Blocking senescence and tolerogenic function of dendritic cells induced by γδ Treg cells enhances tumor-specific immunity for cancer immunotherapy.

BACKGROUND: Regulatory T (Treg) cells are a key component in maintaining the suppressive tumor microenvironment and immune suppression in different types of cancers. A precise understanding of the molecular mechanisms used by Treg cells for immune suppression is critical for the development of effective strategies for cancer immunotherapy. METHODS: Senescence development and tolerogenic functions of dendritic cells (DCs) induced by breast cancer tumor-derived γδ Treg cells were fully characterized using real-time PCR, flow cytometry, western blot, and functional assays. Loss-of-function strategies with pharmacological inhibitor and/or neutralizing antibody were used to identify the potential molecule(s) and pathway(s) involved in DC senescence and dysfunction induced by Treg cells. Impaired tumor antigen HER2-specific recognition and immune response of senescent DCs induced by γδ Treg cells were explored in vitro and in vivo in humanized mouse models. In addition, the DC-based HER2 tumor vaccine immunotherapy in breast cancer models was performed to explore the enhanced antitumor immunity via prevention of DC senescence through blockages of STAT3 and programmed death-ligand 1 (PD-L1) signaling. RESULTS: We showed that tumor-derived γδ Treg cells promote the development of senescence in DCs with tolerogenic functions in breast cancer. Senescent DCs induced by γδ Treg cells suppress Th1 and Th17 cell differentiation but promote the development of Treg cells. In addition, we demonstrated that PD-L1 and STAT3 signaling pathways are critical and involved in senescence induction in DCs mediated by tumor-derived γδ Treg cells. Importantly, our complementary in vivo studies further demonstrated that blockages of PD-L1 and/or STAT3 signaling can prevent γδ Treg-induced senescence and reverse tolerogenic functions in DCs, resulting in enhanced HER2 tumor-specific immune responses and immunotherapy efficacy in human breast cancer models. CONCLUSIONS: These studies not only dissect the suppressive mechanism mediated by tumor-derived γδ Treg cells on DCs in the tumor microenvironment but also provide novel strategies to prevent senescence and dysfunction in DCs and enhance antitumor efficacy mediated by tumor-specific T cells for cancer immunotherapy.

Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway.

BACKGROUND: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells. METHODS: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors. RESULTS: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4+ and CD8+ T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors. CONCLUSIONS: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy.

Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 µM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

CRISPR-Cas9 applications in T cells and adoptive T cell therapies.

T cell immunity is central to contemporary cancer and autoimmune therapies, encompassing immune checkpoint blockade and adoptive T cell therapies. Their diverse characteristics can be reprogrammed by different immune challenges dependent on antigen stimulation levels, metabolic conditions, and the degree of inflammation. T cell-based therapeutic strategies are gaining widespread adoption in oncology and treating inflammatory conditions. Emerging researches reveal that clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR-Cas9) genome editing has enabled T cells to be more adaptable to specific microenvironments, opening the door to advanced T cell therapies in preclinical and clinical trials. CRISPR-Cas9 can edit both primary T cells and engineered T cells, including CAR-T and TCR-T, in vivo and in vitro to regulate T cell differentiation and activation states. This review first provides a comprehensive summary of the role of CRISPR-Cas9 in T cells and its applications in preclinical and clinical studies for T cell-based therapies. We also explore the application of CRISPR screen high-throughput technology in editing T cells and anticipate the current limitations of CRISPR-Cas9, including off-target effects and delivery challenges, and envisioned improvements in related technologies for disease screening, diagnosis, and treatment.

Control of adaptive immunity by pattern recognition receptors.

One of the most significant conceptual advances in immunology in recent history is the recognition that signals from the innate immune system are required for induction of adaptive immune responses. Two breakthroughs were critical in establishing this paradigm: the identification of dendritic cells (DCs) as the cellular link between innate and adaptive immunity and the discovery of pattern recognition receptors (PRRs) as a molecular link that controls innate immune activation as well as DC function. Here, we recount the key events leading to these discoveries and discuss our current understanding of how PRRs shape adaptive immune responses, both indirectly through control of DC function and directly through control of lymphocyte function. In this context, we provide a conceptual framework for how variation in the signals generated by PRR activation, in DCs or other cell types, can influence T cell differentiation and shape the ensuing adaptive immune response.

B cells and the coordination of immune checkpoint inhibitor response in patients with solid tumors.

Immunotherapy profoundly changed the landscape of cancer therapy by providing long-lasting responses in subsets of patients and is now the standard of care in several solid tumor types. However, immunotherapy activity beyond conventional immune checkpoint inhibition is plateauing, and biomarkers are overall lacking to guide treatment selection. Most studies have focused on T cell engagement and response, but there is a growing evidence that B cells may be key players in the establishment of an organized immune response, notably through tertiary lymphoid structures. Mechanisms of B cell response include antibody-dependent cellular cytotoxicity and phagocytosis, promotion of CD4+ and CD8+ T cell activation, maintenance of antitumor immune memory. In several solid tumor types, higher levels of B cells, specific B cell subpopulations, or the presence of tertiary lymphoid structures have been associated with improved outcomes on immune checkpoint inhibitors. The fate of B cell subpopulations may be widely influenced by the cytokine milieu, with versatile roles for B-specific cytokines B cell activating factor and B cell attracting chemokine-1/CXCL13, and a master regulatory role for IL-10. Roles of B cell-specific immune checkpoints such as TIM-1 are emerging and could represent potential therapeutic targets. Overall, the expanding field of B cells in solid tumors of holds promise for the improvement of current immunotherapy strategies and patient selection.

Proximity-Based Labeling Identifies MHC Class II and CD37 as B Cell Receptor-Proximal Proteins with Immunological Functions.

The BCR allows for Ag-driven B cell activation and subsequent Ag endocytosis, processing, and presentation to recruit T cell help. Core drivers of BCR signaling and endocytosis are motifs within the receptor's cytoplasmic tail (primarily CD79). However, BCR function can be tuned by other proximal cellular elements, such as CD20 and membrane lipid microdomains. To identify additional proteins that could modulate BCR function, we used a proximity-based biotinylation technique paired with mass spectrometry to identify molecular neighbors of the murine IgM BCR. Those neighbors include MHC class II molecules, integrins, various transporters, and membrane microdomain proteins. Class II molecules, some of which are invariant chain-associated nascent class II, are a readily detected BCR neighbor. This finding is consistent with reports of BCR-class II association within intracellular compartments. The BCR is also in close proximity to multiple proteins involved in the formation of membrane microdomains, including CD37, raftlin, and Ig superfamily member 8. Known defects in T cell-dependent humoral immunity in CD37 knockout mice suggest a role for CD37 in BCR function. In line with this notion, CRISPR-based knockout of CD37 expression in a B cell line heightens BCR signaling, slows BCR endocytosis, and tempers formation of peptide-class II complexes. These results indicate that BCR molecular neighbors can impact membrane-mediated BCR functions. Overall, a proximity-based labeling technique allowed for identification of multiple previously unknown BCR molecular neighbors, including the tetraspanin protein CD37, which can modulate BCR function.

Role of Natural Killer Cells as Cell-Based Immunotherapy in Oral Tumor Eradication and Differentiation Both In Vivo and In Vitro.

Despite advancements in the field of cancer therapeutics, the five-year survival rate remains low in oral cancer patients. Therefore, the effective therapeutics are needed against oral cancer. Also, several studies including ours, have shown severely suppressed function and number of NK cells in oral cancer patients. In this review, we discuss the approach to inhibit the tumor growth and metastasis by direct killing or NK cell-mediated tumor differentiation. This review also provides an overview on supercharging NK cells using osteoclasts and probiotic bacteria, and their efficacy as cancer immunotherapeutic in humanized-BLT mice.

Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV.

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

The potential role of CMC1 as an immunometabolic checkpoint in T cell immunity.

T cell immunity is critical for human defensive immune response. Exploring the key molecules during the process provides new targets for T cell-based immunotherapies. CMC1 is a mitochondrial electron transport chain (ETC) complex IV chaperon protein. By establishing in-vitro cell culture system and Cmc1 gene knock out mice, we evaluated the role of CMC1 in T cell activation and differentiation. The B16-OVA tumor model was used to test the possibility of targeting CMC1 for improving T cell anti-tumor immunity. We identified CMC1 as a positive regulator in CD8+T cells activation and terminal differentiation. Meanwhile, we found that CMC1 increasingly expressed in exhausted T (Tex) cells. Genetic lost of Cmc1 inhibits the development of CD8+T cell exhaustion in mice. Instead, deletion of Cmc1 in T cells prompts cells to differentiate into metabolically and functionally quiescent cells with increased memory-like features and tolerance to cell death upon repetitive or prolonged T cell receptor (TCR) stimulation. Further, the in-vitro mechanistic study revealed that environmental lactate enhances CMC1 expression by inducing USP7, mediated stabilization and de-ubiquitination of CMC1 protein, in which a mechanism we propose here that the lactate-enriched tumor microenvironment (TME) drives CD8+TILs dysfunction through CMC1 regulatory effects on T cells. Taken together, our study unraveled the novel role of CMC1 as a T cell regulator and its possibility to be utilized for anti-tumor immunotherapy.

Regulation of pulmonary plasma cell responses during secondary infection with influenza virus.

During secondary infection with influenza virus, plasma cells (PCs) develop within the lung, providing a local source of antibodies. However, the site and mechanisms that regulate this process are poorly defined. Here, we show that while circulating memory B cells entered the lung during rechallenge and were activated within inducible bronchus-associated lymphoid tissues (iBALTs), resident memory B (BRM) cells responded earlier, and their activation occurred in a different niche: directly near infected alveoli. This process required NK cells but was largely independent of CD4 and CD8 T cells. Innate stimuli induced by virus-like particles containing ssRNA triggered BRM cell differentiation in the absence of cognate antigen, suggesting a low threshold of activation. In contrast, expansion of PCs in iBALTs took longer to develop and was critically dependent on CD4 T cells. Our work demonstrates that spatially distinct mechanisms evolved to support pulmonary secondary PC responses, and it reveals a specialized function for BRM cells as guardians of the alveoli.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation.

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

The functional role of L-fucose on dendritic cell function and polarization.

Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from melanoma- and breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

A comprehensive analysis of the KLRB1 expression and its clinical implication in testicular germ cell tumors: A review.

Testicular germ cell tumors (TGCT) are the most common testicular malignancies. KLRB1 is considered to influence the development and progression of a number of cancers. However, it is unclear how the KLRB1 gene functions in TGCT. First, it was determined the expression level of KLRB1 in TGCT using The Cancer Genome Atlas (TCGA) (The Cancer Genome Atlas) dataset and GTEx (Genotype-Tissue Expression) dataset. The clinical significance and biological functions of KLRB1 were explored using the TCGA dataset, and we analyzed the correlation of the KLRB1 gene with tumor immunity and infiltrating immune cells using gene set variation analysis and the TIMER database. We found that the expression level of KLRB1 was upregulated in TGCT malignant tissues with the corresponding normal tissues as controls, and KLRB1 expression correlated with clinicopathologic features of TGCT. Functional enrichment analysis suggested that KLRB1 might be involved in immune response and inflammatory response. KLRB1 was highly positively correlated with natural killer cell activation in immune response and positively correlated with tumor-infiltrating immune cells. This study demonstrated for the first time the role of KLRB1 in TGCT, which may serve as a new biomarker associated with immune infiltration and provide a potential therapeutic target for the treatment of TGCT.

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation.

The T cell membrane is studded with >104 T cell receptors (TCRs) that are used to scan target cells to identify short peptide fragments associated with viral infection or cancerous mutation. These peptides are presented as peptide-major-histocompatibility complexes (pMHCs) on the surface of virtually all nucleated cells. The TCR-pMHC complex forms at cell-cell junctions, is highly transient, and experiences mechanical forces. An important question in this area pertains to the role of the force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune the tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how the accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F > 7.1 piconewtons (pN) between TCRs and pMHCs. This rate of disruption, or force lifetime (τF), is tunable from tens of minutes down to 1.9 min. T cells challenged with FUSE probes with F > 7.1 pN presenting cognate antigens showed up to a 23% decrease in markers of early activation. FUSE probes with F > 17.0 pN showed weaker influence on T cell triggering further showing that TCR-pMHC with F > 17.0 pN are less frequent compared to F > 7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement boosting TCR activation.

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

SUMF1 overexpression promotes tumorous cell growth and migration and is correlated with the immune status of patients with glioma.

BACKGROUND: Glioma is a prevalent type of malignant tumor. To date, there is a lack of literature reports that have examined the association between sulfatase modifying factor 1 (SUMF1) and glioma. METHODS: The levels of SUMF1 were examined, and their relationships with the diagnosis, prognosis, and immune microenvironment of patients with glioma were investigated. Cox and Lasso regression analysis were employed to construct nomograms and risk models associated with SUMF1. The functions and mechanisms of SUMF1 were explored and verified using gene ontology, cell counting kit-8, wound healing, western blotting, and transwell experiments. RESULTS: SUMF1 expression tended to increase in glioma tissues. SUMF1 overexpression was linked to the diagnosis of cancer, survival events, isocitrate dehydrogenase status, age, and histological subtype and was positively correlated with poor prognosis in patients with glioma. SUMF1 overexpression was an independent risk factor for poor prognosis. SUMF1-related nomograms and high-risk scores could predict the outcome of patients with glioma. SUMF1 co-expressed genes were involved in cytokine, T-cell activation, and lymphocyte proliferation. Inhibiting the expression of SUMF1 could deter the proliferation, migration, and invasion of glioma cells through epithelial mesenchymal transition. SUMF1 overexpression was significantly associated with the stromal score, immune cells (such as macrophages, neutrophils, activated dendritic cells), estimate score, immune score, and the expression of the programmed cell death 1, cytotoxic T-lymphocyte associated protein 4, CD79A and other immune cell marker. CONCLUSION: SUMF1 overexpression was found to be correlated with adverse prognosis, cancer detection, and immune status in patients with glioma. Inhibiting the expression of SUMF1 was observed to deter the proliferation, migration, and invasion of cancer cells. The nomograms and risk models associated with SUMF1 could predict the prognosis of patients with glioma.