The steroidal lactone withaferin A impedes T-cell motility by inhibiting the kinase ZAP70 and subsequent kinome signaling.

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 µM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.

Depletion of T cells via Inducible Caspase 9 Increases Safety of Adoptive T-Cell Therapy Against Chronic Hepatitis B.

T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo, induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity.

Characterization of a Cohort of Patients With LIG4 Deficiency Reveals the Founder Effect of p.R278L, Unique to the Chinese Population.

DNA ligase IV (LIG4) deficiency is an extremely rare autosomal recessive primary immunodeficiency disease caused by mutations in LIG4. Patients suffer from a broad spectrum of clinical problems, including microcephaly, growth retardation, developmental delay, dysmorphic facial features, combined immunodeficiency, and a predisposition to autoimmune diseases and malignancy. In this study, the clinical, molecular, and immunological characteristics of 15 Chinese patients with LIG4 deficiency are summarized in detail. p.R278L (c.833G>T) is a unique mutation site present in the majority of Chinese cases. We conducted pedigree and haplotype analyses to examine the founder effect of this mutation site in China. This suggests that implementation of protocols for genetic diagnosis and for genetic counseling of affected pedigrees is essential. Also, the search might help determine the migration pathways of populations with Asian ancestry.

A combined treatment regimen of MGMT-modified γδ T cells and temozolomide chemotherapy is effective against primary high grade gliomas.

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug's concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O6-alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach.

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis.

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α-/-ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α-/-ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4+ subpopulations; and an increased number of CXCR5+ T cells in the draining lymph nodes of the p110α-/-ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α-/-ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α-/-ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.

Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release.

Human neutrophils constitutively express high amounts of arginase-1, which depletes arginine from the surrounding medium and downregulates T-cell activation. Here, we have found that neutrophil arginase-1, released from activated human neutrophils or dead cells, induced apoptosis in cancer cells through an endoplasmic reticulum (ER) stress pathway. Silencing of PERK in cancer cells prevented the induction of ER stress and apoptosis. Arginase inhibitor Nω-hydroxy-nor-arginine inhibited apoptosis and ER stress response induced by conditioned medium from activated neutrophils. A number of tumor cell lines, derived from different tissues, were sensitive to neutrophil arginase-1, with pancreatic, breast, ovarian and lung cancer cells showing the highest sensitivity. Neutrophil-released arginase-1 and arginine deprivation potentiated the antitumor action against pancreatic cancer cells of the ER-targeted antitumor alkylphospholipid analog edelfosine. Our study demonstrates the involvement of neutrophil arginase-1 in cancer cell killing and highlights the importance and complex role of neutrophils in tumor surveillance and biology.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation.

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.

Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells.

BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively.

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-ß) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

A most versatile kinase: The catalytic subunit of PKA in T-cell biology.

The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.

Mesenchymal stem cells alleviate experimental immune-mediated liver injury via chitinase 3-like protein 1-mediated T cell suppression.

Liver diseases with different pathogenesis share common pathways of immune-mediated injury. Chitinase-3-like protein 1 (CHI3L1) was induced in both acute and chronic liver injuries, and recent studies reported that it possesses an immunosuppressive ability. CHI3L1 was also expressed in mesenchymal stem cells (MSCs), thus we investigates the role of CHI3L1 in MSC-based therapy for immune-mediated liver injury here. We found that CHI3L1 was highly expressed in human umbilical cord MSCs (hUC-MSCs). Downregulating CHI3L1 mitigated the ability of hUC-MSCs to inhibit T cell activation, proliferation and inflammatory cytokine secretion in vitro. Using Concanavalin A (Con A)-induced liver injury mouse model, we found that silencing CHI3L1 significantly abrogated the hUC-MSCs-mediated alleviation of liver injury, accompanying by weakened suppressive effects on infiltration and activation of hepatic T cells, and secretion of pro-inflammatory cytokines. In addition, recombinant CHI3L1 (rCHI3L1) administration inhibited the proliferation and function of activated T cells, and alleviated the Con A-induced liver injury in mice. Mechanistically, gene set enrichment analysis showed that JAK/STAT signalling pathway was one of the most significantly enriched gene pathways in T cells co-cultured with hUC-MSCs with CHI3L1 knockdown, and further study revealed that CHI3L1 secreted by hUC-MSCs inhibited the STAT1/3 signalling in T cells by upregulating peroxisome proliferator-activated receptor δ (PPARδ). Collectively, our data showed that CHI3L1 was a novel MSC-secreted immunosuppressive factor and provided new insights into therapeutic treatment of immune-mediated liver injury.

Endothelium-restricted endothelin-1 overexpression in type 1 diabetes worsens atherosclerosis and immune cell infiltration via NOX1.

AIMS: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing human ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe-/-) mice enhanced high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We tested the hypothesis that ET-1 overexpression in the endothelium would worsen atherosclerosis in type 1 diabetes through a mechanism involving NOX1 but not NOX4. METHODS AND RESULTS: Six-week-old male Apoe-/- and eET-1/Apoe-/- mice with or without Nox1 (Nox1-/y) or Nox4 knockout (Nox4-/-) were injected intraperitoneally with either vehicle or streptozotocin (55 mg/kg/day) for 5 days to induce type 1 diabetes and were studied 14 weeks later. ET-1 overexpression increased 2.5-fold and five-fold the atherosclerotic lesion area in the aortic sinus and arch of diabetic Apoe-/- mice, respectively. Deletion of Nox1 reduced aortic arch plaque size by 60%; in contrast, Nox4 knockout increased lesion size by 1.5-fold. ET-1 overexpression decreased aortic sinus and arch plaque alpha smooth muscle cell content by ∼35% and ∼50%, respectively, which was blunted by Nox1 but not Nox4 knockout. Reactive oxygen species production was increased two-fold in aortic arch perivascular fat of diabetic eET-1/Apoe-/- and eET-1/Apoe-/-/Nox4-/- mice but not eET-1/Apoe-/-/Nox1y/- mice. ET-1 overexpression enhanced monocyte/macrophage and CD3+ T-cell infiltration ∼2.7-fold in the aortic arch perivascular fat of diabetic Apoe-/- mice. Both Nox1 and Nox4 knockout blunted CD3+ T-cell infiltration whereas only Nox1 knockout prevented the monocyte/macrophage infiltration in diabetic eET-1/Apoe-/- mice. CONCLUSION: Endothelium ET-1 overexpression enhances the progression of atherosclerosis in type 1 diabetes, perivascular oxidative stress, and inflammation through NOX1.

Concomitant use of a dual Src/ABL kinase inhibitor eliminates the in vitro efficacy of blinatumomab against Ph+ ALL.

Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome-positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Cellular and Mathematical Analyses of LUBAC Involvement in T Cell Receptor-Mediated NF-κB Activation Pathway.

The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, stimulates the canonical nuclear factor-κB (NF-κB) activation pathways through its Met1-linked linear ubiquitination activity. Here we performed cellular and mathematical modeling analyses of the LUBAC involvement in the T cell receptor (TCR)-mediated NF-κB activation pathway, using the Jurkat human T cell line. LUBAC is indispensable for TCR-induced NF-κB and T cell activation, and transiently associates with and linearly ubiquitinates the CARMA1-BCL10-MALT1 (CBM) complex, through the catalytic HOIP subunit. In contrast, the linear ubiquitination of NEMO, a substrate of the TNF-α-induced canonical NF-κB activation pathway, was limited during the TCR pathway. Among deubiquitinases, OTULIN, but not CYLD, plays a major role in downregulating LUBAC-mediated TCR signaling. Mathematical modeling indicated that linear ubiquitination of the CBM complex accelerates the activation of IκB kinase (IKK), as compared with the activity induced by linear ubiquitination of NEMO alone. Moreover, simulations of the sequential linear ubiquitination of the CBM complex suggested that the allosteric regulation of linear (de)ubiquitination of CBM subunits is controlled by the ubiquitin-linkage lengths. These results indicated that, unlike the TNF-α-induced NF-κB activation pathway, the TCR-mediated NF-κB activation in T lymphocytes has a characteristic mechanism to induce LUBAC-mediated NF-κB activation.

NOX2-Derived Reactive Oxygen Species in Cancer.

The formation of reactive oxygen species (ROS) by the myeloid cell NADPH oxidase NOX2 is critical for the destruction of engulfed microorganisms. However, recent studies imply that ROS, formed by NOX2+ myeloid cells in the malignant microenvironment, exert multiple actions of relevance to the growth and spread of neoplastic cells. By generating ROS, tumor-infiltrating myeloid cells and NOX2+ leukemic myeloid cells may thus (i) compromise the function and viability of adjacent cytotoxic lymphocytes, including natural killer (NK) cells and T cells, (ii) oxidize DNA to trigger cancer-promoting somatic mutations, and (iii) affect the redox balance in cancer cells to control their proliferation and survival. Here, we discuss the impact of NOX2-derived ROS for tumorigenesis, tumor progression, regulation of antitumor immunity, and metastasis. We propose that NOX2 may be a targetable immune checkpoint in cancer.

Human Adipose Tissue-Derived Stromal Cells Suppress Human, but Not Murine Lymphocyte Proliferation, via Indoleamine 2,3-Dioxygenase Activity.

Over recent years, mesenchymal stromal cells (MSC) have gained immense attraction in immunotherapy, regenerative medicine and tissue engineering. MSC microenvironment modulation occurs through synergy of direct cell-cell contact, and secreted soluble factors and extracellular vesicles (EV). MSC-derived EV have been suggested as cell-free immunomodulatory alternative to MSC; however, previous findings have challenged this. Furthermore, recent data suggest that evaluating the mechanism of action of human MSC (hMSC) in animal models might promote adverse immune reactions or lack of functionality due to xeno-incompatibilities. In this study, we first assessed the immunomodulatory strength of different human MSC sources on in vitro stimulated T cells and compared this to interferon-gamma (IFNγ) primed MSC conditioned medium (CM) and EV. Second, we addressed the main molecular mechanisms, and third, we assessed the MSC in vitro immunosuppressive effect across interspecies barriers. We identified human adipose tissue-derived stromal cells (ASC) with strongest immunomodulatory strength, followed by bone marrow (BM) and cord blood-derived MSC (CB). Whilst CM from primed ASC managed to exert analogous effects as their cellular counterpart, EV derived thereof did not, reproducing previous findings. IFNγ-induced indoleamine 2,3-dioxygenase (IDO) activity was identified as key mechanism to suppress human lymphocyte proliferation, as in the presence of the IDO inhibitor epacadostat (Epac) a stimulation of proliferation was seen. In addition, we revealed MSC immunosuppressive effects to be species-specific, because human cells failed to suppress murine lymphocyte proliferation. In summary, ASC were the strongest immunomodulators with the IDO-kynurenine pathway being key within the human system. Importantly, the in vitro lack of interspecies immunomodulatory strength suggests that preclinical data need to be carefully interpreted especially when considering a possible translation to clinical field.

Control of T lymphocyte fate decisions by PI3K signaling.

Virtually all aspects of T and B lymphocyte development, homeostasis, activation, and effector function are impacted by the interaction of their clonally distributed antigen receptors with antigens encountered in their respective environments. Antigen receptors mediate their effects by modulating intracellular signaling pathways that ultimately impinge on the cytoskeleton, bioenergetic pathways, transcription, and translation. Although these signaling pathways are rather well described at this point, especially those steps that are most receptor-proximal, how such pathways contribute to more quantitative aspects of lymphocyte function is still being elucidated. One of the signaling pathways that appears to be involved in this "tuning" process is controlled by the lipid kinase PI3K. Here we review recent key findings regarding both the triggering/enhancement of PI3K signals (via BCAP and ICOS) as well as their regulation (via PIK3IP1 and PHLPP) and how these signals integrate and determine cellular processes. Lymphocytes display tremendous functional plasticity, adjusting their metabolism and gene expression programs to specific conditions depending on their tissue of residence and the nature of the infectious threat to which they are responding. We give an overview of recent findings that have contributed to this model, with a focus on T cells, including what has been learned from patients with gain-of-function mutations in PI3K as well as lessons from cancer immunotherapy approaches.

Protein Arginine Methyltransferase 5 in T Lymphocyte Biology.

Protein arginine methyltransferase 5 (PRMT5) is the major methyltransferase (MT) catalyzing symmetric dimethylation (SDM). PRMT5 regulates developmental, homeostatic and disease processes in vertebrates and invertebrates, and a carcinogenic role has been observed in mammals. Recently, tools generated for PRMT5 loss of function have allowed researchers to demonstrate essential roles for PRMT5 in mouse and human lymphocyte biology. PRMT5 modulates CD4+ and CD8+ T cell development in the thymus, peripheral homeostasis, and differentiation into CD4+ helper T lymphocyte (Th)17 cell phenotypes. Here, we provide a timely review of the milestones leading to our current understanding of PRMT5 in T cell biology, discuss current tools to modify PRMT5 expression/activity, and highlight mechanistic pathways.

Involvement of heme oxygenase-1 in suppression of T cell activation by quercetin.

AIM: Acute rejection is still a major problem in transplantation and one of the most important causes of late graft loss. Cyclosporine and tacrolimus are widely used for suppression of T cell function to avoid graft rejection, but long-term use of these compounds is associated with serious toxicities. Quercetin, a flavonoid found in fruits and vegetables, has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO) -1, an enzyme involved in heme catabolism. We hypothesized that quercetin induces HO-1 in T cells and suppresses T cell function via HO-1. In the present study, we showed that quercetin suppressed the A23187-mediated expression of interleukin (IL) -2 in T cells. METHODS: Mouse splenocytes, enriched T cells, and EL4 cells, a mouse T cell line, were treated with quercetin, and then stimulated with A23187, a calcium ionophore, concanavalin A, or anti-CD3ε and anti-CD28 antibodies. Cell proliferation, expression of IL-2, calcium mobilization, apoptosis, cell cycle, and phosphorylation of extracellular signal-regulated kinase (ERK) were investigated. RESULTS: Quercetin induced HO-1, and this induction of HO-1 was implicated in the suppression of IL-2 production. Furthermore, the induction of HO-1 by quercetin suppressed the influx of calcium ions, a known trigger of IL-2 production. Additionally, quercetin suppressed T cell proliferation through promotion of cell cycle arrest via HO-1 induction, but quercetin did not induce apoptosis. To investigate the role of the signal transduction pathway in quercetin's effect on cell proliferation, we evaluated the phosphorylation of ERK in T cells. Quercetin suppressed the A23187-mediated stimulation of ERK, an effect that was mediated through HO-1. These results suggested that HO-1 is involved in the suppressive effects of quercetin on T cell activation and proliferation. CONCLUSION: Our findings indicate that the quercetin may be a promising candidate for inducing HO-1 in T cells, thereby facilitating immunosuppressive effects.