LILRB2 promotes immune escape in breast cancer cells via enhanced HLA-A degradation.

PURPOSE: Increased expression of leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is associated with immune evasion in breast cancer (BC). The aim of this study to elucidate the role of LILRB2 in BC progression. METHODS: LILRB2 expression in tumor tissues was detected by immunohistochemical staining. Human leukocyte antigen A (HLA-A) expression in BC cells was detected by Western blotting, and HLA-A ubiquitination was detected by immunoprecipitation and histidine pulldown assay. An in-situ tumor model was established in nude BALB/c mice to verify the role of LILRB2 in immune escape. Finally, the functions and potential mechanisms of LILRB2 in BC progression were explored using in silico data. RESULTS: LILRB2 was upregulated in BC tissues and cells, and correlated positively with poor prognosis. LILRB2 promoted BC progression by downregulating HLA-A expression. Mechanistically, LILRB2 facilitates the ubiquitination and subsequent degradation of HLA-A by promoting the interaction between the ubiquitin ligase membrane-associated ring finger protein 9 (MARCH9) and HLA-A. In syngeneic graft mouse models, LILRB2-expressing BC cells evaded CD8 + T cells and inhibited the secretion of cytokines by the cytotoxic CD8 + T cells. CONCLUSION: LILRB2 downregulates HLA-A to promote immune evasion in BC cells and is a promising new target for BC treatment.

Endothelial Response to Type I Interferon Contributes to Vasculopathy and Fibrosis and Predicts Disease Progression of Systemic Sclerosis.

OBJECTIVE: Interferon (IFN)-1 signatures are a hallmark of patients with systemic sclerosis (SSc). However, its significance in clinical stratification and contribution to deterioration still need to be better understood. METHODS: For hypothesis generation, we performed single-cell RNA sequencing (scRNA-seq) on skin biopsies (four patients with SSc and two controls) using the BD Rhapsody platform. Two publicly available data sets of skin scRNA-seq were used for validation (GSE138669: 12 patients with diffuse cutaneous SSc [dcSSc] and 10 controls; GSE195452: 52 patients with dcSSc and 41 patients with limited cutaneous SSc [lcSSc] and 54 controls). The IFN-1 signature was mapped, functionally investigated in a bleomycin plus IFNα-2 adenovirus-associated virus (AAV)-induced model and verified in an SSc cohort (n = 61). RESULTS: The discovery and validation data sets showed similar findings. Endothelial cells (ECs) had the most prominent IFN-1 signature among dermal nonimmune cells. The EC IFN-1 signature was increased both in patients with SSc versus controls and in patients with dcSSc versus those with lcSSc. Among EC subclusters, the IFN-1 signature was statistically higher in the capillary ECs of patients with dcSSc, which was higher than those in patients with lcSSc, which in turn was higher than those in healthy controls (HCs). Endothelial-to-mesenchymal transition (EndoMT) scores increased in parallel. Deteriorated bleomycin-induced dermal fibrosis, EndoMT, and perivascular fibrosis and caused blood vessel loss with EC apoptosis. Vascular myxovirus resistance (MX) 1, an IFN-1 response protein, was significantly increased both in total SSc versus HC skin and in dcSSc versus lcSSc skin. Baseline vascular MX1 performed similarly to skin score in predicting disease progression over 6 to 34 months in total SSc and was superior in the dcSSc subpopulation. CONCLUSION: The EC IFN-1 signature distinguished SSc skin subtypes and disease progression and may contribute to vasculopathy and fibrosis.

Characterization of age-related immune features after autologous NK cell infusion: Protocol for an open-label and randomized controlled trial.

Background: Aging is usually accompanied by functional declines of the immune system, especially in T-cell responses. However, little is known about ways to alleviate this. Methods: Here, 37 middle-aged healthy participants were recruited, among which 32 were intravenously administrated with expanded NK cells and 5 with normal saline. Then, we monitored changes of peripheral senescent and exhausted T cells within 4 weeks after infusion by flow cytometry, as well as serum levels of senescence-associated secretory phenotype (SASP)-related factors. In vitro co-culture assays were performed to study NK-mediated cytotoxic activity against senescent or exhausted T cells. Functional and phenotypic alteration of NK cells before and after expansion was finally characterized. Results: After NK cell infusion, senescent CD28-, CD57+, CD28-CD57+, and CD28-KLRG1+ CD4+ and CD8+ T-cell populations decreased significantly, so did PD-1+ and TIM-3+ T cells. These changes were continuously observed for 4 weeks. Nevertheless, no significant changes were observed in the normal saline group. Moreover, SASP-related factors including IL-6, IL-8, IL-1α, IL-17, MIP-1α, MIP-1ß, and MMP1 were significantly decreased after NK cell infusion. Further co-culture assays showed that expanded NK cells specifically and dramatically eliminated senescent CD4+ T cells other than CD28+CD4+ T cells. They also showed improved cytotoxic activity, with different expression patterns of activating and inhibitory receptors including NKG2C, NKG2A, KLRG1, LAG3, CD57, and TIM3. Conclusion: Our findings imply that T-cell senescence and exhaustion is a reversible process in healthy individuals, and autologous NK cell administration can be introduced to alleviate the aging. Clinical Trial Registration: ClinicalTrials.gov, ChiCTR-OOh-17011878.

Gallic acid induces T-helper-1-like Treg cells and strengthens immune checkpoint blockade efficacy.

BACKGROUND: Foxp3+ regulatory T (Treg) cells facilitate tumor immune evasion by forming a suppressive tumor microenvironment. Therefore, immune therapies promoting Treg fragility may greatly enhance immune checkpoint blockade (ICB) efficacy in cancers. METHODS: We have screened 2640 compounds and identified the gut microbial metabolite gallic acid, which promotes Foxp3 degradation and Treg instability by repressing Usp21 gene transcription. In vivo and in vitro experiments have been performed to explore the roles of Usp21 in Treg cells. Importantly, we treated tumor-bearing mice with gallic acid and anti-PD-1 antibody to explore the potential therapeutic value of gallic acid in clinical cancer immunotherapy. RESULTS: Mechanistically, gallic acid prevents STAT3 phosphorylation and the binding of phosphorylated STAT3 to Usp21 gene promoter. The deubiquitinated Usp21 and stabilized PD-L1 proteins boost the function of Treg cells. Combination of gallic acid and anti-PD-1 antibody, in colorectal cancer (CRC) treatment, not only significantly dampen Treg cell function by impairing PD-L1/PD-1 signaling and downregulating Foxp3 stability, but also promote CD8+ T cells' production of IFN-γ and limited tumor growth. CONCLUSION: Our findings have implications for improving the efficacy of ICB therapy in CRC by inducing T-helper-1-like Foxp3lo Treg cells.

TGF-ß-Induced FLRT3 Attenuation Is Essential for Cancer-Associated Fibroblast-Mediated Epithelial-Mesenchymal Transition in Colorectal Cancer.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. The effects of CAFs on the progression of colorectal cancer remain controversial. In this study, we found the ectopic overexpression of Fibronectin leucine-rich transmembrane protein 3 (FLRT3) inhibited the process of epithelial-mesenchymal transition (EMT), as well as the proliferation, migration, invasion, and promote apoptosis of colorectal cancer cells, whereas silencing FLRT3 expression resulted in the opposite phenomenon. FLRT3 downregulation was associated with a poor prognosis in colorectal cancer. Also, FLRT3 expression was significantly related to some clinicopathologic factors, including T stage (P = 0.037), N stage (P = 0.042), and E-cadherin (P = 0.002) level. Via univariate and multivariate analyses, M stage (P < 0.0001), FLRT3 (P = 0.044), and E-cadherin (P = 0.003) were associated with overall survival and were independent prognostic factors for it. Mechanistically, CAFs secreted TGF-ß, which downregulated FLRT3 expression by activating SMAD4 to promote aggressive phenotypes in colorectal cancer cells. Moreover, FLRT3 repressed tumorigenesis and lung metastasis, which could be reversed by LY2109761, a dual inhibitor of TGF-ß receptor type I and II. Treatment with LY2109761 increased IFN-γ expression in CD8+ T cells and reduced the number of regulatory T cells in the tumor microenvironment. Taken together, we revealed the metastasis-suppressive function of FLRT3, which was attenuated during the CAFs-mediated activation of the TGF-ß/SMAD4 signaling pathway to promote EMT in colorectal cancer. LY2109761 that significantly inhibited metastasis could be a new treatment option for advanced colorectal cancer. IMPLICATIONS: CAFs enhance colorectal cancer aggressiveness by reducing FLRT3 expression through activating TGF-ß/SMAD4 signaling pathway. CAF-targeted therapy and/or LY2109761 were promising treatments for colorectal cancer.

hnRNPA1 enhances FOXP3 stability to promote the differentiation and functions of regulatory T cells.

Regulatory T cells (Tregs) are indispensable for the maintenance of immunological self-tolerance and homeostasis. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is required for optimal Treg induction. Here, we reveal that human-induced Tregs (iTregs) lacking hnRNPA1 show reduced expression of the transcription factor FOXP3, increased ubiquitination level of FOXP3, and impaired suppressive abilities. Human naïve CD4 T cells with hnRNPA1 knockdown show a decreased Treg differentiation ratio. hnRNPA1 could interact with FOXP3 as well as with the E3 ligase Stub1. The phosphorylation at hnRNPA1 S199 could increase both interactions. The overexpression of FOXP3 in Tregs containing shhnRNPA1 could not recover the phenotype caused by hnRNPA1 knockdown. Therefore, there might be multiple essential pathways regulated by hnRNPA1 in Tregs. In conclusion, we present a new role of hnRNPA1 in promoting Treg function, indicating it as a promising target for tumor therapies.

Targeting FOXP3 complex ensemble in drug discovery.

Forkhead Box P3 (FOXP3) is a key transcriptional regulator of regulatory T cells (Tregs), especially for its function of immune suppression. The special immune suppression function of Tregs plays an important role in maintaining immune homeostasis, and is related to several diseases including cancer, and autoimmune diseases. At the same time, FOXP3 takes a place in a large transcriptional complex, whose stability and functions can be controlled by various post-translational modification. More and more researches have suggested that targeting FOXP3 or its partners might be a feasible solution to immunotherapy. In this review, we focus on the transcription factor FOXP3 in Tregs, Treg functions in diseases and the FOXP3 targets.

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance.

CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G2 phase.

Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.

Protein expression, crystallization and preliminary X-ray crystallographic studies on HSCARG from Homo sapiens.

Human HSCARG has been annotated as a possible cancer related protein. Amino acid homology, although at a low percentage, suggested that HSCARG contains NmrA domain and might be a member of short chain dehydrogenase reductase superfamily. In order to investigate its structure and function, HSCARG gene has been successfully expressed and purified in E. coli. HSCARG was crystallized and diffracted to a resolution of 2.4 A on Mar225 CCD Detector at SER-CAT 22BM synchrotron source. The crystals belong to F23 space group, with unit cell parameters a=b=c=223.30A, alpha=beta=gamma=90 degrees . There are two molecules per asymmetry unit.