Immunotherapy has emerged as the new therapeutic frontier of cancer treatment, showing enormous survival benefits in multiple tumor diseases. Although undeniable success has been observed in clinical trials, not all patients respond to treatment. Different concurrent conditions can attenuate or completely abrogate the usefulness of immunotherapy due to the activation of several escape mechanisms. Indeed, the tumor microenvironment has an almost full immunosuppressive profile, creating an obstacle to therapeutic treatment. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) governs a plethora of cellular processes, including maintenance of genomic stability, cell survival/apoptosis, migration, and metabolism. The repertoire of PTEN functions has recently been expanded to include regulation of the tumor microenvironment and immune system, leading to a drastic reevaluation of the canonical paradigm of PTEN action with new potential implications for immunotherapy-based approaches. Understanding the implication of PTEN in cancer immunoediting and immune evasion is crucial to develop new cancer intervention strategies. Recent evidence has shown a double context-dependent role of PTEN in anticancer immunity. Here we summarize the current knowledge of PTEN’s role at a crossroads between tumor and immune compartments, highlighting the most recent findings that are likely to change future clinical practice. PTEN has been identified as a lost or mutated driver gene in numerous sporadic and heritable tumors [Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a major nonredundant, dose-dependent tumor-suppressor gene able to act in both a phosphatidylinositol 3-kinase (PI3K)-dependent and -independent manner . Since ie tumors ,5,6,7,8 e tumors . ConsistIncreasing studies have investigated the potential prognostic and predictive role of PTEN in cancer. However, due to its complex regulation, the mere evaluation of gene mutations is not sufficient to fully uncover the broad range of activity loss status . Indeed,Notably, the repertoire of PTEN functions has recently been expanded to include regulation of the tumor microenvironment and immune system, thus changing the canonical paradigm of PTEN action with new potential implications for immunotherapy-based approaches. Here we summarize the current knowledge of the role of PTEN at a crossroads between tumor and immune compartments.Tumor-associated stromal cells, such as fibroblasts and endothelial cells, cooperate with cancer cells to promote proliferation, invasion, and metastasis to distant sites. The immune system orchestrates a primary protective antitumor response; however, tumors often foster a tolerant microenvironment switch, inducing immunosuppressive signals to reduce this protective mechanism.The basis of tumor-induced anergy has been widely investigated in the last decades, and several studies highlighted the role of T cell unresponsiveness in the early events of tumor progression ,16,17. IThe first evidence of PTEN involvement in immunity emerged in a study correlating its germline deletion with autoimmune disorders , followe+ and CD103+ subpopulations [+ T cells enhanced their helper function, with augmented activation and production of cytokines [The emerging literature suggests a central role of PTEN in both innate and adaptive immunity. Myeloid PTEN-deficient mice exhibited augmented collagen deposition, dysregulation of macrophage polarization, and secretion of proinflammatory and profibrotic factors after induction of pulmonary fibrosis . Myeloidulations . PTEN loytokines .Further studies demonstrated that PTEN controlled cancer cell secretome, avoiding secretion of cytokines, with immunosuppressive potential. In a PTEN-defective melanoma cell line engineered to express PTEN under a tetracycline-responsive promoter, PTEN negatively regulated the expression of the immunosuppressive cytokines interleukin (IL)-10, IL-6, and vascular endothelial growth factor (VEGF) by inhibiting signal transducer and activator of transcription (STAT)3.The addition of conditioned media from established melanoma cell lines and patient-derived short-term melanoma cultures lacking PTEN to monocyte-derived dendritic cells inhibited the ability of these cells to secrete the inflammatory cytokine IL-12. Blockage of IL-10, IL-6 and VEGF by using neutralizing antibodies partially restored IL-12 production, thus confirming the role of these secreted cytokines in mediating PTEN loss-dependent immunosuppression . An analpc−/− mice) developed premalignant prostatic lesions characterized by a strong senescence response that counteracted tumor progression. At this stage, PTEN-null lesions showed a concomitant presence of both senescent and proliferative cells; however, the massive infiltration of MDSCs induced secretion of interleukin-1 receptor antagonist (IL-1RA), which hampered the senescence response, thus sustaining tumor growth [Furthermore, mice bearing PTEN-null senescent prostate tumors displayed a massive tumor infiltration of granulocytic myeloid-derived suppressor cells (MDSCs) recruited in tumor bulk by the unrestricted activation of the Janus kinase (JAK)2/STAT3 pathway and the consequent secretion of chemoattractant molecules. Consistently, treatment with Jak2/Stat3 inhibitors reprogrammed the secreted cytokine network, leading to antitumor immune response and enhanced chemotherapy efficacy . In agrer growth .Moreover, in mouse models of pancreatic ductal adenocarcinoma, PTEN loss influenced the frequency of intratumoral neutrophils, monocytes, and regulatory T cells (Tregs) through activation of NFkB and expression of chemoattractant cytokines such as CXCL1, G-CSF, and IL-23 .Other deletions may also cooperate with PTEN loss to modify the immune compartment of the tumor microenvironment. For instance, combined deletion of PTEN and Zbtb7a in prostate tumors promoted tumor progression through MDSC recruitment and NF-κB signaling activation, whereas compound loss of PTEN and p53 were associated with an immunosuppressive phenotype . A recent preclinical study on gastric cancer models demonstrated that cancer-secreted exosomes were able to deliver specific PTEN-regulating microRNA (miRNA) to MDSCs, inducing their expansion and activation .PTEN loss in fibroblast showed extended gene expression reprogramming and massive remodeling of the tumor microenvironment, with increased extracellular matrix (ECM) deposition, innate immune cell infiltration, and increased angiogenesis [Notably, a bidirectional regulation mechanism by which PTEN may suppress pro-oncogenic secretome and stroma immune tolerance has been postulated; on the contrary, stroma cells may secrete miRNA-containing exosomes to target PTEN expression in cancer cells. This epigenetic downregulation occurs only at specific metastatic sites and is strictly linked to the specific local microenvironment. More specifically, it has been demonstrated in both human samples and mouse models that astrocyte-derived exosomes mediate an intercellular transfer of PTEN-miRNAs to brain metastatic tumor cells to simulate transient PTEN loss status, which in turn leads to CC-motif chemokine ligand 2 (CCL2) secretion with recruitment of ionized calcium-binding adapter molecule 1 (IBA1)-expressing myeloid cells, thus further enhancing metastatic outgrowth . The finogenesis ,36.PTEN is frequently mutated in sporadic cancers as well as hereditary tumor predisposition syndromes, such as PTEN hamartoma tumor syndrome (PHTS), which increases the risk of benign and malignant tumors, including thyroid cancer. A recent work demonstrated that co-culture of macrophages with a PTEN-deficient thyroid cancer cell line induced a strong proinflammatory phenotype compared to PTEN wild-type ones [+ T cells, leading to impaired tumor eradication [ype ones . Furtherdication .These findings indicate that PTEN may also have a critical role in immunity, thus demonstrating a functional link between its activities as a tumor suppression driver gene and tumor immune microenvironment regulator gene .+ FOXP3+ regulatory T cells (Tregs) are a fundamental component of the adaptive immune system, able to mediate contact inhibition of effector T cells and deputed to the release of suppressive cytokines such as TGF-β and IL-10 [+ T cells in Tregs, and several Treg cell functions appear to rely on an intact PTEN/PI3K/AKT pathway [CD4nd IL-10 . Cytokin pathway .+ T cells into Tregs and enhances their suppressive function [Tumor cells may avoid immune surveillance through upregulation of specific proteins deputed to the maintenance of peripheral immune tolerance, such as programmed cell death ligand-1 (PD-L1), which binds the immune receptor programmed cell death-1 (PD-1) on immune cells. The activated downstream pathway leads to inhibition of cytotoxic T lymphocytes and reduction of tumor-infiltrating T cells; on the other hand, PD-1/PD-L1 interaction induces the differentiation of CD4function . This efTregs increase their regulatory function in response to inflammatory stimuli via induction of PTEN expression . ActivatFurthermore, it has been demonstrated that cell contact–dependent potentiation of Treg stability and function is mediated by the interaction between semaphorin-4a, expressed on T effector cells and dendritic cells, and NRP-1, expressed at high levels on Tregs, via activation of the PTEN axis .+CD11b+ myeloid dendritic cells, which expressed more CD86 and less PD-L1 [Indoleamine 2,3-dioxigenase (IDO) is an enzyme involved in immunosuppressive metabolism, catalyzing the rate-limiting step of L-tryptophan (Trp) conversion into L-kynurenine (Kyn) . It exerss PD-L1 .Therefore, PTEN may act in two opposite ways: as a powerful tumor suppressor if expressed in tumor cells, and as an immune suppressor and tumor escape enhancer if expressed in Tregs .+CD103+ myeloid response and elicited no FOXO3a+PD-1+ Treg recruitment. Similar results were obtained with pharmacological PTEN inhibition [Genetically modified mice with specific deletion of PTEN in Tregs (PTENTreg-KO mice) showed slow melanoma and Lewis lung carcinoma (LLC) tumor growth and a high grade of inflammation and were not able to create an immunosuppressive tumor microenvironment. Moreover, injection of apoptotic cells in PTENTreg-KO tumor-free mice induced an inflammatory CD11bhibition .PTEN acts as a negative regulator of IL-2-mediated expansion of Tregs. Indeed, Treg cells lacking PTEN were increased in number . FurtherOverall, these findings demonstrate that PTEN is highly expressed in Tregs and this is essential to retain the characteristic immunosuppressive phenotype. In conflict with these data, it has been shown that mice with a deletion of PTEN in the T cell compartment developed normal Tregs that responded to IL-2 proliferative stimulation while retaining their ability to suppress effector T cells . Moreove+ Treg population. In melanoma and LLC mouse models, VO-OHpic in combination with low doses of chemotherapy showed a marked synergistic antitumor effect with rapid immune system activation [In recent years, PTEN inhibitor drugs have been developed and evaluated for their neuroprotective and proregeneration properties, and subsequently for their anticancer function . The orttivation ,58. In eRegarding the “tumor side” of the problem, recent studies have proposed new interesting strategies to reactivate antitumor PTEN function in tumor cells. For instance, the natural compound indole-3-carbinol has been proposed as a potential therapeutic strategy for cancer prevention and treatment thanks to its induction of PTEN reactivation . Indole-Another proposed strategy to restore functional PTEN is cell-mediated therapy, in which specific cells with tumor-homing ability, such as neural stem cells for glioblastoma, are genetically modified to express a particular protein with therapeutic potential. An alternative PTEN isoform produced by translation from an alternative upstream start codon called PTEN long (PTEN-L) has been described. Unlike conventional PTEN, this isoform can be secreted and then captured by neighboring cells in the intracellular compartment thanks to a specific signal sequence. An engineered version of PTEN-L with leader sequence from human light-chain immunoglobulin G has been proposed to arm neural stem cells. Interestingly, this modified PTEN-L showed enhanced secretion capacity and an increased propensity to be transferred between cells . FinallyImmunotherapy springs from the primary need to revert T cell tolerance toward tumor antigens. Much effort has been invested in target discovery and immunomodulating strategies, and today cancer immunotherapy represents one of the major breakthroughs of cancer treatment.The landscape of immunotherapy approaches comprises a plethora of targeting strategies, such as tumor-targeting monoclonal antibodies, adoptive cell transfers, oncolytic viruses, cancer vaccines, dendritic cell-based immunotherapies, immunostimulatory cytokines, immunomodulatory monoclonal antibodies, immunosuppressive metabolism inhibitors, pattern recognition receptor agonists, and immunogenic cell death inducers . Among tSimilarly, the PD-1 blockade restarts an effective antitumor immune response and reverses Treg-mediated suppression of effector T cells. In physiological conditions, PD-1 activation inhibits phosphorylation of TCR downstream molecules, thus reducing TCR signaling after extensive activation. In pathological conditions, constitutive hyperactivation of PD-1 increases the number of exhausted T cells, resulting in altered functionality and poor control of infections and tumors ,76. CTLASince the first success of the anti-CTLA-4 ipilimumab in metastatic melanoma, several practice-changing clinical studies have established the usefulness of such treatments in many solid tumor types ,78. ActuThe tumor microenvironment has a nearly full immunosilencing profile, creating a main obstacle to immunotherapy treatment combined with immunogenic chemoradiotherapy. Indeed, although a massive release of tumor antigens occurs following cytotoxic treatment, the recognition machinery frequently fails to cross-present them in an immunogenic fashion.Studies in the literature report a different context-related role of PTEN in immunity according to cell type. Indeed, the PTEN pathway in Tregs drives stabilization of the suppressive immune cell population and tolerance toward apoptotic cells, thus revealing Treg-specific PTEN targeting as a previously unsuspected strategy for tumor treatment. Despite its pro-tumor immunosuppressive role in Tregs, specific loss of PTEN in cancer cells has been linked to reduction of antitumor immunity, with induction of PD-L1 expression on glioma cells through a translational regulation mechanism . SimilarConsistently, activation of the PI3K-AKT-S6K1 pathway induces immune escape and apoptosis of activated T cells, impairing tumor-specific T cell killing through upregulation of PD-L1 on colorectal , lung 8, renal , breast,Interestingly, a metastatic non-small-cell lung cancer (NSCLC) case harboring PTEN mutation, PD-L1 positivity, and high tumor mutation burden showed a lack of durable response to PD-1 inhibitor . An analMoreover, Peng et al. demonstrated that PTEN knockout in melanoma cells determined protection from cell lysis when co-cultured in vitro with tumor-reactive T cells and decreased T cell trafficking in tumor bulk in adoptive T cell therapy mouse models . More imThese findings led to the design of a Phase I/II clinical trial that will test a combination of immunotherapy and targeted therapy with a selective PI3K-beta inhibitor in patients with metastatic melanoma who lack the PTEN gene (NCT03131908).A recent analysis of whole-exome sequencing data of 110 pretreatment melanoma tumor biopsies with matching germline tissue samples and RNA-seq data from a subset of 42 patients revealed that a lower burden of copy number loss was significantly associated with clinical benefit to CTLA-4 blockade. Among the regions associated with recurrent copy number loss, chromosome 10q emerges as being rich in tumor suppressor genes such as PTEN. In a further analysis, the authors highlighted a closer relationship between PTEN loss and CTLA-4 blockade response with an odds of resistance 5.58 times greater than PTEN proficient tumors .Consistent with these observations, a case study reported that in a patient with metastatic uterine leiomyosarcoma who had an exceptional response to anti-PD-1 pembrolizumab, PTEN was revealed as the principal actor of anti-PD-1 checkpoint blockade response . The autA deconvolution analysis of genome-wide DNA methylation data from the complex cellular mixtures of a wide spectrum of solid cancers demonstrated the existence of “immune hot” and “immune cold” tumors with distinctive prognoses, genomic alterations, cytokine pathway activation, and oncogenic drivers . Moreoven = 45) showed that PTEN somatic mutations were significantly associated with immunosuppressive expression signatures in non-responder tumors, suggesting that PTEN may play a role in the establishment of a specific tumor-immune microenvironment. Indeed, PTEN mutation was found in 23 out of 32 non-responders and only three out of 13 responders. Conversely, mutations in the mitogen-activated protein kinase (MAPK) pathway are significantly enriched in responders. Of note, the immunosuppressive signature was most associated with the tumor subpopulation expressing the migration marker CD44 in PTEN-mutated tumors rather than Tregs, revealing a central role of PTEN-mutated tumor cells in immune regulation. To further explore the connection between PTEN and immunological features, the authors examined RNA-sequencing data from 172 TCGA samples, demonstrating a significant correlation between PTEN mutation and FOXP3-related transcriptional signature, together with a higher percentage of macrophages, microglia, and neutrophils in the tumor microenvironment. Finally, quantitative multiplex immunofluorescence of matched pre- and post-anti-PD-1 treatment samples showed that PTEN-mutated tumors had a significantly higher level of CD68+HLA−DR− macrophages, which was previously linked to poor survival in melanoma [+ T cells were enriched only in PTEN wild-type tumor, while PTEN-mutated tumors displayed reduced immune infiltration. The authors speculate that this phenomenon may be due to the changing spatial structure in PTEN-mutant glioblastoma, characterized by increased clustering of tumor cells, which physically hampers immune infiltration. More interestingly, immunosuppression gene sets were elevated in non-responders before immunotherapy, but also in responders following immunotherapy, highlighting the fundamental concept of primary resistance in non-responder tumors, probably led by PTEN mutation, and acquired resistance in initial responders under treatment selection pressure [Consistent with these observations, PTEN loss of function seems to be the driver of resistance to anti-PD-1 checkpoint blockade also in glioblastoma . In detamelanoma . In postpressure . A stillAnother study on primary human glioblastoma cell lines derived from resected patients and co-cultured with matched autologous T cells demonstrated high T cell apoptosis upon contact with PTEN-deficient cancer cells, indicating that PTEN-deficient glioblastoma patients are suboptimal candidates for immunotherapy. Pretreatment of these tumor cell lines with a specific PI3K inhibitor reverses the massive apoptotic effect, suggesting the future exploration of immunotherapy in combination with drugs targeting the PI3K/AKT/mTOR pathway . SimilarAdoptive cell transfer therapy (ACT) is a promising cellular immunotherapy approach based on administering ex vivo expanded tumor-specific immune cells to cancer patients. Different cell types can be administered for ACT, such as chimeric antigen receptor (CAR) T cells, TCR transduced T cells, natural killer (NK) cells, and TILs. There are currently two CAR T cell therapies that are approved by the FDA for the treatment of pediatric patients and young adults with refractory or relapsing B cell precursor acute lymphoblastic leukemia and adult patients with refractory or relapsing large B cell lymphoma. Despite encouraging clinical benefits of ACT, a main obstacle for long-lasting therapeutic effects is the insufficient persistence of immune cells after adoptive transfer. Of note, constitutive activation of PI3K impairs immunity response, promoting the development of short-lived terminally differentiated effector T cells at the expense of long-lived memory T cells . ConversClinical trials are currently under way to investigate a potential connection between the PTEN pathway and immunotherapy. An ongoing Phase I study of targeted therapy plus anti-PD-L1 durvalumab in patients with advanced or metastatic solid tumors (MEDIPAC) is evaluating the relationship between mutations in the AKT/PIK3CA/PTEN pathway and response to combination therapy (NCT03772561). Another recent Phase Ib study (not yet recruiting) aims to evaluate the safety and antitumor activity of targeted therapy in combination with durvalumab in patients with advanced solid tumors selected for specific molecular alterations, including PTEN mutation (NCT03842228).Moreover, several studies are investigating the feasibility and antitumor effect of PI3K-targeting drugs in combination with immunotherapy in patients with metastatic melanoma who lack the PTEN gene (NCT03131908), colorectal cancer (NCT03711058), large B cell lymphoma (NCT03484819), lung cancer (NCT03257722), breast cancer, and gynecologic malignancies (NCT03719326). Currently ongoing clinical trials are schematically summarized in PTEN is a well-known tumor suppressor gene able to block the proto-oncogenic PI3K/AKT pathway in tumor cells. Notably, the repertoire of PTEN functions has been expanded to include regulation of the tumor microenvironment and the immune system .Moreover, a controversial function has been reported in Treg-associated PTEN expression with the growing idea of a new paradoxical pro-tumor-like skill. This evidence suggests a double context-dependent role of PTEN according to the specific cell type. Indeed, PTEN expression is crucial to prevent malignant transformation in normal and precancerous cells, and its loss both drives cancer progression and induces a cascade of events that influence the microenvironment toward an immunosuppressive profile. Contrary to its normal role as a tumor suppressor, PTEN-specific expression in Tregs stabilizes these cells, preventing them from switching to an inflamed T helper-like differentiation, thus contributing to the creation of a Treg-dependent immune-suppressive milieu, which finally allows tumor immune escape and survival. Conversely, Treg decrement causes a deep change of tumor microenvironment, with the acquisition of strong immunogenic features that would be suitable for immunotherapy efficacy. A constitutively high level of PTEN protein has been shown in tumor-associated Treg but not in effector T cells. Specifically, it has been demonstrated that Treg cells require PTEN to sustain their suppressive function, supporting the expression of IL-2Rα, a negative modulator of local immune response, and FOXP3 transcription factor. Indeed, mice bearing Treg-specific deletion of PTEN lose Treg homeostasis and stability, gain Th1 response and B cell activation, and are prone to developing autoimmune disease ,62. On tGiven the central role of PTEN in tumor growth and antitumor immune response, interesting approaches have been proposed to target PTEN expression in Tregs or induce its expression in tumor cells. However, the therapeutic potential of each PTEN-modulating approach may be mitigated by the counterpart side effects, raising important concerns. Indeed, systemic administration of PTEN-inhibiting drugs could hit not only PTEN activity in Tregs, but also residual PTEN function in tumor and stroma, with potential pro-tumor consequences. Conversely, enhancing PTEN function could reduce tumor growth while increasing the stability of the immunosuppressive Treg population. A possible strategy to overcome the problems and increase the feasibility of these approaches could be to use more selective modulating strategies or to explore complementary two-step double hit approaches to induce PTEN depletion followed by cell-specific restoration of its pathway. Furthermore, interfering with AKT downstream pathway in adoptively transferred T cells seems to be a promising strategy to promote features of memory T cells and a long-lasting anti-tumor effect.In recent years, the emerging field of oncoimmunology has fostered an increased understanding of cancer biology, the tumor-immune interrelationship, and the fundamental balance between pro- and antitumor forces. Strong efforts in this direction have guaranteed the development of several immune-modulating strategies able to induce a lasting response in cancer patients. However, different concurrent conditions can attenuate or completely abrogate the usefulness of immunotherapy due to numerous escape mechanisms, such as a lack of immune cell infiltration, poor antigen expression or presentation, tumor-mediated immune suppression, and release of immunomodulatory cytokines. PTEN seems to play a pivotal, context-dependent role in antitumor immune regulation and thus needs to be further investigated.


Allele-specific transcriptional regulation, including of imprinted genes, is essential for normal mammalian development. While the regulatory regions controlling imprinted genes are associated with DNA methylation (DNAme) and specific histone modifications, the interplay between transcription and these epigenetic marks at allelic resolution is typically not investigated genome-wide due to a lack of bioinformatic packages that can process and integrate multiple epigenomic datasets with allelic resolution. In addition, existing ad-hoc software only consider SNVs for allele-specific read discovery. This limitation omits potentially informative INDELs, which constitute about one fifth of the number of SNVs in mice, and introduces a systematic reference bias in allele-specific analyses.Here, we describe MEA, an INDEL-aware Methylomic and Epigenomic Allele-specific analysis pipeline which enables user-friendly data exploration, visualization and interpretation of allelic imbalance. Applying MEA to mouse embryonic datasets yields robust allele-specific DNAme maps and low reference bias. We validate allele-specific DNAme at known differentially methylated regions and show that automated integration of such methylation data with RNA- and ChIP-seq datasets yields an intuitive, multidimensional view of allelic gene regulation. MEA uncovers numerous novel dynamically methylated loci, highlighting the sensitivity of our pipeline. Furthermore, processing and visualization of epigenomic datasets from human brain reveals the expected allele-specific enrichment of H3K27ac and DNAme at imprinted as well as novel monoallelically expressed genes, highlighting MEA’s utility for integrating human datasets of distinct provenance for genome-wide analysis of allelic phenomena.https://github.com/julienrichardalbert/MEA.Our novel pipeline for standardized allele-specific processing and visualization of disparate epigenomic and methylomic datasets enables rapid analysis and navigation with allelic resolution. MEA is freely available as a Docker container at The online version of this article (10.1186/s12864-018-4835-2) contains supplementary material, which is available to authorized users. BWA and Bowtie2 allele-specific alignment results. (XLSX 49 kb)Additional file 3: Table S2. Human RNAseq, ChIPseq and WGBS allele-specific alignment results. (TXT 6957 kb)Additional file 4:Table S3. List of datasets used in this study and their source. (XLSX 11 kb)


Cancer immunotherapy aims to utilize the host immune system to kill cancer cells. Recent representative immunotherapies include T-cell transfer therapies, such as chimeric antigen receptor T cell therapy, antibody-based immunomodulator therapies, such as immune checkpoint blockade therapy, and cytokine therapies. Recently developed therapies leveraging engineered cells for immunotherapy against cancers have been reported to enhance antitumor efficacy while reducing side effects. Such therapies range from biologically, chemically and physically -engineered cells to bioinspired and biomimetic nanomedicines. In this review, advances of engineering cells for cancer immunotherapy are summarized, and prospects of this field are discussed. Immunotherapies that either induce or inhibit the host immune response have been used in the clinical treatment of many diseases including infections ®) is the first cell-based therapeutic vaccine approved by the U.S. Food and Drug Administration (FDA) to treat prostate cancer ex vivo expanded TILs ®, the first CAR-T cell therapy approved by the FDA, has demonstrated effective clinical therapeutic outcomes for the treatment of refractory or recrudescent B-cell precursor acute lymphoblastic leukemia Cancer immunotherapy, also referred to as immuno-oncology, aims to induce the immune system of host to identify and eliminate cancers Figure 1) To this end, many novel strategies from the perspectives of drug discovery and drug delivery have been developed. Among them, cell-based drug delivery systems provide a promising platform to enhance delivery efficiency, increase therapeutic efficacy, and reduce off-target and side effects of cancer immunotherapy. By utilizing recent advances in immunotechnology, micro/nanotechnology and molecular pharmaceutics, such cellular systems range from biologically, chemically, and physically-engineered cells to bioinspired and biomimetic nanomedicines /interleukin-2 (IL-2) and cytotoxic proteins, such as perforin/granzyme TCRs are a characteristic biomolecule of T cells, and consist of et al. developed TCR-T cells capable of specifically binding to Wilms tumor 1 (WT1) peptide, a specific epitope on leukemic cells of acute leukemia and myelodysplastic syndrome  ex vivo peptide-specific immune reactivity in the peripheral blood of patients. Hematopoietic function recovery was observed in 40% of patients after treatment. Additionally, TCR-T therapy can also be used for treatment of solid tumors such as melanoma et al. identified that the tumor-associated antigen, preferentially expressed antigen in melanoma (PRAME) was a specific epitope on medulloblastoma cells correlated with poor overall survival in vitro and in vivo anticancer activities were observed after treatment with the PRAME-specific TCR-T cells. Meanwhile, lower toxicity of these TCR-T cells introduced with an inducible caspase 9 gene was observed compared with the untransduced control T cells TCR-T therapy is often utilized as a therapy for hematological malignancies ζ-chain of the CD3 complex and the γ-chain of the high-affinity receptor for immunoglobulin E (FcεRI) et al. constructed an anti-CD19 CAR molecule (CD19-BBz(86)) with intracellular 4-1BB co-stimulatory and CD3ζ signaling domains Recently, two FDA-approved CAR-T cell-based therapies, Kymriah and Yescarta are being utilized for the treatment of patients with acute lymphoblastic leukemia and non-Hodgkin lymphoma, respectively ex vivo expansion of T cells is limited by low efficiency and limited functionality of the T cell products. Cheung et al. developed APC-mimic scaffolds (APC-ms) composed of lipid membrane-coated mesoporous silica micro-rods in vivo. Due to costly and time-consuming processes of ex vivo preparation of CAR-T cells, in situ programming of T cells with nanoparticles was proposed by Smith et al.89. The CAR gene-encoded plasmid DNA was mixed with a cationic polymer to form nanosized complexes, followed by modification with the T-cell-targeting anti-CD3e f(ab')2 fragments that mediate endocytosis by lymphocytes. When administered to the mice bearing B-cell acute lymphoblastic leukemia, the nanoparticles programmed the circulating T cells and induced tumor regression equivalent to the traditional CAR-T cell therapy.Efficient activation and expansion of T cells is of the essence in enhancing immunotherapy. The use of commercial expansion beads (Dynabeads) for et al. developed lymph node-targeted amphiphile CAR-T cell ligands (amph-ligands) to directly promote donor cells via their chimeric receptor in vivo for enhanced efficacy of the CAR-T cell therapy against solid tumors  et al. developed CAR-T cells expressing both IL-7 and CCL19, which could significantly augment the DC and T cell infiltration into the solid tumor compared with the traditional counterpart without cytokine expression et al. applied photothermal pre-treatment to disrupt extracellular matrix (ECM) for enhanced tumor penetration of CAR-T cells  co-glycolic) acid (PLGA) nanoparticles, which were intratumorally injected into the tumor tissue. Upon light irradiation, mild heating generated by the ICG-loaded nanoparticles resulted in the disruption of the ECM followed by decreased interstitial fluid pressure and increased blood perfusion. This photothermal pre-treatment significantly improved the tumor penetration of subsequent intravenously-injected CAR-T cells, leading to enhanced antitumor efficacy in the solid tumors. Cho et al. reported that addition of a pair of leucine zippers between the scFv and the intracellular domain controlled the recognition of different antigens by T cells by altering the structure of the leucine zipper-scFv, thereby increasing the functionality of T cells Although effective in treating hematological malignancies, the utilization of CAR-T cells for the treatment of solid tumors is more challenging, which is due in part to limited expansion, poor penetration, and decreased viability of administered CAR-T cells. Recently, Ma via membrane protein, such as natural killer group 2 member D (NKG2D) receptor or DNAX accessory molecule-1 (DNAM-1) et al. genetically modified NK-92MI cells with a CAR containing anti-CD138 fragment, which showed significantly enhanced cytotoxicity toward the CD138-positive multiple myeloma cells compared with the CD138-negative counterparts et al. developed CS1-specific CAR-expressing NK cells for immunotherapy of multiple myeloma In addition to T and B cells, NK cells are another type of lymphocyte, which are critical to the innate immune system and defend the human body against cancer. By secreting cytokines, NK cells regulate immune response and promote maturation of APCs et al. engineered platelets decorated with PD1 for preventing post-surgical cancer recurrence  in vitro large-scale production of platelets were genetically modified using lentivirus encoding PD1. The obtained PD1-presenting MKs was able to generate mature platelets with PD1, which increased the accumulation of PD1 to surgical wounds by relying on the physiological function of platelets as monitors of vascular injury. PD1 was released via platelet-derived microparticles from cell membranes upon platelet activation and blocked PDL1 on tumor cells to reinvigorate the exhausted CD8+ T lymphocytes. Cyclophosphamide (CP), an immunosuppressant, was simultaneously delivered by the PD1-expressing platelets to exhaust the regulatory T cells (Tregs) and enhance the cytotoxic effects of the CD8+ T cells. Xue et al. conjugated granulocyte macrophage-colony stimulating factor (GM-CSF) mRNAs to polypeptide linker covalently to construct fusion gene GC2A, which was further inserted into the adenovirus vector to transfect human embryonic kidney 293T (HEK293T) cells Zhang in vitro, followed by reinfusion to the body. Depending on the phagocytosed substances, the representative approaches include vaccine endocytosis for antigen presentation and nanoparticle endocytosis for drug delivery Endocytosis is an approach for cell engineering in which autologous cells are treated with proteins, drugs or nanoparticles Figure 5) + and CD8+ T cells. The CD8+ T cells induce cell lysis of the prostate tumor cells with the help of cytokines secreted by CD4+ T cells. Sipuleucel-T showed prolonged median survival of 4.1 months with mild side effects Research of cancer vaccines reached a milestone with the development of Sipuleucel-T for treating patients with metastatic castration-resistant prostate cancer in 2010  Like fusion proteins, nanoparticles can also be internalized by cells to construct a cell-based drug delivery system to combine the advantages of nanoparticles and cells for augmented immunotherapeutic efficacy et al. developed platelets conjugated with anti-PDL1 (P-aPDL1) that could travel to the surgical site in order to inhibit post-surgical tumor recurrence et al. applied P-aPDL1 to inhibit tumor relapse and metastasis after thermal ablation Endocytosis may involve concerns about the instability of nanoparticles and unexpected release of drugs by endocytic vesicle-mediated degradation et al. reported nanoparticle-conjugated T cells for enhanced targeting of chemotherapeutics to disseminated tumor cells in lymph nodes et al. developed a cytokine nanogel-decorated T cell-mediated delivery system for enhanced T cell function and cancer immunotherapy  N-hydroxy succinimide crosslinker to form a “carrier-free” protein nanogel, which was later decorated with poly(ethylene glycol)-co-poly(lysine) (PEG-b-PLL) and anti-CD45 antibodies. The cationic PEG-b-PLL polymer facilitated electrostatic absorption of nanogels onto T cells, while anti-CD45 antibody increased cell surface retention of nanogels by preventing internalization. Elevation of surface reduction potential of T cells following antigen recognition in lymph node and tumor tissues led to nanogel collapse and IL-15 release. This controlled manner brought about an 8-fold higher maximum tolerated dosage of IL-15 and a 16-fold amplification of T cells. The mouse T cells and human CAR-T cells backpacking the nanogels with large quantities of IL-15 presented superior antitumor effects in mouse melanoma and glioblastoma models. Apart from IL-15, similar strategy was adopted for delivery of IL-2 to overcome the IL-2-induced vascular leak syndrome. A sustained and slow release of IL-2 was achieved by the IL-2 nanogel backpacks, leading to more CD8+ memory precursor differentiation and less T-cell exhaustion compared with free IL-2 Cellular backpacks using nanoparticles without being internalized also hold promise of targeted drug delivery and enhanced cancer immunotherapy, which integrates the respective merits of nanoparticles as drug depots and cells with natural directional migration potency as active carriage et al. conjugated anti-PD1 antibody (aPD1)-decorated platelets to hematopoietic stem cells (HSCs) to enhance the delivery of aPD1 to the leukemia site in order to inhibit leukemia growth and relapse  In addition to modification with antibodies or nanoparticles for cell engineering, Hu et al. used cationic polymers to condense the DNA plasmid encoding VEGFR2 and Salmonella, a low-cost live attenuated bacteria as a carrier for oral delivery of DNA-based vaccines  + and CD8+ T cells to eradicate the VEGFR2-expressing tumor vascular endothelial cells. This bacteria-mediated DNA-based vaccine revealed higher effect in inhibiting tumor growth due to increased angiogenesis suppression and tumor necrosis.Non-covalent physical modification is a convenient strategy for engineering cells, often enabling high activity of cargoes linked to the cellular carriers compared to covalent bioconjugation. Hu et al. prepared nanovaccines with notable antivirulence efficacy by absorbing intact pore-forming toxins on RBC membrane-coated nanoparticles, which laid a foundation for new vaccine design et al. reported a RBC membrane-based core-shell drug delivery system for melanoma immunotherapy  via mannose receptor-mediated interaction, in which hgp100 was released due to the high intracellular level of glutathione et al. prepared NK cell membrane-camouflaged nanoparticles (NK-NPs) by disguising photosensitizer-loaded nanoparticles with the NK cell membranes  via the receptor-mediated interaction. Upon near-infrared (NIR) irradiation, a build-up of ROS generated by tetra(4-carboxyphenyl)porphine (TCPP) as a photosensitizer induced apoptosis of tumor cells. Antigens on dead tumor cells were presented to the T cells by APCs, which activated the T cells to kill remnant tumor cells. On the other hand, proteins on the NK cell membranes stimulated M1-type polarization of macrophages, which further secreted pro-inflammation cytokines to activate APCs for a durable immune response. Xie et al. reported cancer cell membrane-coated glucose oxidase-loaded mesoporous silica nanoparticles (MSNs) for cancer immunotherapy via DCs, which were activated by the proteins presented on the cancer cell membrane. This starvation strategy combined with immunotherapy resulted in a synergistic anti-tumor effect. Apart from coating nanoparticles with one kind of cell membrane, mixed cell membrane decorated nanoparticles were also investigated to enhance immune response. Liu et al. prepared a cytomembrane consisting of fused cells from DCs and cancer cells to coat nanoparticles (NP@FM). NP@FM presented whole tumor antigens from DC membrane and endogenous tumor antigens from cancer cell membrane, and enhanced the activation of T cells In addition to living cells, cell membranes have been widely used to develop biomimetic drug delivery systems. Preserving cell membrane integrity and a significant portion of membrane proteins facilitates protecting drugs from degradation or activating an immune response et al. transfected HEK293 cells with the DNA plasmid encoding three different proteins: CD3 antibody for T cell recognition, endothelial growth factor receptor (EGFR) antibody for breast cancer cell targeting and membrane protein for surface anchoring  In vitro cell experiments verified the T cell activation ability of SMART-Exo and in vivo studies revealed the engineered exosomes significantly suppressed tumor growth in the xenograft TNBC mouse models. Morishita et al. isolated murine melanoma cell-derived exosomes that contain cancer cell antigens and can activate APCs to provoke antitumor immunity via the biotin-streptavidin interaction. The functionalized exosomes exhibited superior antitumor efficacy in tumor-bearing mice in comparison to co-administration of exosomes and CpG DNA.Exosomes, which are membrane vesicles with a size of 30-100 nm derived from various cell types and resemble cell membranes, are vital for cell communication and take along a collection of biological information from the donor cells In order to enhance the efficacy and reduce the adverse effects of cancer immunotherapy, cell-based immunotherapies have attracted considerable attention today, mainly because of their potential biocompatibility and dynamic physiological functions involving immune interactions. In addition to genetically engineered cell therapies such as CAR-T and TCR-T cell therapies, other kinds of cell-based delivery systems have also been utilized for cancer immunotherapy. Augmenting the antitumor immune response and overcoming immunosuppression are the two main goals for cancer immunotherapy. The combination of immunotherapeutic drugs with cell-based delivery systems can potentially enhance the efficiency or efficacy of cancer immunotherapy, mainly reflects in protecting the drug from unexpected degradation, increasing accumulation in target sites, such as tumor and lymph node and/or boosting the immune response and overcoming the tumor immunosuppression to combat with tumor growth, recurrence or metastasis.in vivo. Strict criteria for quality control should be established in order to achieve reproducible engineering approaches with quality assurance.Although remarkable advantages have been validated when combining immunotherapy with cells or cell-derived delivery systems, many issues should be taken into account for accelerating clinical translation. For example, preparation processes of these engineered cells or cell-based delivery systems are often complicated


Converging behavioral and functional neuroimaging evidence indicates that East Asian and Western individuals have different orientations for processing information that may stem from contrasting cultural values. In this cross-cultural magnetic resonance imaging (MRI) study, we used voxel-based morphometry (VBM) approach to investigate culture-related and individual differences of independent-interdependent orientation in structural brain volume between 57 Taiwanese and 56 Western participants. Each participant’s degree of endorsement of independent and interdependent cultural value was assessed by their self-report on the Singelis Self-Construal Scale (SCS). Behaviorally, Taiwanese rated higher SCS scores than Westerners in interdependent value and Westerners rated higher SCS scores than Taiwanese in independent value. The VBM results demonstrated that Western participants showed greater gray matter (GM) volume in the fronto-parietal network, whereas Taiwanese participants showed greater regional volume in temporal and occipital regions. Our findings provide supportive evidence that socio-cultural experiences of learned independent-interdependent orientations may play a role in regional brain volumes. However, strategic differences in cognition, genetic variation, and/or modulations of other environmental factors should also be considered to interpret such culture-related effects and potential individual differences. In contrast, individuals with an interdependent, collectivistic, and group-based focus of their sociocultural values are more sensitive to contextual information of the environment  and to relations among people and social harmony  who were born and raised in Taiwan. Fifty-six participants were WesternersThere is cultural psychological evidence demonstrating that nationality  and cultural affiliation  may not necessarily be reliable predictors of independent and interdependent cultural values  − (mean of agreement for interdependent items)]  with a 20-channel head coil at the Taiwan Mind and Brain Imaging Center in National Chengchi University, Taipei, Taiwan was used for all MRI data acquisition. High-resolution, three-dimensional, sagittal-oriented, T1-weighted anatomical images were collected using a three-dimensional (3D) ultrafast magnetization-prepared rapid acquisition with gradient echo (MPRAGE) imaging sequence with the following parameters: FOV: 256 × 256 mmAll MRI data were preprocessed and analyzed in Statistical Parametric Mapping SPM12. The VBME = 0.5 and H = 2. We consequently established a conservative family-wise error (FWE)-corrected threshold of p < 0.05.Two major statistical analyses were performed in this cross-cultural VBM study. To examine culture-related differences in regional gray-matter volumes, the voxel-wise generalized linear modeling (GLM) was applied within the MNI space mask across the whole brain. Voxel-wise parametric statistical tests, which directly compare the smoothed MRI images from two cultural groups, was then performed to identify specific regions in which GM areas showed significantly volumetric differences between Taiwanese and Westerners participants, controlling for global intracranial volume (ICV)p < 0.05.To investigate the relationship between individual differences in independence-interdependence orientations and cross-culture gray-matter volume, we then performed the multiple regression analyses by using the TFCE to identify specific brain regions in which gray-matter correlated with the SCS scores and the two subscale scores  separately across the whole brain, controlling for global ICV, age, and gender. Statistical maps were assessed at FWE-corrected threshold of p = 0.00000523). There were no significant differences in age (p = 0.50) and educational levels (p = 0.91) between Taiwanese and Westerners.p < 0.05. The Taiwanese participants showed greater regional gray-matter volume in left and right middle temporal gyrus, right inferior temporal gyrus, left middle occipital gyrus, right calcarine sulcus, and right superior occipital gyrus. In contrast, the Western participants showed greater volume in frontal-parietal network, including left superior medial frontal gyrus , left superior frontal gyrus (superior PFC), and left postcentral gyrus. Significant culture-related differences in brain regions between Taiwanese and Western groups are illustrated in To examine culture-related differences in regional gray-matter volume, we first performed a whole-brain VBM analysis to identify the significant differences in the gray-matter volume of brain regions between two cultural groups using TFCE method with FWE-corrected threshold of p < 0.05 to identify specific brain regions whose volume is predicted by the SCS scores, adjusted for global ICV, age, and gender. However, no cluster was found with a significant positive or negative correlation with regional gray-matter volume for the SCS score, independence subscale score and interdependence subscale score. Additionally, when an extent-threshold  and larger frontal and parietal regions, and cerebellum for individuals from a Western culture. Taiwanese participants showed significantly higher SCS scores than Westerners in interdependent cultural value, whereas Westerners rated higher SCS scores than Taiwanese in independent cultural value. However, individuals with the spectrum of independent-interdependent orientation did not show positive or negative significant association between regional gray-matter volume and the SCS scores. Our findings provide neuroimaging evidence that culture-related experience may influence regional brain structures.Culture-related structural differences in regional brain volume observed in our results between Taiwanese and Westerners are consistent with previous structural neuroimaging findings using various data analysis methods, including VBM, cortical thickness measures, and pattern classification approaches (Chee et al., Moreover, the similar results that Western participants showed greater volume in frontal regions, including left medial frontal gyrus and left superior frontal gyrus compared with Taiwanese participants have been reported in previous structural neuroimaging studies (Chee et al., The effect of culture reported in this study, however, is dissimilar to some of the previous studies (Kochunov et al., p < 0.05 was applied in this study. We employed the TFCE method to correct for multiple comparisons, which is different from the previous studies using either an extent-threshold (Wang et al., p < 0.005 and cluster-extent threshold of FWE p < 0.05 used in Wang et al. (p < 0.05 used in Kitayama et al. (There is no positive or negative statistically significant associations between regional gray-matter volume and the SCS scores when TFCE method with FWE-corrected threshold of g et al.  and a voa et al.  were fura et al. . Given tDespite the similarities between the previous and the current MRI studies, however, we should note that there are several discrepancies between studies that may be driven by other unknown genetic diversity, environmental biases, and/or gene-environment interaction across multiple time scales (Chee et al., This study was approved by the National Taiwan University Institutional Review Board and all participants gave informed consent prior to their participations.C-MH, RD, CW, H-WH and Y-PC contributed to the conception and experiment design. C-MH, RD, CW, H-WH and Y-PC contributed to preparation and revision of the manuscript. C-MH, CW and Y-PC contributed to data analysis. All authors reviewed and approved the manuscript.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.


Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores – non-bilaterian basal animals – typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected.Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported.We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.The online version of this article (10.1186/s12862-019-1418-z) contains supplementary material, which is available to authorized users. The origin of animal multicellularity and complexity appears to have required the evolution of elaborate internally-regulated intercellular signalling , 2. ConsAlthough metazoans have a number of unique intercellular signalling systems, including Wnt and TGF-β (transforming growth factor-beta) pathways, they also use signalling pathways that are more ancient, such as the receptor tyrosine kinase (RTK) pathway –12. ThesAmongst the bilaterian RTK families identified in sponges and ctenophores, the Eph-receptors are of particular interest in the context of understanding the origins of metazoan intercellular communication. The Eph-receptors comprise the largest RTK subfamily in bilaterians and, unlike the other RTK families that bind secreted diffusible ligands, they interact with cell surface-associated ephrin ligands on neighbouring cells Fig. a 3, 16,,16, 17. Despite this promiscuity, all bilaterian Eph receptors have an identical domain architecture that includes an extracellular globular ligand binding domain (LBD), a cysteine rich region (containing sushi and EGF-like motifs), a variable number of fibronectin domains, a single-pass transmembrane domain that connects to intracellular kinase, and SAM  and PDZ domains  searches of a wide range of publicly available genomes and transcriptomes, and unpublished transcriptome datasets, with a greater focus towards non-bilaterian and non-metazoan genomes. This survey of 104 unikont species included in particular a comprehensive collection of non-bilaterian metazoans  and 28 unicellular holozoans , comprising all recently published transcriptomic and genomic datasets  do contain a N-terminal domain that is not readily detectable by Pfam/CDD domain search engines. Manual inspection of an alignment with Eph-LBD of metazoan counterparts hinted at the presence of an Eph-LBD domain in these choanoflagellates, and thus each case was further investigated by searching against a database of HMM profiles constructed from individual Protein Databank (PDB) entries with the HHpred program. HHpred searches yielded significant similarity to the ephrin-binding (EPH-LBD) ectodomain of bona fide EPH receptors , 100 (15 full-length), and 9 (3 full-length), respectively Fig. . Across Mnemiopsis leidyi , and in this case the receptor lacks the intracellular Tyr-kinase domain. Thus, based on currently available genome and transcriptome data, there is no evidence for functional Eph-ephrin signalling outside of the Metazoa, even though components are present in some choanoflagellates.Taken together, our iterative HMM search results retrieved Eph-ephrin receptor-ligand pairs in most metazoans, including sponges and ctenophores, suggesting that Eph-ephrin signalling was present in the last common ancestor to all contemporary animals. Our results also raise the possibility that the origin of Eph-ephrin signalling predated the divergence of metazoan and choanoflagellate lineages, as we recovered putative Eph-like receptors with intact ligand binding domains in some choanoflagellates, and ephrins in others. Notably, however, we found only a single instance where both an Eph-like receptor and an ephrin ligand was found in the same choanoflagellate . In general, variations of this highly-conserved domain organisation are limited to the number of fibronectin repeats, differences in the length of the cysteine rich regions and absence of the intracellular SAM domains  for structure-based sequence comparisons.To further investigate the ligand-receptor recognition and binding in the newly identified Ephs and ephrins, we analysed the sequence conservation of the Eph-LBD and ephrin-RBD domains. The interaction of Eph-LBD and ephrin-RBD is the first critical step for the initiation of Eph-ephrin signalling via the formation of heterodimers and tetrameric complexes , 18, 24.Alignment of Eph-LBDs revealed a high degree of conservation, including invariant cysteine residues forming disulphide bridges  and conserved blocks of beta sheets forming the antiparallel beta-sandwich jellyroll fold of the Eph ectodomains   anchors that are characteristic of ephrin-A see Fig. . We founAs classified by the SCOP database, and examined previously , 44, 45 To examine the relationship of ephrins to cupredoxins and the SRS superfamily, we performed sequence-structure-based comparisons and phylogenetic reconstructions. Using Pfam HMM models Copper-bind PF00127), Cu_bind_like (PF02298) and Cupredoxin_1 (PF13473), we identified cupredoxin homologues in a diversity of organisms, including representative prokaryotes, non-metazoan eukaryotes and non-bilaterian metazoans. This survey recovered a range of cupredoxins, including the large multidomain enzymes comprising cupredoxin domain as one of their subunits. We restricted further analyses to the monodomain cupredoxins. Several of these were identified in sponges, ctenophores, unicellular holozoans and fungi  . Similarly, the Pfam domain Ephrin (PF00812), archetypal for ephrin-A and ephrin-B was used to search for ephrin orthologues. Novel putative Ephs and ephrins initially identified in non-bilaterian metazoans were aligned with representative full-length sequences from bilaterians to build new, distinct HMM profiles that were used in the second iteration. This iterative approach using domain specific HMM profiles was repeated for two rounds by including newly identified sequences to previous alignments. This approach maximised the chances of identifying distant homologues in basal metazoans and non-metazoans, and identified partial sequences, which may contain only one of these domains. After removal of redundant sequences using CD-HIT software, a final list of Ephs and ephrins were used in further sequence and phylogenetic analysis. For display purposes, we built heap maps using pheapmaps package in R. Absolute protein counts of Eph and ephrins, identified from each analysed species were plotted as heat maps , Cu_bind_like (PF02298) and Cupredoxin_1 (PF13473). These HMMER searches retrieved several cupredoxin domain containing sequences including monodomain and multidomain cupredoxins, however, we restricted our analysis to monodomain cupredoxins, as mutlidomain cupredoxins contain the same domain in additional copies. Our strategy to search for cupredoxins in metazoans and closest unicellular relatives was to verify if non-bilaterian metazoans containing ephrins also encode genes for monodomain cupredoxins with copper binding centres. This allowed us to examine possible scenarios of the diversification of ephrins from cupredoxins during the eukaryotic evolution. We also retrieved the SRS superfamily members, which are specific to apicomplexans, to resolve the overall phylogenetic relationships with the closely related ephrins and cupredoxins. SRS superfamily members were recovered through BLASTP searches against the NCBI NR database using representatives  from several SRS families including S48/45 that constitutes the same SRS- structural fold. Such search strategy recovered the large expansions of SRS superfamily, and thus the overall dataset was clustered based on 40% identity threshold CD-HIT  clusteriMultiple full-length sequence alignments were computed using MAFFT software (Version 7), utilising the E-INS-I algorithm, optimised for sequences with conserved motifs and carrying multiple domains, with BLOSUM62 as the scoring matrix and default parameters . Domain To analyse the potential loss of copper binding sites in ephrins, we generated a structure-based sequence alignment using the T-Coffee Expresso alignment server . With nuhttp://tree.bio.ed.ac.uk/software/figtree/).Maximum-likelihood phylogenetic trees were estimated by RAxML using PROTGAMMAAUTO to automatically determine the best protein substitution model for the given dataset . StatistTo distinguish between membrane-anchored (GPI- linked) ephrin-A or a transmembrane type ephrin-B we predicted potential TM regions and GPI anchoring sites in all newly identified ephrins from non-bilaterian metazoans. We utilised Topcons single (a reliable approach that engage a consensus predictive methodology derived from outputs of five different TM prediction tools)  for predInferred protein domain organisations were obtained using HMMER searches against Pfam database and Reverse Position-Specific BLAST searches against the pre-calculated Position-Specific Score Matrix (PSSMs) of the Conserved Domain Database (CDD) version 3.14. The cysteine rich regions of Eph-receptors, flanked between the Eph-LBD and the fibronectin domains are generally informed as either Sushi, EGF-like and other cysteine rich domains in Pfam and CDD searches. To be consistent, we uniformly denote the region between Eph-LBD and fibronectin as CRD (Cysteine rich domain). Also, as several of identified sequences lack one or more domains, we consider Eph-receptors as to be potentially full-length receptor, only if both the N-terminal ephrin-ligand binding domain, and C-terminal tyrosine kinase domain were present.Additional file 1:Figure S1. Domain architectures of Eph receptors identified across all analyzed taxa. Figure S2. Multiple sequence alignment containing selected representatives of the Eph-receptor extracellular Ligand Binding Domain (LBD) animals and choanoflagellates. Figure S3. Multiple sequence alignment of the Ephrin ligand Receptor Binding Domain (RBD) containing representatives from animals and choanoflagellates. Figure S4. Evolutionary relationships of metazoan Eph receptors inferred using the canonical intracellular Pkinase-Tyr domain. Figure S5. Evolutionary relationships of metazoan Eph receptors inferred using the extracellular ligand binding domain (LBD). Figure S6. Evolutionary relationships among metazoan ephrin ligands, highlighting multiple expansions in the phylum Cnidaria. Figure S7. Cartoon rendering of representative structures of cupredoxins, ephrins and the SRS superfamily. Figure S8. Structure based sequence alignment of ephrins, monodomain cupredoxins and the SRS superfamily from diverse taxa. Figure S9. Phylogenetic relationships between ephrins, monodomain cupredoxins and SRS superfamily. Table S1. List of analyzed taxa and database sources. Table S2. Dataset utilized to plot the heat map shown in Fig. Table S3. Distribution of Eph, ephrin and monodomain cupredoxins. Table S4. Mapping of Eph-LBD and Ephrin-RBD interaction interface residues. Table S5. Predicted TM regions and lipid anchoring sites in ephrins from non-bilaterian metazoans. Accompanying note. Genome/transcriptome datasets used for sequence search. Eph-LBD and ephrin hits in choanoflagellates. (PDF 47231 kb)Additional file 2:Complete FASTA sequences of all Eph receptors, ephrin ligands and cupredoxins identified new, or used for new analyses, in this study. (PDF 1079 kb)Additional file 3:Raw tree files of all phylogenetic trees reported in the manuscript. (PDF 271 kb)


The data presented in this article are related to the research article entitled “Physicochemical and functional properties of coconut (Cocos nucifera L) cake dietary fibres: Effects of cellulase hydrolysis, acid treatment and particle size distribution”  Specifications TableValue of the data•The data provide the potential re-use of coconut cake or other plants by-products.•The data provide information on how to improve some functional properties of defatted coconut cake dietary fiber.•This data allows other researchers to extend the statistical analyses.1The 2The experiments was carried out to determine the relationship between cellulase hydrolysis, acid treatment and particle size distribution and the monosaccharide composition, X-ray diffraction, Fourier-transformed infrared and spectroscopy surface area of coconut cake dietary fiber. Firstly, defatted coconut cake dietary fiber (DCCDF) was prepared from defatted coconut cake with α-amylase, papain and glucoamylase. Then DCCDF was subjected to acidic treatment and cellulase hydrolysis respectively, and defatted coconut cake dietary fiber treated by acid (DCCDF-A) and defatted coconut cake dietary fiber treated by cellulase hydrolysis (DCCDF-C) were obtained. Secondly, the chemical composition, particle size distribution, color, surface and microstructure, X-ray diffraction and Fourier-transformed infrared spectroscopy of DCCDF, DCCDF-A, DCCDF-C were determined


Moreover, the efficiency of their crosstalk, and the frequency of intermediary players have a considerable role in determining the disease severity. Single cell and next generation sequencing technologies are revealing that numerous immune subtypes including previously uncharacterized subsets are affected in these diseases. Further, layers of underlying immune resistance and escape mechanisms interfere with the clinical outcomes, indicating our poor understanding of disease microenvironments and immune cell networkings.In pathologies of cancer, autoimmunity, chronic infections with prolonged inflammation, or tissue damage, lymphocytes are either compromised or they respond overtly. Immune dysfunction is linked to skewed immune cell distribution across several subtypes, including CD8This Research Topic was developed to gain an overview of these timely issues in lymphocyte biology with a particular emphasis on functional crosstalk and its regulation. In this topic, a series of articles ranging from basic science to translational and clinical reports, including a few very insightful reviews, provides meaningful insights toward this interesting field. These articles strongly support the premise that harnessing the immune cell crosstalk in immune disorders and cancer may uncover novel strategies to cure these diseases more effectively.+ Treg-cells  is another crucial immunoregulatory receptor. Yu et al. discussed BTLA and its ligand HVEM (herpes virus entry mediator) signaling pathways, and highlighted chronological research needed in BTLA field. The widespread expression of BTLA across T, B, NKT, and DC indicates potential role of BTLA in immune crosstalk that could be harnessed to balance lymphocyte function and counteract immune disorders. The intracellular second messenger cyclic-AMP (cAMP) that acts in immunosuppressive signaling in T-cells is upregulated by multiple tumor-derived suppressive factors, such as prostaglandin E2 and adenosine. Schmetterer et al. show that ectopic overexpression of phosphodiesterase 4A (PDE4A) in T-cells leads to efficient degradation of cAMP. They suggest that PDE4A can be exploited as an immune checkpoint inhibitor against multiple suppressive factors.To improve the outcome of T-cell immunotherapy in cancer patients, immune checkpoint inhibitors targeting PD-1/PD-L1, or CTLA-4 pathways received a lot of excitement in the past decade. Checkpoint antibodies were developed and approved by the Food and Drug Administration (FDA) in the USA and Europe. A blockade of T-cell exhaustion or contraction molecules, PD-1/PD-L1 and CTLA-4, respectively, should reactivate and expand cancer antigen-specific T-cells. However, clinical responses were heterogeneous to none, with several resistance mechanisms identified across cancer types. Surprisingly, a hyperprogression was also reported recently in multiple cancer types, including melanoma and non-small cell lung cancer, possibly mediated by antibody-Fc/FcR interaction and amplification of PD-1eg-cells –27. Selioutcomes . Limitedoutcomes . IncludeDwivedi et al. reviewed the strategies that regulate CAR T-cell efficacy and persistence with focus on roles of different structural component of CAR construct.Recently, the FDA approved chimeric antigen receptor (CAR) T-cell therapy for relapse and refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma following their success in multiple phase-I/II clinical trials. While CAR T-cells are considered as major breakthrough in the field of cancer immunotherapy, the regulation of CAR T-cells remains poorly understood. Vigano et al. presented an illustrious overview of several pre-clinical and clinical strategies to inhibit the negative effects of adenosine for superior cancer control. They described several mechanisms to intersect adenosine effects impacting T-cells, DC, macrophages, NK, neutrophils, MDSC, stromal cells, and tumor cells. Further enhancing the cross-talk among various immune cells in a hostile tumor microenvironment, Karan introduced the emerging concept of targeting the inflammasome to improve antitumor immunity. Since a multitude of cytokines play a pivotal role in the regulation of immune cell function, inflammasome targeting would likely modulate the profile of inflammatory cytokines reducing immunosuppression at tumor sites. It would be interesting to see how ongoing studies on inflammasomes would benefit the field of cancer immunotherapy.Tumor microenvironment exhibits hypoxic and non-hypoxic areas heterogeneously. Hypoxic area is immune-deserted and appears to be resistant to immune cell attacks. Hypoxia is often associated with high amounts of adenosine, which is generated following ATP degradation. Adenosine restrains the effector function of T and NK cells. Several reports have indicated that interference with the adenosine signaling may improve anti-tumor immunity, especially in conjunction with immune checkpoint inhibitors. In a comprehensive review directed toward targeting adenosine in cancer immunotherapy, Kotlan et al. investigated tumor-infiltrating B-cells. They performed antibody repertoire analysis at the genetic level and identified disialylated glycosphingolipid as tumor antigens in melanoma. They generated a novel tool, single-chain variable (ScFV) antibody, and performed several other tests to confirm that tumor-infiltrated B-cells can recognize tumor-associated glycosphingolipid on melanoma and other solid cancers. Since tumor-derived ganglioside can suppress B-cell antibody production, this B cell-cancer cell crosstalk could be utilized as a therapeutic target.Several groups have detected B-cell infiltration into solid cancers. Uzhachenko and Shanker presented an emerging perspective that bidirectional crosstalk, and membranous reorganization between these CTL and NK cells may enable them to effectively eradicate cancer and infections. From human immune network proteome database, it is apparent that NK and CTL can profusely interact with DC to prime and optimize their effector function. Moreover, DC, being the professional antigen-presenting cells, have been tested as antigen delivery vehicle in several clinical trials. Vujanovic et al. interrogated the immunological effects of autologous DC transduced with MART-1, tyrosinase, and MAGE-A6 melanoma tumor antigens in a phase-I clinical trial. Interestingly, they identified that systemic high dose IFN-α2β after DC vaccination modulates a unique CD56dimCD16negative non-cytolytic NK subset in melanoma patients. This dominant immunoregulatory NK subset in tumor microenvironment appears to contribute to better clinical outcomes.NK cells and T-cells recognize tumor cells with distinct types of receptors; however, they utilize common effector molecules, such as TNFα, perforin, and granzymes to exert their cytotoxic action. in vitro CTL activation model, Wu et al. report that CTL-derived exosomes can increase the effector function and proliferation of CTL activated by low affinity peptides. The exosomes appear to mediate crosstalk among high and low-affinity CTLs at the beginning of CTL response. This may increase the repertoire of low affinity CTL and afford their long-term sustenance, thereby significantly improving T-cell mediated anti-cancer responses. In a mouse model, Kim et al. demonstrated that IL-7 and IL-15 cytokine-based crosstalk regulates the proportion and survival of naïve and memory T-cell populations. In the context of spontaneous proliferation of T-cells, Kim et al. showed an antigen-independent but IL-2-dependent crosstalk between CD4+ and CD8+T-cells.Exosomes mediate several immunoregulatory mechanisms. In an Wanjalla et al. correlated subcutaneous adipose tissue with significantly increased frequency of CD4+ and CD8+ T-effector-memory and effector-memory-RA+ cells. Adipose tissue from HIV-infected individuals showed a higher expression of TLR2, TLR8, and multiple chemokines relevant to immune cell homing compared to HIV-negative controls with similar glucose tolerance.T-cells also play a central role in modulating adipose tissue inflammation. In people living with HIV infection, Bak et al. showed significantly improved CMV-specific effector-memory T-cell function following inhibition of mammalian target of rapamycin (mTOR) with sirolimus. Monitoring of TCR-repertoire dynamics by next generation sequencing confirmed that the increased functionality was not related to sirolimus-resistant CTL-clones. Instead, environmental cues during CMV-CTL development via IL-2 receptor-driven signal transducer and activator of transcription-5 (STAT-5) signaling under mTOR inhibition allowed fine-tuning of T-cell programming for enhanced antiviral responses with stable TCR-repertoire dynamics. In a non-interventional prospective clinical trial in patients with multiple trauma, Hefele et al. assessed the role of platelets, Treg and Th17 cells in the post-traumatic immune response. They observed increased IL-17A expression in Th17 and Treg during the first 10 days following trauma. Moreover, despite a rising number of platelets, their analysis showed post-traumatic platelet dysfunction. Further studies are necessary to understand the underlying functional crosstalk between T-cells and platelets.The metabolic reprogramming of T cells by immunosuppressive drugs controls several inflammatory disorders . In humaRoy and Awasthi presented evidence that extracellular ATP promotes the differentiation of Th9 cells. They proposed a “feed-forward loop model” where nitric oxide production in Th9 cells enhances the mTOR-HIF-1-α pathway that further culminates in Th9 cell differentiation. This finding may have major implications in several cancer types. The role of Th9 cells in cancer is currently under investigation in several studies. Gaudino and Kumar described the crosstalk of T-cells and APC at multiple layers and presented a schematic of dysbiosis and gut microbiome. They highlighted how intestinal IL-17 receptor signaling and reciprocal crosstalk with gut microbiota can regulate autoimmunity. A comprehensive understanding of molecular interactions of immune cells and cytokines is important to refine appropriate immunotherapies. Dayakar et al. summarized the current understanding of a wide spectrum of cytokines and their interaction with immune cells that determine the clinical outcome of visceral leishmaniasis. They also highlighted opportunities for the development of novel diagnostics and intervention therapies for leishmaniasis.Recently, IL-9-producing Th9 cells have been identified in several disorders including cancer. Upadhyay describes the novel role of Ly6 gene family in cancer immune regulation. Ly6 genes are scattered in various chromosomes in human genome and have similarity to stem cell antigen-1 gene, a well-known cancer stem cell marker. The overexpression of this class of genes in solid cancers leads to poor survival. Some of these genes are expressed on both cancer cells and innate immune cells. Further research is necessary to understand if the Ly6 family genes could play a role in innate and adaptive immune crosstalk in the context of solid tumor microenvironments. Macrophage polarization is involved in many pathologies such as anti-cancer immunity and autoimmune diseases. Polarized macrophages exhibit plasticity when M2 macrophages are reprogrammed into an M1-like phenotype following treatment with IFNγ and/or LPS. At the same time, M1 macrophages are resistant to reprogramming in the presence of M2-like stimuli (IL-4). Veremeyko et al. explored the role of early growth response (Egr) family of transcriptional regulators in the induction and maintenance of M1 and M2 polarization. They demonstrated that a molecular crosstalk between Egr2 and CEBPβ transcription factors regulated macrophage polarization under distinct inflammatory conditions. An original study by Khare et al. investigated a switch of proximal and distal promoters fine-tuning the expression of genome organizer, special AT-rich sequence-binding protein (SATB1), which plays a crucial role in expression of multiple genes in a cell type-specific manner during the thymic development and peripheral differentiation and polarization of T-helper cells. Cytokine and TCR signaling crosstalk impacts SATB1 alternative promoter usage.Collectively, the articles contained within the Research Topic highlight the leading concept of lymphocyte functional crosstalk and its underlying complexity that impacts disease pathology and outcomes. Further research into the functional dynamics of immune networks is essential and timely for advancing our understanding of the immunological basis of diseases and the design of preventive or therapeutic approaches. The knowledge acquired from the published articles will contribute to meaningful insights for the development of more refined and novel immune strategies.RS and AS conceived, designed, and wrote the manuscript. FM provided substantial intellectual feedback. All authors read and approved the final manuscript for publication.TM Hospital, Playas Tijuana, Baja California, Mexico. 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.FM is employed by the company Refuge Biotechnologies Inc., Menlo Park, CA. FM is also on the Advisory Boards of Calidi Biotherapeutics Inc., San Diego, CA and CHIPSA


Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of  Drosophila, piRNA precursors are derived from distinct genomic regions termed piRNA clusters, which are enriched in TE fragments  (according to the dm3 genome assembly). The transgenic strain KG02245 (Bloomington Drosophila Stock Centre #12975) carries a P{SUPor-P} insertion in the 49E locus. Transheterozygous V/su(Hw)fsu(Hw) flies were used in the study.The transgenic strain eviously . Transgehttp://www.fruitfly.org/p_disrupt/TE.html) or to the piRNA clusters [Small RNAs 19–29 nt in size from total ovarian RNA extracts were cloned as previously described . The libclusters .KG10047;+/+, KG10047;V/su(Hw)fsu(Hw), KG09351;+/+; KG09351;V/su(Hw)fsu(Hw) ; KG02245;+/+ and KG02245;V/su(Hw)fsu(Hw) were deposited at Gene Expression Omnibus (GEO), accession number GSE125173.Ovarian small RNA-seq data for rp49 gene as a control genomic region. Standard error of mean (SEM) of triplicate PCR measurements for three biological replicates was calculated.ChIP was performed according to the published procedure . Chromatrp49. The primers used are described in RNA was isolated from the ovaries of three-day-old females. cDNA was synthesized using random hexamers or strand-specific primers and SuperScriptII reverse transcriptase . cDNA samples were analysed by real-time quantitative PCR using SYTO-13 dye on a LightCycler96 . Values were averaged and normalized to the expression level of the ribosomal protein gene p-value 1 × 10−5 was used as the threshold level.To estimate the frequency of the Su(Hw) insulator sites, the corresponding PWM profile (MA0533.1 from JASPAR_2016 database) was searched against the dm6 genome assembly or piRNA cluster regions . The p-vgypsy insulators and located within endogenous piRNA clusters. KG10047 and KG09351 transgenes were inserted into the 3′UTR of telomeric retrotransposon HeT-A and the major pericentromeric piRNA cluster in the 42AB locus, respectively fsu(Hw) mutations. These mutations cause the loss of Su(Hw) binding to the gypsy insulator [Both integrated loci were previously described as potent piRNA clusters able to adapt new insertions for piRNA production ,15,16,32th cases B. We sugnsulator ,34 and, nsulator   mutants are 21-nt endogenous small interfering RNAs (endo-siRNAs) C. Indeedgypsy insulators inserted in Drosophila subtelomeric and telomeric piRNA clusters are incorporated in piRNA production and acquire chromatin properties of their surrounding regions [gypsy insulators prevent piRNA production from P{SUPor-P} transgenic sequences, even if the latter are inserted into endogenous piRNA-producing regions.Previously, it was reported that P{lArB}, pW8-hsp-pA and P{EPgy2} transgenes lacking  regions ,18,32. S regions . We showV null allele and the Su(Hw)f, which carry a defective zinc finger 10 [f protein demonstrated the loss of binding to gypsy insulator and the reduced occupancy of many non-gypsy Su(Hw)-binding sites [V/su(Hw)fsu(Hw) ovaries, shows a dramatic decrease of Su(Hw) binding to the gypsy insulator and P{SUPor-P} transgenes. As it was reported previously, mutant Su(Hw)f is retained at some insulators including site located in 62D locus [To learn more about the mechanism of the Su(Hw)-mediated prevention of piRNA production, we compared the chromatin state of transgenes in the wild type and Su(Hw)-mutant backgrounds. In our experiments, we used transheterozygous flies with two Su(Hw) alleles: the Su(Hw)inger 10 . Su(Hw)fng sites ,38. ChIP2D locus ,34. Inde42AB locus and telomeric retroelement HeT-A lack these chromatin hallmarks in the presence of Su(Hw) protein (in the wild type genetic background) but acquire them in Su(Hw)-mutant ovaries  mutation in KG09351 and only 1.5-fold increases in KG10047;V/fsu(Hw) ovaries  was not affected by Su(Hw) mutation (gypsy insulator complex is established upstream of the Piwi-dependent chromatin formation of piRNA clusters and Su(Hw) binding mostly affects local chromatin conformation.The data from ChIP using Rhi, HP1 and H3K9me3 antibodies show that P{SUPor-P} transgenes inserted in both the  ovaries C–E. At tmutation . Therefomini-white gene of the transgenes inserted into HeT-A (KG10047 strain) and 42AB (KG09351 strain) in the wild-type and mutant background using transgene-specific primers  mutants for both insertions  binding  and Modifier of mdg4 (Mod67.2) [Our data suggest that Su(Hw) binding to P{SUPor-P} transgenes inserted into piRNA clusters should block transcription of long piRNA precursors. To verify this suggestion, we compared expression of  primers A. It shosertions A. In addsertions A. Strandspecific B. We thespecific C. These  binding D. HoweveMod67.2) ,40, the gypsy insulators might impair the functioning of the cluster regions in the close vicinity of transgenic insertion. To verify this suggestion, we estimated piRNA production in 42AB regions located around the KG09351 insertion.Given the functional integrity of piRNA clusters, we suggested that 42AB locus  mutations restored the level of transgene-flanking piRNAs in the 7 strain A. We alsransgene C. This f1 strain B. Thus, in  binding results in a local decrease in small RNA production not only from the HeT-A 3′UTR at 2R telomere because the unique mapping of small RNAs to poorly assembled and highly repetitive telomeric regions was technically impossible.We could not perform the analysis of flanking piRNAs near the KG10047 transgene inserted in the 42AB was helpful for understanding of the transcription regulation of the major uni-strand flamenco (flam) locus that controls TE expression in ovarian follicular cells [flam locus produces primary piRNAs from single strand precursors [gypsy, ZAM and Idefix [flam transcription. It is believed that piRNAs complementary to the coding transcripts of these TEs are produced from the single strand precursors transcribed by the flam sense strand [gypsy, Zam and Idefix insulators do not interfere with the transcription of the flam piRNA precursors, likely due to a strand-specific mode of insulator influence on transcription.To a certain extent, insertion of P{SUPor-P} transgene in ar cells ,44. In cd Idefix ,23,24, aHeT-A, is a non-autonomous retroelement and reverse transcriptase (RT) activity is likely to be provided by TART or TAHRE telomeric retrotransposons. In Drosophila germline, telomeric regions are organized in the piRNA clusters, although telomeric elements are heterogeneous in piRNA production and Rhi binding: TART retrotransposons are less susceptible to piRNA production and Rhi deposition than HeT-A [TART transcripts may be protected by an unknown mechanism from piRNA biogenesis machinery to provide stable expression of RT essential for telomere elongation in the germline. Here, we present the data suggesting a role for Su(Hw) in this mechanism.The main structural telomeric element, an HeT-A . This imUsing small RNAseq data, we studied the genome-wide impact of Su(Hw) depletion on transposon-derived piRNA production. The mapping of small RNAs to a canonical set of TEs does not demonstrate global changes caused by Su(Hw) mutations .TART-B and TART-C subfamilies of telomeric retrotransposons fsu(Hw) strains. To this end, we performed PCR on genomic DNA and showed that the relative copy numbers of HeT-A, TAHRE, TART-A, TART-B and TART-C are very similar in both strains (TART-B/TART-C transcripts are protected by Su(Hw)-dependent border elements from the piRNA production. Next, we examined the RNA levels of telomeric retroelements in the ovaries of Su(Hw) mutants by RT-qPCR. We found an increased level of TART-A transcripts in the ovaries of Su(Hw) mutants, while HeT-A and TAHRE expression was not affected  mutants (TART-encoding transcripts in telomeres. Su(Hw) serves as a transcriptional repressor of coding genes in the ovary [TART transcripts are less abundant than upon Su(Hw) depletion. Thus, Su(Hw) likely mediates transcriptional repression of TART in the germline.However, piRNA production from few TEs was strongly affected . In partnsposons . To excl strains C. These affected D. Howeve mutants E. These he ovary . Notably42AB cluster -binding sites in the whole genome and in piRNA clusters . We found that the Su(Hw) site density was lower in piRNA clusters (one site per 45 kb) than in the genome . It is tempting to speculate that the spectrum of regulatory sequences associated with TEs in piRNA clusters is subjected to strict selection to provide functional integrity of piRNA-producing loci.What could be happened if the insulator is inserted into the piRNA cluster? To some extent, this situation is simulated by the insertion of P{SUPor-P} transgene into the  cluster . Interesactivity ,46. The TART and TAHRE retrotransposons  failed to identify the Su(Hw) binding sites in canonical copies of telomeric retrotransposons. Thus, the nature of Su(Hw) binding sites in telomeres is still to be determined.Indeed, mapping of small RNAseq reads to the annotated piRNA clusters  did not nsposons . HoweverAlthough the loss of Su(Hw) causes female sterility, its particular role in the female germline development is not well understood ,38. Su(HV/Su(Hw)fSu(Hw) mutations on the abundance of piRNAs derived from coding genes (V/Su(Hw)fSu(Hw) ovaries, suggesting that Su(Hw) might prevent the spread of piRNA production from piRNA clusters/TEs to neighbouring genes. Indeed, Su(Hw) depletion causes the appearance of piRNAs antisense to the transcripts of the Rab8 gene located next to the cluster #48  in the germline. We revealed a genome-wide effect of ng genes . Most ofster #48 B. PCR byster #48 . At leasTART and coding gene transcripts from spurious piRNA production in the germline.Genomic regions containing damaged transposon fragments were for a long time considered as waste dumps. Most of these regions are piRNA-producing loci that play an essential role in antitransposon defence. The complex regulation of piRNA clusters has evolved to provide piRNA production from the entire piRNA cluster. This mechanism ensures an adaptive response to insertions of alien transposons in a piRNA cluster. We show that Su(Hw) binding sites disrupt the integrity of endogenous piRNA clusters, indicating that the assembly of insulator complexes occurs upstream of cluster-specific chromatin formation. Considering that distinct TE families comprise insulator-binding sites and other regulatory sequences, the TE content of the piRNA clusters should be under strict evolutionary constraints. Moreover, Su(Hw)-mediated complexes likely protect telomeric retrotransposon


OBJECTIVES/SPECIFIC AIMS: Immune-based therapies hold great promise for treatment of refractory tumors. However, development is limited by a lack of identified immune correlates to vaccination. We recently showed that dendritic cells (DCs) prolong progression-free survival (PFS) and overall survival (OS) in patients with glioblastoma, and that DC migration to site draining lymph nodes robustly correlates with both PFS and OS. While this appears to be a reliable immune correlate, the complexity of routine labeling for PET and SPECT prohibits validation in a large clinical study. We therefore seek to develop a safe, translatable reporter that can be imaged with a widely available imaging modality. METHODS/STUDY POPULATION: The cationic liposome 1,2-doleoyl-3-trimethylammonium-propane (DOTAP) was loaded with MRI-imageable iron oxide nanoparticles (IONPs) with or without the neutral molecules PEG and cholesterol. The resulting nanoparticles were loaded with RNA to form RNA-NPs that were characterized with transmission electron microscopy (TEM) and used to transfect DCs in vitro; 4.7 T MRI was then used to image particles or cells in agarose gel phantoms. RESULTS/ANTICIPATED RESULTS: TEM images of RNA-NPs indicate the presence of IONP-loaded liposomes. In vitro transfection experiments demonstrate that iron oxide does not reduce RNA-NP-mediated transfection of DCs. Additionally, small amounts of either PEG or cholesterol within RNA-NPs increased transfection of DC2.4s and enhanced T-cell priming by bone marrow-derived dendritic cells. A series of 4.7 T MRI images of particles in cells, spleens, and LNs demonstrated quantifiable differences in particle density between groups. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that IONP-loaded RNA-NPs can be imaged with MRI and manipulated to augment DC function. Future work will include in vivo imaging in mice and safety studies to facilitate translation into first-in-human studies. Successful completion of this project would provide a powerful clinical tool to improve and track patient responses to immune therapy.