogen synthase kinase-3 beta and cellular accumulation of β-catenin. This study investigated whether Gαq-mediated cellular accumulation of β-catenin could affect the transcriptional activity of this protein. HEK-293T and HT-29 cells were used for cell culture and transfection. Protein localization and quantification were assessed by using immunofluorescence microscopy, cell fractionation assay, and Western blotting analysis. Gene expression at the transcription level was examined by quantitative reverse transcriptase/real-time PCR method. Transcription of two cellular β-catenin target genes (c-MYC and CCND1) and the β-catenin/ T-cell factor reporter luciferase gene (TopFlash plasmid) significantly increased by Gαq activation. The Gαq-mediated increase in the expression level of the β-catenin-target genes was sensitive to the expression of a minigene encoding a specific Gαq blocking peptide. The results of cell fractionation and Western blotting experiments showed that activation of Gαq signaling increased the intracellular β-catenin protein level, but it blocked its membrane localization. Our results reveal that the Gαq-dependent cellular accumulation of β-catenin can enhance β-catenin transcriptional activity.

Retraction Note: Forkhead box (FOX) G1 promotes hepatocellular carcinoma epithelial-Mesenchymal transition by activating Wnt signal through forming T-cell factor-4/Beta-catenin/FOXG1 complex

Retraction Note: Forkhead box (FOX) G1 promotes hepatocellular carcinoma epithelial-Mesenchymal transition by activating Wnt signal through forming T-cell factor-4/Beta-catenin/FOXG1 complex

EIF3a sustains non-small cell lung cancer stem cell-like properties by promoting YY1-mediated transcriptional activation of β-catenin

Cancer stem cells (CSCs) are the leading cause of recurrence and poor prognosis in non-small cell lung cancer (NSCLC). Eukaryotic translation initiation factor 3a (eIF3a) participates in many tumor development processes, such as metastasis, therapy resistance, and glycolysis, all of which are closely associated with the presence of CSCs. However, whether eIF3a maintains NSCLC-CSC-like properties remains to be elucidated. In this study, eIF3a was highly expressed in lung cancer tissues and was linked to poor prognosis. eIF3a was also highly expressed in CSC-enriched spheres compared with adherent monolayer cells. Moreover, eIF3a is required for NSCLC stem cell-like traits maintenance in vitro and in vivo. Mechanistically, eIF3a activates the Wnt/β-catenin signaling pathway, promoting the transcription of cancer stem cell markers. Specifically, eIF3a promotes the transcriptional activation of β-catenin and mediates its nuclear accumulation to form a complex with T cell factor 4 (TCF4). However, eIF3a has no significant effect on protein stability and translation. Proteomics analysis revealed that the candidate transcription factor, Yin Yang 1 (YY1), mediates the activated effect of eIF3a on β-catenin. Overall, the findings of this study implied that eIF3a contributes to the maintenance of NSCLC stem cell-like characteristics through the Wnt/β-catenin pathway. eIF3a is a potential target for the treatment and prognosis of NSCLC.

Memory profiles distinguish cross-reactive and virus-specific T cell immunity to mpox

Mpox represents a persistent health concern with varying disease severity. Reinfections with mpox virus (MPXV) are rare, possibly indicating effective memory responses to MPXV or related poxviruses, notably vaccinia virus (VACV) from smallpox vaccination. We assessed cross-reactive and virus-specific CD4+ and CD8+ Tcells in healthy individuals and mpox convalescent donors. Cross-reactive Tcells were most frequently observed in healthy donors over 45 years. Notably, long-lived memory CD8+ Tcells targeting conserved VACV/MPXV epitopes were identified in older individuals more than four decades after VACV exposure and exhibited stem-like characteristics, defined by Tcell factor-1 (TCF-1) expression. In mpox convalescent donors, MPXV-reactive CD4+ and CD8+ Tcells were more prevalent than incontrols, demonstrating enhanced functionality and skewing toward effector phenotypes, which correlated with milder disease. Collectively, we report robust effector memory MPXV-specific Tcell responses in mild mpox and long-lived TCF-1+ VACV/MPXV-specific CD8+ Tcells decades after smallpox vaccination.

Abstract 5870: T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells

Abstract Background: We previously identified 14 isoforms of T-cell factor (TCF)-4, a key transcriptional factor in the Wnt signaling pathway, from human hepatocellular carcinoma (HCC) cell lines (Exp Cell Res 2011). The TCF-4J and K pair has been characterized based on the presence (K) or absence (J) of a conserved SxxSS motif at the head of exon 9. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012); however, resistance to anti-cancer agents was more prominent in K cells than J cells. Another analysis demonstrated that loss of exon 4 increased resistance to chemotherapeutic agents and induced epithelial-mesenchymal transition (EMT) (Liver Int 2013). EMT and the expression of programmed cell death 1-ligand 1 (PD-L1) is closely associated with each other, and our preliminary study revealed that EMT was regulated by TCF-4 variants, suggesting possible involvement of the variants in the regulation of PD-L1 expression. In general, PD-L1 is known to be upregulated by hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Therefore, if the TCF-4 variants regulate PD-L1 expression under normoxia, it would be a unique mechanism bridging the Wnt signaling pathway and the tumor immune microenvironment (TIME). Aim: To assess whether or not the TCF-4 variants regulate the expression of PD-L1 in cancer cells under normoxia. Methods: HAK-1A, a well-differentiated HCC cell line (Hepatology 1993), was used in this study. TCF-4K mutants (S269A, S272A, and S273A) were prepared with the conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (S269A, S272A, and S273A cells, respectively) were established. Empty vector-transfected mock cells (EV cells) were used as a control. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Results: J cells and K cells expressed PD-L1 mRNA 2-fold and 4-fold of that in the EV cells, respectively. Of note, the PD-L1 expression mRNA was enhanced up to 11-fold in S269A cells, 11-fold in S272A cells, and 17-fold in S273A cells. The increased mRNA expressions were verified in protein levels. Conclusion: PD-L1 expression was regulated by TCF-4 variants in liver cancer cells under normoxic conditions. The finding suggests that the Wnt signaling pathway fine-tunes the TIME by regulating the expression levels of PD-L1 in a HIF-1α-independent manner. Citation Format: Hironori Koga, Yasuko Imamura, Toshimitsu Tanaka, Hideki Iwamoto, Toru Nakamura, Atsutaka Masuda, Takahiko Sakaue, Hiroyuki Suzuki, Hirohisa Yano, Takumi Kawaguchi. T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5870.

Abstract P3-08-07: Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer

Abstract Background: Stable humanized immune mice transplanted with peripheral blood mononuclear cells (PBMCs) derived from breast cancer patients are important models for assessing the tumor immune responses and the tumor immune microenvironment in breast cancer, helping to advance both pre-clinical and clinical research. The PBMCs of breast cancer patients exhibit various differences from those of healthy individuals depending on the stage of cancer progression, subtype, and type of treatment. Recent studies indicate an association of the self-renewal signatures of T cells with successful generation of a humanized mouse model; however, the optimal T cell signature for the successful generation of a humanized mouse model derived from cancer patient cells is poorly understood. Therefore, we examined the relationship between the signature of T cell subsets, focusing on the self-renewal signatures, in PBMCs derived from patients with breast cancer and the successful generation of a humanized immune mouse model. Materials and Methods: We collected PBMCs from 12 patients with breast cancer. All samples were stimulated with interleukin-2 and beads coated with CD3 and CD28 agonist antibodies to expand T cells. After washing, 1 × 10^7 cells/mouse were intraperitoneally injected into NOD/Shi-scid IL2rgamma (null) (NOG) mice. Transplants were performed on three mice per case. Successful engraftment of immune cells into NOG mice was defined by the presence of human CD45+ cells in one or more mice. Self-renewal signatures of in vitro expanded T cells before injection into NOG mice, including the markers T cell factor-1 (TCF-1), CD45RA, CCR7, CD95, and CXCR3, were determined using flow cytometry, mass cytometry and quantitative RT-PCR. Comparisons between groups of data were evaluated by t-test. Results: The success rate of engraftment of immune cells derived from breast cancer patients into NOG mice was 66.7% (8 out of 12 patients). After expansion, the magnitude of the CD8+ stem cell memory subset and the TCF-1 expression level on the CD4+ and CD8+ T cells in the engrafted group were significantly higher than those in non-engrafted group. TCF-1 and CCR7 mRNA levels in the engrafted group were upregulated compared with those in non-engrafted group. The success of engraftment was not associated with clinical characteristics such as breast cancer progression, subtype, or prior chemotherapy treatment. Conclusion: The self-renewal signatures of T cells from breast cancer patients were associated with successful engraftment of a humanized mouse model. These results suggest that prior identification of the self-renewal signatures of the T-cell population is of direct relevance to the appropriate design of a pre-clinical model for testing immunotherapies and for elucidating the characteristics of the tumor immune microenvironment. Citation Format: Yukiko Fukui, Kosuke Kawaguchi, Ryuji Murakami, Wei Li, Yuki Nakamura, Yurina Maeshima, Sunao Tanaka, Shinpei Kawaoka, Eiji Suzuki, Masakazu Toi. Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-07.

WNT3a Signaling Inhibits Aromatase Expression in Breast Adipose Fibroblasts—A Possible Mechanism Supporting the Loss of Estrogen Responsiveness of Triple-Negative Breast Cancers

Estrogen-dependent breast cancers rely on a constant supply of estrogens and expression of estrogen receptors. Local biosynthesis, by aromatase in breast adipose fibroblasts (BAFs), is their most important source for estrogens. Triple-negative breast cancers (TNBC) rely on other growth-promoting signals, including those from the Wnt pathway. In this study, we explored the hypothesis that Wnt signaling alters the proliferation of BAFs, and is involved in regulation of aromatase expression in BAFs. Conditioned medium (CM) from TNBC cells and WNT3a consistently increased BAF growth, and reduced aromatase activity up to 90%, by suppression of the aromatase promoter I.3/II region. Database searches identified three putative Wnt-responsive elements (WREs) in the aromatase promoter I.3/II. In luciferase reporter gene assays, promoter I.3/II activity was inhibited by overexpression of full-length T-cell factor (TCF)-4 in 3T3-L1 preadipocytes, which served as a model for BAFs. Full-length lymphoid enhancer-binding factor (LEF)-1 increased the transcriptional activity. However, TCF-4 binding to WRE1 in the aromatase promoter, was lost after WNT3a stimulation in immunoprecipitation-based in vitro DNA-binding assays, and in chromatin immunoprecipitation (ChIP). In vitro DNA-binding assays, ChIP, and Western blotting revealed a WNT3a-dependent switch of nuclear LEF-1 isoforms towards a truncated variant, whereas β-catenin levels remained unchanged. This LEF-1 variant revealed dominant negative properties, and most likely recruited enzymes involved in heterochromatin formation. In addition, WNT3a induced the replacement of TCF-4 by the truncated LEF-1 variant, on WRE1 of the aromatase promoter I.3/II. The mechanism described here may be responsible for the loss of aromatase expression predominantly associated with TNBC. Tumors with (strong) expression of Wnt ligands actively suppress aromatase expression in BAFs. Consequently a reduced estrogen supply could favor the growth of estrogen-independent tumor cells, which consequently would make estrogen receptors dispensable. In summary, canonical Wnt signaling within (cancerous) breast tissue may be a major factor controlling local estrogen synthesis and action.

TCF-1 Is Required for CD4 T Cell Persistence Functions during AlloImmunity

The transcription factor T cell factor-1 (TCF-1) is encoded by Tcf7 and plays a significant role in regulating immune responses to cancer and pathogens. TCF-1 plays a central role in CD4 T cell development; however, the biological function of TCF-1 on mature peripheral CD4 T cell-mediated alloimmunity is currently unknown. This report reveals that TCF-1 is critical for mature CD4 T cell stemness and their persistence functions. Our data show that mature CD4 T cells from TCF-1 cKO mice did not cause graft versus host disease (GvHD) during allogeneic CD4 T cell transplantation, and donor CD4 T cells did not cause GvHD damage to target organs. For the first time, we showed that TCF-1 regulates CD4 T cell stemness by regulating CD28 expression, which is required for CD4 stemness. Our data showed that TCF-1 regulates CD4 effector and central memory formation. For the first time, we provide evidence that TCF-1 differentially regulates key chemokine and cytokine receptors critical for CD4 T cell migration and inflammation during alloimmunity. Our transcriptomic data uncovered that TCF-1 regulates critical pathways during normal state and alloimmunity. Knowledge acquired from these discoveries will enable us to develop a target-specific approach for treating CD4 T cell-mediated diseases.

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells invivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRβ-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRβ-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRβ-sufficient Tfh cells. Loss of LXRβ confers inactivation of GSK3β induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/β-catenin pathway, leading to elevated TCF-1 expression in CD4+ Tcells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ Tcells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3β-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells.

Mutations in components of the Wnt/β-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.

MiR-872-5p/FOXO3a/Wnt signaling feed-forward loop promotes proliferation of endogenous neural stem cells after spinal cord ischemia-reperfusion injury in rats.

The activation of endogenous neural stem cells (NSCs) is considered an important mechanism of neural repair after mechanical spinal cord injury; however, whether endogenous NSC proliferation can also occur after spinal cord ischemia-reperfusion injury (SCIRI) remains unclear. In this study, we aimed to verify the existence of endogenous NSC proliferation after SCIRI and explore the underlying molecular mechanism. NSC proliferation was observed after SCIRI in vivo and oxygen-glucose deprivation and reperfusion (OGD/R) in vitro, accompanied by a decrease in forkhead box protein O 3a (FOXO3a) expression. This downward trend was regulated by the increased expression of microRNA-872-5p (miR-872-5p). miR-872-5p affected NSC proliferation by targeting FOXO3a to increase the expression of β-catenin and T-cell factor 4 (TCF4). In addition, TCF4 in turn acted as a transcription factor to increase the expression level of miR-872-5p, and knockdown of FOXO3a enhanced the binding of TCF4 to the miR-872-5p promoter. In conclusion, SCIRI in vivo and OGD/R in vitro stimulated the miR-872-5p/FOXO3a/β-catenin-TCF4 pathway, thereby promoting NSC proliferation. At the same time, FOXO3a affected TCF4 transcription factor activity and miR-872-5p expression, forming a positive feedback loop that promotes NSC proliferation.

Transcriptional activity mediated by β-CATENIN and TCF/LEF family members is completely dispensable for survival and propagation of multiple human colorectal cancer cell lines

Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10% of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Survival of these cells and the ability to propagate them demonstrate their complete β-CATENIN- and TCF/LEF-independence. Nonetheless, one β-CATENIN-deficient cell line eventually became senescent, and absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β-CATENIN and TCF7L2 independently control sizeable fractions of their target genes. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.

Expression dynamics of lymphoid enhancer-binding factor 1 in terminal Schwann cells, dermal papilla, and interfollicular epidermis.

Transcription factor lymphoid enhancer-binding factor 1 (LEF1) is a downstream mediator of the Wnt/β-catenin signaling pathway. It is expressed in dermal papilla and surrounding cells in the hair follicle, promoting cell proliferation, and differentiation. Here, we report that LEF1 is also expressed all through the hair cycle in the terminal Schwann cells (TSCs), a component of the lanceolate complex located at the isthmus. The timing of LEF1 appearance at the isthmus coincides with that of hair follicle innervation. LEF1 is not found at the isthmus in the aberrant hair follicles in nude mice. Instead, LEF1 in TSCs is found in the de novo hair follicles reconstituted on nude mice by stem cells chamber graft assay. Cutaneous denervation experiment demonstrates that the LEF1 expression in TSCs is independent of nerve endings. At last, LEF1 expression in the interfollicular epidermis during the early stage of skin development is significantly suppressed in transgenic mice with T-cell factor 3 (TCF3) overexpression. We reveal the expression dynamics of LEF1 in skin during development and hair cycle. LEF1 expression in TSCs indicates that the LEF1/Wnt signal might help to establish a niche at the isthmus region for the lanceolate complex, the bulge stem cells and other neighboring cells.

β-Catenin-treated peptides effectively inhibit the proliferation of colorectal cancer.

To verify the inhibitory mechanism of β-catenin-designed peptides in colorectal cancer(CRC) tumors, the following experiments were performed. In vitro colony formation, Transwell assays, and flow cytometry were performed to assess the biological effects of designed peptides (F18KD, F20A4-7k, F20A4-10k, and F20A3-9k+F20A4-10k+F20A5-9k) in HT-29 cells. In vivo xenograft experiments were performed and treated with peptides. Next, tumors were subjected to Hematoxylin and eosin staining (HE), immunohistochemical, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays to evaluate the inhibitory effect of peptides on tumors. β-Catenin levels were quantified via western blotting (WB) and quantitative real-time polymerase chain reaction, and β-catenin was located using confocal laser scanning microscopy. T-cell factor-4 (TCF-4), C-myc, and CCND1 levels were quantified via WB. Results were obtained as following. First, the peptides reduced viability, migration, and invasion; promoted apoptosis; and stabilized the S phase of HT-29 cells. Second, peptides suppressed tumor growth and downregulated the expression of CD34, vascular endothelial growth factor, and β-catenin in tumors. Furthermore, we found that peptides downregulated β-catenin expression in both the cytoplasm and nucleus; TCF-4, C-myc, and CCND1 expression was also downregulated. Notably, β-catenin-targeting peptides had a better inhibitory effect on CRC than non-β-catenin-target peptides, and a combination of peptides exerted a more potent inhibitory effect on CRC than single peptides. It suggested that β-Catenin-targeting peptides promote apoptosis in CRC tumors by inhibiting activation of the Wnt/β-catenin pathway.

LncRNA TCF7 contributes to high glucose-induced damage in human podocytes by up-regulating SEMA3A via sponging miR-16-5p.

Long non-coding RNAs (lncRNAs) exert essential functions in the pathogenesis of diabetic nephropathy (DN). LncRNA T-cell factor 7 (TCF7) and semaphorin-3A (SEMA3A) have been found to be involved in the progression of diabetic nephropathy. However, whether the effect of TCF7 on the pathogenesis of diabetic nephropathy is mediated by SEMA3A remains unclear. TCF7, miR-16-5p, and SEMA3A were quantified by a qRT-PCR or immunoblotting method. A CCK-8 assay gauged the cell viability. Measurement of cell apoptosis was done using flow cytometry. RNA immunoprecipitation (RIP), dual-luciferase reporter, and RNA pull-down assays were utilized to assay the targeted interactions among the variables. The TCF7 and SEMA3A levels were elevated in serum from patients with diabetic nephropathy. TCF7 silencing or SEMA3A depletion ameliorated high glucose (HG)-induced podocyte injury. Moreover, TCF7 silencing protected against HG-induced podocyte injury by down-regulating SEMA3A. TCF7 targeted miR-16-5p, and miR-16-5p targeted SEMA3A. Furthermore, TCF7 affected the expression of SEMA3A by competing specifically for shared miR-16-5p. These findings suggested that TCF7 silencing attenuated high glucose-induced podocyte damage partially through the miR-16-5p/SEMA3A regulation cascade.

Self-Renewing CD8+ T-cell Abundance in Blood Associates with Response to Immunotherapy.

Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non-small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7-) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7-CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7-CD8+ T cells. The observed correlation of TCF1 frequency in CCR7-CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.

METTL14 upregulates TCF1 through m6A mRNA methylation to stimulate osteogenic activity in osteoporosis.

Alteration of N6-methyladenosine (m6A) is closely linked to spanning biological processes including osteoporosis (OP) development. This research focuses on the function of methyltransferase like 14 (METTL14) in bone turnover and its interaction with T cell factor 1 (TCF1). A mouse model of OP was established by ovariectomy (OVX). The bone mass parameters were evaluated by micro-CT analysis. Mouse MC3T3-E1 cells and mouse bone marrow macrophages (BMMs) were induced for osteogenic or osteoclastic differentiation, respectively, for in vitro experiments. The osteogenesis or osteoclasis activity was analyzed by measuring the biomarkers such as OPG, ALP, NFATC1, CTSK, RANKL, and TRAP. RT-qPCR and IHC assays identified reduced METTL14 expression in bone tissues of osteoporotic patients and ovariectomized mice. Artificial METTL14 overexpression increased bone mass of mice and promoted osteogenesis whereas suppressed osteoclasis both in vivo and in vitro. METTL14 promoted TCF1 expression through m6A mRNA methylation, and TCF1 increased the osteogenic activity by elevating the protein level of RUNX2, a key molecule linked to bone formation. In rescue experiments, TCF1 restored the RUNX2 level and osteogenic activity of cells suppressed by METTL14 silencing. In summary, this research demonstrates that METTL14 plays a protective role against OP by promoting the TCF1/RUNX2 axis.

Modulatory Effect of Chlorogenic Acid and Coffee Extracts on Wnt/β-Catenin Pathway in Colorectal Cancer Cells.

The Wnt/β-Catenin pathway alterations present in colorectal cancer (CRC) are of special interest in the development of new therapeutic strategies to impact carcinogenesis and the progression of CRC. In this context, different polyphenols present in natural products have been reported to have modulatory effects against the Wnt pathway in CRC. In this study, we evaluate the effect of two polyphenol-rich coffee extracts and chlorogenic acid (CGA) against SW480 and HT-29 CRC cells. This involved the use of MTT and SRB techniques for cell viability; wound healing and invasion assay for the evaluation of the migration and invasion process; T cell factor (TCF) reporter plasmid for the evaluation of transciption factor (TCF) transcriptional activity; polymerase chain reaction (PCR) of target genes and confocal fluorescence microscopy for β-Catenin and E-Cadherin protein fluorescence levels; and subcellular localization. Our results showed a potential modulatory effect of the Wnt pathway on CRC cells, and we observed a reduction in the transcriptional activity of β-catenin. All the results were prominent in SW480 cells, where the Wnt pathway deregulation has more relevance and implies a constitutive activation of the signaling pathway. These results establish a starting point for the discovery of a mechanism of action associated with these effects and corroborate the anticancer potential of polyphenols present in coffee, which could be explored as chemopreventive molecules or as adjunctive therapy in CRC.

The outcome and risk factor of refractory T-cell–mediated rejection on renal allograft transplantation based on the Korean Organ Transplantation Registry

Eun-Jeong Kwon, Gi-Ae Yun, Seokwoo Park, Sejoong Kim, Ho Jun Chin, Ki Young Na, Dong-Wan Chae, Hyung Sub Park, Taeseung Lee, Jong Cheol Jeong. Korean J Transplant 2022;36:314. https://doi.org/10.4285/ATW2022.F-4537

Alnuctamab (ALNUC; BMS-986349; CC-93269), a B-Cell Maturation Antigen (BCMA) x CD3 T-Cell Engager (TCE), in Patients (pts) with Relapsed/Refractory Multiple Myeloma (RRMM): Results from a Phase 1 First-in-Human Clinical Study

Introduction Triple-class-exposed pts with MM typically have a short duration of response (DOR) to subsequent therapies. ALNUC, a 2+1 BCMA x CD3 TCE with bivalent binding to BCMA, has demonstrated clinical activity in pts with RRMM treated with ≥ 3 prior lines of therapy (LOT) in an open-label phase 1 study (NCT03486067) when administered intravenously (Costa LJ et al. Blood. 2019;134[Suppl 1]:143). However, IV ALNUC was associated with a high rate of cytokine release syndrome (CRS; any grade, 89%; grade ≥ 3, 5%). Based on preclinical data suggesting subcutaneous (SC) administration may improve tolerability, we evaluated the safety and efficacy of SC ALNUC. Here, we present long-term follow-up data for IV ALNUC and preliminary data for SC ALNUC in pts with RRMM treated in the phase 1 study. Methods Pts with MM who had received ≥ 3 prior LOT, including an immunomodulatory drug (IMiD®), a proteasome inhibitor, and an anti-CD38 therapy, were eligible. IV ALNUC was administered as previously reported at target doses of 0.15-10 mg, with both fixed and step-up dosing. SC ALNUC was given on day 1 (D1), 4, 8, 15, and 22 of cycle 1 (C1), QW in C2-3, Q2W in C4-6, and Q4W in C7 and beyond (28-d cycles). Pts received 2 step-up doses (3 mg on C1D1 and 6 mg on C1D4) and a ≥ 10 mg target dose on C1D8 and thereafter. Target doses tested in SC dose escalation were 10, 15, 30, and 60 mg, with 10 and 30 mg evaluated in dose expansion. Primary objectives included assessments of ALNUC safety and tolerability. Minimal residual disease (MRD) was assessed from bone marrow aspirate by Next Generation Flow using the EuroFlow panel. Results At data cutoff on May 31, 2022, 70 pts have received IV ALNUC, 39% (27/70) achieved an objective response, and median progression-free survival was 13.3 wks (95% CI, 8.1-23.9). Median DOR in pts achieving a response with IV ALNUC was 146.1 wks (95% CI, 40.6-not reached). Forty-seven pts were treated with SC ALNUC in dose escalation (10 mg: n = 6; 15 mg: n = 4; 30 mg: n = 6; 60 mg: n = 3) and dose expansion (10 mg: n = 19; 30 mg: n = 9). Median age was 63 yrs; 55% were female. Pts received a median of 4 prior regimens; 98% had myeloma refractory to the last LOT, 96%/62% had triple-class exposed/refractory myeloma, and 60%/21% had penta-drug exposed/refractory myeloma. At the May 31, 2022 data cutoff, median duration of follow-up was 2.6 mo (range, 0-11.4), and 68% (n = 32) of pts were continuing SC ALNUC treatment. Any-grade/grade 3-4 treatment-emergent adverse events occurred in 89%/62% of treated pts; the most common were CRS (53%/0%), neutropenia (34%/30%), and anemia (34%/17%). All CRS events were limited to grade 1 (21 pts; 45%) or grade 2 (4 pts; 9%); 20 pts received ≥ 1 concomitant medication for CRS, including tocilizumab (n = 12) and/or corticosteroids (n = 8). Median time to CRS onset was 3 d (range, 1-20); median duration was 2 d (range, 1-11). In 16 pts with grade 3-4 neutropenia, median time to resolution (grade ≤ 2) was 6 d (range, 1-36). There was one grade 1 immune effector cell-associated neurotoxicity event. No pts discontinued treatment due to adverse events; no treatment-related deaths occurred. Preliminary population pharmacokinetic analysis estimated SC ALNUC bioavailability of ~70%; 30 mg SC achieved similar concentrations observed with 10 mg IV Cmax by end of C1; baseline body weight was not a significant covariate of exposure. Hallmark pharmacodynamic effects of TCEs were observed with SC and IV ALNUC (peripheral blood immune cell redistribution, transient cytokine release, and induction of T-cell factors associated with antitumor activity). Among 41 efficacy-evaluable (EE) pts treated with SC ALNUC (received ≥ 1 dose and had ≥ 1 post-baseline disease assessment), ORR was 51% (21/41 pts) across all dosing regimens and 77% (10/13 pts) in pts receiving target doses ≥ 30 mg (Figure). Among the 21 pts who achieved a response, 14 pts had evaluable MRD samples, and all (100%) were MRD negative (10-5 sensitivity) at C2D1 or C4D1. Median time to response was 4.3 wks (range, 4.1-17.4), and all 21 responses (100%) were ongoing. Conclusions IV administration of ALNUC was associated with durable responses in heavily pretreated pts with RRMM. SC administration widened the therapeutic index with an improved safety profile compared with IV; CRS was limited to grade 1-2 events. SC ALNUC exhibited promising dose-dependent antitumor activity with a high proportion of MRD responses. Enrollment in the phase 1 study is ongoing and updated data will be presented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal