ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

A milder form of NSRP1-associated neurodevelopmental disorder, caused by a missense variant in the nuclear localization signal.

Nuclear Speckle Splicing Regulator Protein 1 (NSRP1) is a splice factor found in nuclear speckles, which are small membrane-free organelles implicated in epigenetic regulation, chromatin organization, DNA repair, and RNA modification. Bi-allelic loss-of-function variants in NSRP1 have recently been identified in patients suffering from a severe neurodevelopmental disorder, presenting with neurodevelopmental delay, epilepsy, microcephaly, hypotonia, and spastic cerebral palsy. Described patients acquired neither independent walking nor speech and often showed anomalies on cerebral MRI. Here we describe the case of a 14-year-old girl with motor and language delay as well as intellectual disability, who presents an ataxic gait but walks without assistance and speaks in short sentences. Whole-genome sequencing revealed the compound heterozygous NSRP1 variants c.114 + 2T > G and c.1595T > A (p.Val532Glu). Functional validation using HEK293T cells transfected with either wild-type or mutated GFP-tagged Nsrp1 suggests that the Val532Glu variant interferes with the function of the nuclear localization signal, and leads to mislocalization of NSRP1 in the cytosol, thus confirming the pathogenicity of the observed variant. This case helps to expand the phenotypic and genetic spectrum associated with pathogenic NSRP1 variants and indicates that this diagnosis should also be suspected in patients with milder phenotypes.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

NFAT1 Regulates Systemic Autoimmunity through the Modulation of a Dendritic Cell Property.

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Swiprosin-1: Its Expression and Diverse Biological Functions.

Swiprosin-1/EFhd2 is a Ca2+ binding adapter protein involved in the various cellular functions. Swiprosin-1 is significantly upregulated in a number of pathological conditions of inflammation, neurodegeneration, and cancer. Swiprosin-1 associated with actin and its expression level amplifies the production of proinflammatory mediators and modulates the activation of transcription factor during immune cells activation. This review aims at providing an overview of the expression and function of swiprosin-1/EFhd2 in various pathophysiological conditions. We also discussed the key role of swiprosin-1 in immune cell activation, cell migration, apoptosis, humoral immunity, cancer invasion and metastasis, neuronal transport, and major signaling cascades. J. Cell. Biochem. 119: 150-156, 2018. © 2017 Wiley Periodicals, Inc.

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity.

Nuclear speckles are subnuclear storage sites containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as serine/arginine-rich splicing factor 1 (SRSF1 or ASF/SF2) and SRSF2 (SC35). NSrp70 mediates alternative splice site selection, targeting several pre-mRNAs, including CD44 exon v5. Here we demonstrated that NSrp70 interacts physically with two SR proteins, SRSF1 and SRSF2, and reverses their splicing activity in terms of CD44 exon v5 as exon exclusion. The NSrp70 RS-like region was subdivided into three areas. Deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins and to target mRNA and also failed to induce splicing of CD44 exon v5, suggesting that RS1 is critical for NSrp70 functioning. Interestingly, RS1 deletion also resulted in the loss of NSrp70 and SR protein speckle positioning, implying a potential scaffolding role for NSrp70 in nuclear speckles. NSrp70 contains an N-terminal coiled-coil domain that is critical not only for self-oligomerization but also for splicing activity. Consistently, deletion of the coiled-coil domain resulted in indefinite formation of nuclear speckles. Collectively, these results demonstrate that NSrp70 acts as a new molecular counterpart for alternative splicing of target RNA, counteracting SRSF1 and SRSF2 splicing activity.

Structural mechanism underlying regulation of human EFhd2/Swiprosin-1 actin-bundling activity by Ser183 phosphorylation.

EF-hand domain-containing protein D2/Swiprosin-1 (EFhd2) is an actin-binding protein mainly expressed in the central nervous and the immune systems of mammals. Intracellular events linked to EFhd2, such as membrane protrusion formation, cell adhesion, and BCR signaling, are triggered by the association of EFhd2 and F-actin. We previously reported that Ca2+ enhances the F-actin-bundling ability of EFhd2 through maintaining a rigid parallel EFhd2-homodimer structure. It was also reported that the F-actin-bundling ability of EFhd2 is regulated by a phosphorylation-dependent mechanism. EGF-induced phosphorylation at Ser183 of EFhd2 has been shown to inhibit F-actin-bundling, leading to irregular actin dynamics at the leading edges of cells. However, the underlying mechanism of this inhibition has remained elusive. Here, we report the crystal structure of a phospho-mimicking mutant (S183E) of the EFhd2 core domain, where the actin-binding sites are located. Although the overall structure of the phospho-mimicking mutant is similar to the one of the unphosphorylated form, we observed a conformational transition from ordered to disordered structure in the linker region at the C-terminus of the mutant. Based on our structural and biochemical analyses, we suggest that phosphorylation at Ser183 of EFhd2 causes changes in the local conformational dynamics and the surface charge distribution of the actin-binding site, resulting in a re-coordination of the actin-binding sites in the dimer structure and a reduction of F-actin-bundling activity without affecting the F-actin-binding capacity.

NFAT1 and JunB cooperatively regulate IL-31 gene expression in CD4+ T cells in health and disease.

IL-31 is a key mediator of itching in atopic dermatitis (AD) and is preferentially produced by activated CD4(+) T cells and Th2 cells. Although pathophysiological functions of IL-31 have been suggested in diverse immune disorders, the molecular events underlying IL-31 gene regulation are still unclear. In this study we identified the transcription start site and functional promoter involved in IL-31 gene regulation in mouse CD4(+) T cells. TCR stimulation-dependent IL-31 expression was found to be closely linked with in vivo binding of NFAT1 and JunB to the IL-31 promoter. Although NFAT1 alone enhanced IL-31 promoter activity, it was further enhanced in the presence of JunB. Conversely, knockdown of either NFAT1 or JunB resulted in reduced IL-31 expression. NFAT1-deficient CD4(+) T cells showed a significant defect in IL-31 expression compared with wild-type CD4(+) T cells. In agreement with these findings, mice subjected to atopic conditions showed much higher levels of IL-31, which were closely correlated with a significant increase in the number of infiltrated NFAT1(+)CD4(+) T cells into the AD ears. Amelioration of AD progression by cyclosporin A treatment was well correlated with downregulation of IL-31 expressions in CD4(+) T cells and total ear residual cells. In summary, our results suggest a functional cooperation between NFAT1 and JunB in mediating IL-31 gene expression in CD4(+) T cells and indicate that interference with this interaction or their activity has the potential of reducing IL-31-mediated AD symptoms.

Four New Lignans and IL-2 Inhibitors from Magnoliae Flos.

Four new lignans, a furofuran lignan medioresinol B (10) and three tetrahydrofuran lignans kobusinol C (16), 7'-methoxy magnostellin A (21), and mangnostellin D (23), along with 19 known lignans, were isolated from the flower buds of Magnolia biondii PAMP. The structures of the isolates were elucidated using spectroscopic analysis, mainly one- and two-dimensional NMR, high resolution-MS, and circular dichroism techniques as well as Mosher's esterification method. The anti-allergic effects of the isolated compounds were evaluated by analyzing the inhibition of interleukin-2 (IL-2) expression in Jurkat T-cells. Compounds 11-14 reduced IL-2 expression in a dose-dependent manner.

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae.

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.

Cation-Selective and Anion-Controlled Fluorogenic Behaviors of a Benzothiazole-Attached Macrocycle That Correlate with Structural Coordination Modes.

We report how the metal cation and its counteranions cooperate in the complexation-based macrocyclic chemosensor to monitor the target metal ion via the specific coordination modes. The benzothiazolyl group bearing NO2S2-macrocycle L was synthesized, and its mercury(II) selectivity (for perchlorate salt) as a dual-probe channel (UV-vis and fluorescence) chemosensor exhibiting the largest blue shift and the fluorescence turn-off was observed. In the mercury(II) sensing with different anions, except ClO4(-) and NO3(-), no responses for mercury(II) were observed with other anions such as Cl(-), Br(-), I(-), SCN(-), OAc(-), and SO4(2-). A crystallographic approach for the mononuclear mercury(II) perchlorate complex [Hg(L)(ClO4)2]·0.67CH2Cl2 (1) and polymeric mercury(II) iodide complex [Hg(L)I2]n (2) revealed that the observed anion-controlled mercury(II) sensing in the fluorescence mainly stems from the endo- and exocoordination modes, depending on the anion coordinating ability, which induces either the Hg-Ntert bond formation or not. The detailed complexation process with mercury(II) perchlorate associated with the cation sensing was also monitored with the titration methods by UV-vis, fluorescence spectroscopy, and cold-spray ionization mass spectrometry.

6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation.

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell-based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4(+) T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.

Label-free quantitative phosphorylation analysis of human transgelin2 in Jurkat T cells reveals distinct phosphorylation patterns under PKA and PKC activation conditions.

BACKGROUND: Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms. RESULTS: Here, a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and label-free relative quantification analysis was used to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signalling pathways of immune cell activation and homeostasis. A total of six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS. CONCLUSIONS: Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes. Accordingly, different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signalling events under immune response activation or homeostasis conditions.

Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid.

Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.

Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin.

Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them in accessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells over expressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.

NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo.

Nuclear speckles are known to be the storage sites of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its subcellular localization and apparent molecular weight. This protein was first identified as CCDC55 by the National Institutes of Health Mammalian Gene Collection, although its function has not been assigned. NSrp70 was colocalized and physically interacted with SC35 and ASF/SF2 in speckles. NSrp70 has a putative RNA recognition motif, the RS-like region, and two coiled-coil domains, suggesting a role in RNA processing. Accordingly, using CD44, Tra2ß1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal 10 amino acids (531-540), including (536)RD(537), were identified as a novel nuclear localization signal, and the region spanning 290-471 amino acids was critical for speckle localization and binding to SC35 and ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice led to a lack of progeny, including fetal embryos. Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.

The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption.

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

T Cell Immunological Synaptosomes: Definition and Isolation.

In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.

T Cell Microvilli: Finger-Shaped External Structures Linked to the Fate of T Cells.

Microvilli are outer membrane organelles that contain cross-linked filamentous actin. Unlike well-characterized epithelial microvilli, T-cell microvilli are dynamic similar to those of filopodia, which grow and shrink intermittently via the alternate actin-assembly and -disassembly. T-cell microvilli are specialized for sensing Ags on the surface of Ag-presenting cells (APCs). Thus, these finger-shaped microprotrusions contain many signaling-related proteins and can serve as a signaling platforms that induce intracellular signals. However, they are not limited to sensing external information but can provide sites for parts of the cell-body to tear away from the cell. Cells are known to produce many types of extracellular vesicles (EVs), such as exosomes, microvesicles, and membrane particles. T cells also produce EVs, but little is known about under what conditions T cells generate EVs and which types of EVs are released. We discovered that T cells produce few exosomes but release large amounsts of microvilli-derived particles during physical interaction with APCs. Although much is unanswered as to why T cells use the same organelles to sense Ags or to produce EVs, these events can significantly affect T cell fate, including clonal expansion and death. Since TCRs are localized at microvilli tips, this membrane event also raises a new question regarding long-standing paradigm in T cell biology; i.e., surface TCR downmodulation following T cell activation. Since T-cell microvilli particles carry T-cell message to their cognate partner, these particles are termed T-cell immunological synaptosomes (TISs). We discuss the potential physiological role of TISs and their application to immunotherapies.