Cell Receptor Research Articles

Multi-omic characteristics of longitudinal immune profiling after breakthrough infections caused by Omicron BA.5 sublineages

Omicron sub-variants breakthrough infections (BTIs) have led to millions of coronavirus disease 2019 (COVID-19) cases worldwide. The acute-phase immune status is critical for prognosis, however, the dynamic immune profiling of COVID-19 during the first month after BTIs remains unclear. In this study, we monitored the immune dynamics at various timepoints in a longitudinal cohort during the first month post-BTIs through clinical evaluation, single-cell RNA sequencing (scRNA-seq), T cell receptor (TCR)/B cell receptor (BCR) sequencing, and antibody mass spectrometry. Serological analysis revealed limited impairment to functions of major organs, active cellular and humoral immunity at 2 weeks post-BTI, with significant increases in cytokines (CKs) and neutralizing antibody levels. However, 1 month post-BTI, organ function parameters and CK levels reverted to pre-infection levels, whereas neutralizing antibody levels remained high. Notably, scRNA-seq showed that lymphocytes maintained strong antiviral activity and cell depletion at 2 weeks and 1 month post-BTI, with genes CD81, ABHD17A, CXCR4, DUSP1, etc. upregulated, and genes PFDN5, DYNLRB1, CD52, etc. downregulated, indicating that lymphocytes status take longer to recover to normal levels than that routine blood tests revealed. Additionally, T cell-exhaustion associated genes, including LAG3, TIGIT, PDCD1, CTLA4, HAVCR2, and TOX, were upregulated after BTI. TCRs and BCRs exhibited higher clonotypes, mainly in CD8Tem or plasmablast cells, at 2 weeks post-BTI comparing 1 month. More IgG and IgA-type BCRs were found in the groups of 1 month post-BTI, with higher somatic hypermutation, indicating greater maturity. Verification of monoclonal antibodies corresponding to amplified BCRs highlighted the antigen-specific and broad-spectrum characteristics. Our study elucidated the dynamic immune profiling of individuals after Omicron BA.5 sublineages BTI. Strong immune activation, antiviral response, antibody maturation and class transition at 2 weeks and 1 month after BTI may provide essential insights into pathogenicity, sequential immune status, recovery mechanisms of Omicron sublineage BTI. This study was supported by the National Key R&D Program of China, the China Postdoctoral Science Foundation, Guangdong Basic and Applied Basic Research Foundation, the National Natural Science Foundation of China, CAS Project for Young Scientists in Basic Research, and the Air Force Special Medical Center Science and Technology Booster Program.

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and ResultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

Pharmacological characterization of prostaglandin E2 EP2 and EP4 receptors in male rat locus coeruleus neurons ex vivo

The inflammatory mediator prostaglandin E2 (PGE2) binds to Gs-coupled EP2 and EP4 receptors. These receptors are located in the locus coeruleus (LC), the principal noradrenergic nucleus in the brain, but their functional role remains unknown. In this study, the PGE2 EP2 and EP4 receptors in LC cells from male rat brain slices were pharmacologically characterized by single-unit extracellular electrophysiology. The EP2 receptor agonists butaprost (0.01–10 μM) and treprostinil (0.03–10 µM) and the EP4 receptor agonists rivenprost (0.01 nM–1 µM) and TCS2510 (0.20 nM–2 µM) increased the firing rate of LC neurons in a concentration-dependent manner. The EP2 receptor antagonist PF-04418948 (10 nM) hindered the excitatory effect of butaprost and treprostinil while the EP4 receptor antagonist L-161,982 (30 and 300 nM) blocked the excitatory effect caused by rivenprost and TCS2510. The effects of butaprost and rivenprost were prevented by extracellular sodium replacement but were not modified by the protein kinase A (PKA) activator 8-Br-cAMP (1 mM) or the inhibitor H-89 (10 μM). However, the Gβγ subunit blocker gallein (20 μM) hindered the stimulatory effect of butaprost while the Gαs subunit inhibitor NF449 (10 µM) prevented that of rivenprost. Finally, rivenprost-induced stimulation (30 nM) was not occluded by butaprost (1 µM).In conclusion, activation of EP2 and EP4 receptors excites LC noradrenergic neurons through sodium-dependent currents via different G protein subunits in male rat brain slices. EP2 and EP4 in the LC may constitute pharmacological targets for the treatment of pain, fever, drug addiction, anxiety and neuroinflammatory diseases.

Discovery of a Potential Allosteric Site in the SARS-CoV-2 Spike Protein and Targeting Allosteric Inhibitor to Stabilize the RBD Down State using a Computational Approach.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide public health crisis. At present, the development of effective drugs and/or related therapeutics is still the most urgent and important task for combating the virus. The viral entry and associated infectivity mainly rely on its envelope spike protein to recognize and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through a conformational switch of the spike receptor binding domain (RBD) from inactive to active state. Thus, it is of great significance to design an allosteric inhibitor targeting spike to lock it in the inactive and ACE2-inaccessible state. This study aims to discover the potential broad-spectrum allosteric inhibitors capable of binding and stabilizing the diverse spike variants, including the wild type, Delta, and Omicron, in the inactive RBD down state. In this work, we first detected a potential allosteric pocket within the SARS-CoV-2 spike protein. Then, we performed large-scale structure-based virtual screening by targeting the putative allosteric pocket to identify allosteric inhibitors that could stabilize the spike inactive state. Molecular dynamics simulations were further carried out to evaluate the effects of compound binding on the stability of spike RBD. Finally, we identified three potential allosteric inhibitors, CPD3, CPD5, and CPD6, against diverse SARS-CoV-2 variants, including Wild-type, Delta, and Omicron variants. Our simulation results showed that the three compounds could stably bind the predicted allosteric site and effectively stabilize the spike in the inactive state. The three compounds provide novel chemical structures for rational drug design targeting spike protein, which is expected to greatly assist in the development of new drugs against SARS-CoV-2.

411 T cell Receptor Repertoire Analysis for Early Diagnosis of Mycosis Fungoides in CTCL Patients

411 T cell Receptor Repertoire Analysis for Early Diagnosis of Mycosis Fungoides in CTCL Patients

Single-cell analysis reveals expanded CD8+ GZMKhigh T cells in CSF and shared peripheral clones in sporadic amyotrophic lateral sclerosis

Abstract Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the brain and spinal cord. Despite the crucial role of aberrant immune responses in ALS pathogenesis, studies investigating immunological profiles in the cerebrospinal fluid (CSF) of ALS patients have reported inconsistent findings. Herein, we explored the intrathecal adaptive immune response and features of circulating T cells between CSF and blood of ALS patients, using single-cell RNA and T cell receptor (TCR) sequencing. This study comprised a total of 11 patients with apparently sporadic ALS and three controls with non-inflammatory diseases. We collected CSF from all participants, and for three ALS patients, we additionally obtained paired samples of peripheral blood mononuclear cells (PBMCs). Utilizing droplet-based single-cell RNA and TCR sequencing, we analyzed immunological profiles, gene expression characteristics, and clonality. Furthermore, we examined T cell characteristics in both PBMC and CSF samples, evaluating the shared T cell clones across these compartments. In the CSF, ALS patients exhibited a lower proportion of CD4+ T cells (45.2% vs. 61.2%, p = 0.005), and a higher proportion of CD8+ GZMKhi effector memory T cells (TEMs) than controls (21.7% vs. 16.8%, p = 0.060). Higher clonality was observed in CD8+ TEMs in ALS patients compared to controls. In addition, CSF macrophages of ALS patients exhibited a significant increase in chemokines recruiting CD8+ TEMs. Immunohistochemical analysis showed slightly higher proportions of T cells in the perivascular and parenchymal spaces in ALS patients than in controls, and CD8+ TEMs co-localized with neurons or astrocytes in the motor cortices of ALS patients. Clonally expanded CD8+ GZMKhi TEMs primarily comprised shared T cell clones between CSF and PBMCs. Moreover, the shared CD8+ TEMs of PBMCs exhibited gene expression profiles similar to CSF T cells. ALS patients showed an increase in proportion and clonality of CD8+ GZMKhi TEMs and activated features of macrophages in CSF. The shared T cell clone between CSF and blood was mainly composed of expanded CD8+ GZMKhi TEMs. In conclusion, single-cell immune profiling provided novel insights into the pathogenesis of ALS, characterized by activated macrophages and clonally expanded CD8+ T cells potentially communicate between the central nervous system and peripheral circulation.

MR1 presents vitamin B6–related compounds for recognition by MR1-reactive T cells

The major histocompatibility complex class I related protein (MR1) presents microbially derived vitamin B2 precursors to mucosal-associated invariant T (MAIT) cells. MR1 can also present other metabolites to activate MR1-restricted T cells expressing more diverse T cell receptors (TCRs), some with anti-tumor reactivity. However, knowledge of the range of the antigen(s) that can activate diverse MR1-reactive T cells remains incomplete. Here, we identify pyridoxal (vitamin B6) as a naturally presented MR1 ligand using unbiased mass spectrometry analyses of MR1-bound metabolites. Pyridoxal, and the related compound, pyridoxal 5-phosphate bound to MR1 and enabled cell surface upregulation of wild type MR1*01 and MR1 expressing the Arg9His polymorphism associated with the MR1*04 allotype in a manner dependent on Lys43-mediated Schiff-base formation. Crystal structures of MR1*01 in complex with pyridoxal and pyridoxal 5-phosphate showed how these ligands were accommodated within the A-pocket of MR1. T cell lines transduced with the 7.G5 TCR, which has reported "pan-cancer" specificity, were specifically activated by pyridoxal presented by antigen-presenting cells expressing MR1*01 and MR1 allotypes bearing the less common Arg9His polymorphism. 7.G5 T cells also recognized, to a lesser extent, pyridoxal 5-phosphate and, importantly, recognition of both vitamers was blocked by an anti-MR1 antibody. 7.G5 TCR reactivity toward pyridoxal was enhanced when presented by the Arg9His polymorphism-bearing MR1 allotypes. Vitamin B6, and vitamers thereof, have been associated with various cancers, and here we describe a link between this ligand, MR1, and its allomorphs, and the pan-cancer 7.G5 TCR. This work identifies an MR1 ligand that can activate a diverse MR1-restricted TCR.

Investigating the Role of B Cell Receptors in Pathogen Recognition and Immune Response Activation: Implications for Infection Control and Therapeutic Development

Investigating the Role of B Cell Receptors in Pathogen Recognition and Immune Response Activation: Implications for Infection Control and Therapeutic Development

STAT5 activation enhances adoptive therapy combined with peptide vaccination by preventing PD-1 inhibition.

Adoptive cell therapy (ACT) using retrovirally transduced T cells represents a promising strategy for enhancing antitumor responses. When used with TriVax, a peptide vaccination strategy, this approach synergistically expands antigen-specific cell populations. STAT5 plays a vital role as a transcription factor in regulating T cell proliferation and their differentiation into effector and memory T cells. We aimed to explore the combination therapy using CD8 T cells engineered to express constitutively active STAT5 (CA-STAT5) with vaccines. CD8 T cells were transduced with a retrovirus (RV) encoding the mouse gp100 T cell receptor (TCR). In certain treatment groups, cells were also co-transduced with RV encoding CA-STAT5. We assessed transduction efficiency and functional activity through flow cytometry and various functional assays. B16F10 tumor-bearing mice were treated with ACT using RV-transduced CD8 T cells and subsequently vaccinated with TriVax. We demonstrate that TriVax selectively enhanced the expansion of ACT cell populations bearing gp100-specific TCRs. T cells engineered to express CA-STAT5 showed not only increased expansion and polyfunctionality but also reduced PD-1 expression, leading to decreased cellular exhaustion. In a B16F10 melanoma mouse model, our approach yielded a potent antitumor effect, with CA-STAT5 further amplifying this response. We found that CA-STAT5 improved antitumor activities, in part, by attenuating the PD-1/PD-L1 inhibitory pathway. These findings indicate that TCR-transduced CD8 T cells can undergo antigen-dependent expansion when exposed to TriVax. Additionally, the expression of CA-STAT5 enhances T cell proliferation and persistence, partly by promoting resistance to PD-1/PD-L1-mediated inhibition in antitumor T cells.

Multi-Omic Data Integration Suggests Putative Microbial Drivers of Aetiopathogenesis in Mycosis Fungoides

Background: Mycosis fungoides (MF) represents the most prevalent entity of cutaneous T cell lymphoma (CTCL). The MF aetiopathogenesis is incompletely understood, due to significant transcriptomic heterogeneity and conflicting views on whether oncologic transformation originates in early thymocytes or mature effector memory T cells. Recently, using clinical specimens, our group showed that the skin microbiome aggravates disease course, mainly driven by an outgrowing, pathogenic S. aureus strain carrying the virulence factor spa, which was shown by others to activate the T cell signalling pathway NF-κB. Methods: To explore the role of the skin microbiome in MF aetiopathogenesis, we here performed RNA sequencing, multi-omic data integration of the skin microbiome and skin transcriptome using Multi-Omic Factor Analysis (MOFA), virome profiling, and T cell receptor (TCR) sequencing in 10 MF patients from our previous study group. Results: We observed that inter-patient transcriptional heterogeneity may be largely attributed to differential activation of T cell signalling pathways. Notably, the MOFA model resolved the heterogenous activation pattern of T cell signalling after denoising the transcriptome from microbial influence. The MOFA model suggested that the outgrowing S. aureus strain evoked signalling by non-canonical NF-κB and IL-1B, which in turn may have fuelled the aggravated disease course. Further, the MOFA model indicated aberrant pathways of early thymopoiesis alongside enrichment of antiviral innate immunity. In line with this, viral prevalence, particularly of Epstein–Barr virus (EBV), trended higher in both lesional skin and the blood compared to nonlesional skin. Additionally, TCRs in both MF skin lesions and the blood were significantly more likely to recognize EBV peptides involved in latent infection. Conclusions: First, our findings suggest that S. aureus with its virulence factor spa fuels MF progression through non-canonical NF-κB and IL-1B signalling. Second, our data provide insights into the potential role of viruses in MF aetiology. Last, we propose a model of microbiome-driven MF aetiopathogenesis: Thymocytes undergo initial oncologic transformation, potentially caused by viruses. After maturation and skin infiltration, an outgrowing, pathogenic S. aureus strain evokes activation and maturation into effector memory T cells, resulting in aggressive disease. Further studies are warranted to verify and extend our data, which are based on computational analyses.

Single-Cell Multiomics Identifies Glycan Epitope LacNAc as a Potential Cell-Surface Effector Marker of Peripheral T Cells in Bladder Cancer Patients.

Cancer is a systemic disease continuously monitored and responded to by the human global immune system. Peripheral blood immune cells, integral to this surveillance, exhibit variable phenotypes during tumor progression. Glycosylation, as one of the most prevalent and significant post-translational modifications of proteins, plays a crucial role in immune system recognition and response. Glycan analysis has become a key method for biomarker discovery. LacNAc, a prominent glycosylation modification, regulates immune cell activity and function. Therefore, we applied our previously developed single-cell glycomic multiomics to analyze peripheral blood in cancer patients. This platform utilizes chemoenzymatic labeling with DNA barcodes for detecting and quantifying LacNAc levels at single-cell resolution without altering the transcriptional status of immune cells. For the first time, we systematically integrated single-cell transcriptome, T cell receptor (TCR) repertoire, and glycan epitope LacNAc analyses in tumor-patient-derived peripheral blood. Our integrated analysis reveals that lower-stage bladder cancer patients showed significantly higher levels of LacNAc in peripheral T cells, and peripheral T cells with high levels of cell-surface LacNAc exhibit higher cytotoxicity and TCR clonal expansion. In summary, we identified LacNAc as a potential cell-surface effector marker for peripheral T cells in bladder cancer patients, which enhances our understanding of peripheral immune cells and offers potential advancements in liquid biopsy.

Single-cell sequencing reveals PTX3 involvement in ovarian cancer metastasis

BackgroundPentraxin 3 (PTX3) has been associated with the development and progression of various malignant tumors. However, its roles and the mechanisms underlying its involvement in ovarian cancer (OC) peritoneal metastasis remain unclear.MethodsSingle-cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC) were conducted to determine the expression profiles, potential functionalities, and underlying mechanisms of PTX3 within the context of OC. To assess the proliferative response of OC cells, we utilized both EdU (5-ethynyl-2’ -deoxyuridine) and CCK8 assays. The role of PTX3 in facilitating cell migration and invasion was quantified through the use of Transwell assays. The protein expression levels were meticulously analyzed via Western blotting. Furthermore, to explore the interactions between proteins, we conducted immunofluorescence (IF) staining and co-immunoprecipitation (Co-IP) experiments. To determine the factors responsible for the upregulation of PTX3, we performed both coculture and suspension assays, providing a comprehensive approach to understanding the regulatory mechanisms involved.ResultsThis study confirmed, for the first time, that the expression of PTX3 in OC metastatic lesions is greater than that in primary lesions and that tumor cells with high PTX3 expression have greater metastatic ability. PTX3 can activate the EMT and NF-κB signaling pathways in OC cells and can interact with the TLR4 and CD44 receptors in OC cells. Additionally, PTX3’s modulation of the EMT and NF-κB pathways is partially dependent on its interaction with TLR4. Furthermore, this study revealed the intercellular regulatory network related to PTX3 in OC cells via bioinformatic analysis. High levels of PTX3 in OC cells potentially enhance the attraction of dendritic cells (DCs) and CD4 + T cells while diminishing the recruitment of B cells and CD8 + T cells. Finally, this study indicated that PTX3 upregulation was driven by multiple factors, including specific transcription factors (TFs) and modifications within the tumor microenvironment (TME).ConclusionsOur research revealed the contribution of PTX3 to the peritoneal dissemination process in OC patients, identifying a novel potential biomarker and therapeutic target for this disease.

Structural characterization and AlphaFold modeling of human T cell receptor recognition of NRAS cancer neoantigens.

T cell receptors (TCRs) that recognize cancer neoantigens are important for anticancer immune responses and immunotherapy. Understanding the structural basis of TCR recognition of neoantigens provides insights into their exquisite specificity and can enable design of optimized TCRs. We determined crystal structures of a human TCR in complex with NRAS Q61K and Q61R neoantigen peptides and HLA-A1 major histocompatibility complex (MHC), revealing the molecular underpinnings for dual recognition and specificity versus wild-type NRAS peptide. We then used multiple versions of AlphaFold to model the corresponding complex structures, given the challenge of immune recognition for such methods. One implementation of AlphaFold2 (TCRmodel2) with additional sampling was able to generate accurate models of the complexes, while AlphaFold3 also showed strong performance, although success was lower for other complexes. This study provides insights into TCR recognition of a shared cancer neoantigen as well as the utility and practical considerations for using AlphaFold to model TCR-peptide-MHC complexes.

Dynamic transition of Tregs to cytotoxic phenotype amid systemic inflammation in Graves' ophthalmopathy.

Graves' disease (GD) is an autoimmune condition that can progress to Graves' ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, patients with GD without GO, newly diagnosed patients with GO, and treated patients with GO. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated Treg (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yin yang 1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of the GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results reveal the dynamic changes in immune landscape during GO progression and provide direct insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.

An early, novel arginine methylation of KCa3.1 attenuates subsequent T cell exhaustion.

T cell receptor (TCR) engagement initiates the activation process, and this signaling event is regulated in multifaceted ways. Nutrient availability in the immediate niche is one such mode of regulation 1-3 . Here, we investigated how the availability of an essential amino acid methionine (Met) and TCR signaling might interplay in the earliest events of T cell activation to affect subsequent T cell fate and function. We found that limiting Met during only the initial 30 minutes of CD8 + T cell activation increased Ca 2+ influx, Ca 2+ -mediated NFAT1 ( Nfatc2 ) activation, NFAT1 promoter occupancy, and T cell exhaustion. We identified changes in the protein arginine methylome during the initial 30 min of TCR engagement and discovered a novel arginine methylation of a Ca 2+ -activated potassium transporter, KCa3.1, which regulates Ca 2+ -mediated NFAT1 signaling to ensure optimal activation. Ablation of arginine methylation in KCa3.1 led to increased NFAT1 activation, rendering T cells dysfunctional in murine tumour and infection models. Furthermore, acute Met supplementation at early stages reduced nuclear NFAT1 in tumour-infiltrating T cells and augmented their anti-tumour activity. Our findings identify a metabolic event occurring early after T cell activation that influences the subsequent fate of the cell.

Differential Effector Function of Tissue-Specific Natural Killer Cells Against Lung Tumors.

Natural killer (NK) cells are innate lymphoid cells capable of directly killing target cells while modulating immune effector responses. Despite their multifunctional capacities, a limited understanding of their plasticity and heterogeneity has impeded progress in developing effective NK cell-based cancer therapies. In this study, we investigated NK cell tissue heterogeneity in relation to their phenotype and effector functions against lung tumors. Using hanging-drop tumor spheroid and subcutaneously established LL/2 lung tumor models, we examined NK cell receptor diversity and its correlation with tissue-specific cytotoxicity through multiparametric flow cytometry, fluorescence imaging, and cytotoxicity assays. We identified distinct patterns of cell surface receptor expression on tissue-specific NK cells that are crucial for antitumor activity. Linear regression mathematical analyses further revealed significant positive correlations between activation-associated cell surface receptors and cytotoxic capacity in NK cells from tissues such as the liver and bone marrow. These findings underscore the differential effector capacities of NK cells from distinct tissues, even prior to exposure to LL/2 tumor cells. This highlights the significance of tissue-specific NK cell heterogeneity and its impact on their antitumor cytotoxicity. Recognizing these distinct tissue-specific receptor expression patterns will be instrumental in developing more efficacious NK cell-based cancer treatments.

Arachidonic acid impairs natural killer cell functions by disrupting signaling pathways driven by activating receptors and reactive oxygen species

BackgroundHigh levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied.MethodsWe employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA’s impact on NK cell functions.ResultsAA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity.ConclusionOur findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression.

Roles of TULA-family proteins in T cells and autoimmune diseases.

The T cell Ubiquitin Ligand (TULA) protein family contains two members, UBASH3A and UBASH3B, that display similarities in protein sequence and domain structure. Both TULA proteins act to repress T cell activation via a combination of overlapping and nonredundant functions. UBASH3B acts mainly as a phosphatase that suppresses proximal T cell receptor (TCR) signaling. In contrast, UBASH3A acts primarily as an adaptor protein, interacting with other proteins (including UBASH3B) in T cells upon TCR stimulation and resulting in downregulation of TCR signaling and NF-κB signaling. Human genetic and functional studies have revealed another notable distinction between UBASH3A and UBASH3B: numerous genome-wide association studies have identified statistically significant associations between genetic variants in and around the UBASH3A gene and at least seven different autoimmune diseases, suggesting a key role of UBASH3A in autoimmunity. However, the evidence for an independent role of UBASH3B in autoimmune disease is limited. This review summarizes key findings regarding the roles of TULA proteins in T cell biology and autoimmunity, highlights the commonalities and differences between UBASH3A and UBASH3B, and speculates on the individual and joint effects of TULA proteins on T cell signaling.

Salmonella cancer therapy metabolically disrupts tumours at the collateral cost of T cell immunity.

Bacterial cancer therapy (BCT) is a promising therapeutic for solid tumours. Salmonella enterica Typhimurium (STm) is well-studied amongst bacterial vectors due to advantages in genetic modification and metabolic adaptation. A longstanding paradox is the redundancy of T cells for treatment efficacy; instead, STm BCT depends on innate phagocytes for tumour control. Here, we used distal T cell receptor (TCR) and IFNγ reporter mice (Nr4a3-Tocky-Ifnγ-YFP) and a colorectal cancer (CRC) model to interrogate T cell activity during BCT with attenuated STm. We found that colonic tumour infiltrating lymphocytes (TILs) exhibited a variety of activation defects, including IFN-γ production decoupled from TCR signalling, decreased polyfunctionality and reduced central memory (TCM) formation. Modelling of T-cell-tumour interactions with a tumour organoid platform revealed an intact TCR signalosome, but paralysed metabolic reprogramming due to inhibition of the master metabolic controller, c-Myc. Restoration of c-Myc by deletion of the bacterial asparaginase ansB reinvigorated T cell activation, but at the cost of decreased metabolic control of the tumour by STm. This work shows for the first time that T cells are metabolically defective during BCT, but also that this same phenomenon is inexorably tied to intrinsic tumour suppression by the bacterial vector.

An Insulin Upstream Open Reading Frame (INSU) Is Present in Skeletal Muscle Satellite Cells: Changes with Age.

Insulin resistance, stem cell dysfunction, and muscle fiber dystrophy are all age-related events in skeletal muscle (SKM). However, age-related changes in insulin isoforms and insulin receptors in myogenic progenitor satellite cells have not been studied. Since SKM is an extra-pancreatic tissue that does not express mature insulin, we investigated the levels of insulin receptors (INSRs) and a novel human insulin upstream open reading frame (INSU) at the mRNA, protein, and anatomical levels in Baltimore Longitudinal Study of Aging (BLSA) biopsied SKM samples of 27-89-year-old (yrs) participants. Using RT-qPCR and the MS-based selected reaction monitoring (SRM) assay, we found that the levels of INSR and INSU mRNAs and the proteins were positively correlated with the age of human SKM biopsies. We applied RNAscope fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to SKM cryosections and found that INSR and INSU were co-localized with PAX7-labeled satellite cells, with enhanced expression in SKM sections from an 89 yrs old compared to a 27 yrs old. We hypothesized that the SKM aging process might induce compensatory upregulation of INSR and re-expression of INSU, which might be beneficial in early embryogenesis and have deleterious effects on proliferative and myogenic satellite cells with advanced age.