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Chemotherapy resistance remains a significant obstacle that limits the long-term efficacy of cancer therapy, necessitating further investigations into the underlying mechanisms. Here, we find that DNA fragments induced by chemotherapeutic agents trigger the degradation of cGAS, a potent double-strand DNA (dsDNA) sensor, by lysosomes. Mechanically, the lysosome-localized protein LAMTOR1 is up-regulated, and the interaction between LAMTOR1 and cGAS is enhanced upon exposure to DNA fragments, boosting the accumulation and digestion of cGAS in lysosomes through the receptor protein p62. LAMTOR1 deficiency increases cGAS abundance and promotes activation of the cGAS-STING pathway, leading to subsequent production of type I interferons induced by cytosolic DNA stimulation. Loss of LAMTOR1 synergizes with immunotherapy and chemotherapy to inhibit tumor growth and prolong the survival time of tumor-bearing mice by promoting the infiltration of effective T lymphocytes. Thus, our study reveals a regulation of cGAS abundance and provides a potential strategy to overcome chemotherapy resistance by targeting LAMTOR1. [ABSTRACT FROM AUTHOR]

The CD4 T cell, when engineered with a chimeric antigen receptor (CAR) containing specific intracellular domains, has been transformed into a zero-order drug-delivery platform. This introduces the capability of prolonged, disease-specific engineered protein biologics production, at the disease site. Experimental findings demonstrate that CD4 T cells offer a solution when modified with a CAR that includes 4-1BB but excludes CD28 intracellular domain. In this configuration, they achieve ~3X transduction efficiency of CD8 T cells, ~2X expansion rates, generating ~5X more biologic, and exhibit minimal cytolytic activity. Cumulatively, this addresses two main hurdles in the translation of cell-based drug delivery: scaling the production of engineered T cell ex vivo and generating sufficient biologics in vivo. When programmed to induce IFNß upon engaging the target antigen, the CD4 T cells outperforms CD8 T cells, effectively suppressing cancer cell growth in vitro and in vivo. In summary, this platform enables precise targeting of disease sites with engineered protein-based therapeutics while minimizing healthy tissue exposure. Leveraging CD4 T cells' persistence could enhance disease management by reducing drug administration frequency, addressing critical challenges in cell-based therapy. [ABSTRACT FROM AUTHOR]

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a-and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID. [ABSTRACT FROM AUTHOR]

Mechanical force has repeatedly been highlighted to be involved in T cell activation. However, the biological significance of mechanical force for T cell receptor signaling remains under active consideration. Here, guided by theoretical analysis, we provide a perspective on how mechanical forces between a T cell and an antigen-presenting cell can influence the bond of a single T cell receptor major histocompatibility complex during early T cell activation. We point out that the lifetime of T cell receptor bonds and thus the degree of their phosphorylation which is essential for T cell activation depends considerably on the T cell receptor rigidity and the average magnitude and frequency of an applied oscillatory force. Such forces could be, for example, produced by protrusions like microvilli during early T cell activation or invadosomes during full T cell activation. These features are suggestive of mechanical force being exploited by T cells to advance self-nonself discrimination in early T cell activation. [ABSTRACT FROM AUTHOR]

Developing an effective Staphylococcus aureus (S. aureus) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus. Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates. [ABSTRACT FROM AUTHOR]

We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors. [ABSTRACT FROM AUTHOR]

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development. [ABSTRACT FROM AUTHOR]

Although many cytokine pathways are important for dendritic cell (DC) development, it is less clear what cytokine signals promote the function of mature dendritic cells. The signal transducer and activator of transcription 4 (STAT4) promotes protective immunity and autoimmunity downstream of proinflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), Stat4-/-mice are resistant to the development of inflammation and paralysis. To define whether STAT4 is required for intrinsic signaling in mature DC function, we used conditional mutant mice in the EAE model. Deficiency of STAT4 in CD11c-expressing cells resulted in decreased T cell priming and inflammation in the central nervous system. EAE susceptibility was recovered following adoptive transfer of wild-type bone marrow-derived DCs to mice with STAT4-deficient DCs, but not adoptive transfer of STAT4-or IL-23R-deficient DCs. Single-cell RNA-sequencing (RNA-seq) identified STAT4-dependent genes in DC subsets that paralleled a signature in MS patient DCs. Together, these data define an IL-23-STAT4 pathway in DCs that is key to DC function during inflammatory disease. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection can induce multisystem inflammatory syndrome in children, which resembles superantigen-induced toxic shock syndrome. Recent work has suggested that the SARS-CoV-2 spike (S) protein could act as a superantigen by binding T cell receptors (TCRs) and inducing broad antigen-independent T cell responses. Structure-based computational modeling identified potential TCR-binding sites near the S receptor-binding domain, in addition to a site with homology to known neurotoxins. We experimentally examined the mechanism underpinning this theory--the direct interaction between the TCR and S protein. Surface plasmon resonance of recombinantly expressed S protein and TCR revealed no detectable binding. Orthogonally, we pseudotyped lentiviruses with SARS-CoV-2 S in both wild-type and prefusion-stabilized forms, demonstrated their functionality in a cell line assay, and observed no transduction, activation, or stimulation of proliferation of CD8+ T cells. We conclude that it is unlikely that the SARS-CoV-2 spike protein engages nonspecifically with TCRs or has superantigenic character. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) represents a challenge in oncology, with limited treatment options for advanced-stage patients. Chimeric antigen receptor T cell (CAR T) therapy targeting mesothelin (MSLN) shows promise, but challenges such as the hostile immunosuppressive tumor microenvironment (TME) hinder its efficacy. This study explores the synergistic potential of combining proton radiation therapy (RT) with MSLN-targeting CAR T therapy in a syngeneic PDAC model. Proton RT significantly increased MSLN expression in tumor cells and caused a significant increase in CAR T cell infiltration into tumors. The combination therapy reshaped the immunosuppressive TME, promoting antitumorigenic M1 polarized macrophages and reducing myeloid-derived suppressor cells (MDSC). In a flank PDAC model, the combination therapy demonstrated superior attenuation of tumor growth and improved survival compared to individual treatments alone. In an orthotopic PDAC model treated with image-guided proton RT, tumor growth was significantly reduced in the combination group compared to the RT treatment alone. Further, the combination therapy induced an abscopal effect in a dual-flank tumor model, with increased serum interferon-γ levels and enhanced proliferation of extratumoral CAR T cells. In conclusion, combining proton RT with MSLN-targeting CAR T therapy proves effective in modulating the TME, enhancing CAR T cell trafficking, and exerting systemic antitumor effects. Thus, this combinatorial approach could present a promising strategy for improving outcomes in unresectable PDAC. [ABSTRACT FROM AUTHOR]

Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. In this manuscript, we show that miR-155 targets transcripts encoding IcosL, the ligand for Inducible T-cell costimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. We specifically found that overexpression of miR-155 in B cells of Eµ-miR-155 mice causes loss of IcosL expression as they progress toward malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking IcosL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates composed of IcosL-positive B cells and Icos-positive T cells forming immunological synapses. Therefore, we next engineered malignant cells to express IcosL, in order to determine whether IcosL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an IcosL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intratumor CD8+ T cell infiltration, that formed synapses with IcosL-expressing MC38 cells. Our results underscore the fact that by targeting IcosL transcripts, miR-155 impairs the infiltration of tumors by cytotoxic T cells, as well as the importance of IcosL on enhancing the immune response against malignant cells. These findings should lead to the development of more effective anticancer treatments based on maintaining, increasing, or restoring IcosL expression by malignant cells, along with impairing miR-155 activity. [ABSTRACT FROM AUTHOR]

Cerebral (Aβ) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aβ/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aβ/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aβ/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aβ/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aβ/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD. [ABSTRACT FROM AUTHOR]

The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/-autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity. [ABSTRACT FROM AUTHOR]

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases. [ABSTRACT FROM AUTHOR]

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression. [ABSTRACT FROM AUTHOR]

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance. [ABSTRACT FROM AUTHOR]

The current paradigm about the function of T cell immune checkpoints is that these receptors switch on inhibitory signals upon cognate ligand interaction. We here revisit this simple switch model and provide evidence that the T cell lineage protein THEMIS enhances the signaling threshold at which the immune checkpoint BTLA (B- and T-lymphocyte attenuator) represses T cell responses. THEMIS is recruited to the cytoplasmic domain of BTLA and blocks its signaling capacity by promoting/stabilizing the oxidation of the catalytic cysteine of the tyrosine phosphatase SHP-1. In contrast, THEMIS has no detectable effect on signaling pathways regulated by PD-1 (Programmed cell death protein 1), which depend mainly on the tyrosine phosphatase SHP-2. BTLA inhibitory signaling is tuned according to the THEMIS expression level, making CD8+ T cells more resistant to BTLA-mediated inhibition than CD4+ T cells. In the absence of THEMIS, the signaling capacity of BTLA is exacerbated, which results in the attenuation of signals driven by the T cell antigen receptor and by receptors for IL-2 and IL-15, consequently hampering thymocyte positive selection and peripheral CD8+ T cell maintenance. By characterizing the pivotal role of THEMIS in restricting the transmission of BTLA signals, our study suggests that immune checkpoint operability is conditioned by intracellular signal attenuators. [ABSTRACT FROM AUTHOR]

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population. [ABSTRACT FROM AUTHOR]

Here, we report recurrent focal deletions of the chr14q32.31- 32 locus, including TRAF3, a negative regulator of NF- kB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF- kB kinase, and increased NC NF- kB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF- kB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3- deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco- addiction to NC NF- kB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL- 10. Coculturing of TRAF3- deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL- 10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF- kB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL. [ABSTRACT FROM AUTHOR]

Class II histone deacetylases (HDACs) are important in regulation of gene transcription during T cell development. However, our understanding of their cell- specific functions is limited. In this study, we reveal that class IIa Hdac4 and Hdac7 (Hdac4/7) are selectively induced in transcription, guiding the lineage- specific differentiation of mouse T- helper 17 (Th17) cells from naive CD4+ T cells. Importantly, Hdac4/7 are functionally dispensable in other Th subtypes. Mechanistically, Hdac4 interacts with the transcription factor (TF) JunB, facilitating the transcriptional activation of Th17 signature genes such as Il17a/f. Conversely, Hdac7 collaborates with the TF Aiolos and Smrt/Ncor1- Hdac3 corepressors to repress transcription of Th17 negative regulators, including Il2, in Th17 cell differentiation. Inhibiting Hdac4/7 through pharmacological or genetic methods effectively mitigates Th17 cell--mediated intestinal inflammation in a colitis mouse model. Our study uncovers molecular mechanisms where HDAC4 and HDAC7 function distinctively yet cooperatively in regulating ordered gene transcription during Th17 cell differentiation. These findings suggest a potential therapeutic strategy of targeting HDAC4/7 for treating Th17- related inflammatory diseases, such as ulcerative colitis. [ABSTRACT FROM AUTHOR]

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals. [ABSTRACT FROM AUTHOR]

In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state. [ABSTRACT FROM AUTHOR]

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes. [ABSTRACT FROM AUTHOR]

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo. [ABSTRACT FROM AUTHOR]

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases. [ABSTRACT FROM AUTHOR]

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the micro-biota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene--related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP--RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell--specific deletion of CPT1A, combined with stable 13C isotope tracing, we observe reduced mitochondrial function and fatty acid--derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell--based immunotherapies. [ABSTRACT FROM AUTHOR]

T cells help orchestrate immune responses to pathogens, and their aberrant regulation can trigger autoimmunity. Recent studies highlight that a threshold number of T cells (a quorum) must be activated in a tissue to mount a functional immune response. These collective effects allow the T cell repertoire to respond to pathogens while suppressing autoimmunity due to circulating autoreactive T cells. Our computational studies show that increasing numbers of pathogenic peptides targeted by T cells during persistent or severe viral infections increase the probability of activating T cells that are weakly reactive to self-antigens (molecular mimicry). These T cells are easily re-activated by the self-antigens and contribute to exceeding the quorum threshold required to mount autoimmune responses. Rare peptides that activate many T cells are sampled more readily during severe/persistent infections than in acute infections, which amplifies these effects. Experiments in mice to test predictions from these mechanistic insights are suggested. [ABSTRACT FROM AUTHOR]

Identification of mechanisms that program early effector T cells to either terminal effector T (Teff) or memory T (Tm) cells has important implications for protective immunity against infections and cancers. Here, we show that the cytosolic transcription factor aryl hydrocarbon receptor (AhR) is used by early Teff cells to program memory fate. Upon antigen engagement, AhR is rapidly up-regulated via reactive oxygen species signaling in early CD8+ Teff cells, which does not affect the effector response, but is required for memory formation. Mechanistically, activated CD8+ T cells up-regulate HIF-1α to compete with AhR for HIF-1β, leading to the loss of AhR activity in HIF-1αhigh short-lived effector cells, but sustained in HIF-1αlow memory precursor effector cells (MPECs) with the help of autocrine IL-2. AhR then licenses CD8+ MPECs in a quiescent state for memory formation. These findings partially resolve the long-standing issue of how Teff cells are regulated to differentiate into memory cells. [ABSTRACT FROM AUTHOR]

PD1 blockade therapy, harnessing the cytotoxic potential of CD8+ T cells, has yielded clinical success in treating malignancies. However, its efficacy is often limited due to the progressive differentiation of intratumoral CD8+ T cells into a hypofunctional state known as terminal exhaustion. Despite identifying CD8+ T cell subsets associated with immunotherapy resistance, the molecular pathway triggering the resistance remains elusive. Given the clear association of CD38 with CD8+ T cell subsets resistant to anti-PD1 therapy, we investigated its role in inducing resistance. Phenotypic and functional characterization, along with single-cell RNA sequencing analysis of both in vitro chronically stimulated and intratumoral CD8+ T cells, revealed that CD38-expressing CD8+ T cells are terminally exhausted. Exploring the molecular mechanism, we found that CD38 expression was crucial in promoting terminal differentiation of CD8+ T cells by suppressing TCF1 expression, thereby rendering them unresponsive to anti-PD1 therapy. Genetic ablation of CD38 in tumor-reactive CD8+ T cells restored TCF1 levels and improved the responsiveness to anti-PD1 therapy in mice. Mechanistically, CD38 expression on exhausted CD8+ T cells elevated intracellular Ca2+ levels through RyR2 calcium channel activation. This, in turn, promoted chronic AKT activation, leading to TCF1 loss. Knockdown of RyR2 or inhibition of AKT in CD8+ T cells maintained TCF1 levels, induced a sustained anti-tumor response, and enhanced responsiveness to anti-PD1 therapy. Thus, targeting CD38 represents a potential strategy to improve the efficacy of anti-PD1 treatment in cancer. [ABSTRACT FROM AUTHOR]

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Al33p53a lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy. [ABSTRACT FROM AUTHOR]

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stagespecific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors. [ABSTRACT FROM AUTHOR]

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality. [ABSTRACT FROM AUTHOR]

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies. [ABSTRACT FROM AUTHOR]

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells. [ABSTRACT FROM AUTHOR]

Integrating multimodal neuro-and nanotechnology-enabled precision immunotherapies with extant systemic immunotherapies may finally provide a significant breakthrough for combatting glioblastoma (GBM). The potency of this approach lies in its ability to train the immune system to efficiently identify and eradicate cancer cells, thereby creating anti-tumor immune memory while minimizing multi-mechanistic immune suppression. A critical aspect of these therapies is the controlled, spatiotemporal delivery of structurally defined nanotherapeutics into the GBM tumor microenvironment (TME). Architectures such as spherical nucleic acids or poly(beta-amino ester)/dendrimer-based nanoparticles have shown promising results in preclinical models due to their multivalency and abilities to activate antigen-presenting cells and prime antigen-specific T cells. These nanostructures also permit systematic variation to optimize their distribution, TME accumulation, cellular uptake, and overall immunostimulatory effects. Delving deeper into the relationships between nanotherapeutic structures and their performance will accelerate nano-drug development and pave the way for the rapid clinical translation of advanced nanomedicines. In addition, the efficacy of nanotechnology-based immunotherapies may be enhanced when integrated with emerging precision surgical techniques, such as laser interstitial thermal therapy, and when combined with systemic immunotherapies, particularly inhibitors of immune-mediated checkpoints and immunosuppressive adenosine signaling. In this perspective, we highlight the potential of emerging treatment modalities, combining advances in biomedical engineering and neurotechnology development with existing immunotherapies to overcome treatment resistance and transform the management of GBM. We conclude with a call to action for researchers to leverage these technologies and accelerate their translation into the clinic. [ABSTRACT FROM AUTHOR]

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes. [ABSTRACT FROM AUTHOR]

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer. [ABSTRACT FROM AUTHOR]

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) infection has long been associated with multiple sclerosis (MS), but the role of EBV in the pathogenesis of MS is not clear. Our hypothesis is that a major fraction of the expanded clones of T lymphocytes in the cerebrospinal fluid (CSF) are specific for autologous EBV-infected B cells. We obtained blood and CSF samples from eight relapsing-remitting patients in the process of diagnosis. We stimulated cells from the blood with autologous EBV-infected lymphoblastoid cell lines (LCL), EBV, varicella zoster virus, influenza, and candida and sorted the responding cells with flow cytometry after 6 d. We sequenced the RNA for T cell receptors (TCR) from CSF, unselected blood cells, and the antigen-specific cells. We used the TCR Vβ CDR3 sequences from the antigen-specific cells to assign antigen specificity to the sequences from the CSF and blood. LCL-specific cells comprised 13.0 ± 4.3% (mean ± SD) of the total reads present in CSF and 13.3 ± 7.5% of the reads present in blood. The next most abundant antigen specificity was flu, which was 4.7 ± 1.7% of the reads in the CSF and 9.3 ± 6.6% in the blood. The prominence of LCL-specific reads was even more marked in the top 1% most abundant CSF clones with statistically significant 47% mean overlap with LCL. We conclude that LCL-specific sequences form a major portion of the TCR repertoire in both CSF and blood and that expanded clones specific for LCL are present in MS CSF. This has important implications for the pathogenesis of MS. [ABSTRACT FROM AUTHOR]

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity. [ABSTRACT FROM AUTHOR]

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts. [ABSTRACT FROM AUTHOR]

The perinatal period is a critical time window in establishing T cell tolerance. Regulatory T cells (Tregs) made during the first 2 wk of life are key drivers of perinatal tolerance induction, but how these cells are generated and operate has not been established. To elucidate the unique environment murine perinatal Tregs encounter within the lymph nodes (LNs) as they first emerge from the thymus, and how it evolves over the succeeding days, we employed single-cell RNA sequencing to generate an atlas of the early LN niche. A highly dynamic picture emerged, the stromal cell compartment showing the most striking changes and putative interactions with other LN cell compartments. In particular, LN stromal cells showed increasing potential for lymphocyte interactions with age. Analogous studies on mice lacking α:β T cells or enriched for autoreactive α:β T cells revealed an acute stromal cell response to α:β T cell dysfunction, largely reflecting dysregulation of Tregs. Punctual ablation of perinatal Tregs induced stromal cell activation that was dependent on both interferon-gamma signaling and activation of conventional CD4+ T cells. These findings elucidate some of the earliest cellular and molecular events in perinatal induction of T cell tolerance, providing a framework for future explorations. [ABSTRACT FROM AUTHOR]

Injuries to the retinal pigment epithelium (RPE) and outer retina often result in the accumulation of retinal microglia within the subretinal space. These subretinal microglia play crucial roles in inflammation and resolution, but the mechanisms governing their functions are still largely unknown. Our previous research highlighted the protective functions of choroidal γδ T cells in response to RPE injury. In the current study, we employed single-cell RNA sequencing approach to characterize the profiles of immune cells in mouse choroid. We found that γδ T cells were the primary producer of interleukin-17 (IL-17) in the choroid. IL-17 signaled through its receptor on the RPE, subsequently triggering the production of interleukin-6. This cascade of cytokines initiated a metabolic reprogramming of subretinal microglia, enhancing their capacity for lipid metabolism. RPE-specific knockout of IL-17 receptor A led to the dysfunction of subretinal microglia and RPE pathology. Collectively, our findings suggest that responding to RPE injury, the choroidal γδ T cells can initiate a protective signaling cascade that ensures the proper functioning of subretinal microglia. [ABSTRACT FROM AUTHOR]

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vß3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vß3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vß3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vß repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders. [ABSTRACT FROM AUTHOR]

This article explores the potential of the TP53 gene, specifically the Δ133p53α isoform, in enhancing CAR-T cell therapy for cancer. The Δ133p53α isoform has been found to increase the efficiency of tumor cell killing in laboratory and animal studies. It works by shutting down genes that inhibit cell growth and division, leading to increased cellular processes and energy production. While there are still questions about how it works and potential risks, this research shows promise for improving CAR-T cell therapy for chronic lymphocytic leukemia. [Extracted from the article]

Many autoimmune diseases are characterized by the activation of autoreactive T cells. The T cell repertoire is established in the thymus; it remains uncertain whether the presence of disease-associated autoreactive T cells reflects abnormal T cell selection in the thymus or aberrant T cell activation in the periphery. Here, we describe T cell selection, activation, and T cell repertoire diversity in female mice deficient for B lymphocyte-induced maturation protein (BLIMP)-1 in dendritic cells (DCs) (Prdm1 CKO). These mice exhibit a lupus-like phenotype with an expanded population of T follicular helper (Tfh) cells having a more diverse T cell receptor (TCR) repertoire than wild-type mice and, in turn, develop a lupus-like pathology. To understand the origin of the aberrant Tfh population, we analyzed the TCR repertoire of thymocytes and naive CD4 T cells from Prdm1 CKO mice. We show that early development and selection of T cells in the thymus are not affected. Importantly, however, we observed increased TCR signal strength and increased proliferation of naive T cells cultured in vitro with antigen and BLIMP1-deficient DCs compared to control DCs. Moreover, there was increased diversity in the TCR repertoire in naive CD4+ T cells stimulated in vitro with BLIMP1-deficient DCs. Collectively, our data indicate that lowering the threshold for peripheral T cell activation without altering thymic selection and naive T cell TCR repertoire leads to an expanded repertoire of antigen-activated T cells and impairs peripheral T cell tolerance. [ABSTRACT FROM AUTHOR]