Supplementary Figures 1 - 6, Table 1 from The SIL Gene Is Essential for Mitotic Entry and Survival of Cancer Cells

The positive-sense single-stranded (ss) RNA viruses of the Betacoronavirus (beta-CoV) genus can spillover from mammals to humans and are an ongoing threat to global health and commerce, as demonstrated by the current zoonotic pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current anti-viral strategies focus on vaccination or targeting key viral proteins with antibodies and drugs. However, the ongoing evolution of new variants that evade vaccination or may become drug-resistant is a major challenge. Thus, antiviral compounds that circumvent these obstacles are needed. Here we describe an innovative antiviral modality based on in silico designed fully synthetic mRNA that is replication incompetent in uninfected cells (termed herein PSCT: parasitic anti-SARS-CoV-2 transcript). The PSCT sequence was engineered to include key untranslated cis-acting regulatory RNA elements of the SARS-CoV-2 genome, so as to effectively compete for replication and packaging with the standard viral genome. Using the Vero E6 cell-culture based SARS-CoV-2 infection model, we determined that the intracellular delivery of liposome-encapsulated PSCT at 1 hour post infection significantly reduced intercellular SARS-CoV-2 replication and release into the extracellular milieu as compared to mock treatment. In summary, our findings are a proof-of-concept for the therapeutic feasibility of in silico designed mRNA compounds formulated to hinder the replication and packaging of ssRNA viruses sharing a comparable genomic-structure with beta-CoVs.

Objective RasGTPases are master regulators of multiple intracellular signaling cascades. Perturbation of this pathway has been implicated in the pathogenesis of rheumatoid arthritis (RA). In this study we aimed to define the therapeutic potential of a novel RasGTPases inhibitor, farnesylthiosalicylate (FTS), in the preclinical mouse model of collagen‐induced arthritis (CIA) and better delineate its immunomodulatory effects both ex vivo and in the mouse. Methods We analyzed in vitro the immunomodulatory effects of FTS on various CD4 + T‐cell functions such as activation, proliferation, T‐helper polarization, and production of proinflammatory cytokines. Using the CIA model, we further determined the efficacy of FTS to inhibit clinical, histopathologic, and diverse immunological outcomes of arthritis. Results FTS treatment of CD4 + T cells in vitro effectively targeted distinct kinases (extracellular signal‐regulated kinase 1/2, p38, protein kinase B/AKT, and mammalian target of rapamycin), the production of interleukin (IL)‐17A, IL‐22, and granulocyte‐macrophage colony‐stimulating factor, and Th17 polarization. FTS therapy in the mouse CIA model significantly reduced clinical disease severity and joint inflammation/damage by histology. Importantly, FTS suppressed the in vivo induction of splenic IL‐17 + IL‐22 + Th17 cells and the secretion of proinflammatory cytokines. The production of pathogenic autoantibodies and their abnormal hyposialylation was significantly attenuated by FTS therapy. Importantly, in vivo generation of collagen type‐II specific effector CD4 + T cells was likewise repressed by FTS therapy. Conclusion The RasGTPases inhibitor FTS attenuates the production of proinflammatory cytokines by in vitro–activated T cells and is a potent immunomodulatory compound in the CIA model, primarily targeting the generation of autoreactive Th17 cells and the production of autoantibodies and their subsequent pathogenic hyposialylation.

Background: The Ras family of GTPases plays an important role in signaling nodes downstream to T cell antigen receptor (TCR) and CD28, potentially lowering the threshold for TCR activation by autoantigens [1]. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from Rheumatoid Arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) interferes with the interaction between RasGTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization and effective downstream signaling. Previous studies in the Lewis rat adjuvant induced arthritis show that FTS attenuates arthritis development and that the inhibition of pathogenic Th17-type cells is a central mechanism of action of this compound [2]. Objectives: To further study the therapeutic efficacy and molecular mechanisms that mediate the immunomodulatory effects of FTS in DBA/1 mouse collagen type-II induced arthritis (CIA) the pre-clinical model. Methods: Arthritis was induced in 8-10 week old male DBA/1 mice by immunization with collagen type-II (CII) and complete Freund's adjuvant. Animals were treated semi-prophylactically with daily oral FTS (100 mg/kg); weekly i.p. injection of MTX (0.5 mg/kg); or daily 0.5% CMC vehicle solution (control treatment). Arthritis severity was graded daily by a validated clinical score (0-16 scale), starting at disease onset till study termination. In addition, multiple relevant immunological and molecular biomarkers were analyzed. Results: We found that the clinical scores of mice in the FTS and MTX arms was significantly reduced (by ∼80%, area under curve) compared to the control arm. Accordingly, FTS therapy significantly reduced joint pathology scores for inflammation, pannus formation, bone resorption, and cartilage damage. FTS also significantly inhibited the production of pathogenic anti-CII autoantibodies, anti–citrullinated peptide antibodies, and notably the de-sialylation of these autoantibodies as compared to control mice (Figure 1). The analysis of the effect of FTS on the T cell response to CII immunization, revealed strong attenuation of IL-22, IL-17, IL-9, GM-CSF, TNF, and IFN-gamma producing pro-inflammatory CD4+ Th cells. Importantly, we found that in vitro FTS treatment during TCR-stimulation (anti-CD3/CD28 mAbs) significantly inhibited the ensuing phosphorylation of multiple critical MAP kinases such as Erk, Akt, p38, and mTOR. Conclusion: We determined in the preclinical CIA model that FTS, a first-in-class oral Ras-GTPases inhibitor, is a potent immune modulator, via the inhibition of TCR/CD28/Ras-dependent activation of critical MAPKs, consequently attenuating the generation of pro-inflammatory autoreactive T cells. References [1] Singh K, et al. (2012) K-RAS GTPase- and B-RAF kinase-mediated T-cell tolerance defects in rheumatoid arthritis. Proc Natl Acad Sci USA109(25):E1629-1637. [2] Zayoud M, et al. (2017) Ras Signaling Inhibitors Attenuate Disease in Adjuvant-Induced Arthritis via Targeting Pathogenic Antigen-Specific Th17-Type Cells. Frontiers in immunology8:799. Acknowledgement: This work was supported by the U.S Department of Defense Grant number AMRMC W81XWH-14-1-0609/10 Disclosure of Interests: None declared

The Ras family of GTPases plays an important role in signaling nodes downstream to T cell receptor and CD28 activation, potentially lowering the threshold for T-cell receptor activation by autoantigens. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from rheumatoid arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule farnesylthiosalicylic acid (FTS) interferes with the interaction between Ras GTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization. In the present study, we tested the therapeutic and immunomodulatory effects of FTS and its derivative 5-fluoro-FTS (F-FTS) in the rat adjuvant-induced arthritis model (AIA). We show that AIA severity was significantly reduced by oral FTS and F-FTS treatment compared to vehicle control treatment. FTS was as effective as the mainstay anti-rheumatic drug methotrexate, and combining the two drugs significantly increased efficacy compared to each drug alone. We also discovered that FTS therapy inhibited both the CFA-driven in vivo induction of Th17 and IL-17/IFN-γ producing "double positive" as well as the upregulation of serum levels of the Th17-associated cytokines IL-17A and IL-22. By gene microarray analysis of effector CD4+ T cells from CFA-immunized rats, re-stimulated in vitro with the mycobacterium tuberculosis heat-shock protein 65 (Bhsp65), we determined that FTS abrogated the Bhsp65-induced transcription of a large list of genes (e.g., Il17a/f, Il22, Ifng, Csf2, Lta, and Il1a). The functional enrichment bioinformatics analysis showed significant overlap with predefined gene sets related to inflammation, immune system processes and autoimmunity. In conclusion, FTS and F-FTS display broad immunomodulatory effects in AIA with inhibition of the Th17-type response to a dominant arthritogenic antigen. Hence, targeting Ras signal-transduction cascade is a potential novel therapeutic approach for RA.

Ras-GTPases are molecular switches that regulate key cellular processes, such as proliferation, differentiation, apoptosis, and motility. In T cells, Ras-family GTPases (e.g. K/N-Ras) are crucial for proper TCR-dependent activation following antigen recognition. Defective Ras GTPases signaling has been associated with T cell anergy, and accordingly increased expression of active Ras was shown to reverse anergy and to restore IL-2 production. Importantly, T cells from patients with Rheumatoid Arthritis (RA) display augmented activation of the Ras/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS in normal CD4+ T cells has been shown to promote T cells reactivity to relevant autoantigen in RA. Thus, Ras GTPases appear to be a promising molecular target for inhibiting T cell activation in RA. Based on an innovative concept Kloog (the partnering PI) and colleagues discovered a potent non-toxic inhibitor of Ras, Farnesylthiosalicylic acid (FTS). This small molecule does not belong to the class of farnesyltransferase inhibitors (FTIs) that failed in clinical trials. It interferes with the interactions between Ras and distinct prenyl-binding chaperone proteins that are vital for the proper plasma membrane (PM) localization and signaling dynamics of RasGTPases, and indeed FTS dislodges the classical H/N/K-Ras GTPases from the PM and inhibits their effective downstream signaling. In multiple preclinical animal studies it has been shown that FTS effectively inhibited in vivo tumor growth of oncogenic K/N-Ras-dependent cancers. Thus, in collaboration with Concordia Pharmaceuticals Inc., FTS was developed into and oral drug, Salirasib®. The drug has been already tested in the clinic for the treatment of cancers with oncogenic mutations in KRAS and NRAS. No dose-limiting toxicities or major adverse events were reported during Salirasib® treatment, in phase I clinical trials of patients with advanced pancreatic cancer, hematolological malignancies (NCT00867230; M.D. Anderson Cancer Center, TX), and in phase II clinical study in non-small-cell lung cancer patients (NCT00531401; Memorial Sloan Kettering Cancer Center, NY). Thus, Salirasib® is the only available successful Ras GTPases inhibitor that reached Phase II clinical trials, and moreover received an orphan drug designation by the FDA for the treatment of pancreatic cancer. Importantly, we have extensively studied the effects of FTS and related derivatives (e.g., FTSAmide and FTS-methoxymethylester), in multiple pre-clinical animal models of autoimmune inflammation, such as: experimental autoimmune encephalomyelitis; Type-1 diabetes; colitis and others. More recently, our preliminary studies in the adjuvant-induced arthritis (AIA) rat model − a classical animal model for RA − imply that FTS attenuates disease manifestation, as assessed by: clinical scores; MRI imaging; histopathology; and serum levels of pro-inflammatory cytokines. Thus, our working hypothesis is that Salirasib® has a good potential to be a “silver bullet” drug for RA and other T cell-dependent autoimmune disorders. Our objectives are to test further this hypothesis in the AIA model as well as in another established animal model of RA, the collagen type-II induced arthritis (CIA) in DBA/1 (H-2q) mice. In parallel we will study in vitro, the effects of FTS and its different newer derivatives on a wide range of T cell functions and signaling networks implicated in RA. For the therapeutic treatment protocol, we will be administer FTS orally by gavage, once daily, starting immediately after disease initiation. Multiple modalities will be used to assess joint inflammation/damage and the immune response, as follows: arthritis clinical scores; MRI scans, micro-CT; histopathology examination by a blinded pathologist; serum cytokine profiles; T cell subset analysis (e.g., Foxp3+ Treg, Th1, Th17, etc.) by polychromatic flow cytometry. Additionally, we will analyzed the activation of different Ras downstream effectors and relevant cellular programs by Western blotting, Affymetrix GeneChip® whole-transcript arrays, and quantitative real-time PCR analysis. The proposed project is highly relevant to the FY13 PRMRP topic area of Rheumatoid Arthritis (RA). The short-term impact of our research will be an improved understanding of the role of Ras GTPases in shaping, tuning, and regulating the autoimmune T cell response, and the effects of Ras inhibitors on the pathogenesis of the inflammatory arthritis in two relevant preclinical models of RA. We envision that the long-term impact of our proposed research plan will be the introduction of a new class of synthetic drugs, orally available small molecule Rasinhibitors such as Salirasib®, to advance the clinical management of RA patients with conceivably fewer side effects and reduced healthcare system costs compared to biologic drugs.

Abstract Background Ras proteins are vital for normal T cell activation, and downstream effectors of Ras include the MEK/ERK, PI3-kinase/AKT, and NF-kB pathways. T cells from Rheumatoid Arthritis patients exhibit abnormal activation of the Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) blocks the interaction between Ras proteins and their prenyl binding chaperones, attenuating plasma membrane localization and signaling. Objectives To investigate the immunomodulatory effect of FTS alone or combined with methotrexate (MTX) in the DBA/1 mouse collagen type-II induced arthritis (CIA) model. Methods Arthritis was induced in 8–10 week old male DBA/1 mice by immunization with collagen type-II (CII) and complete Freund’s adjuvant (CFA). Animals were treated semi-prophylactically with once daily oral FTS (100 mg/kg); weekly i.p injection of MTX (0.5 mg/kg), FTS combined with MTX, or daily oral vehicle solution (control). Arthritis severity was scored daily from disease onset until study termination and multiple immunological biomarkers of inflammation were analyzed. Results Our data from the mouse CIA model show that the therapeutic efficacy of FTS was similar to MTX, and both drugs significantly reduced arthritis severity compared to CMC controls. Importantly, FTS significantly inhibited the production of pathogenic anti-CII autoantibodies and upregulation of serum IL-6 and IL-17A compared to control arthritic mice. The in depth, multiplex, analysis of the effect of FTS on the T cell cytokine response to CII, revealed strong suppression of IL-22, IL-17, IL-9, GM-CSF and TNF production. Importantly, FTS therapy positively correlated with reduced p-ERK1/2 and p-AKT levels in splenic lymphocytes.

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

Background Ras-GTPases are vital for normal T cell activation, and downstream effectors of Ras include the MEK/ERK, PI3-kinase/AKT, mTOR/p70S6-kinase, and NF-kB pathways. Somatic mutations in NRAS cause an autoimmune lymphoproliferative disorder and T cells from Rheumatoid Arthritis (RA) patients exhibit perturbation of the Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) inhibits the interaction between Ras-GTPases and prenyl-binding chaperones vital for proper plasma membrane localization and downstream signaling [1]. Previous pre-clinical studies suggest that FTS has an immunomodulatory effect in various animal models of autoimmunity [2]. Objectives To test in the Lewis rat adjuvant induced arthritis (AIA) and in the DBA/1 mouse collagen type-II induced arthritis (CIA) models the therapeutic immunomodulatory effect of FTS alone or combined with methotrexate (MTX). Methods Arthritis was induced in 8–12 week old male Lewis rats by complete Freund9s adjuvant (CFA) injection and in male DBA/1 mice by collagen type-II (CII) immunization. Animals were treated prophylactically with once daily oral FTS (100 mg/kg); weekly i.p injection of MTX (0.5 mg/kg), oral FTS combined with MTX, or daily oral vehicle solution (0.5% carboxy methyl cellulose; CMC). Arthritis severity was scored daily from disease onset until study termination. In addition, we measured multiple disease- and drug-related immunological/molecular biomarkers. Results AIA severity was significantly reduced by FTS treatment compared to CMC controls (Figure 1A, P<0.001). Combining FTS and low dose MTX significantly increased its therapeutic efficacy compared to each drug alone (Figure 1A, P<0.05). FTS or FTS+MTX treatment also suppressed the upsurge in serum IL-17 and CRP compared to ailing controls. Global gene expression analysis of relevant splenic CD4+ T cells revealed that FTS is a potent inhibitor of pro-inflammatory and TH17 related gene networks. Next, our data from the mouse CIA model show that the therapeutic efficacy of FTS was non-inferior to MTX and it significantly reduced arthritis severity compared to controls (Figure 2, P<0.001). Importantly, FTS significantly inhibited the production of pathogenic anti-CII autoantibodies and upregulation of serum IL-6 and IL-17A compared to control arthritic mice. The in depth, multiplex, analysis of the effect of FTS on the T cell cytokine response to CII, revealed strong suppression of IL-22, IL-17, IL-9, GM-CSF and TNF production. Noteworthy, FTS therapy positively correlated with reduced Ras-GTP, p-ERK and p-AKT levels in splenic lymphocytes (drug related biomarkers). Conclusions FTS, a first-in-class oral selective Ras-GTPases inhibitor, exhibits a potent immunomodulatory effect in two classical murine model of arthritis, coupled with the inhibition of the TH17 response to relevant arthritogenic-antigens. Thus, Ras-signaling-blockade is a promising novel therapeutic approach for RA. References Kloog Y, Cox AD. Prenyl-binding domains: potential targets for Ras inhibitors and anti-cancer drugs. Semin Cancer Biol. 2004 Aug; 14(4):253–261. Mor A, Aizman E, Chapman J, Kloog Y. Immunomodulatory properties of farnesoids: the new steroids? Curr Med Chem. 2013; 20(10):1218–1224. Disclosure of Interest None declared

The two human lymphotrophic γ-herpesviruses, Kaposi's sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV), are a recognized cause of human cancer, encoding multiple miRs that are major players in carcinogenesis. Previously, we discovered that EBV-encoded miRs transfer between infected B and T lymphocytes. To further explore the biological significance of the spreading of γ-herpesvirus-encoded miRs on carcinogenesis, we focused on KSHV-miR-K12-11 (miR-K12-11) that is unique in having an identical seed sequence with the oncomiR hsa-miR-155, implicated in B cell lymphomas development. Here, we show for the first time that miR-K12-11 transfers in vitro from KSHV-infected BCBL-1 and BC-1 lymphoma lines to T cells. The transferred miR-K12-11 is active in the adopting T cells and binds its canonical target, the 3′-UTR of BACH1. Importantly, we show that the transfer of miR-K12-11 from BCBL-1 to Jurkat cells correlates with inhibition of the innate type-I interferons response to viral dsRNAs downstream of IKKε, a validated miR-K12-11 target. Finally, we show that miR-K12-11 spreading is not reduced by blocking the classical ceramide-dependent exosome secretion pathway. In summary, we report for the first time that intercellular viral oncomiR spreading is an additional mechanism employed by KSHV to inhibit host anti-viral immunity and consequently promote oncogenesis.

Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.

Adenosine deaminase acting on RNA (ADAR) 1 is the master editor of the transcriptome, catalyzing the conversion of adenosine to inosine (A-to-I). RNA transcripts fold into a variety of secondary structures including long intramolecular RNA duplexes that are the major substrate of ADAR1. Most A-to-I editing sites occur within RNA duplexes formed by complementary pairing of inverted retrotransposable elements interspersed within noncoding regions of transcripts. This catalytic activity of ADAR1 most likely prevents the abnormal activation of cytosolic nucleic acid sensors by self-dsRNAs. Homozygous disruption of mouse Adar is embryonic lethal due to a toxic type-I interferons response and correspondingly biallelic missense mutations in human ADAR1 cause a severe congenital interferonopathy. Here, we report that Cd19-Cre-mediated Adar gene ablation in the mouse causes a significant defect in the final stages of B cell development with an almost complete absence of newly formed immature and CD23+ mature recirculating B cells in the BM. Adar ablation in pre-B cells induced upregulation of typical interferon-stimulated genes (ISGs) and apoptosis upon further maturation. ADAR1 deficiency also inhibited the in vitro, IL-7-mediated, differentiation of BM-derived B cell precursors. In summary, ADAR1 is required, non-redundantly, for normal B lymphopoiesis in the BM and peripheral maintenance.

BACKGROUND:A study was designed to identify the source of fever in a patient with post-polycythemia myelofibrosis, associated with clonal Janus Kinase 2 (JAK2) mutation involving duplication of exon 12. The patient presented with 1-2 day long self-limited periodic episodes of high fever that became more frequent as the hematologic disease progressed.METHODS:After ruling out other causes for recurrent fever, analysis of the pyrin encoding Mediterranean fever gene (MEFV) was carried out by Sanger sequencing in peripheral blood DNA samples obtained 4 years apart, in buccal cells, laser dissected kidney tubular cells, and FACS-sorted CD3-positive or depleted mononucleated blood cells. Hematopoeitc cells results were validated by targeted deep sequencing. A Sanger sequence based screen for pathogenic variants of the autoinflammatory genes NLRP3, TNFRSF1A and MVK was also performed.RESULTS:A rare, c.1955G>A, p.Arg652His MEFV gene variant was identified at negligible levels in an early peripheral blood DNA sample, but affected 46 % of the MEFV alleles and was restricted to JAK2-positive, polymorphonuclear and CD3-depleted mononunuclear DNA samples obtained 4 years later, when the patient experienced fever bouts. The patient was also heterozygous for the germ line, non-pathogenic NLRP3 gene variant, p.Q705K. Upon the administration of colchicine, the gold standard treatment for familial Mediterranean fever (FMF), the fever attacks subsided.CONCLUSIONS:This is the first report of non-transmitted, acquired FMF, associated with a JAK2 driven clonal expansion of a somatic MEFV exon 10 mutation. The non-pathogenic germ line NLRP3 p.Q705K mutation possibly played a modifier role on the disease phenotype.

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Anti-CD3 (aCD3) nanoarrays fabricated by self-assembled nanopatterning combined with site-directed protein immobilization techniques represent a novel T cell stimulatory platform that allows tight control over ligand orientation and surface density. Here, we show that activation of primary human CD4+ T cells, defined by CD69 upregulation, IL-2 production and cell proliferation, correlates with aCD3 density on nanoarrays. Immobilization of aCD3 through nanopatterning had two effects: cell activation was significantly higher on these surfaces than on aCD3-coated plastics and allowed unprecedented fine-tuning of T cell response.

Lymphocytes form cell–cell connections by various mechanisms, including intercellular networks through actin-supported long-range plasma membrane (PM) extensions, termed tunneling nanotubes (TNTs). In this study, we tested in vitro whether TNTs form between human antigen-presenting B cells and T cells following cell contact and whether they enable the transfer of PM-associated proteins, such as green fluorescent protein (GFP)-tagged H-Ras (GFP-H-Ras). To address this question, we employed advanced techniques, including cell trapping by optical tweezers and live-cell imaging by 4D spinning-disk confocal microscopy. First, we showed that TNTs can form after optically trapped conjugated B and T cells are being pulled apart. Next, we determined by measuring fluorescence recovery after photobleaching that GFP-H-Ras diffuses freely in the membrane of TNTs that form spontaneously between B and T cells during coculturing. Importantly, by 4D time-lapse imaging, we showed that GFP-H-Ras-enriched PM patches accumulate at the junction between TNTs and the T-cell body and subsequently transfer to the T-cell surface. Furthermore, the PM patches adopted by T cells were enriched for another B-cell-derived transmembrane receptor, CD86. As predicted, the capacity of GFP-H-Ras to transfer between B and T cells, during coculturing, was dependent on its normal post-transcriptional lipidation and consequent PM anchorage. In summary, our data indicate that TNTs connecting B and T cells provide a hitherto undescribed route for the transfer of PM patches containing, for example, H-Ras from B to T cells.

Lymphocytes establish dynamic cell–cell interactions with the cells they scan. Previous studies show that upon cell contact, various membrane-associated proteins, such as Ras-family proteins, transfer from B to T and NK lymphocytes. Mutations in RAS genes that encode constitutively active, GTP-bound, oncoproteins are rather common in human cancers; for instance, melanoma. Cancer immunoediting has been postulated to contribute to the elimination of malignant melanoma. Thus, we asked whether Ras oncoproteins can transfer from melanoma to T cells, including tumor-infiltrating lymphocytes (TILs), and subsequently induce functional effects in the adopting T cells. To explore this issue, we genetically engineered an HLA-A2+ melanoma cell line, MEL526, to express GFP or GFP-tagged H-Ras mutants stably. In this study, we show by an in vitro coculture system that GFP-tagged H-Ras, but not GFP, transfers from MEL526 to T cells and localizes to the inner aspect of their plasma membrane. This cell-contact-dependent process was increased by TCR stimulation and did not require strict Ag specificity. Importantly, we found a positive correlation between the levels of the acquired constitutively active H-RasG12V and ERK1/2 phosphorylation within the adopting TILs. We also show a significant increase in IFN-γ production and cytotoxic activity in TILs that acquired H-RasG12V compared to TILs that acquired a different H-Ras mutant. In conclusion, our findings demonstrate a hitherto unknown phenomenon of intercellular transfer of Ras oncoproteins from melanoma to TILs that consequently augments their effector functions.

Introduction . Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Methods . Here we compared T-cell functions including the number of circulating CD3 + T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. Results . MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. Conclusion . SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9+Vδ2+ (Vγ9+) T cells, in JIA. We found that as opposed to CD4+ T cells, equally high percentages (∼35%) of Vγ9+ T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2+ T cells, similarly amplified cytokine secretion by SF and PB Vγ9+ T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9+ T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9+ T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4+CD25+FOXP3+ cells (regulatory T cells). Furthermore, coculture with the Vγ9+ T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9+ T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.