Thymocyte Activation Research Articles

Sema3e/Plexin D1 Modulates Immunological Synapse and Migration of Thymocytes by Rap1 Inhibition.

Regulation of thymocyte trafficking plays an important role during thymic selection, but our understanding of the molecular mechanisms underlying these processes is limited. In this study, we demonstrated that class III semaphorin E (sema3e), a guidance molecule during neural and vascular development, directly inhibited Rap1 activation and LFA-1-dependent adhesion through the GTPase-activating protein activity of plexin D1. Sema3e inhibited Rap1 activation of thymocytes in response to chemokines and TCR stimulation, LFA-mediated adhesion, and T cell-APC interactions. Immunological synapse (IS) formation in mature thymocytes on supported lipid bilayers was also attenuated by sema3e. Impaired IS formation was associated with reduced Rap1 activation on the contact surface and cell periphery. Moreover, a significant increase of CD4(+) thymocytes was detected in the medulla of mice with T cell lineage-specific deletion of plexin D1. Two-photon live imaging of thymic explants and slices revealed enhanced Rap1 activation and migration of CD69(+) double-positive and single-positive cells with plexin D1 deficiency. Our results demonstrate that sema3e/plexin D1 modulates IS formation and Ag-scanning activities of thymocytes within thymic tissues.

Curcumin attenuates Mancozeb-induced toxicity in rat thymocytes through mitochondrial survival pathway

The widely used fungicide Mancozeb (Man) has been shown to cause genotoxic effects in rodents and toxicological manifestations in different cells, mainly by altering the antioxidant defense in cells. On the other hand, curcumin (Cur), a natural phenolic compound, is thought to possess anti-inflammatory and antioxidant properties. Here, we investigated the possible protective role of Cur on Man-induced toxicity in rat thymocytes and potential mechanism involved. Rat thymocytes were treated with Man(0.01 μg/ml) and/or increasing Cur(0.3, 1, 3 μM) concentrations and levels of cell viability, apoptosis, mitochondrial membrane potential (MMP),Bcl-2, Bax protein expression, caspase-3 and -9 activity and p38 MAPK signaling involvement were examined. Cells treated with Man displayed increased cell toxicity, hypodiploid cells, caspase-3 and -9 activity, Bax protein expression, followed with decreased MMP and Bcl-2 protein expression. Inhibition of p38 MAPK signaling pathway markedly reduced apoptosis rate and caspase-3 activity in thymocytes exposed to Man. Application of increasing Cur (1, 3 μM) concentrations resulted with significantly reduced cytotoxicity, apoptosis, caspase-3, -9 activity, Bax protein expression, together with increased MMP and Bcl-2 protein expression in rat thymocytes. These result suggest that certain Cur concentrations may mediate Man-induced rat thymocytes toxicity through mitochondrial survival pathway, which may be useful in preventing possible secondary immunological consequences induced by Man.

Mechanisms of diabetic autoimmunity: I--the inductive interface between islets and the immune system at onset of inflammation.

The mechanisms of autoimmune reactivity onset in type 1 diabetes (T1D) remain elusive despite extensive experimentation and discussion. We reconsider several key aspects of the early stages of autoimmunity at four levels: islets, pancreatic lymph nodes, thymic function and peripheral immune homeostasis. Antigen presentation is the islets and has the capacity to provoke immune sensitization, either in the process of physiological neonatal β cell apoptosis or as a consequence of cytolytic activity of self-reactive thymocytes that escaped negative regulation. Diabetogenic effectors are efficiently expanded in both the islets and the lymph nodes under conditions of empty lymphoid niches during a period of time coinciding with a synchronized wave of β cell apoptosis surrounding weaning. A major drive of effector cell activation and expansion is inherent peripheral lymphopenia characteristic of neonates, though it remains unclear when is autoimmunity triggered in subjects displaying hyperglycemia in late adolescence. Our analysis suggests that T1D evolves through coordinated activity of multiple physiological mechanisms of stimulation within specific characteristics of the neonate immune system.

The sensitivity of cells with the various level of NAD(P)H:quinone oxidoreductase 1

The effects of α-tocopherol with shortened to 6 carbon atoms side chain (α-Toc-C6), α-tocopherol succinate (α-TS) and quinonimine 2,6-dichlorophenolindophenol (DCPIP) on DT-diaphorase activity and viability of rat thymocytes, splenocytes and hepatocytes were investigated. It was shown that the lowest basal activity of the enzyme is inherent in splenocytes. In comparison to splenocytes, DT-diaphorase activity was 1.4 and 5 times higher in thymocytes and hepatocytes, respectively. It was found that the sensitivity of cells to the cytotoxic effect of DCPIP was inversely proportional to the basal level of DT-diaphorase activity and accompanied by its activation with subsequent inhibition at non-toxic and toxic concentrations, respectively. Hepatocytes were least sensitive to the cytotoxic effect of α-Toc-C6. In thymocytes and splenocytes α-Toc-C6 exerts inhibitory effects on DT-diaphorase, whereas in hepatocytes an increased activity of the enzyme was observed, which probably caused their high survival rate. Simultaneous induction of cytochrome P450 enzyme expression by α-Toc-C6 in hepatocytes is also possible. Cytotoxic effect of α-TS does not depend on the basal level of DT-diaphorase activity in cells, is not accompanied by its induction and it is most likely determined by the non-specific esterase activity.

Studies on antioxidative and immunostimulating fucogalactan of the edible mushroom Macrolepiota dolichaula

A water soluble fucogalactan (PS-II) of an average molecular weight ∼1.2×105 Da was isolated from the aqueous extract of an edible mushroom Macrolepiota dolichaula. It was composed of fucose, galactose and 3-O-methyl galactose in a molar ratio of nearly 1:4:1. Structural characterization of PS-II was carried out using total hydrolysis, methylation analysis, Smith degradation, and 1D/2D NMR experiments. These results indicated that the proposed repeating unit of the PS-II had a backbone chain consisting of four (1→6)- linked α-d-Galp residues, one residue methylated at O-3, and another one substituted at O-2 by (1→2)-α-d-Galp residue, which is terminated with a α-l-Fucp moiety. The PS-II exhibited the antioxidant properties in different in vitro test systems, and also showed in vitro macrophage activation in RAW 264.7 cell line as well as splenocyte and thymocyte activation in mouse cell culture medium.

Graves' disease TSHR-stimulating antibodies (TSAbs) induce the activation of immature thymocytes: a clue to the riddle of TSAbs generation?

Graves' disease (GD) is an autoimmune thyroid disease defined by the production of stimulating autoantibodies to the thyroid-stimulating hormone receptor (TSHR) (TSAbs) that induce a sustained state of hyperthyroidism in patients. We previously demonstrated that TSHR, the target of this autoimmune response, is also a key susceptibility gene for GD, probably acting through thymic-dependent central tolerance. We also showed that TSHR is, unexpectedly, expressed in thymocytes. In this report, we confirm the expression of TSHR in thymocytes by protein immunoblotting and quantitative PCR, and show that expression is confined to maturing thymocytes. Using functional assays, we show that thymic TSHR is functional and that TSAbs can stimulate thymocytes through this receptor. This new activity of TSAbs on thymocytes may: 1) explain GD-associated thymic enlargement (hyperplasia), and 2) suggest the provocative hypothesis that the continuous stimulation of thymocytes by TSAbs could lead to a vicious cycle of iterative improvement of the affinity and stimulating capability of initially low-affinity antibacterial (e.g., Yersinia) Abs cross-reactive with TSHR, eventually leading to TSAbs. This may help to fill one of the gaps in our present understanding of unusual characteristics of TSAbs.

Long-Term Changes in Proliferative Capacity of Thymocytes and Splenocytes in Mouse Progeny after Stimulation of Maternal Lymphocyte Proliferation at Early Terms of Pregnancy.

We studied the possibility of prenatal programming of proliferative potential of thymocytes and splenocytes in the offspring of C57Bl/6 mice via concanavalin A mitogen stimulation of lymphocyte proliferation in maternal organism at the early terms of pregnancy before the formation of immune system organs in the embryo. The offspring demonstrated elevated proliferative activity of thymocytes and splenocytes, enhanced lymphocyte proliferation, and increased production of IL-2 in in vitro response to concanavalin A during the prepubertal period and puberty. Mitogen stimulation of the maternal immune system in early pregnancy results in long-term enhancement of the proliferative potential of thymus and splenic cells in the offspring.

Changes in cellular thiol content and intracellular Zn2+ level by 1,4-naphthoquinone in rat thymocytes

1,4-Naphthoquinone is an active metabolite of naphthalene and it is also found in diesel exhaust particles. It is known to cause oxidative stress. In this study, we characterized 1,4-naphthoquinone-mediated cytotoxicity and its effects on the levels of non-protein thiols and intracellular Zn2+ in rat thymocytes (thymic lymphocytes) by using 5-chloromethylfluorescein (5-CMF) fluorescence and FluoZin-3 fluorescence, respectively. Low concentrations of 1,4-naphthoquinone (0.3μM) increased the intensity of 5-CMF fluorescence, which is used to measure non-protein thiols. In contrast, 5-CMF intensity decreased at higher concentrations (1–3μM) of 1,4-naphthoquinone. Removal of intracellular Zn2+ attenuated the 1,4-naphthoquinone-induced augmentation of 5-CMF fluorescence. Additionally, 1,4-naphthoquinone (0.3–3μM) increased FluoZin-3 fluorescence, which is used to assess intracellular Zn2+, in a concentration-dependent manner. The augmentation of FluoZin-3 fluorescence by 1,4-naphthoquinone was due to the release of intracellular Zn2+, because the removal of extracellular Zn2+ did not affect the augmentation of FluoZin-3 fluorescence. These results suggest that sublethal concentrations of 1,4-naphthoquinone (0.3–1μM) affect the cellular levels of non-protein thiols and intracellular Zn2+. The difference in the observed decrease in cellular thiol content due to 1,4-naphthoquinone treatment and increase due to Zn2+ release following 1,4-naphthoquinone treatment likely confers the change in cellular thiol content. Further, the increase in intracellular Zn2+ concentration after 1,4-naphthoquinone exposure may change the activity of thymocytes because thymulin, a thymus-specific hormone, requires Zn2+ for its biological activity.

Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (INM2P.434)

Abstract NK cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their limited efficacy requires a better understanding of the molecular mechanisms dampening their effector functions. NK cells recognize and kill pathogen-infected or tumor cells through invariant NK cell receptors (NKR). Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble IP4. We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product PIP3 and control PIP3-mediated protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

Cognate CD40-CD40L interaction is important for maintenance of thymic B cells. (IRM8P.705)

Abstract B cells are present in the thymus in numbers similar to dendritic cells and epithelial cells, but the functional roles of thymic B cells and the mechanisms that support this population are unclear. Previously, we showed B cells are essential for endogenous superantigen-mediated thymocyte negative selection. In this study, we investigated mechanisms regulating thymic B cells. Thymic B cell frequency and absolute number are markedly lower in TCRa KO mice, indicating the necessity of SP-T cells for maintenance of thymic B cells. Thymic B cell numbers are also reduced in mice expressing a TCR Tg in absence of antigenic ligand, but dramatically increased in mice expressing both TCR and corresponding ligand, demonstrating TCR recognition and thymocyte activation strongly influence thymic B cells. To elucidate molecular interactions that underlie the requirement for activated T cells, we tested the role of CD40L expressed on activated SP-T cells interacting with CD40 to mediate thymic B cell maintenance. In CD40L and CD40 KO, thymic B cell frequency is substantially reduced. In mixed BM-chimeras in which CD40 KO and WT donor-derived cells co-exist, cell autonomous CD40 expression is important for thymic B cell maintenance, whereas mixed BM-chimeras using MHCII KO and WT showed that cell autonomous MHCII expression plays no detectable role. Thus, CD40L-CD40 cognate interaction, but not MHCII-TCR cognate interaction, between T cells and B cells supports the thymic B cell population.

Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (1013.1)

NK cells are important for cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. Overcoming their limited efficacy in cancer immunotherapies requires a better understanding of the mechanisms limiting NK cell effector functions. NK cells recognize and kill pathogen‐infected or tumor cells through NK cell receptors (NKR). Two second‐messenger downstream pathways are required for NK cell maturation and function: PIP3‐generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 has a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). IP4 can act as a soluble analog of the PI3K lipid‐product phosphatidylinositol(3,4,5)trisphosphate (PIP3) and control PIP3‐mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show published and unpublished data which suggest that IP4 limits NKR induced IFNγ secretion, granule exocytosis and target‐cell clearance, in part by inhibiting the PIP3 effector‐kinase Akt in NK cells. This identifies IP4 as an important novel regulator of NK cell function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.Grant Funding Source: Supported by NIH grants AI070845, GM088647, AI089805 & Leukemia & Lymphoma Society Award 1440‐11

TCR signaling: the barrier within

Acute inhibition of the regulatory kinase Csk reveals additional checkpoints for full activation of thymocytes via the T cell antigen receptor.

CHANGES IN RAT THYMUS MORPHOLOGY AND FUNCTION AFTER CHRONIC EXPOSURE TO LOW DOSES OF DDT

There is increasing evidence that chronic environmental exposure to DDT and other pesticides play an important role in disregulation of the immune system and increased incidence of autoimmune and allergic diseases. The aim of the research was to investigate whether consumption of low doses of DDT may affect the immune system and change morphology and function of thymus. The research was performed on 42 Wistar rats 80-100gg body weight. Rats were exposed to 1.89±0.086μg/kg DDT per os for 6 and 10 weeks. Histological examination found time-depending acceleration of apoptosis both of lymphocytes and reticuloepitelial cells. After 10 weeks of exposure proliferation of thymocytes was significantly decreased. Concanavalin A-induced mitogenic activity of thymocytes after 10 weeks of exposure to DDT was 6 times higher than the control values. The data obtained showed that even low doses of DDT accelerated thymus aging and modulated proliferation of thymocytes.

Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A

A water-soluble heteropolysaccharide (PS) was isolated from extracellular polymeric substances (EPS) produced by a novel metal tolerant bacterium, Acinetobacter junii BB1A. Sugar analysis showed that the PS was composed of mannose, galactose and arabinose in a molar ratio of nearly 3:1:1. Structural characterization of the PS was carried out using methylation analysis, periodate oxidation, Smith degradation and 1D/2D NMR experiments. Methylation analysis revealed that the PS was consisted of 2,4-linked-mannopyranosyl, 3,4-linked-mannopyranosyl, 2-linked-galactopyranosyl, terminal mannopyranosyl and arabinopyranosyl residues in a relative proportion of nearly 1:1:1:1:1. Smith degradation of the PS showed the presence of hydrated glyceraldehyde containing disaccharide unit consisting of α-d-Manp-(1→and→4)-α-d-Manp-(1→residues where the later was directly attached to a hydrated glyceraldehyde moiety. This polysaccharide showed significant in vitro splenocyte, thymocyte, and macrophage activations with optimum dose of 100μg/mL for macrophage and 25μg/mL both for the splenocyte and thymocyte.

Structural characterization of an immunoenhancing glucan isolated from a mushroom Macrolepiota dolichaula

A water soluble branched glucan (PS-I) was isolated from aqueous extract of the fruit bodies of an edible mushroom Macrolepiota dolichaula, having average molecular weight ∼2.02×105Da. The structure of this PS-I was determined using total hydrolysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR experiments. Total hydrolysis and methylation analysis results showed the presence of (1→3, 6)-, (1→6)-, (1→4)-, (1→3)-linked and terminal β-d-glucopyranosyl residues in a relative proportion of nearly 1:2:1:1:1. All the chemical and NMR results indicated that the PS-I was a branched glucan, and the repeating unit of this glucan consisted of a backbone chain of three (1→6)-linked-β-d-glucopyranosyl residues where one of the backbone residues is branched at O-3 with (1→3)- moiety which is further attached to another (1→4)- residue and terminated with a non-reducing β-d-glucopyranosyl residue. The PS-I exhibited in vitro macrophage activation in RAW 264.7 cell line as well as splenocyte and thymocyte activation in mouse cell culture medium.

Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (P1198)

Abstract NK cells have important functions in cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize pathogen-infected or tumor cells through invariant NK cell receptors (NKR), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product phosphatidylinositol(3,4,5) trisphosphate (PIP3) and control PIP3-mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

Structural characterization of an immunoenhancing heteroglycan of a hybrid mushroom (pfls1h) of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer

An immunostimulating water-soluble heteroglycan (PS-II) was isolated from an aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h produced by intergeneric protoplast fusion between Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-II was carried out using sugar analysis, methylation experiment, periodate oxidation and 1D/2D NMR studies. Sugar analysis indicated the presence of glucose, mannose and galactose in a molar ratio of 1:1:2. Methylation analysis revealed that PS-II was composed of (1→6)- and (1→2,4,6)-α-d-galactopyranosyl, terminal β-d-mannopyranosyl and terminal β-d-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. On the basis of chemical analysis and NMR studies, the structure of the repeating unit of the heteroglycan was established to consist of a backbone chain of two (1→6)-α-d-galactopyranosyl residues, one of which is branched at O-2 with terminal β-d-Manp and at O-4 with terminal β-d-Glcp. This heteroglycan (PS-II) showed in vitro splenocyte, thymocyte and macrophage activations.

Structure of full-lengthToxascaris leoninagalectin with two carbohydrate-recognition domains

The full-length crystal structure of Toxascaris leonine galectin (Tl-gal), a galectin-9 homologue protein, was determined at a resolution of 2.0 Å. Galectin-9 exhibits a variety of biological functions, including cell aggregation, eosinophil chemoattraction, activation and apoptosis of murine thymocytes, T cells and human melanoma cells. Similar to this galectin, Tl-gal may function as a regulatory molecule in the host immune system; however, no molecular or structural information has been reported for Tl-gal. Moreover, until now, there have been no reports of a full-length galectin structure. There are two molecules of Tl-gal per asymmetric unit in space group P2(1)2(1)2(1), and the N-terminal and C-terminal carbohydrate-recognition domains (NCRD and CCRD) of Tl-gal are composed of six-stranded β-sheets and five-stranded β-sheets with a short α-helix. The NCRD of Tl-gal resembles that of human galectin-7 and its CCRD resembles human galectin-9, but the residues in the interface and loop regions of the NCRD and CCRD are flexible and are related to interaction. Engagement of the T-cell immunoglobulin mucin-3 (Tim-3) immunoglobulin variable (IgV) domain by a galectin-9 ligand is known to be important for appropriate termination of T-helper 1 immune responses. To investigate the binding site of Tl-gal, the interaction between Tl-gal and Tim-3 was modelled. Tim-3 is docked into a major groove of the Tl-gal structure, which is larger and deeper than the minor groove. The structural information presented here will provide insight into the development of novel anti-inflammatory agents or selective modulators of immune response.

Вплив мультипробіотика “Апібакт®” на продукцію інтерферону й активність 2',5'-олігоаденілат-синтетази в лімфоцитах тимусу щурів за умов гіпоацидності, викликаної омепразолом

The interferon (IFN) production by rat thymus lymphocytes and the activity of IFN-induced 2′,5′-oligoadenylate synthetase (2′,5′-OA-synthetase) in these cells were stu­died at hypoacidity of gastric juice evoked by omeprazole injection and also at combined omeprazol and multiprobiotic “Apibact®” treatment. The IFN production by thymocytes of rats with hypoacidity of gastric juice was shown to increase by 2.5±1.2 times in comparison to control (intact animals), the 2′,5′-OAS activity decreased by 28±1.34%. Both the IFN production and enzyme activity were increased comparative to unstimulated cells in response to IFN inducers – phytohemagglutinin A (PHA) (20 µg/ml) and cycloferon (50 µg/ml) treatment in vitro. Administration of “Apibact®” in parallel with omeprazole administration, led to amplification of the PHA action on IFN production and 2′,5′-OAS activity in thymocytes. Probably, the IFN synthesis by thymocytes is stimulated under hypoacidity of gastric juice. It may be a result of activation of immune response to opportunistic microflora colonization of stomach. “Apibact®” exhibit immunomodulatory properties, amplifying the ability of thymocytes to synthesize IFN in response to exoge­nous stimulation in rats with hypoacidity of gastric juice. Keywords: hypoacidity, omeprazol, multiprobiotics, “Apibact®”, thymocytes, 2′,5′-oli­goadenylate synthetase, interferon inducers.

Structural elucidation of a biologically active heteroglycan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes

A water soluble heteroglycan isolated from hot aqueous extract of fruit bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes consists of d-glucose, d-mannose, and d-galactose residues in a molar ratio of nearly 1:1:1. This polysaccharide showed macrophage, splenocyte, and thymocyte activation. On the basis of sugar hydrolysis, methylation, periodate oxidation study, and NMR studies (1H, 13C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as:▪