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Memory B cells (MBCs) differentiate into plasma cells (PCs) or germinal centers (GCs) upon antigen recall. How this decision is programmed is not understood. We found that the relative strength between two antagonistic transcription factors, B lymphocyte-induced maturation protein 1 (BLIMP1) and BTB domain and CNC homolog 2 (BACH2), progressively increases in favor of BLIMP1 in antigen-responding B cells through the course of primary responses. MBC subsets that preferentially produce secondary GCs expressed comparatively higher BACH2 but lower BLIMP1 than those predisposed for PC development. Skewing the BLIMP1-BACH2 balance in otherwise fate-predisposed MBC subsets could switch their fate preferences. Underlying the changing BLIMP1-over-BACH2 balance, we observed progressively increased accessibilities at chromatin loci that are specifically opened in PCs, particularly those that contain interferon-sensitive response elements (ISREs) and are controlled by interferon regulatory factor 4 (IRF4). IRF4 is upregulated by B cell receptor, CD40 or innate receptor signaling and it induces graded levels of PC-specifying epigenetic imprints according to the strength of stimulation. By analyzing history-stamped GC B cells, we found progressively increased chromatin accessibilities at PC-specific, IRF4-controlled gene loci over time. Therefore, the cumulative stimulation history of B cells is epigenetically recorded in an IRF4-dependent manner, determines the relative strength between BLIMP1 and BACH2 in individual MBCs and dictates their probabilities to develop into GCs or PCs upon restimulation., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation., (© 2024. The Author(s).)

Background: Trichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2 , a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown., Objective: The aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1., Methods: Cellular immune phenotype was investigated using multicolor flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients., Results: We investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harbored novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of γ-H2AX levels, decreased proliferation activity and reduced cell viability following UV-irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo ., Conclusion: In summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired BCR-mediated B-cell activation and activation-induced gene transcription., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rossmanith, Sauerwein, Geier, Leiss-Piller, Stemberger, Sharapova, Gruber, Bergler, Verbsky, Csomos, Walter and Wolf.)

The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation, and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers., (© 2024. The Author(s).)

RHOH, an atypical small GTPase predominantly expressed in hematopoietic cells, plays a vital role in immune function. A deficiency in RHOH has been linked to epidermodysplasia verruciformis, lung disease, Burkitt lymphoma and T cell defects. Here, we report a novel germline homozygous RHOH c.245G > A (p.Cys82Tyr) variant in a 21-year-old male suffering from recurrent, invasive, opportunistic infections affecting the lungs, eyes, and brain. His sister also succumbed to a lung infection during early adulthood. The patient exhibited a persistent decrease in CD4 + T, B, and NK cell counts, and hypoimmunoglobulinemia. The patient's T cell showed impaired activation upon in vitro TCR stimulation. In Jurkat T cells transduced with RHOH C82Y , a similar reduction in activation marker CD69 up-regulation was observed. Furthermore, the C82Y variant showed reduced RHOH protein expression and impaired interaction with the TCR signaling molecule ZAP70. Together, these data suggest that the newly identified autosomal-recessive RHOH variant is associated with T cell dysfunction and recurrent opportunistic infections, functioning as a hypomorph by disrupting ZAP70-mediated TCR signaling., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8 + single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4 + recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling., Competing Interests: AB is a cofounder of Datirium, the developer of Scientific Data Analysis Platform https://SciDAP.com used here for processing RNA-Seq data. AB and MK developed CWL-Airflow licensed to Datirium, LLC. AL is an employee of Amgen Inc. HB is a consultant for Mahzi therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Potter, Zhang, Kotliar, Wu, Schafer, Stefan, Boukas, Qu’d, Bodamer, Simpson, Barski, Lindsley and Bjornsson.)

Vγ9Vδ2 T cells are specialized effector cells that have gained prominence as immunotherapy agents due to their ability to target and kill cells with altered pyrophosphate metabolites. In our effort to understand how cancer cells evade the cell-killing activity of Vγ9Vδ2 T cells, we performed a comprehensive genome-scale CRISPR screening of cancer cells. We found that four molecules belonging to the butyrophilin (BTN) family, specifically BTN2A1, BTN3A1, BTN3A2, and BTN3A3, are critically important and play unique, nonoverlapping roles in facilitating the destruction of cancer cells by primary Vγ9Vδ2 T cells. The coordinated function of these BTN molecules was driven by synchronized gene expression, which was regulated by IFN-γ signaling and the RFX complex. Additionally, an enzyme called QPCTL was shown to play a key role in modifying the N-terminal glutamine of these BTN proteins and was found to be a crucial factor in Vγ9Vδ2 T cell killing of cancer cells. Through our research, we offer a detailed overview of the functional genomic mechanisms that underlie how cancer cells escape Vγ9Vδ2 T cells. Moreover, our findings shed light on the importance of the harmonized expression and function of gene family members in modulating T-cell activity., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for the survival of cells that encounter DNA damage during replication. The Rad6/18 pathway is instrumental in this process, mediating damage bypass by ubiquitination of proliferating cell nuclear antigen. Previous studies have shown different roles of Rad18 in vivo and in tumorigenesis. Here, we show that B cells induce Rad18 expression upon proliferation induction. We have therefore analysed the role of Rad18 in B cell activation as well as in B cell lymphomagenesis mediated by an Eµ-Myc transgene. We find no activation defects or survival differences between Rad18 WT mice and two different models of Rad18 deficient tumour mice. Also, tumour subtypes do not differ between the mouse models. Accordingly, functions of Rad18 in B cell activation and tumorigenesis may be compensated for by other pathways in B cells., (© 2024. The Author(s).)

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a pivotal immune checkpoint receptor, playing a crucial role in modulating T-cell activation. In this study, we delved into the underlying mechanism by which a common mutation, G199R, in the cytoplasmic domain of CTLA-4 impacts its inhibitory function. Utilizing nuclear magnetic resonance (NMR) spectroscopy and biochemical techniques, we mapped the conformational changes induced by this mutation and investigated its role in CTLA-4 activity. Our findings reveal that this mutation leads to a distinct conformational alteration, enhancing protein-membrane interactions. Moreover, functional assays demonstrated an improved capacity of the G199R mutant to downregulate T-cell activation, underscoring its potential role in immune-related disorders. These results not only enhance our understanding of CTLA-4 regulatory mechanisms but also provide insights for targeted therapeutic strategies addressing immune dysregulation linked to CTLA-4 mutations.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors., Competing Interests: Conflict of interest C. R. B. is a cofounder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), InterVenn Biosciences, Lycia Therapeutics, Virsti Therapeutics, OliLux Biosciences, and GanNA Bio. S. W. and C. R. B. are coinventors on a patent application related to work reviewed in this article held by Stanford University (grant no.: PCT/US2020/041603)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Welsh, Song, Park, Peske and Sadegh-Nasseri.)

Background: Circular RNAs are involved in autoimmune disease pathogenesis. Our previous study indicated that circPTPN22 is involved in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the underlying mechanisms remain unclear., Methods: First, the expression of circPTPN22 was detected by real-time PCR and western blotting. After overexpression or knockdown of circPTPN22, the proliferation of Jurkat cells was detected by the CCK-8 assay, and the apoptosis of Jurkat cells was detected by flow cytometry. In addition, the relationship between circPTPN22-miR-4689-S1PR1 was confirmed by bioinformatic analyses, fluorescence in situ hybridization assays, RNA-binding protein immunoprecipitation, and dual luciferase reporter assays., Results: We found that circPTPN22 expression was downregulated in the PBMCs of SLE patients compared to those of healthy controls. Overexpression of circPTPN22 increased proliferation and inhibited apoptosis of Jurkat T cells, whereas knockdown of circPTPN22 exerted the opposite effects. CircPTPN22 acts as a miR-4689 sponge, and S1PR1 is a direct target of miR-4689. Importantly, the circPTPN22/miR-4689/S1PR1 axis inhibited the secretion of TNF-α and IL-6 in Jurkat T cells., Conclusions: CircPTPN22 acts as a miR-4689 sponge to regulate T-cell activation by targeting S1PR1, providing a novel mechanism for the pathogenesis of SLE., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Genetic engineering of immune cells has opened new avenues for improving their functionality but it remains a challenge to pinpoint which genes or combination of genes are the most beneficial to target. Here, we conduct High Multiplicity of Perturbations and Cellular Indexing of Transcriptomes and Epitopes (HMPCITE-seq) to find combinations of genes whose joint targeting improves antigen-presenting cell activity and enhances their ability to activate T cells. Specifically, we perform two genome-wide CRISPR screens in bone marrow dendritic cells and identify negative regulators of CD86, that participate in the co-stimulation programs, including Chd4, Stat5b, Egr2, Med12, and positive regulators of PD-L1, that participate in the co-inhibitory programs, including Sptlc2, Nckap1l, and Pi4kb. To identify the genetic interactions between top-ranked genes and find superior combinations to target, we perform high-order Perturb-Seq experiments and we show that targeting both Cebpb and Med12 results in a better phenotype compared to the single perturbations or other combinations of perturbations., (© 2023. Springer Nature Limited.)

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial metabolites through a semi-invariant T cell receptor (TCR). Major questions remain regarding the extent of human MAIT cell functional and clonal diversity. To address these, we analyzed the single-cell transcriptome and TCR repertoire of blood and liver MAIT cells and developed functional RNA-sequencing, a method to integrate function and TCR clonotype at single-cell resolution. MAIT cell clonal diversity was comparable to conventional memory T cells, with private TCR repertoires shared across matched tissues. Baseline functional diversity was low and largely related to tissue site. MAIT cells showed stimulus-specific transcriptional responses in vitro, with cells positioned along gradients of activation. Clonal identity influenced resting and activated transcriptional profiles but intriguingly was not associated with the capacity to produce IL-17. Overall, MAIT cells show phenotypic and functional diversity according to tissue localization, stimulation environment and clonotype., (© 2023. The Author(s).)

Childhood is a critical period of immune development. During this time, naïve CD4 (nCD4) T cells undergo programmed cell differentiation, mediated by epigenetic changes, in response to external stimuli leading to a baseline homeostatic state that may determine lifelong disease risk. However, the ontogeny of epigenetic signatures associated with CD4 T cell activation during key developmental periods are yet to be described. We investigated genome-wide DNA methylation (DNAm) changes associated with nCD4 T activation following 72 h culture in media+anti-CD3/CD28 beads in healthy infants (aged 12 months, n = 18) and adolescents (aged 10-15 years, n = 15). We integrated these data with transcriptomic and cytokine profiling from the same samples. nCD4 T cells from both age groups show similar extensive epigenetic reprogramming following activation, with the majority of genes involved in the T cell receptor signaling pathway associated with differential methylation. Additionally, we identified differentially methylated probes showing age-specific responses, that is, responses in only infants or adolescents, including within a cluster of T cell receptor (TCR) genes. These encoded several TCR alpha joining (TRAJ), and TCR alpha variable (TRAV) genes. Cytokine data analysis following stimulation revealed enhanced release of IFN-γ, IL-2 and IL-10, in nCD4 T cells from adolescents compared with infants. Overlapping differential methylation and cytokine responses identified four probes potentially underpinning these age-specific responses. We show that DNAm in nCD4T cells in response to activation is dynamic in infancy and adolescence, with additional evidence for age-specific effects potentially driving variation in cytokine responses between these ages., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

CD19 chimeric antigen receptor (CAR) T-cells have demonstrated remarkable outcomes in B-cell malignancies. Recently, the novel CD19CAR-T cells incorporated with B-cell costimulatory molecules of CD79A/CD40 demonstrated superior antitumor activity in the B-cell lymphoma model compared with CD28 or 4-1BB. Here, we investigated the intrinsic transcriptional gene underlying the functional advantage of CD19.79A.40z CAR-T cells following CD19 antigen exposure using transcriptome analysis compared to CD28 or 4-1BB. Notably, CD19.79A.40z CAR-T cells up-regulated genes involved in T-cell activation, T-cell proliferation, and NF-κB signaling, whereas down-regulated genes associated with T-cell exhaustion and apoptosis. Interestingly, CD19.79A.40z CAR- and CD19.BBz CAR-T cells were enriched in almost similar pathways. Furthermore, gene set enrichment analysis demonstrated the enrichment of genes, which were previously identified to correlate with T-cell proliferation, interferon signaling pathway, and naïve and memory T-cell signatures, and down-regulated T-cell exhaustion genes in CD79A/CD40, compared with the T-cell costimulatory domain. The CD19.79A.40z CAR-T cells also up-regulated genes related to glycolysis and fatty acid metabolism, which are necessary to drive T-cell proliferation and differentiation compared with conventional CD19CAR-T cells. Our study provides a comprehensive insight into the understanding of gene signatures that potentiates the superior antitumor functions by CD19CAR-T cells incorporated with the CD79A/CD40 costimulatory domain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ung, Choochuen, Khopanlert, Maneechai, Sangkhathat, Terakura and Julamanee.)

T cells rapidly transition from a quiescent state into active proliferation and effector function upon exposure to cognate antigen. These processes are tightly controlled by signal transduction pathways that influence changes in chromatin remodeling, gene transcription, and metabolism, all of which collectively drive specific T cell memory or effector cell development. Dysregulation of any of these events can mediate disease and the past several years has shown unprecedented novel approaches to understand these events, down to the single-cell level. The massive explosion of sequencing approaches to assess the genome and transcriptome at the single cell level has transformed our understanding of T cell activation, developmental potential, and effector function under normal and various disease states. Despite these advances, there remains a significant dearth of information regarding how these events are translated to the protein level. For example, resolution of protein isoforms and/or specific post-translational modifications mediating T cell function remains obscure. The application of proteomics can change that, enabling significant insights into molecular mechanisms that regulate T cell function. However, unlike genomic approaches that have enabled exquisite visualization of T cell dynamics at the mRNA and chromatin level, proteomic approaches, including those at the single-cell level, has significantly lagged. In this review, we describe recent studies that have enabled a better understanding of how protein synthesis and degradation change during T cell activation and acquisition of effector function. We also highlight technical advances and how these could be applied to T cell biology. Finally, we discuss future needs to expand upon our current knowledge of T cell proteomes and disease., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Solt.)

There are multiple regulatory layers that control intracellular trafficking and protein secretion, ranging from transcriptional to posttranslational mechanisms. Finely regulated trafficking and secretion is especially important for lymphocytes during activation and differentiation, as the quantity of secretory cargo increases once the activated cells start to produce and secrete large amounts of cytokines, cytotoxins, or antibodies. However, how the secretory machinery dynamically adapts its efficiency and specificity in general and specifically in lymphocytes remains incompletely understood. Here we present a systematic bioinformatics analysis to address RNA-based mechanisms that control intracellular trafficking and protein secretion during B-lymphocyte activation, and differentiation, with a focus on alternative splicing. Our in silico analyses suggest that alternative splicing has a substantial impact on the dynamic adaptation of intracellular traffic and protein secretion in different B cell subtypes, pointing to another regulatory layer to the control of lymphocyte function during activation and differentiation. Furthermore, we suggest that NERF/ELF2 controls the expression of some COPII-related genes in a cell type-specific manner. In addition, T cells and B cells appear to use different adaptive strategies to adjust their secretory machineries during the generation of effector and memory cells, with antibody secreting B cell specifically increasing the expression of components of the early secretory pathway. Together, our data provide hypotheses how cell type-specific regulation of the trafficking machinery during immune cell activation and differentiation is controlled that can now be tested in wet lab experiments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ostwaldt, Los and Heyd.)

T cells are key players in our defence against infections and malignancies. When T cells differentiate or become activated, they undergo substantial alterations in gene expression. Even though RNA expression levels are now well documented throughout different stages of T cells, it is not well understood how mRNA expression translates into the protein landscape. By combining paired RNA sequencing and mass spectrometry data of primary human CD8+ T cells, we report that mRNA expression is a poor proxy for the overall protein output, irrespective of the differentiation or activation status. Yet, gene class stratification revealed a function-specific correlation of mRNA with protein expression. This gene class-specific expression pattern associated with differences in gene characteristics such as sequence conservation and untranslated region (UTR) lengths. In addition, the presence of AU-rich elements in the 3'UTR associated with alterations in mRNA and protein abundance T cell activation dependent, gene class-specific manner. In conclusion, our study highlights the role of gene characteristics as a determinant for gene expression in T cells., Competing Interests: The authors have declared that no competing interests exist.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro . Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics., Competing Interests: Authors RM, TF, GT, EC, DR, MP, JL, HW, and HL are currently employed by the Novartis Institutes for Biomedical Research (NIBR). The following authors were previously employed by NIBR when they were involved with the work described herein: MV (currently employed by Arbor Biotechnologies), GE (currently employed by Foghorn Therapeutics), MM (currently employed by Bristol Myers Squibb), and SK (currently employed by 2seventy Bio)., (Copyright © 2022 Molony, Funk, Trabucco, Corcoran, Ruddy, Varadarajan, Elliot, Piquet, Lam, Meyer, Wang, Kurtulus and Lu.)

Neonatal CD4 + T cells have reduced or delayed T-cell receptor (TCR) signaling responses compared with adult cells, but the mechanisms underlying this are poorly understood. This study tested the hypothesis that human neonatal naïve CD4 + TCR signaling and activation deficits are related to differences in H3K4me3 patterning and chromatin accessibility. Following initiation of TCR signaling using anti-CD3/anti-CD28 beads, adult naïve CD4 + T cells demonstrated increased CD69, phospho-CD3ε and interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ and IL-17A compared with neonatal cells. By contrast, following TCR-independent activation using phorbol myristate acetate (PMA)/ionomycin, neonatal cells demonstrated increased expression of CD69, IL-2 and TNF-α and equivalent phospho-ERK compared with adult cells. H3K4me3 chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on separate cohorts of naïve CD4 + T cells from term neonates and adults, and RNA-seq data from neonatal and adult naïve CD4 + T cells were obtained from the Blueprint Consortium. Adult cells demonstrated overall increased chromatin accessibility and a higher proportion of H3K4me3 sites associated with open chromatin and active gene transcription compared with neonatal cells. Adult cells demonstrated increased mRNA expression of the TCR-associated genes FYN, ITK, CD4, LCK and LAT, which was associated with increased H3K4me3 at the FYN and ITK gene loci and increased chromatin accessibility at the CD4, LCK and LAT loci. These findings indicate that neonatal TCR-dependent defects in activation are epigenetically regulated and provide a potentially targetable mechanism to enhance neonatal CD4 + T-cell responses., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Interleukin-2 inducible T cell kinase (ITK) plays a predominant role in the T-cell receptor (TCR) signaling cascade to ensure valid T-cell activation and function. Nevertheless, whether it regulates T-cell response of early vertebrates remains unknown. Herein, we investigated the involvement of ITK in the lymphocyte-mediated adaptive immune response, and its regulation to T-cell activation in the Nile tilapia Oreochromis niloticus. Both sequence and structure of O. niloticus ITK (OnITK) were remarkably conserved with its homologues from other vertebrates, implying its potential conserved function. OnITK mRNA was extensively expressed in lymphoid-related tissues, and with the relative highest level in peripheral blood. Once Nile tilapia was infected by Edwardsiella piscicida, OnITK in splenic lymphocytes was significantly up-regulated on 7-day post infection at both transcription and translation levels, suggesting that OnITK might involve in the primary adaptive immune response of teleost. Furthermore, upon splenic lymphocytes were stimulated by T-cell specific mitogen PHA, OnITK mRNA and protein levels were dramatically elevated. More importantly, treatment of splenic lymphocytes with specific inhibitor significantly crippled OnITK expression, which in turn impaired the inducible expression of T-cell activation markers IFN-γ, IL-2 and CD122, indicating the critical roles of ITK in regulating T-cell activation of Nile tilapia. Taken together, our results suggest that ITK takes part in the lymphocyte-mediated adaptive immunity of tilapia, and is indispensable for T-cell activation of teleost. Our findings thus provide novel evidences for understanding the mechanism regulating T-cell immunity of early vertebrates, as well as the evolution of adaptive immune system., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Deficiencies in Mannosidase β (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3′UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant β-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS.

Common variable immunodeficiency (CVID) is accompanied by various lymphocyte abnormalities believed to be mostly responsible for disease features in patients with no diagnosed monogenic defects. Here, we evaluated the association of B and T lymphocyte abnormalities with the incidence of CVID. Twenty-six genetically unsolved CVID patients were examined for B and T lymphocyte subsets by flow cytometry and CD4 + T-cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) test. We detected a reduction in total, naive, memory B cells and plasmablasts, and also total, naive, central memory and regulatory CD4 + T cells, besides naive CD8 + T cells. There was an increase in CD21 low and transitional B cells, effector memory (EM) and terminally differentiated effector memory (T EMRA ) CD4 + T-cell subsets as well as total, EM, T EMRA , activated and cytotoxic CD8 + T cells among non-monogenic CVID patients. CD4 + T-cell proliferation response was reduced regarding both division index and percent divided. In conclusion, regarding the similarity of lymphocyte abnormalities between patients without genetic defects and those with monogenic defects, genetic mutations are not responsible for these specific lymphocyte changes. However, the novel correlations observed between lymphocyte alterations among genetically unsolved CVID patients may serve as a guide to predict the potential of future CVID development for hypogammaglobulinemia children., (© 2022 The Scandinavian Foundation for Immunology.)

Common variable immunodeficiency (CVID) is a heterogeneous group of inborn errors of immunity characterized by reduced serum concentrations of different immunoglobulin isotypes. CVID is the most prevalent symptomatic antibody deficiency with a broad range of infectious and non-infectious clinical manifestations. Various genetic and immunological defects are known to be involved in the pathogenesis of CVID. Monogenic defects account for the pathogenesis of about 20-50% of CVID patients, while a variety of cases do not have a defined genetic background. Deficiencies in molecules of B cell receptor signaling or other pathways involving B-cell development, activation, and proliferation could be associated with monogenetic defects of CVID. Genetic defects damping different B cell developmental stages can alter B- and even other lymphocytes' differentiation and might be involved in the clinical and immunologic presentations of the disorder. Reports concerning T and B cell abnormalities have been published in CVID patients, but such comprehensive data on monogenic CVID patients is few and no review article exists to describe the abrogation of lymphocyte subsets in these disorders. Hence, we aimed to review the role of altered B- and T-cell differentiation in the pathogenesis of CVID patients with monogenic defects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fekrvand, Khanmohammadi, Abolhassani and Yazdani.)

T cell hyporesponsiveness is crucial for the functional immune system and prevents the damage induced by alloreactive T cells in autoimmune pathology and transplantation. Here, we found low expression of PRDM1 in T cells from donor and recipients both related to the occurrence of acute graft-versus-host disease (aGVHD). Our systematic multiomics analysis found that the transcription factor PRDM1 acts as a master regulator during inducing human primary T cell hyporesponsiveness. PRDM1 -overexpression in primary T cells expanded Treg cell subset and increased the expression level of FOXP3 , while decreased expression had the opposite effects. Moreover, the binding motifs of key T cell function regulators, such as FOS, JUN and AP-1, were enriched in PRDM1 binding sites and that PRDM1 altered the chromatin accessibility of these regions. Multiomics analysis showed that PRDM1 directly upregulated T cell inhibitory genes such as KLF2 and KLRD1 and downregulated the T cell activation gene IL2 , indicating that PRDM1 could promote a tolerant transcriptional profile. Further analysis showed that PRDM1 upregulated FOXP3 expression level directly by binding to FOXP3 upstream enhancer region and indirectly by upregulating KLF2. These results indicated that PRDM1 is sufficient for inducing human primary T cell hyporesponsiveness by transcriptomic and epigenetic manners., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guo, Wang, Wang, Guo, Cheng, Wang, Sun, Wang, Chang and Huang.)

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas., Competing Interests: Declaration of interests E.J.W. is a member of the Parker Institute for Cancer Immunotherapy, which supported the study. E.J.W. is an advisor for Merck, Marengo, Janssen, Related Sciences, Synthekine, and Surface Oncology. E.J.W. is a founder of Surface Oncology, Danger Bio, and Arsenal Biosciences. E.J.W. has a patent on the PD1 pathway. R.M.Y. and C.H.J. are inventors on patents and/or patent applications licensed to Novartis Institutes of Biomedical Research and receive license revenue from such licenses. C.H.J. is a scientific founder of Tmunity Therapeutics and DeCart Therapeutics for which he has founders stock but no income. T.C.M. received honorarium for advisory board service from Merck and BMS., (Copyright © 2022. Published by Elsevier Inc.)

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4 + T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4 + T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4 + T cells and CD8 + T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer 1 . However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions 2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4 + and CD8 + T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-β receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies 5 . Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Metabolomics analyses suggest changes in amino acid abundance, particularly l-arginine (L-ARG), occur in patients with tuberculosis. Immune cells require L-ARG to fuel effector functions following infection. We have previously described an L-ARG synthesis pathway in immune cells; however, its role in APCs has yet to be uncovered. Using a coculture system with mycobacterial-specific CD4 + T cells, we show APC L-ARG synthesis supported T cell viability and proliferation, and activated T cells contained APC-derived L-ARG. We hypothesize that APCs supply L-ARG to support T cell activation under nutrient-limiting conditions. This work expands the current model of APC-T cell interactions and provides insight into the effects of nutrient availability in immune cells., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Foxp3 + T regulatory cells (Tregs), CD4 + Foxp3 - T cells, and CD8 + T cells are composed of naive phenotype (NP) and memory phenotype (MP) subsets. Ten to 20% of each MP T cell population are cycling (Ki-67 + ) in vivo. We investigated the contribution of costimulatory (CD28) and coinhibitory (CTLA-4, PD-1) receptors on MP T cell homeostatic proliferation in vivo in the mouse. Blockade of CD28-CD80/CD86 signaling completely abolished MP Tregs and profoundly inhibited MP CD4 + Foxp3 - T cell proliferation, but it did not affect MP CD8 + T cell proliferation. Marked enhancement of homeostatic proliferation of MP Tregs and MP CD4 + Foxp3 - T cells was seen after blocking CTLA4-CD80/CD86 interactions and PD-1-PD-L1/2 interactions, and greater enhancement was seen with blockade of both pathways. The CD28 pathway also played an important role in the expansion of Tregs and MP T cells after treatment of mice with agonistic Abs to members of the TNF receptor superfamily, which can act directly (anti-GITR, anti-OX40, anti-4-1BB) or indirectly (anti-CD40) on T cells. Induction of a cytokine storm by blocking the interaction of NK inhibitory receptors with MHC class I had no effect on Treg homeostasis, enhanced MP CD4 + proliferation, and expansion in a CD28-dependent manner, but it enhanced MP CD8 + T cell proliferation in a CD28-independent manner. Because MP T cells exert potent biologic effects primarily before the induction of adaptive immune responses, these findings have important implications for the use of biologic agents designed to suppress autoimmune disease or enhance T effector function in cancer that may have negative effects on MP T cells.

Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes., Competing Interests: The authors have declared that no competing interests exist.

We exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4+ and CD8+ T cells. Such systems-level conservation also extended across human and mouse T cells and unexpectedly encompassed protein-protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2022 Nicolas et al.)

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Rauschmeier et al.)

Background: Tumor-associated macrophages (TAMs) are closely related to unfavorable prognosis of patients with clear cell renal cell carcinoma (ccRCC). However, the important molecules in the interaction between ccRCC and TAMs are unclear., Methods: TCGA-KIRC gene expression data of tumor tissues and normal tissues adjacent to tumor were compared to identify differentially expressed genes in ccRCC. TAMs related genes were discovered by analyzing the correlation between these differentially expressed genes and common macrophage biomarkers. Gene set enrichment analysis was performed to predict functions of TAMs related gene. The findings were further validated using RNA sequencing data obtained from the CheckMate 025 study and immunohistochemical analysis of samples from 350 patients with ccRCC. Kaplan-Meier survival curve, Cox regression analysis and Harrell's concordance index analysis were used to determine the prognostic significance., Results: In this study, we applied bioinformatic analysis to explore TAMs related differentially expressed genes in ccRCC and identified 5 genes strongly correlated with all selected macrophage biomarkers: STAC3, LGALS9, TREM2, FCER1G, and PILRA. Among them, FCER1G was abundantly expressed in tumor tissues and showed prognostic importance in patients with ccRCC who received treatment with Nivolumab; however, it did not exhibit prognostic value in those treated with Everolimus. We also discovered that high expression levels of FCER1G are related to T cell suppression. Moreover, combination of FCER1G and macrophage biomarker CD68 can improve the prognostic stratification of patients with ccRCC from TCGA-KIRC. Based on the immunohistochemical analysis of samples from patients with ccRCC, we further validated that FCER1G and CD68 are both highly expressed in tumor tissue and correlate with each other. Higher expression of CD68 or FCER1G in ccRCC tissue indicates shorter overall survival and progression-free survival; patients with high expression of both CD68 and FCER1G have the worst outcome. Combining CD68 and FCER1G facilitates the screening of patients with a worse prognosis from the same TNM stage group., Conclusions: High expression of FCER1G in ccRCC is closely related to TAMs infiltration and suppression of T cell activation and proliferation. Combining the expression levels of FCER1G and macrophage biomarker CD68 may be a promising postoperative prognostic index for patients with ccRCC., (© 2022. The Author(s).)

T helper (Th) cells provide immunity to pathogens but also contribute to detrimental immune responses during allergy and autoimmunity. Th2 cells mediate asthmatic airway inflammation and Th1 cells are involved in the pathogenesis of multiple sclerosis. T cell activation involves complex transcriptional networks and metabolic reprogramming, which enable proliferation and differentiation into Th1 and Th2 cells. The essential trace element zinc has reported immunomodulatory capacity and high zinc concentrations interfere with T cell function. However, how high doses of zinc affect T cell gene networks and metabolism remained so far elusive. Herein, we demonstrate by means of transcriptomic analysis that zinc aspartate (UNIZINK), a registered pharmaceutical infusion solution with high bioavailability, negatively regulates gene networks controlling DNA replication and the energy metabolism of murine CD3/CD28-activated CD4 + T cells. Specifically, in the presence of zinc, CD4 + T cells show impaired expression of cell cycle, glycolytic and tricarboxylic acid cycle genes, which functionally cumulates in reduced glycolysis, oxidative phosphorylation, metabolic fitness and viability. Moreover, high zinc concentrations impaired nuclear expression of the metabolic transcription factor MYC, prevented Th1 and Th2 differentiation in vitro and reduced Th1 autoimmune central nervous system (CNS) inflammation and Th2 asthmatic airway inflammation induced by house dust mites in vivo. Together, we find that higher zinc doses impair the metabolic fitness of CD4 + T cells and prevent Th1 CNS autoimmunity and Th2 allergy., (© 2022. The Author(s).)

Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress., (© 2021 Wiley Periodicals LLC.)

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation., (© 2022. The Author(s).)

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs), the major antigen-presenting cells of the immune system, sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here, we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac 5 3F ax Neu5Ac. Ac 5 3F ax Neu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8 + T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation, cell-adhesion, and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8 + T cells. This increased avidity was detectable in the absence of antigens, but was especially pronounced in antigen-dependent interactions. Together, our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8 + T cell responses., (© 2022. The Author(s).)

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses., Competing Interests: AS reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, FL82, and Dahlia Biosciences unrelated to this work. AS has received research support from Merck, Novartis, Leo Pharma, Janssen, the Bill and Melinda Gates Foundation, the Moore Foundation, the Pew-Stewart Trust, Foundation MIT, the Chan Zuckerberg Initiative, Novo Nordisk and the FDA unrelated to this work. LK is on the scientific advisory board for HiFiBio and Mammoth Biosciences. She reports research funding from Kymab Limited, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals. She reports consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gillead Sciences, Vertex Pharmaceuticals, and Takeda Pharmaceuticals. LK reports grants and personal fees from Bristol Myers Squibb that are managed under an agreement with Harvard Medical School. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gerdemann, Fleming, Kaminski, McGuckin, Rui, Lane, Keskula, Cagnin, Shalek, Tkachev and Kean.)

Intracellular cytokine staining (ICS) is a widely employed ex vivo method for quantitative determination of the activation status of immune cells, most often applied to T cells. ICS test samples are commonly prepared from animal or human tissues as unpurified cell mixtures, and cell-specific cytokine signals are subsequently discriminated by gating strategies using flow cytometry. Here, we show that when ICS samples contain Ly6G + neutrophils, neutrophils are ex vivo activated by an ICS reagent - phorbol myristate acetate (PMA) - which leads to hydrogen peroxide (H 2 O 2 ) release and death of cytokine-expressing T cells. This artifact is likely to result in overinterpretation of the degree of T cell suppression, misleading immunological research related to cancer, infection, and inflammation. We accordingly devised easily implementable improvements to the ICS method and propose alternative methods for assessing or confirming cellular cytokine expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gong, Li, Shi and Ren.)

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events., Competing Interests: ME, RS, NK, PR, VB, DK, MB, NA, GR, JR No competing interests declared, (© 2022, Ecker et al.)

Immunity Related GTPases (IRG) are a family of proteins produced during infection that regulate membrane remodeling events in cells, particularly autophagy and mitophagy. The human IRGM gene has been strongly associated with Crohn's disease and other inflammatory diseases through Genome-Wide Association studies. Absence of Irgm1 in mice prompts intestinal inflammation, autoimmunity, and impaired immune control of pathogenic bacteria and protozoa. Although prior work has focused on a prominent role for IRGM/Irgm1 in regulating macrophage function, the work described here addresses a potential role of Irgm1 in regulating the function of mature T cells. Irgm1 was found to be highly expressed in T cells in a manner that varied with the particular T cell subset and increased with activation. Mice with a complete lack of Irgm1, or a conditional lack of Irgm1 specifically in T cells, displayed numerous changes in T cell numbers and function in all subsets examined, including CD4 + (Th1 and Treg) and CD8 + T cells. Related to changes in T cell number, apoptosis was found to be increased in Irgm1-deficient CD4 + and CD8 + T cells. Altered T cell metabolism appeared to be a key driver of the phenotypes: Glucose metabolism and glycolysis were increased in Irgm1-deficient CD4 + and CD8 + T cells, and muting these effects with glycolytic inhibitors partially restored T cell function and viability., (© 2022. The Author(s).)

T cells must shift their metabolism to respond to infections and tumors and to undergo memory formation. The ATP-binding cassette transporter ABCB10 localizes to the mitochondrial inner membrane, where it is thought to export a substrate important in heme biosynthesis and metabolism, but its role in T cell development and activation is unknown. In this article, we use a combination of methods to study the effect of ABCB10 loss in primary and malignantly transformed T cells. Although Abcb10 is dispensable for development of both CD4 + and CD8 + T cells, it is required for expression of specific cytokines in CD4 + , but not CD8 + , T cells activated in vitro. These defects in cytokine expression are magnified on repeated stimulation. In vivo, CD8 + cells lacking ABCB10 expand more in response to viral infection than their control counterparts, while CD4 + cells show reductions in both number and percentage. CD4 + cells lacking ABCB10 show impairment in Ag-specific memory formation and recall responses that become more severe with time. In malignant human CD4 + Jurkat T cells, we find that CRISPR-mediated ABCB10 disruption recapitulates the same cytokine expression defects upon activation as observed in primary mouse T cells. Mechanistically, ABCB10 deletion in Jurkat T cells disrupts the ability to switch to aerobic glycolysis upon activation. Cumulatively, these results show that ABCB10 is selectively required for specific cytokine responses and memory formation in CD4 + T cells, suggesting that targeting this molecule could be used to mitigate aberrant T cell activation., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulated T and B lymphocytes. Type I interferons (IFN-I) have been shown to play important pathogenic roles in both SLE patients and mouse models of lupus. Recent studies have shown that B cell intrinsic responses to IFN-I are enough to drive B cell differentiation into autoantibody-secreting memory B cells and plasma cells, although lower levels of residual auto-reactive cells remain present. We speculated that IFN-I stimulation of T cells would similarly drive specific T-cell associated lupus phenotypes including the upregulation of T follicular helper cells and Th17, thereby affecting autoantibody production and the development of glomerulonephritis. Using the B6.Nba2 mouse model of lupus, we evaluated disease parameters in T cell specific IFN-I receptor (IFNAR)-deficient mice (cKO). Surprisingly, all measured CD4+ T cell abnormalities and associated intra-splenic cytokine levels (IFNγ, IL-6, IL-10, IL-17, IL-21) were unchanged and thus independent of IFN-I. In contrast B6.Nba2 cKO mice displayed reduced levels of effector CD8+ T cells and increased levels of Foxp3+ CD8+ regulatory T cells, suggesting that IFN-I induced signaling specifically affecting CD8+ T cells. These data suggest a role for both pathogenic and immunosuppressive CD8+ T cells in Nba2 -driven autoimmunity, providing a model to further evaluate the role of these cell subsets during lupus-like disease development in vivo.

Erythroid differentiation regulator 1 (Erdr1) has previously been reported to control thymocyte selection via TCR signal regulation, but the effect of Erdr1 as a TCR signaling modulator was not studied in peripheral T cells. In this report, it was determined whether Erdr1 affected TCR signaling strength in CD4 T cells. Results revealed that Erdr1 significantly enhanced the anti-TCR antibody-mediated activation and proliferation of T cells while failing to activate T cells in the absence of TCR stimulation. In addition, Erdr1 amplified Ca 2+ influx and the phosphorylation of PLCγ1 in CD4 T cells with the TCR stimuli. Furthermore, NFAT1 translocation into nuclei in CD4 T cells was also significantly promoted by Erdr1 in the presence of TCR stimulation. Taken together, our results indicate that Erdr1 positively modulates TCR signaling strength via enhancing the PLCγ1/Ca 2+ /NFAT1 signal transduction pathway.

Protection against pathogen re-infection is mediated, in large part, by two humoral cellular compartments, namely, long-lived plasma cells and memory B cells. Recent data have reinforced the importance of memory B cells, particularly in response to re-infection of different viral subtypes or in response with viral escape mutants. In regard to memory B cell generation, considerable advancements have been made in recent years in elucidating its basic mechanism, which seems to well explain why the memory B cells pool can deal with variant viruses. Despite such progress, efforts to develop vaccines that induce broadly protective memory B cells to fight against rapidly mutating pathogens such as influenza virus and HIV have not yet been successful. Here, we discuss recent advances regarding the key signals and factors regulating germinal center-derived memory B cell development and activation and highlight the challenges for successful vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Inoue, Shinnakasu and Kurosaki.)

Sorting nexin 27 (SNX27) association to the retromer complex mediates intracellular trafficking of cargoes containing PSD95/Dlg1/ZO-1 (PDZ)-binding C-terminal sequences from endosomes to the cell surface, preventing their lysosomal degradation. Antigen recognition by T lymphocyte leads to the formation of a highly organized structure named the immune synapse (IS), which ensures cell-cell communication and sustained T cell activation. At the neuronal synapse, SNX27 recycles PDZ-binding receptors and its defective expression is associated with synaptic dysfunction and cognitive impairment. In T lymphocytes, SNX27 was found localized at recycling endosomal compartments that polarized to the IS, suggesting a function in polarized traffic to this structure. Proteomic analysis of PDZ-SNX27 interactors during IS formation identify proteins with known functions in cytoskeletal reorganization and lipid regulation, such as diacylglycerol (DAG) kinase (DGK) ζ, as well as components of the retromer and WASH complex. In this study, we investigated the consequences of SNX27 deficiency in cytoskeletal reorganization during IS formation. Our analyses demonstrate that SNX27 controls the polarization towards the cell-cell interface of the PDZ-interacting cargoes DGKζ and the retromer subunit vacuolar protein sorting protein 26, among others. SNX27 silencing abolishes the formation of a DAG gradient at the IS and prevents re-localization of the dynactin complex component dynactin-1/p150 Glued , two events that correlate with impaired microtubule organizing center translocation (MTOC). SNX27 silenced cells show marked alteration in cytoskeleton organization including a failure in the organization of the microtubule network and defects in actin clearance at the IS. Reduced SNX27 expression was also found to hinder the arrangement of signaling microclusters at the IS, as well as the polarization of the secretory machinery towards the antigen presenting cells. Our results broaden the knowledge of SNX27 function in T lymphocytes by showing a function in modulating IS organization through regulated trafficking of cargoes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 González-Mancha, Rodríguez-Rodríguez, Alcover and Merida.)

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)