T helper (Th) cell activation is necessary for the adaptive immune response. Formation of an immunological synapse (IS) between Th cells and antigen-presenting cells is the first step in Th cell activation. In vitro studies indicate that formation of the IS induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2 influx across the plasma membrane and Ca2 -dependent Th cell activation. Earlier studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFA suppress CD4 T cell mitochondrial translocation during the early stages of IS formation and down-modulate Ca2 dependent Th cell activation. CD4 cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were co-cultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. fat-1 mice exhibited a significantly (P< 0.05) reduced percentage of CD4 T cells with mitochondria which translocated to the IS; fat-1 (30 percent) versus wild type control (82 percent). With respect to an effect on the mitochondrial-to-cytosolic Ca2 ratio, wild type cells showed significant increases at the IS (71 percent) and total cell (60 percent) within 30 min of IS formation. In contrast, fat-1 CD4 T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2 ratio was observed in wild type cells co-incubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2 (CRAC) channels, BTP2. Together, these observations provide evidence that n-3 PUFA modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2 entry.

For a long time research has focused on the protein-coding mRNA, but there is a complex world of non-coding RNAs regulating the human body that we yet know very little about. Non-coding RNAs (ncRNAs) are involved in modulation of different cell processes including proliferation, differentiation and apoptosis. In the current study the role of ncRNAs in T cell activation and function was investigated. T cells are important mediators of immune responses, for example upon viral infections. The T helper cells (TH or CD4+ cells) are involved in orchestrating immune processes like aiding the activation of macrophages and enhancement of B cell function. The TH1 cell subtype is generally pro-inflammatory and IFNγ-secreting. There are regulatory T (Treg) cells that are involved in downregulation of TH1 cells, to decrease or terminate the immune response. It has been shown that upon repeated stimulation TH1 cells can switch into a Treg-like IL10-secreting anti-inflammatory phenotype. In the IL10-secreting Treg-like cells the microRNA 150 (miR-150) was found upregulated compared to IFNγ-secreting TH1 cells. Thus, miR-150 was believed to be a candidate in key regulation of the switch between the two phenotypes. Predicted target genes of miR-150 were identified using mRNA arrays investigating down-regulated genes in the IL10-secreting Treg-like subpopulation. In this thesis predicted targets of miR-150 were investigated using luciferase assays. Unfortunately no targets were identified. Upon isolation of IFNγ-secreting TH1 cells and Treg-like IL10-secreting cells, it was found that the ncRNA 886 (nc886) was upregulated in these activated cells, compared to resting TH cells. This indicates that nc886 has an important role in T cell activation. Nc886 has been shown to inhibit PKR activation in other cell types. The effect of nc886 on protein kinase R (PKR) was therefore investigated. PKR shuts down translation upon activation in response to viral double-stranded RNA or cellular stress. We showed that in an activated T cell phenotype nc886 is affecting PKR upon activation by dsRNA from HIV or synthetic origin. The PKR activation pattern is reversed in a resting T cell phenotype.

Humans are exposed to high levels of amorphous silica on a daily basis, via the diet and the use of cosmetic and pharmaceutical products. Amorphous silica particles (10-200 nm) have also been developed for use in biomedical applications, including as binding agents in tissue repair, drug and gene therapy delivery agents, coatings for medical contrast agents and as vaccine adjuvants. Numerous studies have already been conducted to evaluate the cellular toxicity of these silica particles but still little is known about their effects both in vitro and in vivo, especially of nanosilica particles under 10 nm in diameter. The aim of this thesis was to investigate the cellular and in vivo activity of < 10 nm diameter nanosilica particles with different properties (e.g., size and dissolution rate in dilute conditions) as it may infer upon safety after exposure via the diet and intravenous administration (biomedical applications). First, the cytotoxicity of sub-10 nm nanosilica particles, fully characterized by size, dissolution rate, zeta-potential and by NMR spectroscopy, on immune cell function was assessed using transformed and cancerous cell lines and primary cells. The particles were toxic to the immune cells in a dose dependent manner and impaired certain cellular functions. Primary cells were most susceptible to nanosilica induced death and, of the primary cells, phagocytes were most susceptible to its cytotoxicity. Further investigations were conducted to assess the effect of nanosilica on T cells, as there was evidence suggesting that nanosilica particles were directly interacting with these cells. Nanosilica particles 3.6 nm in diameter were found to have a significant effect on T cell function. The particles induced numerous markers of T cell activation, including CD25 and CD69 on CD4 T cells, CD8 T cells, gamma-delta T cells and NK/NKT cells, CD95 on CD4 and CD8 T cells, CD40L, FoxP3, LAP, GARP on CD4 T cells, and IFN-gamma production, but it did not induce T cell proliferation. The particles were found to activate T cells regardless of their antigenic specificity. Further investigations showed that nanosilica interacts with the T cell receptor complex, the first documented case of a non MHC-coated nanoparticle directly interacting with this receptor complex. The nanoparticulate induced signalling through Zap70, LAT, and, eventually, through NFAT but not through MAPK. Similar signalling in the literature has been shown to induce a hyporesponsive T cell state (anergy) or activation induced cell death. The induction of the CD25 and CD69 T cell activation markers was limited to nanosilica particles below 10 nm in size, while similarly sized iron hydroxide nanoparticles (3-5 nm) only induced low levels of CD69 expression on T helper cells. Finally, it was shown that nanosilica is capable of inducing T cell activation in whole blood, though the T cell responses were greatly attenuated. Although identification of activation pathway in vivo remains elusive, the nanosilica particles were shown to have therapeutic value, decreasing murine subcutaneous tumour growth rate and significantly reducing the formation of lung metastases. Whether these in vivo responses are related to T cell activation identified in vitro remains unclear.

Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-20Bitstream added on 2014-06-13T21:04:28Z : No. of bitstreams: 1 ramos_op_dr_arafcf.pdf: 948525 bytes, checksum: 0581866624e7c3f57ffffca838856184 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 – Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII – Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... / Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer’s patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 – Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII – Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below)

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Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-induced proteins. The TCR proximal tyrosine kinase ITK simultaneously influences many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, I examined NFAT, NF-κB and MAP kinase pathways during early activation of individual naïve OT-I CD8+ T cells using peptide-loaded antigen presenting cells. Utilizing both measurement of transcription factor translocation in single T cell nuclei and conventional phospho-flow cytometry, I observed digital activation of Erk-MAPK and NFAT1 at all peptide doses and avidities. However, NF-κB activation showed a graded response to variation in TCR signal strength and was more sensitive to treatment with an ITK inhibitor. Inhibitor-treated cells showed poor induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis revealed genomic regions most sensitive to ITK inhibition are enriched for NF-κB and AP-1 motifs. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, I describe a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

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The normal physiological activation of naive T cells requires the engagement of both the T cell receptor (TCR) and the co-stimulatory molecule, CD28. However, a group of monoclonal antibodies (mAbs) have been identified that are able to activate T cells in vitro and in vivo via CD28 engagement alone. Two defining characteristics found in all CD28 superagonist mAbs are their membrane proximal CD28 epitopes and the requirement for mAb immobilisation. To investigate whether agonistic mAbs to similar cell molecules could be identified based on epitope position alone, mAbs to the inhibitory receptor PD-1 were generated and characterised. Using a drastic mutation-based epitope mapping technique, one mAb was identified with a membrane proximal epitope along with two other mAbs with membrane distal epitopes. These mAbs were tested for triggering activity in a hybridoma stimulation assay. mAb stimulation was observed with all three mAbs but only in cells expressing a PD-1 chimera that associated with the TCR and the strength of activation was dependent on epitope location. Cross-linking of a monomeric PD-1/CD28 chimera with a pair of anti-PD-1 mAbs resulted in signalling in this system, however, suggesting a role for ligand aggregation in addition to epitope position in mAb signalling. To further investigate the role of epitope position in CD28 superagonism, a cell line expressing a chimeric form of CD28 was created wherein the superagonistic mAb epitope was moved to a membrane distal position. When stimulated with a CD28 superagonist mAb signalling was no longer observed. However stimulation with another mAb that had an epitope to a membrane proximal location on the chimera resulted in superagonistic effect. These results show that epitope location is the dominant cause of T cell stimulation observed by CD28 superagonist mAbs and that epitope dependent mAb signalling is possible in other T cell surface molecules. The work described in this thesis has implications for both the development of immune modulating mAb therapeutics and for the general mechanism of triggering of cell surface receptors dependent on extrinsic tyrosine kinases.