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The activation of CD4+ T‐cells in a T cell receptor (TCR)‐dependent antigen‐specific manner is a central characteristic of the adaptive immune response. In addition to ensuring that CD4+ T‐cells recognise their cognate antigen during activation, TCR‐mediated signalling can also direct the outcome of differentiation. In both in vivo and in vitro model systems, strong TCR signalling has been demonstrated to drive Th1 differentiation, whereas weak TCR signalling drives Th2 responses. During the process of differentiation, TCR signal strength acts as a quantitative component in combination with the qualitative effects imparted by cytokines to polarise distinct T‐helper lineages. Here, we investigated the role of interleukin 2 (IL‐2) signalling in determining the outcome of TCR‐dependent differentiation. IL‐2 production was initiated as an early response to TCR‐induced activation and was regulated by the strength of TCR signalling initially received. In the absence of IL‐2, TCR dependent differentiation was found to be abolished. However, proliferative responses and early markers of activation were maintained, including the upregulation of GATA3, Tbet and Foxp3 at 24 h post‐stimulation. Demonstrating that IL‐2 signalling has a key role in stabilising and amplifying lineage‐specific transcirption factor expression during differentiation. Further, activation of IL‐2‐deficient T‐cells in the presence of exogenous cytokines was sufficient to restore differentiation whilst maintaining transcriptional signatures imparted during initial TCR signalling. Combined, our data demonstrate that the integration of quantitative TCR‐dependent signalling and qualitative IL‐2 signalling is essential for determining the fate of CD4+ T‐cells during differentiation. [ABSTRACT FROM AUTHOR]

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome. [ABSTRACT FROM AUTHOR]

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy. [ABSTRACT FROM AUTHOR]

Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 34 patients with HA20, we show that heterozygous TNFAIP3 loss skews immune repertoires toward lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas. This skewing was mediated by a feed-forward tumor necrosis factor (TNF)/A20/nuclear factor κB (NF-κB) loop that shaped pre-lymphoma transcriptome signatures in clonally expanded B (CD81, BACH2, and NEAT1) or T (GATA3, TOX, and PDCD1) cells. The skewing was reversed by anti-TNF treatment but could also progress to overt lymphoma. Analysis of conditional TNFAIP3 knock-out mice reproduced the wiring of the TNF/A20/NF-κB signaling axis with permissive antigen receptors and suggested a distinct regulation in B and T cells. Together, patients with the genetic disorder HA20 provide an exceptional window into A20/TNF/NF-κB-mediated control of immune homeostasis and early steps of lymphomagenesis that remain clinically unrecognized. [ABSTRACT FROM AUTHOR]

T cells express an enormous repertoire of T cell receptors, enabling them to recognize any potential antigen. This large repertoire undergoes stringent selections in the thymus, where receptors that react to self- or non-danger-associated-antigens are purged. We know that thymic tolerance depends on signals and antigens presented by the thymic antigen presenting cells, but we still do not understand precisely how many of these cells actually contribute to tolerance. This is especially true for thymic dendritic cells (DC), which are composed of diverse subpopulations that are derived from different progenitors. Although the importance of thymic DCs has long been known, the functions of specific DC subsets have been difficult to untangle. There remains insufficient systematic characterization of the ontogeny and phenotype of thymic APCs in general. As a result, validated experimental models for studying thymic DCs are limited. Recent technological advancement, such as multi-omics analyses, has enabled new insights into thymic DC biology. These recent findings indicate a need to re-evaluate the current tools used to study the function of these cells within the thymus. This review will discuss how thymic DC subpopulations can be defined, the models that have been used to assess functions in the thymus, and models developed for other settings that can be potentially used for studying thymic DCs. [ABSTRACT FROM AUTHOR]

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors. [ABSTRACT FROM AUTHOR]

The combination of radiotherapy/chemoradiotherapy and immune checkpoint blockade can result in poor outcomes in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Here, we show that combining ATR inhibition (ATRi) with radiotherapy (RT) increases the frequency of activated NKG2A+PD-1+ T cells in animal models of HNSCC. Compared with the ATRi/RT treatment regimen alone, the addition of simultaneous NKG2A and PD-L1 blockade to ATRi/RT, in the adjuvant, post-radiotherapy setting induces a robust antitumour response driven by higher infiltration and activation of cytotoxic T cells in the tumour microenvironment. The efficacy of this combination relies on CD40/CD40L costimulation and infiltration of activated, proliferating memory CD8+ and CD4+ T cells with persistent or new T cell receptor (TCR) signalling, respectively. We also observe increased richness in the TCR repertoire and emergence of numerous and large TCR clonotypes that cluster based on antigen specificity in response to NKG2A/PD-L1/ATRi/RT. Collectively, our data point towards potential combination approaches for the treatment of HNSCC. Immune checkpoint blockade can result in good outcomes in patients with metastatic head and neck squamous cell carcinoma (HNSCC), but not those with locally advanced disease. Here, the authors demonstrate that the addition of anti-NKG2A and anti-PD-L1 blockade to ATR inhibition and radiotherapy in the adjuvant setting can induce a robust antitumour immune response in mice. [ABSTRACT FROM AUTHOR]

IgG4-related disease (IgG4-RD) is a systemic immune-mediated fibroinflammatory disease whose pathomechanisms remain poorly understood. Here, we identified gene variants in familial IgG4-RD and determined their functional consequences. All 3 affected members of the family shared variants of the transcription factor IKAROS, encoded by IKZF1, and the E3 ubiquitin ligase UBR4. The IKAROS variant increased binding to the FYN promoter, resulting in higher transcription of FYN in T cells. The UBR4 variant prevented the lysosomal degradation of the phosphatase CD45. In the presence of elevated FYN, CD45 functioned as a positive regulatory loop, lowering the threshold for T cell activation. Consequently, T cells from the affected family members were hyperresponsive to stimulation. When transduced with a low-avidity, autoreactive T cell receptor, their T cells responded to the autoantigenic peptide. In parallel, high expression of FYN in T cells biased their differentiation toward Th2 polarization by stabilizing the transcription factor JunB. This bias was consistent with the frequent atopic manifestations in patients with IgG4- RD, including the affected family members in the present study. Building on the functional consequences of these 2 variants, we propose a disease model that is not only instructive for IgG4-RD but also for atopic diseases and autoimmune diseases associated with an IKZF1 risk haplotype. [ABSTRACT FROM AUTHOR]

Although neutralizing antibody is an established correlate of protection for measles, T cell-mediated responses play at least two critical roles in immunity to measles: first, through provision of 'help' enabling robust humoral immune responses; and second, through clearance of measles virus-infected cells. Previously, we identified 13 measles-derived peptides that bound to human leukocyte antigen (HLA) molecules in Priess cells infected with measles virus. In this study, we evaluated the immunogenicity of these peptides in a transgenic mouse model. Our results demonstrated that these peptides induced Th1-biased immune responses at varying levels. Of the 13 peptides, the top four immunogenic peptides were further selected for a viral challenge study in mice. A vaccine based on a combination of these four peptides reduced morbidity and weight loss after viral challenge compared to placebo. Our results emphasize the potential of T cell-mediated, peptide-based vaccines against measles. [ABSTRACT FROM AUTHOR]

Glycosphingolipids (GSLs) are abundantly expressed in cancer cells. The effects of GSL-targeted immunotherapies are not fully understood. Here, we show that the inhibition of GSL synthesis with the UDP-glucose ceramide glucosyltransferase inhibitor eliglustat can increase the exposure of the major histocompatibility complex (MHC) and tumour antigen peptides, enhancing the antitumour response of CD8+ T cells in a range of tumour models. We therefore conducted a proof-of-concept phase I trial on the combination of eliglustat and an anti-PD-1 antibody for the treatment of advanced cancers (NCT04944888). The primary endpoints were safety and feasibility, and the secondary endpoint was antitumor activity. All prespecified endpoints were met. Among the 31 enrolled patients, only 1 patient experienced a grade 3 adverse event (AE), and no grade 4 AEs were observed. The objective response rate was 22.6% and the disease control rate reached 71%. Of the 8 patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) colorectal cancer, one achieved complete response and two each had partial response and stable disease. In summary, inhibiting the synthesis of GSLs might represent an effective immunotherapy approach. Glycosphingolipids (GSLs) are abnormally expressed in cancer cells. Here, in addition to showing that inhibition of the synthesis of GSLs with eliglustat promotes anti-tumor immune responses in preclinical models, the authors report the results of a phase I trial of eliglustat and anti-PD-1 antibodies for the treatment of advanced cancers. [ABSTRACT FROM AUTHOR]

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5′ single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung–blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs). [ABSTRACT FROM AUTHOR]

Human orthogonal enzymes (HOEs) do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluate a new HOE from Escherichia coli, α-sulfoquinovosidase (α-SQase). We confirmed that the activity of α-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for α-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed a similar reactivity to the p-nitrophenol substrate, a good substrate for α-SQase. [ABSTRACT FROM AUTHOR]

Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells. Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types. Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions. Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism. [ABSTRACT FROM AUTHOR]

Although immune checkpoint blockade has shown initial success for various cancers, only a small subset of patients benefits from this therapy. Some chemotherapeutic drugs have been reported to induce antitumor T cell responses, prompting a number of clinical trials on combination chemoimmunotherapy. However, how to achieve potent immune activation with traditional chemotherapeutics in a manner that is safe, effective, and compatible with immunotherapy remains unclear. We show that high-density lipoprotein-mimicking nanodiscs loaded with doxorubicin (DOX), a widely used chemotherapeutic agent, can potentiate immune checkpoint blockade in murine tumor models. Delivery of DOX via nanodiscs triggered immunogenic cell death of cancer cells and exerted antitumor efficacy without any overt off-target side effects. "Priming" tumors with DOX-carrying nanodiscs elicited robust antitumor CD8+ T cell responses while broadening their epitope recognition to tumor-associated antigens, neoantigens, and intact whole tumor cells. Combination chemoimmunotherapy with nanodiscs plus anti-programmed death 1 therapy induced complete regression of established CT26 and MC38 colon carcinoma tumors in 80 to 88% of animals and protected survivors against tumor recurrence. Our work provides a new, generalizable framework for using nanoparticle-based chemotherapy to initiate antitumor immunity and sensitize tumors to immune checkpoint blockade. [ABSTRACT FROM AUTHOR]

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes. [ABSTRACT FROM AUTHOR]

The CBL (Casitas B-lineage lymphoma) family, as a class of ubiquitin ligases, can regulate signal transduction and activate receptor tyrosine kinases through various tyrosine kinase-dependent pathways. There are three members of the family: c-CBL, CBL-b, and CBL-c. Numerous studies have demonstrated the important role of CBL in various cellular pathways, particularly those involved in the occurrence and progression of cancer, hematopoietic development, and regulation of T cell receptors. Therefore, the purpose of this review is to comprehensively summarize the function and regulatory role of CBL family proteins in different human tumors, as well as the progress of drug research targeting CBL family, so as to provide a broader clinical measurement strategy for the treatment of tumors. [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]

Chimeric antigen receptor (CAR) serves as the foundational element of CAR-T cells. Exogenous CAR molecules can exert functional effects on allogeneic T cells, leading to their activation and subsequent functional alterations. Here we show a new method based on this biological principle: the transfer of CAR molecules from exogenous cells to the membrane of receptor T cells. This process facilitates receptor T cell to recognize target antigens and induces their activation. These patches imbued normal T cells with enhanced tumor targeting capabilities and activated their inherent killing functions. This method's efficacy introduces an approach for constructing non-genetically manipulated CAR-T cells and holds potential for application to other immune cells. [ABSTRACT FROM AUTHOR]

Neurological disorders, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), may be associated with alterations in blood cell composition and phenotype. Here, we focused our attention on circulating mucosal-associated invariant T (MAIT) cells, a CD8+ T cell memory population expressing the invariant Va7.2 region in the T cell receptor and high surface levels of the CD161 marker. Transcriptomics data relative to peripheral blood mononuclear cells (PBMC) highlighted downregulation of CD161 and other MAIT-associated markers in progressive MS and not relapsing remitting (RR)-MS when gene expressions relative to each disease course were compared to those from healthy controls. Multiparametric flow cytometry of freshly isolated PBMC samples from untreated RR-MS, primary or secondary progressive MS (PP-or SP-MS), ALS and age-and sex-matched healthy controls revealed specific loss of circulating CD8+ MAIT cells in PP-MS and no other MS courses or another neurological disorder such as ALS. Overall, these observations point to the existence of immunological changes in blood specific for the primary progressive course of MS that may support clinical definition of disease. [ABSTRACT FROM AUTHOR]

Mammalian dual-function guanine nucleotide exchange factor (GEF) VAV1 regulates T/B cell receptor signaling, T cell activation, T helper cell differentiation, cytokine production, actin polymerization, and cytoskeleton reorganization via GEF-dependent and -independent pathways. Recent CRISPR-Cas9 screening data and animal models (e.g., rodent) of autoimmune and chronic inflammatory disease confirm VAV1 as a positive T cell regulator and support its potential as a candidate therapeutic target in T and T/B cell-mediated diseases. To date, VAV1 has been considered 'undruggable'. Directly targeting GEF domains is challenging, and approaches to inhibit GEF activity, leaving scaffolding functions intact, may lead to inadequate attenuation of VAV1 activity. Preclinical studies exploring a new modality of attenuating the dual function of VAV1 through protein degradation show promise in animal models of some autoimmune/inflammatory diseases. Despite the availability of multiple advanced targeted biologics and oral small molecules to treat certain autoimmune and chronic inflammatory diseases, considerable unmet needs remain, prompting the search for new drug targets. Novel mechanisms of action that target the dual catalytic and scaffolding functions of the GEF VAV1 might optimally 'drug' VAV1 and, therefore, have broad therapeutic potential in T and T/B cell-mediated diseases. The guanine nucleotide exchange factor (GEF) VAV1, a previously 'undruggable' protein integral to T/B lymphocyte antigen–receptor signaling, promotes actin polymerization, immunological synapse formation, T cell activation and differentiation, and cytokine production. With the development of novel modalities for targeting proteins, we hypothesize that interventions targeting VAV1 will have therapeutic potential in T and T/B cell-mediated autoimmune and chronic inflammatory diseases. This opinion is supported by recent CRISPR-Cas9 studies showing VAV1 as a key positive regulator of T cell receptor (TCR) activation and cytokine production in primary human CD4+ and CD8+ T cells; data demonstrating that loss/suppression of VAV1 regulates autoimmunity and inflammation; and promising preclinical data from T and T/B cell-mediated disease models of arthritis and colitis showing the effectiveness of selective VAV1 targeting via protein degradation. [ABSTRACT FROM AUTHOR]

Inflammatory neuropathies, which include chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain Barré syndrome (GBS), result from autoimmune destruction of the PNS and are characterized by progressive weakness and sensory loss. CD4+ T cells play a key role in the autoimmune destruction of the PNS. Yet, key properties of pathogenic CD4+ T cells remain incompletely understood. Here, we used paired single-cell RNA-Seq (scRNA-Seq) and single-cell T cell receptor--sequencing (scTCR-Seq) of peripheral nerves from an inflammatory neuropathy mouse model to identify IL-21-- expressing CD4+ T cells that were clonally expanded and multifunctional. These IL-21--expressing CD4+ T cells consisted of 2 transcriptionally distinct expanded cell populations, which expressed genes associated with T follicular helper (Tfh) and T peripheral helper (Tph) cell subsets. Remarkably, TCR clonotypes were shared between these 2 IL-21--expressing cell populations, suggesting a common lineage differentiation pathway. Finally, we demonstrated that IL-21 receptor--KO (IL-21R--KO) mice were protected from neuropathy development and had decreased immune infiltration into peripheral nerves. IL-21 signaling upregulated CXCR6, a chemokine receptor that promotes CD4+ T cell localization in peripheral nerves. Together, these findings point to IL-21 signaling, Tfh/Tph differentiation, and CXCR6-mediated cellular localization as potential therapeutic targets in inflammatory neuropathies. [ABSTRACT FROM AUTHOR]

Summary: CD8 T cells are crucial adaptive immune cells with cytotoxicity to fight against pathogens or abnormal self-cells via major histocompatibility complex class I-dependent priming pathways. The composition of the memory CD8 T-cell pool is influenced by various factors. Physiological aging, chronic viral infection, and autoimmune diseases promote the accumulation of CD8 T cells with highly differentiated memory phenotypes. Accumulating studies have shown that some of these memory CD8 T cells also exhibit innate-like cytotoxicity and upregulate the expression of receptors associated with natural killer (NK) cells. Further analysis shows that these NK-like CD8 T cells have transcriptional profiles of both NK and CD8 T cells, suggesting the transformation of CD8 T cells into NK cells. However, the specific induction mechanism underlying NK-like transformation and the implications of this process for CD8 T cells are still unclear. This review aimed to deduce the possible differentiation model of NK-like CD8 T cells, summarize the functions of major NK-cell receptors expressed on these cells, and provide a new perspective for exploring the role of these CD8 T cells in health and disease. The differentiation model of NK-like CD8 T cells. The natural killer-associated receptors expressed on NK-like CD8 T cells. Summary of previous studies evaluating the role of NK-like CD8 T cells in diseases. Graphical Abstract [ABSTRACT FROM AUTHOR]

This article discusses the role of B cells in autoimmune kidney diseases and presents new B cell-targeting therapies used in their treatment. B cells are involved in autoimmune diseases as precursors of cells producing pathogenic autoantibodies and also play a role in presenting antigens to T lymphocytes and perpetrating inflammation. The article explores B cell-depleting therapies, such as anti-CD20 monoclonal antibodies, and B cell-modulating therapies that target molecules involved in B cell homeostasis. The efficacy of these therapies varies across diseases and patients, and further research is needed to determine their place in the management of autoimmune kidney diseases. [Extracted from the article]

ABSTRACT The advent of immunological therapies has revolutionized the treatment of solid and haematological cancers over the last decade. Licensed therapies which activate the immune system to target cancer cells can be broadly divided into two classes. The first class are antibodies that inhibit immune checkpoint signalling, known as immune checkpoint inhibitors (ICIs). The second class are cell‐based immune therapies including chimeric antigen receptor T lymphocyte (CAR‐T) cell therapies, natural killer (NK) cell therapies, and tumour infiltrating lymphocyte (TIL) therapies. The clinical efficacy of all these treatments generally outweighs the risks, but there is a high rate of immune‐related adverse events (irAEs), which are often unpredictable in timing with clinical sequalae ranging from mild (e.g. rash) to severe or even fatal (e.g. myocarditis, cytokine release syndrome) and reversible to permanent (e.g. endocrinopathies).The mechanisms underpinning irAE pathology vary across different irAE complications and syndromes, reflecting the broad clinical phenotypes observed and the variability of different individual immune responses, and are poorly understood overall. Immune‐related cardiovascular toxicities have emerged, and our understanding has evolved from focussing initially on rare but fatal ICI‐related myocarditis with cardiogenic shock to more common complications including less severe ICI‐related myocarditis, pericarditis, arrhythmias, including conduction system disease and heart block, non‐inflammatory heart failure, takotsubo syndrome and coronary artery disease. In this scientific statement on the cardiovascular toxicities of immune therapies for cancer, we summarize the pathophysiology, epidemiology, diagnosis, and management of ICI, CAR‐T, NK, and TIL therapies. We also highlight gaps in the literature and where future research should focus. [ABSTRACT FROM AUTHOR]

Background: Uremia-associated immunodeficiency, mainly characterized by T cell dysfunction, exists in patients on maintenance hemodialysis (MHD) and promotes systemic inflammation. However, T cell senescence, one of the causes of T cell dysfunction, has not been clearly revealed yet. In this cross-sectional research, we aimed to study the manifestation of T cell premature senescence in MHD patients and further investigate the associated clinical factors. Methods: 76 MHD patients including 33 patients with cardiovascular diseases (CVD) and 28 patients with arteriovenous fistula (AVF) event history were enrolled in this study. Complementarity determining region 3 (CDR3) of T cell receptor (TCR) was analyzed by immune repertoire sequencing (IR-Seq). CD28- T cell subsets and expression of senescence marker p16 and p21 genes were detected by multicolor flow cytometry and RT-qPCR, respectively. Results: MHD patients had significantly decreased TCR diversity (P < 0.001), increased CDR3 clone proliferation (P = 0.001) and a left-skewed CDR3 length distribution. The proportion of CD4 + CD28- T cells increased in MHD patients (P = 0.014) and showed a negative correlation with TCR diversity (P = 0.001). p16 but not p21 expression in T cells was up-regulated in MHD patients (P = 0.039). Patients with CVD exhibited increased expression of p16 and p21 genes (P = 0.010 and 0.004, respectively), and patients with AVF events showed further TCR diversity and evenness reduction (P = 0.002 and 0.017, respectively) compared to patients without the comorbidities. Moreover, age, average convection volume, total cholesterol, high-density lipoprotein cholesterol and transferrin saturation were associated with TCR diversity or CD4 + CD28- T cell proportion (P < 0.05). Conclusions: MHD patients undergo T cell premature senescence characterized by significant TCR diversity reduction and repertoire skew, as well as accumulation of the CD4 + CD28- subset and up-regulation of p16 gene. Patients with CVD or AVF events show higher level of immunosenescence. Furthermore, T cell senescence in MHD patients is associated with blood cholesterol and uremic toxin retention, suggesting potential intervention strategies in the future. [ABSTRACT FROM AUTHOR]

Deficits in T cell immunity translate into increased risk of severe viral infection in recipients of solid organ and hematopoietic cell transplants. Thus, therapeutic strategies that employ the adoptive transfer of virus-specific T cells are being clinically investigated to treat and prevent viral diseases in these highly immunocompromised patients. Posoleucel is an off-the-shelf multivirus-specific T cell investigational product for the treatment and prevention of infections due to adenovirus, BK virus, cytomegalovirus, Epstein–Barr virus, human herpesvirus 6 or JC virus. Herein we perform extensive characterization of the phenotype and functional profile of posoleucel to illustrate the cellular properties that may contribute to its in vivo activity. Our results demonstrate that posoleucel is enriched for central and effector memory CD4+ and CD8+ T cells with specificity for posoleucel target viruses and expressing a broad repertoire of T cell receptors. Antigen-driven upregulation of cell-surface molecules and production of cytokine and effector molecules indicative of proliferation, co-stimulation, and cytolytic potential demonstrate the specificity of posoleucel and its potential to mount a broad, polyfunctional, and effective Th1-polarized antiviral response upon viral exposure. We also show the low risk for off-target and nonspecific effects as evidenced by the enrichment of posoleucel in memory T cells, low frequency of naive T cells, and lack of demonstrated alloreactivity in vitro. The efficacy of posoleucel is being explored in four placebo-controlled clinical trials in transplant recipients to treat and prevent viral infections (NCT05179057, NCT05305040, NCT04390113, NCT04605484). [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) multigene family encodes key pathogen‐recognition molecules of the vertebrate adaptive immune system. Hyper‐polymorphism of MHC genes is de novo generated by point mutations, but new haplotypes may also arise by re‐shuffling of existing variation through intra‐ and inter‐locus gene conversion. Although the occurrence of gene conversion at the MHC has been known for decades, we still have limited understanding of its functional importance. Here, I took advantage of extensive genetic resources (~9000 sequences) to investigate broad scale macroevolutionary patterns in gene conversion processes at the MHC across nearly 200 avian species. Gene conversion was found to constitute a universal mechanism in birds, as 83% of species showed footprints of gene conversion at either MHC class and 25% of all allelic variants were attributed to gene conversion. Gene conversion processes were stronger at MHC‐II than MHC‐I, but inter‐specific variation at both MHC classes was explained by similar evolutionary scenarios, reflecting fluctuating selection towards different optima and drift. Gene conversion showed uneven phylogenetic distribution across birds and was driven by gene copy number variation, supporting significant role of inter‐locus gene conversion processes in the evolution of the avian MHC. Finally, MHC gene conversion was stronger in species with fast life histories (high fecundity) and in long‐distance migrants, likely reflecting variation in population sizes and host–pathogen coevolutionary dynamics. The results provide a robust comparative framework for understanding macroevolutionary variation in gene conversion at the avian MHC and reinforce important contribution of this mechanism to functional MHC diversity. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), one of the malignancies with a wide expression of stress ligands recognized by Vδ1γδ T cells, has received much attention in adoptive immunotherapy of γδ T cells. In this study, we aimed to identify the potential anti-tumor Vδ1γδ T subpopulations in HCC. Healthy donors (HDs) and HCC patients were recruited from the Affiliated Cancer Hospital of Zhengzhou University. Blood and tumor tissue samples were obtained respectively. Bioinformatics methods were used to analyze total γδ T cells and subsets infiltration, overall survival of HCC patients with high and low infiltration level of Vδ1γδ T cells, and IFNG , granzyme A, granzyme B and perforin expression in TRDV1 high/low CD69 high/low groups. CD69 expression and Vδ1γδT cells infiltration in HCC were detected by immunofluorescence. Phenotypic analysis of Vδ1γδ T cells in blood and tumor tissue samples were performed by flow cytometry. Vδ1γδ T cells infiltrating in HCC were associated with better clinical outcome. Study in tumor micro-environment (TME) of HCC demonstrated that not total Vδ1γδ T but CD69+ Vδ1γδ subset infiltration was associated with smaller tumor volume. Moreover, HCC patients simultaneously with high TRDV1 and CD69 expression produced more effector molecules and had longer survival time. Since Vδ1γδ T cells in the tumor microenvironment were often difficult to access, we demonstrated that CD69+ Vδ1γδ T cells also existed in peripheral blood mononuclear cells (PBMC) of HCC and displayed enhanced cytotoxic potentials than HDs. Finally, we investigated the functions and found that CD69+ Vδ1γδ T cells exhibited stronger tumor reactivities when challenged by tumor cells. CD69+ Vδ1γδ T cells are functional Vδ1γδ T cell subsets in patients with HCC. Circulating CD69+ Vδ1γδ T cell is a promising candidate in immunotherapy of HCC. [Display omitted] • Vδ1γδ T associated with better clinical outcome of HCC. Higher CD69+ Vδ1γδ subset indicated smaller tumor. • CD69+ Vδ1γδ Tfrom PBMCs displayed enhanced cytotoxicity. [ABSTRACT FROM AUTHOR]

Sepsis is a common complication among patients in intensive care units, and has a high mortality rate, with no effective therapies to date. As immunosuppression has become the research focus of sepsis, the regulatory role of dendritic cells (DCs) in the immune response to sepsis has received attention. To investigate the role of the Wnt/β-catenin signaling pathway in inducing the differentiation of splenic DCs in mice with sepsis caused by cecal ligation and puncture (CLP). C57bl/6 mice were randomly divided into three groups, namely the sham, 24 h post-CLP, and 72 h post-CLP groups. Levels of regulatory T cells (Tregs) among splenic mononuclear cells, suppressor T cells (TSs), and surface markers, such as major histocompatibility complex class II (MHC-II), co-stimulatory molecules (CD80 and CD86), negative co-stimulatory molecule death-ligand 1 (PD-L1), CC chemokine receptor-5 (CCR5), and CC chemokine receptor-7 (CCR7), were analyzed via flow cytometry for each group of mice post-surgery. CD11c+ DCs were purified from the splenic mononuclear cells of each group, and the expression of β-catenin, Wnt5a, and Wnt3a was detected using RT-PCR and western blotting.Each group of DCs was incubated with LPS-containing culture solution, and the supernatant of the culture solution was collected after 24 hours to detect the level of Tumor necrosis factor-α(TNF-α), interleukin (IL)-6, IL-12, and IL-10. Compared with that in the sham group, the expression of β-catenin, Wnt5a, and Wnt3a in splenic DCs of the other two groups of mice increased with prolonged CLP exposure (P<0.05). Meanwhile, the proportion of Tregs and TSs increased in the mouse spleens after CLP, and levels of DC surface molecules, such as CCR5, CCR7, CD80, CD86, and MHC-II, decreased to different degrees, whereas those of PD-L1 increased. These results suggested that DCs differentiate towards regulatory DCs (regDCs) after CLP in mice. The results of ELISA showed that the longer the exposure time after CLP, the lower the ability of DCs to secrete TNF-α and IL-12, but the higher the level of IL-10 and IL-6. The Wnt/β-catenin signaling pathway activates and induces regDCs differentiation in the splenic DCs of mice with sepsis and participates in the regulation of immune tolerance in the organism. • Immunosuppression in sepsis. • DCs differentiated into regDCs in sepsis. • The differentiation of DCs in sepsis promotes the differentiation of Tregs and inhibitory T cells. • Activation of Wnt/β-catenin signal in sepsis induces differentiated regDCs. [ABSTRACT FROM AUTHOR]

Our understanding of the immune response is far from complete, missing out on more detailed explanations that could be provided by molecular insights. To bridge this gap, we introduce the quantum model of T‐cell activation. This model suggests that the transfer of energy during protein phosphorylation within T cells is not a continuous flow but occurs in discrete bursts, or 'quanta', of phosphates. This quantized energy transfer is mediated by oscillating cycles of receptor phosphorylation and dephosphorylation, initiated by dynamic 'catch‐slip' pulses in the peptide‐major histocompatibility complex‐T‐cell receptor (pMHC‐TcR) interactions. T‐cell activation is predicated upon achieving a critical threshold of catch‐slip pulses at the pMHC‐TcR interface. Costimulation is relegated to a secondary role, becoming crucial only when the frequency of pMHC‐TcR catch‐slip pulses does not meet the necessary threshold for this quanta‐based energy transfer. Therefore, our model posits that it is the quantum nature of energy transfer—not the traditional signal I or signal II—that plays the decisive role in T‐cell activation. This paradigm shift highlights the importance of understanding T‐cell activation through a quantum lens, offering a potentially transformative perspective on immune response regulation. [ABSTRACT FROM AUTHOR]

The size and condition of the peripheral CD4 T cell population determine the capacity of the immune response. Under homeostatic conditions, the size of the peripheral CD4 T cell population is maintained through turnover and survival. However, the underlying mechanisms remain inadequately understood. Here, we observed a significant decrease in the percentage of CD4 T cells in the periphery following the targeted deletion of the Paxbp1 gene in mouse T cells. In the absence of Paxbp1, naïve CD4 T cells displayed reduced surface interleukin‐7 receptor levels and a decreased capacity to respond to survival signals mediated by interleukin‐7. In addition, naïve CD4 T cells deficient in Paxbp1 demonstrated impaired T cell antigen receptor signalling, compromised cell cycle entry, decreased proliferation, and increased apoptosis following stimulation, all of which contributed to the reduction in the number of peripheral CD4 T cells. Therefore, our study highlights the indispensable role of Paxbp1 in maintaining peripheral CD4 T cell homeostasis. [ABSTRACT FROM AUTHOR]

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy. [ABSTRACT FROM AUTHOR]

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as T cell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic T cells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha‐beta (αβ) T cells and unconventional gamma delta (γδ) T cells. TCEs were built using camelid single‐domain antibodies (VHHs) targeting the tumor‐associated antigen CEACAM5 (CEA), together with T cell receptor chains or a CD3 domain. We show that Vγ9Vδ2 T cells display stronger in vitro antitumor activity than αβ T cells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral T cells to Vγ9Vδ2 T cells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ T cell‐specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies. [ABSTRACT FROM AUTHOR]

Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcgRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcgR fusion CD64/16A consisting of the extracellular region of CD64 (FcgRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Ra fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNKCD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants. [ABSTRACT FROM AUTHOR]

T cell receptors (TCRs) play crucial roles in regulating T cell response by rapidly and accurately recognizing foreign and non-self antigens. The process involves multiple molecules and regulatory mechanisms, forming a complex network to achieve effective antigen recognition. Mathematical modeling techniques can help unravel the intricate network of TCR signaling and identify key regulators that govern it. In this review, we introduce and briefly discuss relevant mathematical models of TCR initial triggering, with a focus on kinetic proofreading (KPR) models with different modified structures. We compare the topology structures, biological hypotheses, parameter choices, and simulation performance of each model, and summarize the advantages and limitations of them. Further studies on TCR modeling design, aiming for an optimized balance of specificity and sensitivity, are expected to contribute to the development of new therapeutic strategies. [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]

This letter, published in the Journal of Translational Medicine, discusses the role of Toll-like receptor 7 (TLR7) in cancer and its impact on immune activation and prognostic evaluation. The authors highlight conflicting reports on TLR7's involvement in tumor development, with some studies suggesting that TLR7 overexpression promotes tumor growth and drug resistance, while others demonstrate that TLR7 agonists inhibit various types of cancer through immune activation. The authors also discuss the expression and prognostic significance of TLR7 in different cancers, as well as its correlation with immune cell distribution and response to immune checkpoint inhibitors. Overall, TLR7 is identified as a promising molecular biomarker in immune therapy. [Extracted from the article]

Lung cancer is the leading cause of cancer‐related deaths, in part due to its late diagnosis. Increased epidermal growth factor receptor (EGFR) expression in cancer cells is associated with a poor prognosis, and EGFR tyrosine kinase inhibitors are widely used in cancer treatment. This study aimed to clarify the relationship between EGFR expression on T cells and cancer prognosis in patients with non‐small cell lung cancer (NSCLC). Forty patients with NSCLC and 40 healthy volunteers were included in this study. Peripheral CD4+T helper (Th1, Th2, Th9, Th17, Th1Th17, follicular and peripheral Th) and cytotoxic T lymphocyte (CD8+follicular and peripheral T) subsets were identified with flow cytometry according to their chemokine receptors. EGFR expression on T lymphocytes in relation to overall survival (OS) was investigated in patients with NSCLC. The patients [mean age (min‐max) = 64.03 (45–83); 20 stage I–III and 20 stage IV] had increased EGFR expression on CD3+T, CD4+Th, Th1, Th2, and Th17 cells compared to the controls (p < 0.05). High EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells was associated with poor OS. Also, PD‐1 expression on lymphocytes, CD3+T, and Th cells was increased in patients with NSCLC compared to controls. The high expression of EGFR and PD‐1 on Th cells and the reduced percentage of lymphocytes and Th cells, especially in stage IV patients with NSCLC, revealed that increased EGFR activity may trigger apoptosis of Th cells and promote the development of metastases, while high EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells may be an independent poor prognostic marker in NSCLC. [ABSTRACT FROM AUTHOR]

Bacterial synthesis of vitamin B2 generates a by‐product, 5‐(2‐oxopropylideneamino)‐d‐ribityl‐aminouracil (5‐OP‐RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water‐stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17 nM), and their potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine‐alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine‐ and serine‐lined cleft in MR1 via specific pi‐interactions and H‐bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs. [ABSTRACT FROM AUTHOR]

Background: The immunogenicity of the antigen-recognition domains of chimeric antigen receptor (CAR)-T cells leads to immune responses that may compromise the antitumor effects of the adoptively transferred T cells. Herein, we attempt to humanize a CD19-specific VHH (named H85) using in silico techniques and investigate the impact of antigen-recognition domain humanization on CAR expression and density, cytokine secretion, and cytolytic reactivity of CAR-T cells based on the humanized VHH. Methods: H85 was humanized (named HuH85), and then HuH85 was compared with H85 in terms of conformational structure, physicochemical properties, antigenicity and immunogenicity, solubility, flexibility, stability, and CD19-binding capacity using in silico techniques. Next, H85CAR-T cells and HuH85CAR-T cells were developed and CAR expression and surface density were assessed via flow cytometry. Ultimately, the antitumor reactivity and secreted levels of IFN-γ, IL-2, and TNF-α were assessed following the co-cultivation of the CAR-T cells with Ramos, Namalwa, and K562 cells. Results: In silico findings demonstrated no negative impacts on HuH85 as a result of humanization. Ultimately, H85CAR and HuH85CAR could be surface-expressed on transduced T cells at comparable levels as assessed via mean fluorescence intensity. Moreover, H85CAR-T cells and HuH85CAR-T cells mediated comparable antitumor effects against Ramos and Namalwa cells and secreted comparable levels of IFN-γ, IL-2, and TNF-α following co-cultivation. Conclusion: HuH85 can be used to develop immunotherapeutics against CD19-associated hematologic malignancies. Moreover, HuH85CAR-T cells must be further investigated in vitro and in preclinical xenograft models of CD19+ leukemias and lymphomas before advancing into clinical trials. [ABSTRACT FROM AUTHOR]

Treating Multiple sclerosis (MS), a well-known immune-mediated disease characterized by axonal demyelination, is challenging due to its complex causes. Naphthalenedione, present in numerous plants, is being explored as a potential medicine for MS due to its immunomodulatory properties. However, its effects on lymphocytes can vary depending on factors such as the specific compound, concentration, and experimental conditions. In this study, we aim to explore the therapeutic potential of 2-bromo-1,4-naphthalenedione (BrQ), a derivative of naphthalenedione, in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and to elucidate its underlying mechanisms. We observed that mice treated with BrQ exhibited reduced severity of EAE symptoms, including lower clinical scores, decreased leukocyte infiltration, and less extensive demyelination in central nervous system. Furthermore, it was noted that BrQ does not directly affect the remyelination process. Through cell-chat analysis based on bulk RNA-seq data, coupled with validation of flow analysis, we discovered that BrQ significantly promotes the expansion of CD8+ T cells and their interactions with other immune cells in peripheral immune system in EAE mice. Subsequent CD8+ T cell depletion experiments confirmed that BrQ alleviates EAE in a CD8+ T cell-dependent manner. Mechanistically, expanded CD8+ cells were found to selectively reduce antigen-specific CD4+ cells and subsequently inhibit Th1 and Th17 cell development in vivo, ultimately leading to relief from EAE. In summary, our findings highlight the crucial role of BrQ in modulating the pathogenesis of MS, suggesting its potential as a novel drug candidate for treating MS and other autoimmune diseases. [ABSTRACT FROM AUTHOR]

Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) is a valuable experimental tool to study the immune state in health and following immune challenges such as infectious diseases, (auto)immune diseases, and cancer. Several tools have been developed to reconstruct B cell and T cell receptor sequences from AIRR-seq data and infer B and T cell clonal relationships. However, currently available tools offer limited parallelization across samples, scalability or portability to high-performance computing infrastructures. To address this need, we developed nf-core/airrflow, an end-to-end bulk and single-cell AIRR-seq processing workflow which integrates the Immcantation Framework following BCR and TCR sequencing data analysis best practices. The Immcantation Framework is a comprehensive toolset, which allows the processing of bulk and single-cell AIRR-seq data from raw read processing to clonal inference. nf-core/airrflow is written in Nextflow and is part of the nf-core project, which collects community contributed and curated Nextflow workflows for a wide variety of analysis tasks. We assessed the performance of nf-core/airrflow on simulated sequencing data with sequencing errors and show example results with real datasets. To demonstrate the applicability of nf-core/airrflow to the high-throughput processing of large AIRR-seq datasets, we validated and extended previously reported findings of convergent antibody responses to SARS-CoV-2 by analyzing 97 COVID-19 infected individuals and 99 healthy controls, including a mixture of bulk and single-cell sequencing datasets. Using this dataset, we extended the convergence findings to 20 additional subjects, highlighting the applicability of nf-core/airrflow to validate findings in small in-house cohorts with reanalysis of large publicly available AIRR datasets. Author summary: We have created nf-core/airrflow, a workflow to help researchers study the immune system in healthy and disease states, such as infections, autoimmunity, and cancer. The adaptive immune system is responsible for the third line of defense responses, specific to each particular threat, after physical barriers have been compromised and the nonspecific innate immune response has failed to clear the danger. Two types of white blood cells are central players in the adaptive response, namely B cells and T cells. These cells have surface receptors that recognize suspicious elements (antigens). Learning what receptors bind to which antigens is of utmost interest to understand immune responses. Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) is a technique that allows to determine the genetic sequence of these receptors. The amount of data generated in these experiments is large, and the analysis complex. nf-core/airrflow simplifies running a comprehensive analysis connecting tools from Immcantation, a specialized software project to analyze AIRR-seq data. The workflow can efficiently process large datasets on multiple computing platforms. We analyzed immune responses to COVID-19 in 97 infected individuals and 99 healthy people, and confirmed previous findings and provided new insights, demonstrating the workflow's applicability to reanalyzing large publicly available datasets. [ABSTRACT FROM AUTHOR]

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack. [ABSTRACT FROM AUTHOR]

This article explores the role of microglia, a type of immune cell in the central nervous system, in viral-induced neuroinflammation. It discusses the different types of viruses that can cause central nervous system infections and how they infiltrate the CNS. The article highlights the responses of glial cells, particularly astrocytes and microglia, to viral infections and suggests that microglia play a crucial role in regulating the immune response and neuroinflammation associated with viral infections. It also mentions the potential implications of viral infections, such as COVID-19, on the function and activation of microglia and astrocytes. The document includes a list of references that can be valuable resources for researchers and library patrons interested in studying these topics. [Extracted from the article]

Antigen-presenting cells (APCs) play a crucial role in the immune system by breaking down antigens into peptide fragments that subsequently bind to major histocompatibility complex (MHC) molecules. Previous studies indicate that stable proteins can impede CD4+ T cell stimulation by hindering antigen processing and presentation. Conversely, certain proteins require stabilization in order to activate the immune response. Several factors, including the characteristics of the protein and the utilization of different adjuvants in animal experiments, may contribute to this disparity. In this study, we investigated the impact of adjuvants on antigen administration in mice, specifically focusing on the stability of the CH2 domain. Consequently, the CH2 domain induced a stronger IgG response in comparison to the stabilized one when using Alum and PBS (without adjuvant). On the other hand, animal experiment using Freund's adjuvant showed the opposite results. These findings indicate the significance of considering the intrinsic conformational stability of a protein when eliciting its immunogenicity, particularly within the context of vaccine development. [ABSTRACT FROM AUTHOR]

The utilization of anti‐CD3/CD28 magnetic beads for T cell expansion in vitro has been investigated for adoptive cell transfer therapy. However, the impact of the CD3/CD28 antibody ratio on T cell differentiation and function remains incompletely elucidated. This study seeks to address this knowledge gap. To begin with, CD3 antibodies with a relatively low avidity for Jurkat cells (Kd = 13.55 nM) and CD28 antibodies with a relatively high avidity (Kd = 5.79 nM) were prepared. Afterwards, anti‐CD3/CD28 antibodies with different mass ratios were attached to magnetic beads to examine the impacts of different antibody ratios on T cell capture, and proliferation. The research demonstrated that the most significant expansion of T cells was stimulated by the anti‐CD3/CD28 magnetic beads with a mass ratio of 2:1 for CD3 antibodies and CD28 antibodies. Moreover, CD25 and PD1 expression of expanded T cells increased and then decreased, with lower CD25 and PD1 expression in the later stages of expansion indicating that T cells were not depleted. These T cells, which are massively expanded in vitro and have excellent expansion potential, can be infused back into the patient to treat tumor patients. This study shows that altering the ratio of anti‐CD3/CD28 antibodies can control the strength of T cell stimulation, thereby leading to the improvement of T cell activation. This discovery can be utilized as a guide for the creation of other T cell stimulation approaches, which is beneficial for the further development of tumor immunotherapy technology. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor. Editor's summary: T cells that express chimeric antigen receptors (CARs) are therapeutically effective against only some types of cancers. Noting that downstream signaling molecules are recruited inefficiently to current CARs, Balagopalan et al. instead incorporated domains from these signaling proteins into the CAR itself. Inclusion of the kinase domain of ZAP70 to generate CAR tyrosine kinases resulted in cells with enhanced antitumor efficacy in mice compared with that of current CAR T cells. Thus, the signaling properties of CAR tyrosine kinases could be explored to enhance antitumor T cell efficacy. —John F. Foley [ABSTRACT FROM AUTHOR]

The balance of proinflammatory T helper type 17 (TH17) and anti-inflammatory T regulatory (Treg) cells is crucial for immune homeostasis in health and disease. The differentiation of naïve CD4+ T cells into TH17 and Treg cells depends on T cell receptor (TCR) signaling mediated, in part, by interleukin-2–inducible T cell kinase (ITK), which stimulates mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. Here, we report that, in the absence of ITK activity, naïve murine CD4+ T cells cultured under TH17-inducing conditions expressed the Treg transcription factor Foxp3 and did not develop into TH17 cells. Furthermore, ITK inhibition in vivo during allergic inflammation increased the Treg:TH17 ratio in the lung. These switched Foxp3+ Treg-like cells had suppressive function, and their transcriptomic profile resembled that of differentiated, induced Treg (iTreg) cells, but their chromatin accessibility profiles were intermediate between TH17 and iTreg cells. Like iTreg cells, switched Foxp3+ Treg-like cells had reductions in the expression of genes involved in mitochondrial oxidative phosphorylation and glycolysis, in the activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and in the abundance of the TH17 pioneer transcription factor BATF. This ITK-dependent switch between TH17 and Treg cells depended on Ca2+ signaling but not on MAPKs. These findings suggest potential strategies for fine-tuning TCR signal strength through ITK to control the balance of TH17 and Treg cells. Editor's summary: The strength of T cell receptor (TCR) signaling determines whether naïve T cells differentiate into proinflammatory TH17 cells or anti-inflammatory Treg cells. Anannya et al. investigated whether the kinase ITK, which is activated by TCR engagement, influenced these cell fates in mice. ITK inhibition increased the Treg:TH17 ratio in the lungs during allergic inflammation. Furthermore, ITK inhibition of naïve T cells cultured under TH17-inducing conditions differentiated into cells that resembled Treg cells functionally and transcriptionally. This switch in cell fate depended on the capacity of ITK to induce Ca2+ signaling. Thus, an ITK-dependent, Ca2+-mediated switch fine-tunes the balance of TH17 and Treg cells. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]

Lymphedema occurs as a result of lymphatic vessel damage or obstruction, leading to the lymphatic fluid stasis, which triggers inflammation, tissue fibrosis, and adipose tissue deposition with adipocyte hypertrophy. The treatment of lymphedema is divided into conservative and surgical approaches. Among surgical treatments, methods like lymphaticovenular anastomosis and vascularized lymph node transfer are gaining attention as they focus on restoring lymphatic flow, constituting a physiologic treatment approach. Lymphatic endothelial cells form the structure of lymphatic vessels. These cells possess button-like junctions that facilitate the influx of fluid and leukocytes. Approximately 10% of interstitial fluid is connected to venous return through lymphatic capillaries. Damage to lymphatic vessels leads to lymphatic fluid stasis, resulting in the clinical condition of lymphedema through three mechanisms: Inflammation involving CD4+ T cells as the principal contributing factor, along with the effects of immune cells on the VEGF-C/VEGFR axis, consequently resulting in abnormal lymphangiogenesis; adipocyte hypertrophy and adipose tissue deposition regulated by the interaction of CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor-γ; and tissue fibrosis initiated by the overactivity of Th2 cells, leading to the secretion of profibrotic cytokines such as IL-4, IL-13, and the growth factor TGF-β1. Surgical treatments aimed at reconstructing the lymphatic system help facilitate lymphatic fluid drainage, but their effectiveness in treating already damaged lymphatic vessels is limited. Therefore, reviewing the pathophysiology and molecular mechanisms of lymphedema is crucial to complement surgical treatments and explore novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Antigen-specific cytotoxic CD8 T cells are extremely effective in controlling tumor growth and have been the focus of immunotherapy approaches. We leverage in silico tools to investigate whether the occurrence of mutations in proteins previously described as immunogenic and highly expressed by glioblastoma multiforme (GBM), such as Epidermal Growth Factor Receptor (EGFR), Isocitrate Dehydrogenase 1 (IDH1), Phosphatase and Tensin homolog (PTEN) and Tumor Protein 53 (TP53), may be contributing to the differential presentation of immunogenic epitopes. We recovered Class I MHC binding information from wild-type and mutated proteins using the Immune Epitope Database (IEDB). After that, we built peptide-MHC (pMHC-I) models in HLA-arena, followed by hierarchical clustering analysis based on electrostatic surface features from each complex. We identified point mutations that are determinants for the presentation of a set of peptides from TP53 protein. We point to structural features in the pMHC-I complexes of wild-type and mutated peptides, which may play a role in the recognition of CD8 T cells. To further explore these features, we performed 100 ns molecular dynamics simulations for the peptide pairs (wt/mut) selected. In pursuit of novel therapeutic targets for GBM treatment, we selected peptides where our predictive results indicated that mutations would not disrupt epitope presentation, thereby maintaining a specific CD8 T cell immune response. These peptides hold potential for future GBM interventions, including peptide-based or mRNA vaccine development applications. [ABSTRACT FROM AUTHOR]