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.

The Dynamic Role of FOXP3+ Tregs and Their Potential Therapeutic Applications During SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) has been raging all around the world since the beginning of 2020, and leads to acute respiratory distress syndrome (ARDS) with strong cytokine storm which contributes to widespread tissue damage and even death in severe patients. Over-activated immune response becomes one of the characteristics of severe COVID-19 patients. Regulatory T cells (Treg) play an essential role in maintaining the immune homeostasis, which restrain excessive inflammation response. So FOXP3+ Tregs might participate in the suppression of inflammation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Besides suppressive function, tissue resident Tregs are also responsible for tissue repair. In this review, we mainly summarize the latest research focusing on the change of FOXP3+ Tregs in the COVID-19 patients, discuss the relationship between disease severity and number change of Tregs and speculate the potential role of FOXP3+ Tregs during SARS-CoV-2 infection. Furthermore, we introduce some potential Treg-based therapies to improve patients' outcomes, which include small molecular drugs, antibody drugs, CAR-Treg and cytokine treatment. We hope to reduce tissue damage of severe COVID-19 patients and offer better prognosis through Treg-based therapy.

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.

Transcriptional and posttranslational regulation of Th17/Treg balance in health and disease.

Regulatory T (Treg) cells and T helper type 17 (Th17) cells play important roles in adaptive immune responses, antagonizing each other in immune disorders. Th17/Treg balance is critical to maintaining the immune homeostasis of human bodies and is tightly regulated under healthy conditions. The transcription factors that are required for driving Th17 and Treg cell lineages differentiation respectively, RORγt and FOXP3 are tightly regulated under different tissue microenvironment, especially the transcriptional induction, posttranslational modifications, and dynamic enzymatic cofactors binding. The imbalance caused by alteration of the quantity or properties of RORγt+ Th17 or FOXP3+ Treg can contribute to inflammatory disorders in humans. Restoring Th17/Treg balance by modifying the enzymatic activities of RORγt and FOXP3 binding partners may be therapeutically applied to treat severe immune disorders. In this review, we focus on the transcriptional and posttranslational regulations of Th17/Treg balance, immune disorders caused by Th17/Treg imbalance, and new therapeutic strategies for restoring immune homeostasis.

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.

DNA helicase RecQ1 regulates mutually exclusive expression of virulence genes in Plasmodium falciparum via heterochromatin alteration.

The Plasmodium falciparum var gene family encodes ∼60 surface antigens by which parasites escape the host immune responses via clonal expression of var genes. However, the mechanism controlling this mutual exclusivity, associated with alterations in chromatin assembly, is not understood. Here, we determined how expression of the var gene family is regulated by two RecQ DNA helicase family members, PfRecQ1 and PfWRN, in P. falciparum Through genetic manipulation, we found that the complete var repertoire was silenced on PfRecQ1 knockout, whereas their expression did not show noticeable changes when PfWRN was knocked out. More important, mutually exclusive expression of var genes could be rescued by complementation of PfRecQ1. In addition, knocking out either of these two helicase genes changed the perinuclear cluster distribution of subtelomeres and subtelomeric var genes. Whereas deletion of PfRecQ1 increased the heterochromatin mark trimethylated (H3K9me3) at the transcription start site (TSS) of the var gene upsC1, that deletion had no effect on the global distribution of H3K9me3 over gene bodies, including those for the var genes. ChIP-seq assay showed that PfRecQ1 was enriched globally at the TSSs of all genes, whereas PfWRN-enriched regions occurred at the gene bodies of the var gene family, but not of other genes or at TSSs of all genes. On PfRecQ1 deletion, the upsC1 var gene moved from the active perinuclear transcription region to a silenced region of the upsC type. These findings imply that PfRecQ1, but not PfWRN, is essential for maintaining the clonal expression of var genes.

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

Plasmodium falciparum var Gene Is Activated by Its Antisense Long Noncoding RNA.

Plasmodium falciparum erythrocyte membrane protein 1, encoded by var gene, is an immunodominant antigen mediating immune evasion in humans. At a given time, only a single var gene is commonly expressed in one parasite. However, the regulation mechanism of var transcription remains largely unknown. In this study, we identified the antisense long non-coding RNA (aslncRNA) derived from var intron as an activation factor for the corresponding var gene. The exogenous artificial var aslncRNA transcribed by T7 RNA polymerase from episome can specifically activate the homologous var gene, and the exogenous aslncRNA activates transcription of both var mRNA and endogenous aslncRNA in a manner independent of the conserved intron sequence within the var gene family. Interestingly, the newly activated var gene and the previously dominant var gene then could be co-expressed in the same parasite nuclei, which suggests that the aslncRNA-mediated var gene activation could escape from the control of mutually exclusively expression of the var gene family. Together, our work shows that var aslncRNA is the activator responsible for var gene transcriptional regulation.

Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9.

BACKGROUND: Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. However, the use of this technique in P. falciparum is still limited to gene knockout, site-specific mutation and generation of green fluorescent protein (GFP) reporter line with disruption of inserted sites. RESULTS: We have adapted the CRISPR/Cas9 system to add commercial tag sequences to endogenous genes of P. falciparum. To add HA or HA-TY1 tags to ck2ß1, ck2α and stk, pL6cs-hDHFR-ck2ß1/ck2α/stk was constructed, which contained sequences of tags, specific homologous arms, and sgRNA. The P. falciparum 3D7 strain was subsequently transfected with pUF1-BSD-Cas9 and pL6cs-hDHFR-ck2ß1/ck2α/stk plasmids via electroporation. After that, BSD and WR99210 drugs were added to the culture to screen parasites containing both plasmids. Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins. CONCLUSIONS: We have improved the method to integrate tags to Plasmodium falciparum genes using the CRISPR/Cas9 method, which lays the foundation for further study of Plasmodium falciparum at the molecular level.

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.

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in.

Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.

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.

Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection.

Telomeric G-overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G-overhang generation at human telomeres is poorly understood. Here, we show that G-overhangs can undergo cell cycle-regulated changes independent of telomerase activity. G-overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C-strand fill-in, whereas the sizes of G-overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C-strands are precisely defined throughout the cell cycle, indicating that C-strand resection is strictly regulated. We demonstrate that C-strand fill-in is mediated by DNA polymerase alpha (polalpha) and controlled by cyclin-dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G-overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G-overhangs and an increase in telomeric DNA damage. Our results suggest that G-overhang generation at human telomeres is regulated by multiple tightly controlled processes and C-strand fill-in is under the control of polalpha and CDK1.

NADPH is an allosteric regulator of HSCARG.

NADP(H) is an important cofactor that controls many fundamental cellular processes. We have determined the crystal structure of HSCARG, a novel NADPH sensor, and found that it forms an asymmetrical dimer with only one subunit occupied by an NADPH molecule, and the two subunits have dramatically different conformations. To study the role of NADPH in affecting the structure and function of HSCARG, here, we constructed a series of HSCARG mutants to abolish NADPH binding ability. Protein structures of two mutants, R37A and Y81A, were solved by X-ray crystallography. The dimerization of wild-type and mutant HSCARG was studied by dynamic light scattering. Differences between the function of wild-type and mutant HSCARG were also compared. Our results show that binding of NADPH is necessary for HSCARG to form a stable asymmetric dimer. The conformation of the monomeric mutants was similar to that of NADPH-bound Molecule I in wild-type HSCARG, although some conformational changes were found in the NADPH binding site. Furthermore, we also noticed that abolition of NADPH binding ability changes the distribution of HSCARG in the cell and that these mutants without NADPH are more strongly associated with argininosuccinate synthetase as compared with wild-type HSCARG. These data suggest that NADPH functions as an allosteric regulator of the structure and function of HSCARG. In response to the changes in the NADPH/NADP(+) ratio within cells, HSCARG, as a redox sensor, associates and dissociates with NADPH to form a new dynamic equilibrium. This equilibrium, in turn, will tip the dimerization balance of the protein molecule and consequently controls the regulatory function of HSCARG.