Biology subjects

August, A.

Publications and source records attributed to August, A..

17 recordsLinked to original sources

bioRxiv2025

Deletion of a KSF Motif Attenuates NSP1 Host Cell Translation Shutoff and Impairs SARS-CoV-2 Virulence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion ({Delta}KSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo, similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same {Delta}KSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. IMPORTANCEUnderstanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the hosts immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.

microbiology↗

bioRxiv2025

Diverse microbial exposure exacerbates the development of allergic airway inflammation in adult mice

BackgroundExposure to a diversity of microbes has been implicated in playing a major role in susceptibility to the development of allergic lung type diseases. The hygiene hypothesis suggests that those exposed to a broad diversity of microbes are more likely to be protected against developing allergic type diseases. However, changes in exposure to microbial diversity can occur in both younger individuals, as well as in adults, and the effects are not always understood. ObjectiveWe investigated the effect of exposure to broad microbial diversity on the airway T cell response in house dust mite (HDM) induced allergic airway disease (AAD, a model of allergic asthma). MethodsWe increased exposure to broad microbial diversity by co-housing specific pathogen free (SPF) adult or newborn mice with pet store mice (PSE or BiPSE, respectively). Mice were then exposed to HDM to induce AAD. ResultsWe found that the effect of increased microbial exposure on the development of allergic airway inflammation differs by age. Increasing exposure to diverse microbes as adults exacerbates the development of allergic airway inflammation, whereas this was not observed when exposure occurred at birth. ConclusionWe suggest that experimental evaluation of the hygiene hypothesis in inflammation, particularly those using mouse models, may need to consider age of the host and time of microbial exposure. Capsule SummaryMouse models of increased exposure to diverse microbial environment shown to differentially affect the development of allergic airway inflammation, depending on the age of microbial exposure.

immunology↗

bioRxiv2025

Programing Immunogenicity of Dengue EDIII Vaccine Antigens Using Engineered Outer Membrane Vesicles (OMVs)

Dengue is a mosquito-borne viral infection and is more prevalent in the world with no therapeutics and suboptimal vaccine performance against all four serotypes of the dengue virus. Hence, there is an urgent requirement for a non-infectious and non-replicative vaccine candidate that can elicit a balanced and serotype-specific immune response. In this study, we have engineered bacterial outer membrane vesicles (rOMVs) that display EDIII antigens (EDIII rOMVs). The current formulation modulates the expression of costimulatory molecules on antigen-presenting cells (APCs) as well as enhances the uptake and presentation. Subsequently, the EDIII rOMVs elicited a strong antigen-specific polyfunctional response from CD4+ and CD8+ T cells. The robust antibody response was facilitated by a germinal center reaction characterized by high T follicular helper (Tfh) and B cell response levels in the mice that received EDIII rOMVs. Notably, the produced antibodies demonstrated the ability to neutralize all four dengue virus serotypes in an in vitro infection model, indicating its potential role in protective immunity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/640071v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@689d81org.highwire.dtl.DTLVardef@3c878aorg.highwire.dtl.DTLVardef@1e6b1aaorg.highwire.dtl.DTLVardef@9a5180_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design

Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.

synthetic biology↗

bioRxiv2023

Th2 and Th17-Associated Immunopathology Following SARS-CoV-2 Breakthrough Infection in Spike-vaccinated ACE2-humanized Mice

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety. Significance statementThis research investigates the safety and efficacy of a Spike protein subunit vaccine adjuvanted with Alum and CpG in an ACE2-humanized mouse model, simulating SARS-CoV-2 breakthrough infections. The study reveals that despite robust protection against severe COVID-19, vaccinated mice exhibit substantial pulmonary immunopathology, including eosinophilia and enhanced Th2 effector immunity, following breakthrough infections. Surprisingly, the study also uncovers a significant systemic Th17 inflammatory response in vaccinated mice. This research sheds light on the potential risks associated with COVID-19 vaccine breakthrough infections and the need for a comprehensive understanding of vaccine-induced immune responses, emphasizing the importance of ongoing research, surveillance, and careful vaccine development for both protection and safety in the fight against the COVID-19 pandemic.

immunology↗

bioRxiv2023

PTEN loss in glioma cell lines leads to increased extracellular vesicle biogenesis and PD-L1 cargo in a PI3K-dependent manner

Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-{gamma} (IFN-{gamma}). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.

cancer biology↗

bioRxiv2023

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

pathology↗

bioRxiv2023

ITK signaling regulates a switch between T helper 17 and T regulatory cell lineages via a calcium-mediated pathway

The balance of pro-inflammatory T helper type 17 (Th17) and anti-inflammatory T regulatory (Treg) cells is crucial in maintaining immune homeostasis in health and disease conditions. Differentiation of naive CD4+ T cells into Th17/Treg cells is dependent upon T cell receptor (TCR) activation and cytokine signaling, which includes the kinase ITK. Signals from ITK can regulate the differentiation of Th17 and Treg cell fate choice, however, the mechanism remains to be fully understood. We report here that in the absence of ITK activity, instead of developing into Th17 cells under Th17 conditions, naive CD4+ T cells switch to cells expressing the Treg marker Foxp3 (Forkhead box P3). These switched Foxp3+ Treg like cells retain suppressive function and resemble differentiated induced Tregs in their transcriptomic profile, although their chromatin accessibility profiles are intermediate between Th17 and induced Tregs cells. Generation of the switched Foxp3+ Treg like cells was associated with reduced expression of molecules involved in mitochondrial oxidative phosphorylation and glycolysis, with reduced activation of the mTOR signaling pathway, and reduced expression of BATF. This ITK dependent switch between Th17 and Treg cells was reversed by increasing intracellular calcium. These findings suggest potential strategies for fine tune the TCR signal strength via ITK to regulate the balance of Th17/Treg cells.

immunology↗

bioRxiv2022

T cell development is regulated by high fidelity replication of mitochondrial DNA

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase {gamma} (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.

immunology↗

bioRxiv2022

APP family member dimeric complexes are formed predominantly in synaptic compartments

The amyloid precursor protein (APP), a key player in Alzheimes disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with coimmunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, about 20% of APP is dimerized in cultured cells while about 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls, suggesting that loss of dimers of full-length APP might correlate with loss of synapses in the process of AD.

neuroscience↗

bioRxiv2021

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortex-- events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1Cre; LSL-hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

microbiology↗

bioRxiv2021

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7. Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8. Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

microbiology↗

bioRxiv2021

Eosinophils are dispensable for development of MOG35-55-induced experimental autoimmune encephalomyelitis in mice

Experimental autoimmune encephalomyelitis (EAE) represents the mouse model of multiple sclerosis, a devastating neurological disorder. EAE development and progression involves the infiltration of different immune cells into the brain and spinal cord. However, less is known about a potential role of eosinophil granulocytes for EAE disease pathogenesis. In the present study, we found enhanced eosinophil abundance accompanied by increased concentration of the eosinophil chemoattractant eotaxin-1 in the spinal cord in the course of EAE induced in C57BL/6 mice by immunization with MOG35-55 peptide. However, the absence of eosinophils did not affect neuroinflammation, demyelination and clinical development or severity of EAE, as assessed in {Delta}dblGATA1 eosinophil-deficient mice. Taken together, despite their enhanced abundance in the inflamed spinal cord during disease progression, eosinophils were dispensable for EAE development.

immunology↗

bioRxiv2020

Inhibition of ITK differentiates GVT and GVHD in allo-HSCT

Allogeneic hematopoietic stem cell transplantation is a life-saving treatment for many malignant and nonmalignant diseases. Donor T cells contained within the graft prevent tumor recurrence via graft-versus-tumor (GVT) effects, however, also cause graft-versus-host disease (GVHD). Novel treatment strategies are therefore needed to allow maintenance of GVT while suppressing GVHD. Here we show using murine models, that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving the beneficial GVT effects. Donor T cells from Itk-/- mice exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs such as liver and small intestine, while maintaining GVT efficacy against primary B-ALL tumors. Itk-/- T cells exhibited reduced expression of IRF4 and decreased JAK/STAT signaling activity, but preserved cytotoxicity, which was accompanied by upregulation of Eomesodermin (Eomes), which was necessary for GVT function. A novel peptide inhibitor ITK signaling is also able to prevent GVHD. This novel peptide inhibitor also reduced cytokine production in mice and human T cells. Altogether, our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVT effects following allo-HSCT treatment. Key PointsO_LIInhibiting ITK by a novel peptide significantly reduces GVHD but retains GVT. C_LIO_LIITK deficient donor T cells exhibit minimal GVHD, but maintain GVT activity. C_LIO_LIITK deficient donor T cells exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs. C_LIO_LIEomes is required for GVT effect. C_LI

immunology↗

bioRxiv2020

Effect of Perfluorooctanesulfonic acid (PFOS) on immune cell development and function in mice

Perfluorinated compounds, such as Perfluoroctane sulfonic acid (PFOS) are compounds containing carbon chains where hydrogens have been replaced with fluorines, and belong to a larger family known as Per- and polyfluoroalkyl substances (PFAS). The strength of the carbon-fluorine bond makes perfluorinated compounds extremely resistant to environmental degradation. Due to the persistent nature of PFOS, research has been directed to elucidating possible health effects of PFOS on humans and laboratory animals. Here we have explored the effects of PFOS exposure on immune development and function in mice. We exposed adult wild-type mice to 3 and 1.5 g/kg/day of PFOS for 2 and 4 weeks, respectively, and examined the effects of PFOS exposure on populations of T cells, B cells, and granulocytes. These doses of PFOS resulted in serum levels of approximately 100 ng/ml with no weight loss during exposure. We find that PFOS does not affect T-cell development during this time. However, while PFOS exposure reduced immune cell populations in some organs, it also led to an increase in the numbers of cells in others, suggesting possible relocalization of cells. We also examined the effect of PFOS on the response to influenza virus infection. We find that exposure to PFOS at 1.5 g/kg/day of PFOS for 4 weeks does not affect weight loss or survival, nor is viral clearance affected. Analysis of antibody and T cell specific antiviral responses indicate that at this concentration, PFOS does not suppress the immune cell development or antigen specific immune response.

pharmacology and toxicology↗

bioRxiv2018

Beyond type 1 regulatory T cells: co-expression of LAG3 and CD49b in IL-10-producing T cell lineages

Type 1 regulatory CD4+ T (Tr1) cells express high levels of the immunosuppressive cytokine IL-10 but not the master transcription factor Foxp3, and can suppress inflammation and promote immune tolerance. In order to identify and obtain viable Tr1 cells for research and clinical applications, co-expression of CD49b and LAG3 has been proposed as a unique surface signature for both human and mouse Tr1 cells. However, recent studies have revealed that this pattern of co-expression is dependent on the stimulating conditions and the differentiation stage of the CD4+ T cells. Here, using an IL-10GFP/Foxp3RFP dual reporter transgenic murine model, we demonstrate that co-expression of CD49b and LAG3 is not restricted to the Foxp3- Tr1 cells, but is also observed in Foxp3+ T regulatory (Treg) cells and CD8+ T cells that produce IL-10. Our data indicate that IL-10-producing Tr1 cells, Treg cells and CD8+ T cells are all capable of co-expressing LAG3 and CD49b in vitro following differentiation under IL-10-inducing conditions, and in vivo following pathogenic insult or infection in the pulmonary mucosa. Our findings urge caution in the use of LAG3/CD49b co-expression to identify Tr1 cells, since it may mark IL-10-producing T cell lineages more broadly, including the Foxp3- Tr1 cells, Foxp3+ Treg cells and CD8+ T cells.

immunology↗

bioRxiv2018

TCR/ITK signaling via mTOR tunes CD8+ T cell homeostatic proliferation, metabolism, and anti-tumor effector function

T cell homeostatic proliferation (HP) is regulated by T cell receptor (TCR) signals and homeostatic cytokines, and suggested to be proportional to TCR signal strength. However, we show here that ITK, a positive regulator of TCR signaling, negatively tunes CD8+ T cell HP, metabolism, and effector function. Under lymphopenic environments, Itk-/- CD8+ T cells exhibit significant increase in T cell-intrinsic HP, which requires mTOR activity and can be driven by T cell-T cell interaction. TCR signals through ITK tune IL-7-mediated CD8+ T cell metabolism and HP in a mTOR-dependent manner. The lack of ITK also resulted in enhanced effector cell fate programming, antigen sensitivity and anti-tumor immunity by HP cells. Thus, TCR signaling via ITK, is a negative tuner of CD8+ T cell homeostasis, metabolism and effector function, and may be a target for clinical benefit in cancer therapy.\n\nOne Sentence SummaryTCR signal strength had been long-thought to be proportional to T cell proliferation and effector function, here we demonstrate a counterintuitive role of the TCR signaling through ITK in negatively tuning proliferation under lymphopenic conditions via regulating mTOR activity, T cell metabolism, proliferation, and effector function.

immunology↗