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Rankin, Alasdair Menzies. "An investigation of CD28/B7 family binding interactions and costimulation, using immunoglobulin fusion proteins." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360469.
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Friedman, Rachel Sharon. "Early T cell activation in the lymphoid milieu." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3251945.
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Thesis (Ph.D.)--University of California, San Francisco, 2007.Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0876. Adviser: Matthew F. Krummel. Includes supplementary digital materials.
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Moldovan, Maria-Cristina. "Role of CD4 dimerization in T cell activation." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82936.
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Key to development of immunotherapies and vaccines is a thorough understanding of the complex pathways that govern T cell activation. On helper T lymphocytes, the CD4 molecule is a critical player in the early stages of activation, serving to enhance recognition of MHC-peptide complexes by the TcR and to facilitate efficient p56lck recruitment in the vicinity of the engaged TcR:CD3 signalling module. Originally, the CD4 molecule was believed to exist as a monomer on the cell surface; however, within the last years, a growing body of crystallographic, computer modeling and functional studies has suggested that CD4 might assume a dimeric/oligomeric state.Here, we provide direct experimental evidence for the existence of CD4 dimers on the surface of transfected cells from haematopoietic and fibroblastic origin, as well as in primary T lymphocytes. Furthermore, we accurately map the dimerization site at residues K318 and Q344 within the fourth extracellular domain of CD4. More importantly, we demonstrate that dimer formation is essential for the coligand and coreceptor functions of CD4 in T cell activation. Specifically, we show that CD4 dimerization is required for efficient IL-2 production, yet appears without effect on early TcR-associated signalling. Using FRET video microscopy to visualize the dynamics of CD4 molecules during T cell activation, we reveal that CD4 dimers only promote immunological synapse formation, then further accumulate within the synapse.
Overall, the study presented in this thesis sheds light on the refined molecular interplay of the various surface receptors and signalling modules that dictates the efficiency of the T lymphocyte antigenic response.
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Poudrier, Johanne. "Contact events in T help for B cell activation." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28517.
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The generation of an Ab response is modulated by contact and cytokine mediated T help for B cells. Here we show that murine splenic small resting B cells do not express mRNA for, or bear IL-2R. Accordingly, these cells do not respond to IL-2. T- contact events induce IL-2R expression on B cells and this is inhibited by blocking of CD40, MHC II or CD54. Although CD40 ligation on its own induces B cell proliferation, it does not confer IL-2 responsiveness. In contrast, signalling through MHC II and CD54 synergizes with IL-5 to induce functional IL-2R on B cells. Moreover, physiological, gp39$ rm sp{low},$ T help for B cell IL-2 responsiveness is equivalently dependent on ligation of CD40, CD54 and MHC II, and requires prior sIg signalling. IL-5 synergizes with either LPS or Th to render B cell responses to IL-2 autonomous of further stimulus. Thus, expression of a functional IL-2R is a marker of B cell activation which appears to be tightly regulated through sIg signals and T-contact events and can be modulated by cytokines.
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Krakowski, Michelle L. "T cell activation and cytokine production in experimental allergic encephalomyelitis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0001/NQ44479.pdf.
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Bonnard, Madeleine. "A novel role for CD4 in antigen-mediated T-cell activation /." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68156.
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A number of T-cell membrane molecules influence the outcome of antigen recognition by TCR/CD3 complex. CD4, by virtue of its non-covalent association with the protein tyrosine kinase lck has the capacity to enhance TCR$ alpha beta$ signalling. The extracellular domain of CD4 interacts with monomorphic determinants of Major Histocompatibility complex class-II molecules such that antigen presented in association with MHC class-II to CD4$ sp+$ T-cells results in the coaggregation of CD4 and TCR/CD3, thus juxtaposing lck and the antigen-receptor complex. Anti-CD4 antibodies abrogate both antigen and anti-TCR-induced T-cell activation. Studies using antigen specific T-cell clones that express either no CD4, wild type CD4 or mutated CD4 that cannot associate with lck (Db CYS) indicate that CD4 sequesters the majority of cellular lck and when not coaggregated with TCR/CD3, prevents the generation of prerequisite signals.Results presented in this thesis indicate that while CD4-associated lck is providing prerequisite signals for TCR/CD3 signalling, the contribution of CD4 must be more than simply providing a shuttle for lck. Specifically, anti-CD4 inhibits the antigen response of Db CYS CD4-expressing clones. This result cannot be accounted for either by CD4 sequestration of lck, or reduction of avidity of the interaction between the T-cell and the antigen presenting cell, since CD4$ sp-$ variants exhibit an antigen response comparable to that of CD4$ sp+$ variants. Rather, they suggest a novel role for the ectodomain of CD4 in antigen-induced T-cell activation.
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Dushek, Omer. "Mathematical modeling in cellular immunology: T cell activation and parameter estimation." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/2894.
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A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR.
To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC.
Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton.
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Morgan, Sara Hannah. "Molecular aspects of antibody mediated T cell activation." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:8c30ca07-b93b-46a7-aa86-01f94ee97e97.
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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.
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Sabbagh, Laurent. "Transcriptional regulation of the murine caspase-3 gene during T cell activation." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84320.
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Caspases play an important role in shaping the developing organism. They are required to eliminate unwanted or damaged cells and therefore are able to prevent disease. Several reports have shown increased caspase-3 expression in different cell types undergoing apoptosis. We undertook to examine the role of T cell activation through the T cell receptor (TCR) in regulating caspase-3 gene expression. The KOX-14 murine T cell hybridoma was initially used as a model for activation-induced cell death. Caspase-3 mRNA levels increased by 3-fold following T cell activation and was independent of STAT1 activation and therefore of IFN-gamma signaling in KOX-14 cells suggesting that the increase occurs early during T cell activation. Naive T cells were then isolated from the lymph nodes of mice to determine the extent of the increase in caspase-3 mRNA levels in cells undergoing proliferation rather than apoptosis. Caspase-3 mRNA levels were selectively induced (13-fold) following TCR triggering. Furthermore, caspase-3 mRNA levels were the highest in effector T cells which are destined to undergo AICD, when compared to long-lived memory T cells. Interestingly, the levels of procaspase-3 were also induced (6-fold) in activated peripheral T cells. In addition, T cells deficient for caspase-3 were more resistant to different apoptosis inducing molecules when compared to T cells containing normal levels of caspase-3. Altogether, these results demonstrate that the levels of caspase-3 must be maintained in a cell to ensure efficient apoptosis. The caspase-3 promoter region was subsequently cloned to identify transcription factors responsible for the observed increased in caspase-3 mRNA levels during T cell activation. Several regions within the promoter had either positive or negative regulatory effects on reporter activity when deleted. TCR stimulation of KOX-14 cells containing the different caspase-3 promoter constructs did not show changes in reporter ac
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Clement, Mathew. "The role of the CD8 co-receptor in CD8+ T-cell activation." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/47019/.
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CD8+ T-cells are essential for the immune control of pathogens and the natural eradication of cancer. CD8+ T-cells also play a major role in the pathogenesis of autoimmunity and alloreactivity. CD8+ T-cells recognize short peptide fragments (8-13 amino acids) presented at the target cell surface bound to Major Histocompatability Class I (MHCI) molecules. Tcell antigen recognition is unique in nature because it involves the binding of a single ligand (peptide–MHC [pMHC]) by two receptors (TCR and CD8). The CD8 glycoprotein, which serves as the coreceptor on MHCI-restricted T-cells, acts to enhance the antigen sensitivity of T-cells by binding to a largely invariant region of MHCI at a site distinct from the TCR docking platform. CD8 has been shown to have multiple roles including enhancing effects on early T-cell activation events and also in controlling the level of T-cell cross-reactivity. The pMHCI/CD8 interaction is classified as having a very weak binding affinity and very fast kinetics. I discovered that this low solution binding affinity is essential in maintaining homeostasis as dramatically increasing the strength of this interaction resulted in total loss of T-cell specificity and activation independent of TCR engagement. This led me to examine the possibility that anti-CD8 antibodies could also bypass the normal requirements for T-cell activation. I identified one specific clonotype of antibody capable of this phenomenon but simultaneously discovered multiple effector phenotypes of other anti-CD8 antibodies. These included both enhancing and inhibitory effects on pMHCI tetramer binding and CD8+ T-cell activation. Subsequently, I explored the possibility of using these inhibitory anti-CD8 antibodies to block T-cell function in systems which are highly dependent on CD8 such as autoreactive CD8+ T-cells. I demonstrated that targeting CD8 can be used as a strategy to block autoreactive CD8+ T-cell activation in the absence of any effect on pathogen specific immunity. This highlights a novel therapeutic strategy that warrants further investigation. Finally, I demonstrated that CD8 can alter the functional avidity of a CD8+ T-cell for its agonists and act to re-arrange the relative potencies of each of its potential agonists, a novel “focussing mechanism” for CD8 in T cell activation. These results provide new insight to the biological role of CD8 in T-cells and even predict a novel mechanism for CD8 in controlling T-cell function. My results also highlight the potential of targeting CD8 for immunotherapeutic design in autoimmune disorders.
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Getahun, Andrew. "Antibody Feedback Regulation : From Epitope Masking to T Helper Cell Activation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4580.
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Mahon, Robert Norman III. "Direct Inhibition of CD4+ T-cell Activation by Mycobacterium tuberculosis Cell Wall Glycolipids." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1275668686.
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Schroder, Paul. "Targeting Signal 1 of T cell Activation to Restore Self Tolerance in Type 1 Diabetes." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1381086555.
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Tao, Xiang. "Mechanisms of T Cell-mediated Macrophage Activation: Role of Antigen Specific and Antigen Nonspecific Cognate Interactions." Digital Commons @ East Tennessee State University, 1993. https://dc.etsu.edu/etd/2803.
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Macrophages play an important role in host antimicrobial immunity and in non-septic inflammatory reactions. Most studies on macrophage activation have focused on the roles of the T cell-produced cytokine, interferon-$\gamma$ (IFN$\gamma)$ and bacterial product, lipopolysaccharide (LPS). T cell-macrophage interaction is a critical step in initiating both specific and nonspecific immune responses to antigenic stimulation. The current study examines the role of cognate T cell-macrophage interaction in activation of macrophage effector functions and induction of macrophage early activation gene expression. Viable resting T$\sb{\rm H}$2 clone cells can activate IFN$\gamma$-primed macrophages to produce reactive nitrogen intermediates (RNI) or express cytostatic activity. The activating signal is mediated by cognate membrane contact between T cells and macrophages as evidenced by the ability of paraformaldehyde fixed anti-CD3-activated T$\sb{\rm H}$2 cells or plasma membranes isolated from the activated T cells to activate the IFN$\gamma$-primed macrophages. In contrast to the antigen-specific interaction of macrophages with viable resting T$\sb{\rm H}$2 cells, the activation of IFN$\gamma$-primed macrophages by fixed activated T$\sb{\rm H}$2 cells or by membranes from activated T$\sb{\rm H}$2 cells does not display antigen specificity. Fixed resting T$\sb{\rm H}$2 cells or plasma membranes isolated from the resting T cells can not activate the IFN$\gamma$-primed macrophages. Similar results are obtained with use of fresh splenic T cells to induce macrophage RNI production and cytostasis. Monoclonal antibody against CD4, which presumably blocks the interaction between CD4 (a co-receptor of T cell receptor) and class II MHC molecules on macrophages, inhibits significantly the activation of IFN$\gamma$-primed macrophages by viable resting T$\sb{\rm H}$2 cells but does not inhibit the ability of fixed activated T$\sb{\rm H}$2 cells to activate the macrophages. To examine the intracellular events in macrophages initiated by the cognate signaling, the expression of a panel of macrophage early activation genes, c-Myc, c-Fos, JE, IP10, D3, TNF$\alpha$ and IL-$\alpha$, are analyzed by dot blot hybridization. Plasma membranes from activated T$\sb{\rm H}$2 cells induce the expression of all these genes in macrophages stimulated for 1-4 hour. In contrast, the plasma membranes from resting T$\sb{\rm H}$2 cells are unable to induce the expression of most of the genes examined. These results suggest that the T cell-macrophage interaction involves reciprocal activation of both cells--an antigen specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen nonspecific activation of the macrophages. The nature of those T cell membrane components involved in cognate signaling of macrophage is currently being investigated.
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Hassanzadeh-Kiabi, Nargess. "The effects of Dectin-1 stimulation by fungal beta-glucans on T cell activation by dendritic cells." Thesis, Cedars-Sinai Medical Center, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10168998.
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Fungal infections can range from mild to severe and affect over a billion people worldwide. People whose immune systems have been compromised due to diseases such as AIDS or cancer, or patients who have been placed on immunosuppressive drugs after receiving stem cell or organ transplants, are more prone to opportunistic fungal infections. An intact immune system is capable of fending off fungal pathogens, therefore we wanted to further tease out the processes that an intact immune system uses to respond to fungi. The most well studied receptor that detects fungal cell wall components (β-glucans) is Dectin-1. We found that fungal β-glucan-stimulation of dendritic cells (DCs) enhanced antigen cross-presentation on MHCI molecules, up-regulated co-stimulatory molecules (CD40 and CD86) and induced pro-inflammatory cytokines (IL-2, IL-6, IL-12, TNF-α, and type I IFNs). Fungal β-glucans also stimulated DCs to induce strong CD8 T cell responses (up-regulation of activation markers CD44 and CD69, and production of IL-2, IFN-γ, TNF-α, and Granzyme B). Some of these responses were dependent on the autocrine action of type I IFNs on DCs, which promoted antigen cross-presentation on MHCI, expression of the co-stimulatory molecule CD86, and production of IL-2, TNF-α and IL- 6. CD8 T cell proliferation and production of IFN-γ and Granzyme B were also dependent on the effects of autocrine type I IFNs on DCs. These studies give us insight into how our immune systems normally respond to fungal infection in the context of Dectin-1 activation. This can provide us with clues about how to develop future therapeutics that promote more robust antifungal responses especially for patients whose immune systems are compromised.
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Patterson, Andrew R. "Gimap5: A Critical Regulator of CD4+ T Cell Homeostasis, Activation, and Pathogenicity." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1544098387129747.
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Wagner, David H. "Role of the Cd40-cd40 Ligand Interaction in Cd4(+) T Cell Activation of Monocyte Interleukin-1 Synthesis." Digital Commons @ East Tennessee State University, 1994. https://dc.etsu.edu/etd/2816.
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Most studies of the induction of cytokine synthesis in monocytes have used an exogenous triggering agent such as Lipolpoysaccharide (LPS). However, during nonseptic chronic inflammatory responses (e.g., rheumatoid arthritis) monocyte activation occurs as a result of T cell generated signals. This report demonstrated that plasma membranes from anti-CD3 activated peripheral CD4$\sp{+}$ T cells (Tm$\sp{\rm A}$) but not from resting CD4$\sp{+}$ cells (Tm$\sp{\rm R}$) induced monocytes to synthesize IL-1 in the absence of costimulatory cytokines. The expression kinetics of the molecule(s) unique to activated T cells which interact with monocyte receptors to induce IL-1 demonstrated that optimal expression occurred at 6h post activation. This matched Lederman's, et al., (1992) previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of IL-1 induction in monocytes. In this work, it was demonstrated that the signal could be reduced up to 85% by addition of 5c8, a monoclonal anti-CD40L antibody. In addition, a monoclonal anti-CD40 IgM (BL-C4) induced resting monocytes to synthesize IL-1. Experiments demonstrated that crosslinking the CD40 molecules on monocytes was critical for IL-1 induction. Tm$\sp{\rm A}$ but not Tm$\sp{\rm R}$ also up-regulated cell surface expression of adhesion/costimulatory molecules on monocytes including CD40, ICAM-1, and LFA-3. Anti-CD40 signaling up-regulated expression of ICAM-1 and LFA-3. Experiments suggested that signaling through CD40 may utilize a protein tyrosine kinase (PTK) mediated pathway but not a protein kinase C mediated pathway and studies using THP-1, a premonocytic cell line, indicated that the transcription factor, NF-$\kappa$B, was activated through anti-CD40 signaling. Since CD40 ligand-transfected cells alone did not induce IL-1 but Tm$\sp{\rm A}$ did, it was considered that an additional costimulatory cell surface molecule was required. Preliminary experiments suggested that CD69 may be required. In summary, these results indicate that contact-dependent T cell-monocyte interactions, alone, can activate inflammatory cytokine production by resting monocytes and that a critical component of this interaction is the CD40-CD40L signaling event.
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Franko, Jennifer Lynne. "Regulation of Effector/Memory T Cell Activation by Inducible Co-Stimulator (ICOS)." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1228358364.
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Taylor, Michelle. "NK-T Cell Activation by Alpha Galactosylceramide (a –Gal Cer): A Model for Adjuvant Activation of Innate Immunity." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1367409525.
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Chapman, Nicole. "The related kinases FAK and Pyk2 serve distinct functions in TCR-mediated T cell activation." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4954.
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T cells are central regulators of adaptive immunity in infectious and pathophysiological diseases. The activation of T cells is regulated by the T cell antigen receptor (TCR) and co-stimulatory receptors like toll-like receptor 2 (TLR2). These receptors activate distinct and overlapping intracellular signaling pathways that ultimately shape T cell responses. Therefore, studies that elucidate the molecular mechanisms of signal transduction downstream of receptors like the TCR and TLR2 will highlight key pathways required for T cell activation. These pathways could then be clinically targeted to alter dysfunctional T cell responses that promote many human diseases.
Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are two tyrosine kinases activated by multiple surface receptors expressed on T cells. FAK and Pyk2 signaling regulate cell morphology, migration, adhesion, proliferation, and survival in other cell types. However, their functions in T cells are not well-described. Because FAK and Pyk2 functions are dysregulated in many disease states, it is important to understand their function in human T cells so that clinicians can safely target these kinases to treat various disorders. The studies described in this dissertation aim to more fully elucidate how FAK and Pyk2 control T cell activation mediated by the TCR.
We used recombinant microRNAs or kinase inhibitors to transiently suppress FAK and Pyk2's expression or enzymatic function in human T cells. In doing so, several novel functions of FAK and Pyk2 were uncovered. In Chapter III, we revealed that FAK is a negative regulator of TCR signal transduction and function. Interestingly, in contrast to its function in other immune cell lineages, the work described in Chapters III and IV demonstrates that FAK is not required to regulate actin cytoskeletal responses downstream of the TCR. The data presented in Chapter IV demonstrate that Pyk2 regulates TCR-mediated actin cytoskeleton reorganization. This function appears to be linked to Pyk2's scaffolding function and not its enzymatic activity. In Chapter V, we demonstrated that the catalytic function of Pyk2 controls phosphatidylinositol-3-kinase (PI3K) activation in human T cells. Together, these data revealed that FAK and Pyk2 serve distinct functions in TCR signal transduction, actin cytoskeletal rearrangement, and effector responses.
TCR-driven cytokine production and proliferation are enhanced when T cells are concurrently activated via TLR2 ligands. In Chapter VI, we describe the signaling pathways that TLR2 activates in human T cells, and we characterize how TCR and TLR2 signals converge to augment T cell responses. In contrast to studies performed using isolated murine T cells, we demonstrated that TLR2 does not activate nuclear-factor kappa B in human T cells. Instead, we found that TCR and TLR2 co-ligation selectively augments extracellular signal-related kinase 1 (Erk1)/Erk2 and Akt activation in human T cells. Thus, TLR2 co-stimulates murine and human T cells via distinct signaling mechanisms.
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Gallagher, Michael P. "Differential TCR signaling dynamics tune graded gene expression in early-activating CD8+ T cells." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1115.
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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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Milhorn, Denise M. "Role of Mitogen-activated Kinases in Cd40-mediated T Cell Activation of Monocyte/macrophage and Vascular Smooth Muscle Cell Cytokine/chemokine Production." Digital Commons @ East Tennessee State University, 1999. https://dc.etsu.edu/etd/2950.
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This dissertation represents efforts to determine the functional consequences acquired by vascular smooth muscle cells (SMC) in response to CD40 ligation by activated CD154+ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC. To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD154 + , CD4+ T cells. The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were shown to present antigen to, and activate T cells. In turn T cell stimulus resulted in the activation of proinflammatory function in SMC initiated through the CD154:CD40 interaction. CD40 stimulation of SMC resulted in the production of the chemokines interleukin 8 (IL-8) and macrophage chemotactic protein-1 (MCP-1), and the upregulation of intercellular adhesion molecule (ICAM). Examination of the intracellular signaling pathways activated through CD40 signaling revealed the involvement of MAPKs in the pathway leading to induction of proinflammatory activity. Evaluation of CD40 signaling in monocytes demonstrated the activation of the MAPK family members ERK1/2, but not the MAPK family members p38 or c-jun-N-terminal kinase (JNK). In contrast, CD40 signaling in SMC was shown to result in ERK1/2 and p38 activation, and both of these kinases were shown to play a critical role in the induction of chemokine synthesis. An examination of the ability of anti-inflammatory cytokines to modulate CD40 signaling in monocytes and SMC demonstrated that the anti-inflammatory cytokines IL-4 and IL-10 abrogate CD40-mediated induction of inflammatory cytokine production by monocytes. This inhibition was shown to be a result of a negative influence of IL-4 and IL-10 on CD40 mediated ERK1/2, activation in monocytes. However, IL-4 and IL-10 did not inhibit SMC proinflammatory responses indicating a difference in the intracellular responses to these cytokines by the two cell types. (Abstract shortened by UMI.)
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Li, Cheng-Rui Michael. "The Role of Tec Kinases in CD4+ T Cell Activation: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/3.
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The Tec family tyrosine kinases Itk, Tec and Rlk are expressed in T cells. Previous studies have established that these kinases are critical for TCR signaling, leading to the activation of PLCγ1. To further understand the functions of Tec kinases in T cell activation, we took three different approaches. First, we performed a thorough analysis of CD28-mediated signaling events and functional responses with purified naïve T cells from Itk-/- mice and a highly controlled stimulation system. Data from this set of studies definitively demonstrate that CD28 costimulation functions efficiently in naïve CD4+ T cells in the absence of Itk. Second, in order to further study the functions of Tec kinases in vivo, we generated transgenic mouse lines expressing a kinase-dead (KD) mutant of Tec on the Itk-/-Rlk-/- background, hoping to study mice that are functionally deficient for all three Tec kinases. The results hint the importance of the Tec kinases in T cell development and/or survival. Finally, in order to identify potential transcriptional targets of Itk, we used microarray technology to compare global gene expression profiles of naïve and stimulated Itk-/- versus Itk+/- CD4+ T cells. This analysis provided a short list of differentially expressed genes in Itk-/- versus Itk+/- CD4 T cells, providing a starting point for further studies of Itk in T cell activation. Collectively, these studies clarified the role of Itk in CD28 signaling, revealed some unexpected aspects of Tec family kinases in T cells, and indicated potential targets of Itk-dependent signaling pathways in T cells.
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Carlin, Lindsey Elizabeth. "Natural killer cell activation, trafficking, and contribution to immune responses to viral pathogens." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1302.
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Natural killer (NK) cells are a critical component of the immune response against viral infections. NK cell depletion prior to murine cytomegalovirus (MCMV) infections results in increased susceptibility to infection in several mouse strains. The mechanism of protection in C57Bl/6 mice is dependent on the activation of NK cells by Ly49H recognition of m157. Our previous studies have examined important residues of m157 for Ly49H recognition, as well as the contribution of m157 glycosylation to NK cell activation. However, what role the glycophosphatidyl inositol (GPI) anchor of m157 plays in Ly49H activation was unknown. Here we demonstrate that the GPI anchor of m157 regulates the surface expression of the protein. While the GPI anchor was not required for recognition of m157 by the activating or inhibitory Ly49 receptors, expression of GPI-anchored m157 resulted in greater receptor downregulation on NK cells, as well as increased NK cell cytotoxicity compared to transmembrane m157.
In addition to MCMV infections, NK cells have been shown to participate in the immune response to influenza A virus (IAV). However the exact role of NK cells in IAV infection is less clear, as some studies have found NK cells to be protective, while others have shown that NK cells cause lethal immunopathology. It is likely that the severity of IAV infection may dictate the NK cell response to IAV infection (i.e. protective vs. immunopathogenic). Herein we show that NK cell accumulation in IAV-infected lungs and lung-draining lymph nodes (DLN) is regulated by the severity of IAV infection, where there is increased NK cell accumulation in the lungs during high dose IAV infection, and greater NK cell accumulation in the DLN in low dose IAV infections. Despite significant NK cell recruitment to the lung during IAV infection, as well as previously published studies demonstrating the importance of NK cells to IAV immunity, NK cell depletion prior to IAV infection did not result in a significant change in morbidity or mortality. Interestingly, NK cell depletion resulted in a significantly greater number of CD4 T cells in the IAV infected lung. Further, both CD4 and CD8 T cells in NK-depleted mice showed increased IFN-Γ production. Finally, while not statistically significant, NK cell depletion resulted in a trend toward greater protection from heterosubtypic IAV challenge infections. Taken together these results suggest that NK cells may either regulate the adaptive immune response to IAV infection through suppression of CD4 and CD8 T cells, or that the T cell response to IAV infection is able to compensate for the loss of NK cells. Moreover, while NK cell suppression of T cell function during a primary IAV infection does not result in increased susceptibility to primary IAV infections, NK cell regulation of adaptive immune responses may suppress the memory T cell response, and therefore leave the host more susceptible to secondary infections.
Overall the studies presented herein demonstrate a complex role for NK cells in the immune response against viral infections. Ly49H+ NK cells directly kill MCMV-infected cells and m157-bearing targets, but NK cell activation is regulated by ligand density, as well as the ligand membrane anchor. Additionally, NK cells suppress adaptive immune responses during a primary IAV infection, resulting in changes to the T cell response during both primary and memory responses.
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DiSano, Krista D. "Factors promoting B cell activation and accumulation in the inflamed CNS." Kent State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=kent1489666546396967.
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Simmons, Daimon P. "Effects of Toll-Like Receptors and Type I Interferon on Dendritic Cell Maturation and Activation of T Cells." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1311278278.
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Gratton, Sophie. "Modulation of T cell activation and human immunodeficiency virus (HIV) infection by CD4 : identification of functional domains and mechanisms involved in CD4 function." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39912.
Full textAbstract:
The CD4 molecule is expressed on a subset of T lymphocytes which recognize their cognate antigen in the context of MHC class II molecules. It is widely accepted that the interaction between CD4 and MHC class II molecules enhances T cell response to specific antigen. CD4 is non-covalently associated with the src-related tyrosine kinase p56$ rm sp{lck}.$ Using the T cell clone 2.10, we have shown that CD4 can sequester lck and inhibit anti-TcR induced proliferation if not co-aggregated with the TcR. Our results suggest that MHC class II molecules through their simultaneous interaction with the TcR and CD4 potentiate T cell activation by bringing the CD4/lck complex to the proximity of the TcR. This cellular system was also used to demonstrate that the extracellular domain of CD4 can also regulate the initiation of T cell activation independently of its interaction with MHC class II molecules. Indeed, cells expressing chimeric molecules composed of the epidermal growth factor receptor (EGFR) extracellular domain and the CD4 cytoplasmic tail were still responding to anti-TcR simulation in the absence of co-aggregation. The role of the extracellular domain of CD4 was further demonstrated in experiments in which the HIV-1 envelope glycoprotein gp120 was used to inhibit antigenic stimulation of CD4-independent T cell responses. This inhibition was occurring whether CD4 is associated with lck or not, suggesting that gp120 is modulating a CD4 function other than association with lck and which is related to its extracellular portion. In addition to its effect on T cell activation, interaction between gp120 and CD4 modulates HIV replication at a post-transcriptional level. The CD4/lck association is required for this effect as the virus replicates much more efficiently in cells bearing a CD4 which is not associated with lck. Activation of lck through the CD4/gp120 interaction may thus be responsible for the induction of latency. Nef, another HIV protein playing a critical r
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Urban, Stina L. "The Role of Signal 3 Cytokine Timing in CD8 T Cell Activation: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/788.
Full textAbstract:
During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation.
In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks).
Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance.
Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function.
This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation.
Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.
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Urban, Stina L. "The Role of Signal 3 Cytokine Timing in CD8 T Cell Activation: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/788.
Full textAbstract:
During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation.
In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks).
Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance.
Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function.
This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation.
Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.
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Nguyen, Lam. "Immune Activation Induces Telomeric DNA Damage, Reduces Memory Precursors, and Promotes Short-lived Effector T Cell Differentiation in Chronic HCV Infection." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/etd/3828.
Full textAbstract:
Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.
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Priyadharshini, Bhavana. "Regulation of Early T Cell Activation by TNF Superfamily Members TNF and FASL: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/494.
Full textAbstract:
The instructive signals received by T cells during the programming stages of activation will determine the fate of effector and memory populations generated during an immune response. Members of the tumor necrosis factor (TNF) superfamily play an essential role in influencing numerous aspects of T cell adaptive immune responses including cell activation, differentiation, proliferation, survival, and apoptosis. My thesis dissertation describes the involvement of two such members of the TNF superfamily, TNF and FasL, and their influence on the fate of T cells early during responses to viral infections and to the induction of transplantation tolerance.
TNF is a pleiotropic pro-inflammatory cytokine that has an immunoregulatory role in limiting the magnitude of T cell responses during a viral infection. Our laboratory discovered that one hallmark of naïve T cells in secondary lymphoid organs is their unique ability to rapidly produce TNF after activation and prior to acquiring other effector functions. I hypothesized that T cell-derived TNF will limit the magnitude of T cell responses. The co-adoptive transfer of wild type (WT) P14 and TNF-deficient P14 TCR transgenic CD8+ T cells, that recognize the GP33 peptide of lymphocytic choriomeningitis virus (LCMV), into either WT or TNF-deficient hosts demonstrated that the donor TNF-deficient P14 TCR transgenic CD8+ T cells accumulate to higher frequencies after LCMV infection. Moreover, these co-adoptive transfer experiments suggested that the effect of T cell-derived TNF is localized in the microenvironment, since the TNF produced by WT P14 TCR transgenic CD8+ T cells did not prevent the accumulation of TNF-deficient P14 TCR transgenic CD8+ T cells. To determine if T cell-produced TNF is acting on professional APC to suppress the generation of virus-specific T cell responses, I performed co-adoptive transfer experiments with WT P14 TCR transgenic CD8+ and TNF-deficient P14 TCR transgenic CD8+ T cells into TNFR1/2 (1 and 2) deficient mice. These experiments demonstrated that the absence of TNFR1/2 signaling pathway in the host cells resulted in a greater accumulation of WT P14 TCR transgenic CD8+ T cells, thereby considerably diminishing the differences between donor WT P14 TCR transgenic CD8+ and donor TNF-deficient P14 TCR transgenic CD8+ T cells. The increased frequency and absolute numbers of WT P14 TCR transgenic CD8+ T cells in TNFR1/R2 deficient recipients suggests that one mechanism for the suppressive effect of T cell-derived TNF on antigen-specific T cells occurs as a result of TNFR signaling in the host cells. However, the donor TNF-deficient P14 TCR transgenic CD8+T cells still accumulated to higher frequency and numbers compared to their donor WT transgenic counterparts. Together, these findings indicate that T cell-produced TNF can function both in an autocrine and a paracrine fashion to limit the magnitude of anti-viral T cell responses.
Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, I questioned at what stage of development do T cells become licensed to produce this cytokine. The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. I hypothesized that naïve T cells emigrating from the thymus will be competent to produce TNF only after undergoing a maturation process in the periphery. To test this hypothesis, I compared cytokine profiles of CD4+ and CD8+single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics with respect to the upregulation of CD25 and CD69 following stimulation. The reduced ability of SP thymocytes to produce TNF correlated with a decreased level of detectable TNF message following stimulation when compared to splenic counterparts. Stimulation of SP thymocytes in the context of a splenic environment did not fully enable TNF production, suggesting an intrinsic defect in their ability to produce TNF as opposed to a defect in antigen presentation. Using a thymocyte adoptive transfer model, I demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs identified by the expression of green fluorescent protein (GFP) (NG-BAC transgenic mice), showed a significantly enhanced ability to express TNF relative to SP thymocytes, but not to the extent of MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs. This highlights the functional heterogeneity of the naïve T cell pool (with respect to varying degrees of TNF production) during early T cell activation that can contribute to the many subsequent events that shape the course of an immune response.
The productive activation of naïve T cells requires at least initial two signals; the first being through the TCR and the second is the engagement of co-stimulatory molecules on the surface of the T cells. T cells activated in the absence of co-stimulation become anergic or undergo cell death. Agents that block co-stimulation of antigen-specific T cells are emerging as an alternative to immunosuppressive drugs to prolong allograft survival in transplant recipients. Targeted blockade of CD154-CD40 interactions using a αCD154 monoclonal antibody (MR1) with a simultaneous transfusion of allogeneic splenocytes (donor specific transfusion or DST) efficiently induces tolerance to allografts. This co-stimulation blockade-induced tolerance is characterized by the deletion of host alloreactive T cells within 24 hours of treatment. Toll-like receptor (TLR) agonists abrogate tolerance induced by co-stimulation blockade by impairing the deletion of host alloreactive T cells and resulting in allograft rejection. The goal of my study was to determine the underlying molecular mechanisms that protect host alloreactive T cells from early deletion after exposure to TLR agonists. I hypothesized that TLR ligands administered during co-stimulation blockade regimen differentially regulate the expression of pro- and anti-apoptotic molecules in alloreactive T cells, during the initial stages of activation thereby preventing deletion.
To test this hypothesis, I used syngeneic bone marrow chimeric mice containing a trace population of alloreactive KB5 TCR transgenic CD8+ T cells (KB5 Tg CD8+ T cells) that recognize H-2Kb as an alloantigen. I show here that KB5-CD8+ T cells downregulate CD127 (IL-7R!) and become apoptotic as early as 12 hrs after co-stimulation blockade. In contrast, KB5 Tg CD8+ T cells from mice treated with bacterial lipopolysaccaride (LPS) during co-stimulation blockade failed to become apoptotic, although CD127 was downregulated. Examination of the mRNA expression profiles of several apoptotic genes in purified KB5 CD8+ T cells from mice treated with DST+anti-CD154 for 12 hrs revealed a significant upregulation of FasL mRNA expression compared to the untreated counterparts. However, in vitro FasL blockade or in vivo cytotoxicity experiments with mice deficient in Fas or FasL indicated that the Fas-FasL pathway might not be crucial for tolerance induction. Another pro-apoptotic molecule BIM was upregulated in alloreactive T cells during co-stimulation blockade. This suggests that both the Fas pathway and BIM may be playing complementary roles in inducing deletional tolerance. Although FasL expression was diminished in alloreactive T cells in the presence of LPS, BIM expression was not diminished, suggesting that alloreactive T cells may still be vulnerable to undergo apoptosis. Concomitantly, I also found that LPS treatment during co-stimulation blockade resulted in non-specific upregulation of Fas expression in alloreactive T cells and non-transgenic T cells (CD4+ and CD8+). I demonstrate here that treatment with Fas agonistic antibody in vitrofor 4 hours can selectively induce apoptosis of alloreactive T cells that were believed to be refractory to apoptosis during LPS treatment. I speculate that under these conditions, deletion may be occurring due to the involvement of both Fas and BIM. Further, the mRNA expression profile revealed interleukin-10 (IL-10) as a molecule induced in alloreactive T cells during LPS treatment. Analysis of serum confirmed the systemic expression of IL-10 protein in mice treated with LPS during co-stimulation blockade. I hypothesized that LPS-induced IL-10 can have an anti-apoptotic role in preventing the deletion of alloreactive T cells and mediating allograft rejection. Contrary to my hypothesis, I found that IL-10 KO mice rejected allogeneic target cells similar to their WT counterparts, suggesting that IL-10 may not be required for LPS-mediated abrogation of tolerance induction. In addition to the systemic induction of IL-10, LPS also induced cytokines such as interleukin-6 (IL-6), TNF and interferon-γ (IFN-γ).
These findings suggest that both Fas-FasL and BIM mediated apoptotic pathways may play complementary roles in inducing the early deletion of activated alloreactive T cells during tolerance induction. On the other hand, the mechanism of LPS mediated abrogation of tolerance induction can not be attributed to IL-10 alone as it may be playing a synergistic role along with other proinflammatory cytokines that may in turn result in the prevention of alloreactive T cell death during this process. Most importantly, these findings indicate that despite emerging from a pro-inflammatory cytokine milieu, alloreactive T cells are still susceptible to undergo Fas-mediated apoptosis during the first 24 hours after co-stimulation blockade and LPS treatment. Therefore, targeting the Fas-FasL pathway to induce deletion of alloreactive T cells during the peri-transplant period may still be a potential strategy to improve the efficacy of co-stimulation blockade induced transplantation tolerance during an environmental perturbation such as inflammation or infection.
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Strainic, Michael George Jr. "THE ABSENCE OF C3AR AND C5AR SIGNAL TRANSDUCTION PROMOTES T REGULATORY CELL DIFFERENTIATION AND REGULATES IMMUNOLOGIC TOLERANCE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1363707372.
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Geoghegan, Vincent L. "The role of protein arginine methylation in T-lymphocyte activation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:2116068d-2a0a-4785-a450-75169da4dc6b.
Full textAbstract:
T-lymphocytes are an essential cell type of the adaptive immune system. Due to their importance in immune responses and disorders, the molecular mechanisms leading to T-lymphocyte activation have been the subject of extensive research which has translated into important therapeutic developments. Early signalling events involving tyrosine phosphorylation are well characterised. However, later events involving other post-translational modifications are less well understood. Several studies have provided evidence suggesting a role for protein arginine methylation in T-lymphocyte activation. Arginine methylation is an essential post-translational modification in mammals and yet has not been extensively studied. No large scale analysis of arginine methylation sites has been performed. To gain insight into the role of protein arginine methylation in T-lymphocyte activation, the aims of this work were to: 1. Establish whether levels of arginine methylation are altered during Tlymphocyte activation 2. Use mass spectrometry based proteomics to identify arginine methylated proteins in the T-lymphocyte proteome 3. Further characterise an arginine methylated protein important to Tlymphocyte activation Arginine methylation was found to be induced after long term (>20 hours) stimulation of primary T-lymphocytes. Large increases in the main protein arginine methyltransferase, PRMT1, were also observed. Enrichment and labelling methods were developed to detect arginine methylated peptides from T-lymphocytes by mass spectrometry. This resulted in the identification of 265 unique arginine methylation sites in 141 proteins. 204 of the methylation sites were novel and 103 of the proteins had not previously been described as arginine methylated. Individual arginine methylation sites were characterised before and after activation of T-lymphocytes, with some sites showing significant changes in abundance. Among the novel arginine methylated proteins discovered were Dynamin II, WASp and WIPF1. These proteins are involved in re-organisation of the actin cytoskeleton at the immunological synapse formed between a Tlymphocyte and an antigen presenting cell. The functional consequences of the arginine methylation sites inWASp were characterised. WASp is essential for T-lymphocyte activation and some initial evidence showed that one of the arginine methylation sites is important for WASp activation.
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Eickmeier, Ira. "Relevance of the activation and migration patterns of CD8 T cells for the development of immune-mediated liver injury." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2014. http://dx.doi.org/10.18452/17032.
Full textAbstract:
Die initialen immunologischen Prozesse, die zur Entwicklung autoimmuner Lebererkrankungen führen, sind weitgehend unbekannt. Deshalb wurden in dieser Arbeit die Antigenpräsentation, die Migration sowie der Phänotyp in vivo aktivierter CD8 T-Zellen in der Leber anhand eines Mausmodells der autoimmunen Hepatitis untersucht. Es konnte gezeigt werden, dass hepatische dendritische Zellen an der Entstehung von CD8 Effektor-T-Zellen und an der Inflammation der Leber beteiligt sind. Kupffer-Zellen dagegen nehmen im autoimmunen Kontext in der Leber eine tolerogene Funktion ein. Die in vivo in der Leber aktivierten CD8 T-Zellen zeigten spezifische Oberflächenmarker und ein ungewöhnliches Migrationsverhalten. So wurde zum einen mit Neuropilin-1 ein weitgehend unbekannter Oberflächenmarker identifiziert, zum anderen spricht die Expression von bekannten Markern, die den Aktivierungsstatus der CD8 T-Zellen definieren, für einen hybriden Phänotyp. Sie besitzen sowohl Charakteristika von naiven CD8 T-Zellen als auch von Effektorzellen, eine Eigenschaft, die auch bei zentralen Gedächtniszellen gefunden wird. In der Leber aktivierte CD8 T-Zellen können nicht nur proinflammatorische Zytokine ausschütten und somit eine Inflammation in der Leber auslösen, sondern sind außerdem in der Lage durch Lymphknoten zu zirkulieren. Dagegen ist ihnen der Zugang zum Darm verwehrt, womit eine direkte regulatorische Funktion im Darm ausgeschlossen werden kann. Obwohl auf in der Leber aktivierten CD8 T-Zellen spezifische Adhäsionsmoleküle identifiziert wurden, existiert keine exklusive gewebespezifische Migration in die Leber, wie sie etwa für im Darm aktivierte CD8 T-Zellen nachgewiesen wurde. Im darmassoziierten lymphatischen Gewebe aktivierte CD8 T-Zellen akkumulieren in der Leber und tragen möglicherweise zur Schädigung der Leber im Rahmen chronisch entzündlicher Darmerkrankungen bei. Diese Arbeit trägt somit zum besseren Verständnis der Entstehung autoimmuner Prozesse in der Leber bei.Initial immunological processes leading to autoimmune liver diseases are largely unknown. Therefore this thesis analyzed the antigen presentation, the migration as well as the phenotype of in vivo activated CD8 T cells in the liver by employing a mouse model for autoimmune hepatitis. It was shown that hepatic dendritic cells are effective antigen-presenting cells, which contribute to the induction of functional effector CD8 T cells in the liver and hepatitis. In contrast, Kupffer cells have a tolerogenic role during autoimmune processes in the liver. CD8 T cells that were in vivo activated in the liver display specific surface markers and unusual migration patterns. On the one hand an unusual surface molecule Neuropilin-1 was identified, on the other hand expression of well-known markers defining the activation-status of CD8 T cells suggests a hybrid phenotype. They reflect aspects of naive and effector T cells, characteristics also found on central memory T cells. Liver-primed CD8 T cells do not only produce pro-inflammatory cytokines leading to hepatitis, but they also retain their ability to circulate through lymph nodes. However, they have no access to the gut, which suggests that a direct regulatory function in the gut can be excluded. Although specific adhesion molecules on CD8 T cells activated in the liver were identified, no exclusive tissue-specific migration into the liver exists, as was shown for CD8 T cells primed in the gut. CD8 T cells activated in the gut-associated lymphoid tissue accumulate in the liver, in principle enabling them to induce liver pathology in the context of inflammatory bowel disease. Thus, the here described findings contribute to the understanding of initial immunological processes in autoimmune liver diseases.
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Horne, Phillip Howard. "Activation and effector function of unconventional acute rejection pathways studied in a hepatocellular allograft model." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1188397900.
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Andersson, Pär. "Immunological Effects of TBE Vaccination : Increased Expression of Transcription factor T-bet Indicates Activation of Th1-like Cellular Immunity." Thesis, Linköpings universitet, Hälsouniversitetets läkarutbildning, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-59537.
Full textAbstract:
Tick-borne encephalitis virus is the cause of much morbidity and sometimes a fatal infection. A vaccine based on formaldehyde inactivated virus is currently the only available way of preventing disease. This vaccine gives a high rate of seroconversion but there are reports of vaccination breakthrough, even in people who have demonstrated a neutralizing antibody response. The T cell response to inactivated TBE vaccine is largely unknown, but could be of importance for the effect of the vaccine. This study characterizes aspects of the T cell response by investigating the expression of two transcription factors, T-bet and GATA-3 with RT-PCR. T-bet is expressed in CD4+ T cells of the Th1 type, while GATA-3 is expressed in CD4+ T cells of the Th2 type. Our data show that vaccination with inactivated TBE vaccine leads to increase in expression of the T-bet gene when cells of vaccinated subjects are cultured with TBE virus. In contrast, the expression of GATA-3 remains unaffected by vaccination. Thus, this study suggests that the inactivated TBE vaccine leads to a Th1-like immune response in humans.
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Auma, Ann Winniefred Nangobi. "THE IMPACT OF DIRECT-ACTING ANTI-VIRAL THERAPY ON NAIVE CD4+ T CELL LYMPHOPENIA AND CELLULAR IMMUNE ACTIVATION IN HCV INFECTION AND HCV/HIV CO-INFECTION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1625764728651756.
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Judge, Chelsey J. "IL-7-MEDIATED CD56BRIGHT NK CELL FUNCTION IS IMPAIRED IN HCV IN PRESENCE AND ABSENCE OF CONTROLLED HIV INFECTION, WHILE CD14BRIGHTCD16- MONOCYTES NEGATIVELY CORRELATE WITH CD4 MEMORY T CELLS AND HCV DECLINE DURING HCV-HIV CO-INFECTION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481187921533387.
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Oyeyemi, Oyebode. "Modelling HIV-1 interaction with the host system." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/modelling-hiv1-interaction-with-the-host-system(41095e34-78dd-4b75-bd25-9695a4cc768f).html.
Full textAbstract:
Human immunodeficiency virus (HIV-1) is the pathogenic agent of HIV infection thatprecedes the total breakdown of cellular immunity, a condition known as acquiredimmunodeficiency syndrome (AIDS). The pandemic nature of the disease has promptedintense research into its biology. Already, much is known about HIV-1 infection, lifecycle,and progression to aids. Systems biology enables the combination of complex data fromthese studies into a framework where their effect on the various levels of cellularorganization (i.e. Pathways, cells, tissues, organs and the whole body) could be studied insilico. In this thesis, first, we reviewed our knowledge of the HIV-1 Human InteractionDatabase. We examined its contents and identified processes that HIV-1 was not previouslyknown to interact with. Then, we attempted an in silico dynamic model of HIV-1 interaction. We built a model of HIV-1 interaction with the CD4 T cell activation pathway comprised of137 nodes (16 HIV-1, 121 human) and 336 interactions. The model reproduced expectedpatterns of T cell activation. Using interaction graph properties, we identified 26 host cellfactors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activationor inhibition of viral proteins. By following a logical Boolean formalism, we identified 9 hostcell factors essential to the functions of viral proteins in the activation pathway. This wasthe first attempt to model dynamic viral-host interaction relationships. Then, we organize HIV-1 interacting host genes into modules to represent cellular processesneeded by the virus. We combined HIV-1 interactions with host gene GO annotations toclassify host genes according to these needed cellular processes. We obtained 201 modulesand found the same set of viral proteins do not interact with host genes having similarmodules suggesting intelligence in its co-ordination of host processes. This work is one of agrowing list that explores coordination of HIV-1 interactions. But more importantly, it would bebeneficial to functionally downsize the large dynamic HIV-1 interaction network. Finally, in our discussion, we discuss our results and suggest possible ways in which our workon dynamic models could be improved. This work is opening up a new field of systems virologythat studies the effect of viruses on the host in terms of its temporal and spatial aspects.
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Chung, Charlotte Yuk-Yan. "Tight Junctions - The Link Between HIV-Associated Intestinal Barrier Dysfunction and Loss of Immune Homeostasis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1417822947.
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Santos, Luis. "Cell Mechanics Regulate Mesenchymal Stem Cell Morphology and T Cell Activation." Thesis, 2014. https://doi.org/10.7916/D8K64G7T.
Full textAbstract:
The work of my thesis is the cumulative result of 6 years of research in Prof. Michael P. Sheetz laboratory at the Biological Sciences Department of Columbia University, within the collaborative framework of the Nanotechnology Center for Mechanobiology, an interdisciplinary and multi-institutional center for the study of cell mechanics, involving, among other institutions, the Applied Physics department at Columbia University, and the Schools of Medicine of University of Pennsylvania, New York University, and Mt Sinai.
In Chapter 1, I provide an overview of the field of mechanobiology, with an emphasis on the implications of cell-extracellular matrix and cell-cell attachment on cell function. In Chapter 2, I present the aims of the thesis, with a focus on the two cell systems used in the projects described: human mesenchymal stem cells, and T cells. Then, Chapters 3-5 represent the main body of my thesis, where I present detailed descriptions of the projects that I worked on and that successfully made it into scientific publications or that are in preparation for publication. In Chapter 3, I analyze how matrix chemistry and substrate rigidity affect human mesenchymal stem cell morphology in the context of lineage differentiation, and speculate on potential mechanisms that cells use to sense local rigidity. In Chapter 4, I present a new substrate design that facilitates live visualization of the interface formed between a T cell and an antigen presenting cell, i.e. the immunological synapse, and discuss the impact of intercellular forces on T cell activation. In Chapter 5, I explore the molecular mechanism of Cas-L mechanical activation at the immunological synapse of T cells, and demonstrate how Cas-L regulates T cell activation in the context of an immune response. Finally, in Chapter 6, I lay down the main conclusions of the thesis, and discuss ongoing projects that directly follow up on the results of this thesis.
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Yuan, Dennis Jinglun. "Mechanical regulation of T cell activation." Thesis, 2021. https://doi.org/10.7916/d8-nrgh-7w56.
Full textAbstract:
Adoptive T cell immunotherapy is emerging as a powerful approach to treat diseases ranging from cancer to autoimmunity. T cell therapy involves isolation, modification, and reintroduction of T cells as “living drugs” to induce a durable response. A key capability to fully realize the potential of T cell therapies is effective manipulation of ex vivo T cell activation, with the aim of increasing T cell production and promoting specific phenotypes. While initial efforts to modulate T cell activation have heavily focused on mimicking biochemical signaling and ligand-receptor interactions between T cells and antigen presenting cells (APCs), there is increasing appreciation for understanding the role of mechanics at this interface and utilizing these insights to improve T cell activation systems. The aims of this dissertation is to contribute to this understanding by elucidating how mechanical properties of an activating surface regulate T cell activation, and apply these insights to generate biomaterial based systems to enhance activation from leukemia patient derived T cells.
We first use a hydrogel system to investigate patterns T cell activation to substrate stiffness, discovering a biphasic response of T cell activation to stiffness that is synergist with ligand density. We then generate electrospun fiber scaffolds as an alternative platform to improve T cell expansion; we discover that 3D geometry in the form of fiber diameter and span lengths affects T cell activation. Lastly, we characterize the starting makeup of T cell populations from leukemia patients to study patterns of T cell exhaustion, utilizing the developed electrospun fiber scaffold system to enhance expansion of exhausted T cells from leukemia patients, and demonstrate patient-specific responses to different scaffold formulations. This approach allows for engineering of biomaterial designs that can leverage T cell mechanobiology to enhance T cell activation, with potential to be tailored to patient-specific expansion conditions and increasing the availability of T cell therapy to a wider range of patients.
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Laws, Amy Marie. "The role of Notch in T cell activation and development." 2004. https://scholarworks.umass.edu/dissertations/AAI3136748.
Full textAbstract:
Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP) to CD8 + single positive (SP) transition, but regulation of Notch activation is not well understood. In this thesis, I explored the potential of p53, endocytosis, and Cbl-b to regulate Notch activation. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. One aim of this thesis was to determine if p53 regulates Notch activity during T cell development. I found that Notch1 expression and activation were negatively regulated by p53 in several thymoma lines. Additionally, NIC was elevated in Trp53 −/− thymocytes as compared to Trp53 +/+ thymocytes. To determine if elevated Notch1 activation in Trp53−/− thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP:CD8+ SP T cell ratio was decreased in Trp53 −/− splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8 + SP T cells in Trp53−/− mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, I suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation. In the second aim of my thesis I present preliminary data showing that endocytosis does not appear to be involved in mammalian Notch activation although there is evidence in Drosophila for a positive endocytic role in Notch activation. The ability of Cbl-b to regulate Notch activation was the final aim of this thesis. Cbl-b, like Notch, has been shown to play a role in regulating the T cell signaling threshold. With this aim, I wanted to address the possibility of Cbl-b regulating T cell signaling via regulating Notch activation. Due to technical difficulties I was only able to obtain preliminary data suggesting that Cbl-b does positively regulate Notch activation in peripheral T cells. In this dissertation I have shown that Notch activation is regulated by controlling the expression of cellular components needed for cleavage, not just by encountering ligand on neighboring cells.
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Chuck, Mariana. "The Function of LAT in T Cell Activation and Autoimmunity." Diss., 2010. http://hdl.handle.net/10161/3028.
Full textAbstract:
LAT (linker for activation of T cells) is an important transmembrane adaptor protein in TCR-mediated signaling. Upon TCR engagement, LAT associates with multiple proteins which allows for the activation of downstream signaling pathways. The interaction between LAT with phospholipase C (PLC-gamma1) is especially critical for T cell receptor (TCR)-mediated Ca2+ signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. The phenotype observed in LAT-/- mice is even more severe.T cells are absent in the periphery of these mice due to a complete block in thymocyte development at the DN3 stage thereby making it difficult to study the physiological role of LAT in the activation and function of mature T cells. In order to bypass the developmental defects exhibited by LAT-/- and LATY136F mice, we developed conditional knock-in lines in which only a nonfunctional (ERCreLATf/-) or a LATY136F-mutated allele (ERCreLATf/m) of LAT is expressed in mature T cells after deletion of the wildtype LAT allele.
Analysis of ERCreLATf/m T cells after LAT deletion indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced the development of the LATY136F lymphoproliferative syndrome in these mice. Although Foxp3+ natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicated that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4+CD25+ regulatory T cells.
We have also performed studies using ERCreLATf/- T cells to demonstrate that total LAT deficiency reduced the expression of Foxp3, CTLA4, and CD25 in peripheral Treg cells. Interestingly, mice with LAT deleted in peripheral T cells developed a lymphoproliferative syndrome similar to that observed in LATY136F mice although the disease caused by the LATY136F mutation was more severe. These data implicate LAT in both the positive and the negative regulation of mature T cells. Moreover, our findings indicate that LAT is essential in the maintenance of the regulatory T cell profile in the periphery, thereby aiding in the prevention of lymphoproliferative autoimmune disease.
Dissertation
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Bashour, Keenan. "Spatial Dynamics and the Mechanoresponse in CD4+ T Cell Activation." Thesis, 2013. https://doi.org/10.7916/D80864PP.
Full textAbstract:
The activation of naïve CD4+ T cells by antigen presenting cells is a critical step in the response of the immune system to foreign pathogens and in its acclimation to host tissues. Activation of naïve T cells proceeds through TCR engagement and is further augmented by CD28 costimulation: ensuring T cell survival and conferring numerous functional capabilities. The work in this dissertation highlights the spatial and temporal dynamics that regulate the initial coupling of CD28 with TCR signaling and also dissects the mechanical properties conferred by downstream effectors that are required to relay CD28 costimulation. A reaction-diffusion model that describes the spatial regulation of costimulation in activating human T cells is developed. The Src kinase Lck, though predominantly cytosolic, is an ideal candidate for the coupling of the TCR and CD28 pathways. Membrane associations bring Lck in contact with these receptors, where mediation of its active state by kinase activity and regulation of its spatial dynamics dictate its capacity to integrate early TCR and CD28 signaling. This developed reaction-diffusion model focusing on Lck is then extrapolated to mouse cells that do not share similar sensitivity to segregation of TCR and CD28 triggering: indicating that while Lck is essential for costimulation, it does not confer spatial sensitivity in activating mouse T cells. A comparison of human and mouse cells demonstrate underlying differences in the diffusivity of Lck across the membrane and the enrichment of the cytoskeleton at the interface. The role of the cytoskeleton in generating TCR-driven contractile forces is then investigated through use of micropillar arrays. This approach also enables the quantification of forces generated by T cells during cellular activation. The impact of CD28 costimulation on TCR-driven force generation is assessed and noted to increase cellular forces by 80% beyond what is induced through TCR triggering. By manipulating the presentation of CD28 activation, CD28 is determined to be a mechanoresponsive receptor that is not directly responsible for mechanosensitivty. Rather, CD28 mediates a change in cellular forces through PI3 kinase, whose inhibition normalizes force generation in T cells activated by TCR and those costimulated with TCR and CD28. Downstream of PI3 kinase, PDK1 is identified as being essential in both TCR and CD28 costimulatory force generation; inhibition of PDK1 fully abrogates cellular forces. Lastly, we qualitatively characterize T cell activation on micropillar arrays, where their complex topology reveals a multiphasic behavior during activation. Whereas T cells activated on planar surfaces are relatively stationary, T cells activated on micropillars slowly migrate towards the base of the array. Forces exerted during this migration are substantially greater than those previously measured, and the slow migration leads to the characterization of multiple phases and the relocalization of key cellular proteins.
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Shin, Hyun Mu. "Notch functions from the cytoplasm to the nucleus during T cell activation." 2007. https://scholarworks.umass.edu/dissertations/AAI3275792.
Full textAbstract:
Notch1 specifically upregulates expression of the cytokine interferon-γ in peripheral T cells through activation of NF-κB. However, how Notch mediates NF-κB activation remains unclear. NF-κB activation occurs within minutes of TCR engagement and this activation is sustained for at least 48 hours following TCR signaling. We used either γ-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members or siRNA against Notch1 to reduce endogenous expression of Notch1 specifically. We demonstrate that GSI blocked the later, sustained NF-κB activation, but did not affect the initial activation of NF-κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-κB and competes with IκBα, leading to retention of NF-κB in the nucleus, and that N1IC can directly regulate IFN-γ expression through complexes formed on the IFN-γ promoter. Additionally, within the immunological synapse, cytosolic Notch1 associates with CARMA1 and BCL10, mediating a direct interaction among them. Upon TCR and CD28 stimulation, Notch1 directly interacts with PKC&thetas; and the IKK complex, leading to IKK-mediated activation of NF-κB. In the absence of Notch1, there is no formation of CARMA1/BCL10/Malt1 complex, which is required for IKK activation. Taken together, these data suggest that Notch1 plays two roles during T cell activation: as an activation scaffold in the cytoplasm and as a transcriptional activator in the nucleus.
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Funatake, Castle J. "The influence of aryl hydrocarbon receptor activation on T cell fate." Thesis, 2006. http://hdl.handle.net/1957/28643.
Full textAbstract:
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are well-recognized
for their immunosuppressive activity, which is mediated through an
intracellular receptor and transcription factor, aryl hydrocarbon receptor (AhR).
Laboratory animals exposed to TCDD are less resistant to infection and have severely
impaired humoral and cell-mediated immune responses. This dissertation addressed
the hypothesis that exposure to TCDD disrupts early events during the activation of
CD4⁺ T cells, leading to their premature loss from the spleen. Initially, ovalbumin
(OVA)-specific CD4⁺ T cells from transgenic DO11.10 mice were used to monitor the
effects of TCDD on activated antigen-specific T cells. A graft-versus-host (GVH)
model, in which T cells from C57B1/6 (B6) mice are injected into C57B1/6 x DBA/2
Fl (Fl) mice, was used to study the role of AhR specifically in the T cells in response
to TCDD. B6 donor T cells (from AhR[superscript +/+] or AhR[superscript -/-] mice) respond to DBA/2 antigens
in Fl mice and a CD4-dependent CTL response is generated. In both models, exposure
to TCDD significantly decreased the number of responding CD4⁺ T cells in the spleen
beginning on day 4 after initiation of the response. Exposure to TCDD altered the
phenotype of OVA-specific CD4⁺ T cells beginning on day 2 after immunization with
OVA. These studies also suggested that apoptosis was not the primary mechanism
responsible for the loss of CD4⁺ T cells from the spleen in TCDD-treated mice.
Exposure to TCDD induced AhR-dependent changes in the phenotype of B6 donor
CD4⁺ T cells such that a subpopulation of CD25⁺ cells was increased in TCDD-treated
Fl mice, and these cells had in vitro functional characteristics consistent with
regulatory T (Treg) cells. Exposure to TCDD increased the frequency of donor CD4⁺
T cells producing interleukin (IL)-2. In addition, increased expression of CD25 in
TCDD-treated mice was correlated with increased signaling through the IL-2 receptor.
However, IL-2 alone was not sufficient to mimic the potent immunosuppressive
effects of TCDD. These results suggest that TCDD suppresses T cell immunity in part
by inducing and/or expanding a subpopulation of Treg cells by a mechanism that may
involve IL-2.Graduation date: 2006
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Steinbuck, Martin. "Novel T-cell receptor mediated mechanisms of Notch activation and signaling." Thesis, 2016. https://hdl.handle.net/2144/19064.
Full textAbstract:
The Notch receptor is an evolutionarily highly conserved transmembrane protein essential to a wide spectrum of cellular systems. Notch is especially important to T-cell development, and its deregulation leads to leukemia. Although not well characterized, Notch signaling continues to play an integral role in peripheral T-cells, in which a unique mode of Notch activation can occur. In contrast to canonical Notch activation initiated by adjacent ligand-expressing cells, T-cell receptor (TCR)-stimulation is sufficient to induce robust Notch signaling. However, the interactions between these two pathways have not been defined.
In this dissertation, we show that Notch activation occurs in peripheral T-cells within a few hours post TCR-stimulation and is required for optimal T-cell activation. Utilizing a panel of inhibitors against components of the TCR signaling cascade, we demonstrate that Notch activation is facilitated through initiation of protein kinase C-induced ADAM-metalloprotease activity. Moreover, internalization of Notch via endocytosis is indispensible for this process. Whereas ligand-mediated Notch stimulation relies on mechanical pulling forces that disrupt the autoinhibitory domain of Notch, we hypothesized that in T-cells in the absence of ligands, these conformational changes are induced through chemical adjustments in the endosome, causing alleviation of autoinhibition and receptor activation. Our data show that endocytosis is not only a prerequisite for TCR-induced Notch processing during normal T-cell function, but is essential even in Notch-mutated T-leukemia cells exhibiting constitutively active Notch signaling.
Our work has also focused on signaling mechanisms of Notch following receptor activation. The Notch signal is transduced via cleavage of the intracellular portion of the receptor that subsequently translocates to the nucleus where it regulates gene transcription via interactions with its DNA-binding partner, RBPJκ. Utilizing RBPJκ-deficient T-cells, we show that, although Notch signaling is required, RBPJκ-dependent signaling is dispensable for peripheral T-cell proliferation and activation. Using retroviral constructs that encode modified, active forms of Notch restricted to the nucleus or cytoplasm, we provide evidence that Notch signaling may utilize RBPJκ-independent pathways for signal transduction.
In conclusion, T-cells have evolved a unique method of Notch receptor activation, described for the first time in this dissertation, as well as novel mechanisms that facilitate downstream signaling.
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Tilahun, Mulualem Enyew. "Cooperative immunological and pharmacological control of SEB-induced T cell activation and subsequent pathology." 2010. https://scholarworks.umass.edu/dissertations/AAI3397749.
Full textAbstract:
Staphylococcal Enterotoxin B (SEB) is one of the potent exotoxins synthesized by Staphylococcus aureus that causes toxic shock, is a primary cause of food poisoning and is a Class B bioterrorism agent. SEB, a superantigen, mediates antigen-independent activation of a major subset of the T-cell population by crosslinking TCRs of T-cells with MHC class II molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. This excessive secretion of cytokines, some of which are inflammatory, causes immune dysregulation, systemic inflammation and disease. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin’s interaction with the TCR or MHC class II and provide protection against the debilitating effects of this superantigen. In a series of experiments, we derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of noncrossreactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically-acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human PBMCs, or splenocytes derived from BALB/c or HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T cell activation and cytokine production. When tested in vivo in HLA-DR3 transgenic mouse TSS model, the two chimeric antibodies acted synergistically and provided full protection against SEB-mediated TSS symptoms and lethality of SEB. Furthermore, combination of chimeric anti-SEB, an extracellular inhibitor of SEB, and pharmacological agents (γ-secretase inhibitors, rapamycin, or lovastatin), an inhibitor of intracellular pathways recruited by SEB, provided significant reduction of SEB-induced T cell activation in cultures of mouse splenocytes and human PBMCs. Combination of chimeric anti-SEB antibody and lovastatin also provided in vivo protection against lethal doses of SEB in HLA-DR3 transgenic TSS model. In this study, we have developed a pair of chimeric anti-SEB antibodies (for the first time) that neutralize SEB efficiently in vitro as well as in vivo. In addition, we demonstrated that in vivo protection against lethal doses of SEB can be achieved by a statin of proven safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. Both these findings have provided potential treatment options for diseases mediated by SEB, as there is no prophylaxis, or therapy against accidental or malicious exposure.
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Li, Chaoran. "MicroRNA and Epigenetic Controls of CD4+ T cells' Activation, Differentiation and Maintenance." Diss., 2014. http://hdl.handle.net/10161/8639.
Full textAbstract:
As a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.
Dissertation