Browsing by Subject "T cell"

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Biomaterial-based ligand presentation to induce notch signaling in vitro
(2013) Mehta, Naveen; Roy, Krishnendu
T cell adoptive transfer is an important procedure in immunotherapy. In immunocompromised patients, T cells may need to be developed from stem cells, requiring the induction of Notch signaling events responsible for native T cell differentiation in the thymus. Current methods to develop T cells in vitro fail to mimic the 3D thymic niche and are not mechanically tunable. In this study, we improve on these approaches with a 2D polymer system with controllable mechanical properties, and with a 3D system that more accurately mimics the thymic niche. 3D inverse opal poly(ethylene glycol) (PEGDA) scaffolds were fabricated with defined pores using poly(methyl methacrylate) microspheres that are soluble in acetic acid, leaving negative space for cell growth. The PEGDA surface was made bioactive via biotinylation, followed by a streptavidin linker attaching biotinylated Notch ligand Delta-Like Ligand 4 (bDLL4). 2D polyacrylamide gels were prepared by sandwiching a pre-polymer droplet between two glass surfaces during redox initiation. Mechanical properties were modified by adjusting the concentrations of acrylamide and bisacrylamide. Gels were functionalized by immobilizing streptavidin via a SANPAH linker and binding bDLL4. After fabricating the systems and demonstrating their chemical and mechanical tunability, RT-PCR was used to detect Hes-1 expression, a downstream target of Notch, and flow cytometry was used to detect T cell differentiation levels following incubation of T cell progenitors with functionalized polymer systems. We show that a) the thymic niche can be effectively mimicked by synthetic systems, b) these systems effectively induce Notch signaling, and c) Notch signaling results in early T cell differentiation. These data are promising and suggest the potential to develop T cell banks from stem cells for the purposes of immunotherapy.
Characterization of NDE1 in T cells
(2016-05) Nath, Shubhankar; Poenie, Martin F.; Tucker, Haley O.; Ehrlich, Lauren; De Lozanne, Arturo; O'Halloran, Theresa
Helper and cytotoxic T cells accomplish focused secretion through the clustering of vesicles around the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site, known as the immunological synapse (IS). Dynein has been implicated in both the processes. Here, using Jurkat cells as a model T cell system, we showed that the dynein-binding proteins NDE1 and p150 [superscript Glued] form mutually exclusive complexes with dynein and exhibit non-overlapping distributions in target-stimulated Jurkat cells. Immunofluorescent staining showed that NDE1, dynein and Lis1 accumulated at the immunological synapse. NDE1 was cloned from Jurkat cDNA and overexpressed in Jurkat cells as a fusion protein with N-terminal EGFP (EGFP-NDE1) tag. These cells were defective in MTOC polarization when activated by SEE-coated Raji B cells. However, Jurkat cells overexpressing NDE1 fused to a C-terminal monomeric EGFP (NDE1-mEGFP) tag did not show any defect in MTOC polarization. Immunofluorescent staining indicated that dynein failed to accumulate at the IS formed by activated Jurkat cells expressing EGFP-NDE1, but not by NDE1-mEGFP expressing cells. To confirm that NDE1 was required for MTOC translocation, we also used siRNA to deplete NDE1 in Jurkat cells. When NDE1 was depleted, dynein failed to accumulate at the IS and MTOC translocation was also greatly inhibited. Similar results were obtained when NDE1 was depleted in primary mouse CTLs. In this case, TcR transgenic OT-1 mouse CTL were transduced with lentiviruses expressing NDE1 shRNA. NDE1 depleted CTLs showed a reduction in MTOC translocation compared to untreated cells. While both of the C-terminal NDE1-GFP fusions accumulated at the IS, only NDE1-mEGFP showed the ability to bind dynein. There was no detectable coimmunoprecipitation of NDE1 and dynein when anti-GFP antibody was used to immunoprecipitate NDE1 or when anti-DIC antibody was used to immunoprecipitate the dynein intermediate chain. These observations lead to the conclusion that NDE1 accumulates at the IS independent of its binding to dynein. The ability of NDE1 to accumulated at the IS independent of dynein suggests that it is likely what anchors dynein at the IS. It also raises the question of how dynein accumulates at the IS. We observed reduction in NDE1 recruitment to the IS when Jurkat cells were treated with colchicine to depolymerize microtubules so recruitment of NDE1 likely requires microtubule motor for transport. Since dynein does not seem to be required, perhaps kinesin transports NDE1 to the IS. Finally, we investigated vesicle movements in Jurkat cells using expressed CTLA4-mCherry to selectively label secretory lysosomes, vesicles that cluster around the MTOC and accumulate at the central contact site of stimulated Jurkat-Raji cell pairs. Depletion of p150 [superscript Glued] prevented this vesicle accumulation but did not affect MTOC translocation. Immunoprecipitation data showing that NDE1 and p150 [superscript Glued] are in different dynein complexes, that they show different spatial distributions in the cell, and show no functional interdependence, we conclude that the NDE1/Lis1 and dynactin complexes separately mediate the two key components of T cell effector functions.
Degradable poly(ethylene glycol) based hydrogels for pulmonary drug delivery and in vitro T cell differentiation applications
(2013-08) Fleury, Asha Tarika; Roy, Krishnendu
Hydrogels, defined as three-dimensional, hydrophilic networks, offer extensive biomedical applications. The areas of application are heavily concentrated in drug delivery and tissue engineering because of the hydrogels’ ability to mimic extracellular matrixes of tissue while maintaining a high level of biocompatibility. Specifically, poly(ethylene glycol) (PEG) is a well-established biomaterial in hydrogel applications due to its high water-solubility, low toxicity, high biocompatibility, and stealth properties. This thesis discusses two applications of PEG-based degradable hydrogels. The first is the targeted, site-specific, controlled release of biologic drugs administered by inhalation. There are many challenges to designing a pulmonary delivery system for inhalation of biologic drugs such as low respirable fractions and short resident time in the lungs. In this report, the hydrogel microcarriers for encapsulated drugs were formed by cross-linked PEG and peptide sequences synthesized during a mild emulsion process. The microgels underwent freeze-drying in the presence of cryoprotectants and formulated for dry powder inhalation. The microgels displayed swelling properties to avoid local macrophage clearance in the lungs and exhibited triggered release and degradation in response to enzyme for disease specific release. Dry formulations were tested for aerosolization properties and indicated ability to be delivered to the deep lung by a dry powder inhaler. Lastly, microgels were successfully delivered to mice lungs via intratracheal aerosol delivery. This thesis also discusses the use of PEG-based hydrogel as a biomaterial microenvironment for encapsulated stem cells as a means of in vitro T cell differentiation. A 3D hydrogel system creates a biomimetic reconstruction of the cell’s natural microenvironment and allows us to adjust factors such as ligand density and mechanical properties of the hydrogel in order to promote cells differentiation. This report utilizes hydrogels of cross-linked hyaluronic acid and PEG to encapsulate mice bone marrow hematopoietic progenitor cells in the presence of notch ligands, displayed through stromal cells, magnetic microbeads, or immobilized within the hydrogel matrix. Mechanical properties of the hydrogels were tested and the release of encapsulated cells was performed by enzymatic degradation or dissolution. The differentiation data obtained indicated successful differentiation of stem cells into early T cells through the hydrogel system.
Determining the relationship between TCR affinity and T cell response
(2018-08-16) Stevens, Christopher A.; Maynard, Jennifer Ann, 1974-; Georgiou, George; Alper, Hal; Ehrlich, Lauren; Jiang, Ning; Upton, Jason
The T-cell response is a critical component of controlling many cancers and viral diseases and recently developed T-cell based therapies have become very popular and effective treatment methods. Adoptive cell therapy involves the isolation of a patient’s or donor’s T-cells, expansion or modification ex vivo, and then administration into the patient. The recent FDA approval of an adoptive cell therapy involving an engineering antigen receptor has solidified this technique from a passing fad to a legitimate and effective technique for treating cancer. T-cells are able to recognize a vast range of target antigens through the use of a T-cell receptor (TCR) which targets small peptide fragments presented on the surface of diseased cells in the form of major histocompatibility complexes (pMHC). Further improvement of these therapies and methods requires a deeper understanding of how these TCR-pMHC interactions relate to functionality. Cytomegalovirus is a persistent viral infection that can reactivate periodically during periods of immunodepression. Viral control is mediated by a strong T-cell response and it can cause life-threatening complications in immunosuppressed individuals.Additionally, the T-cell response after chronic CMV reactivation becomes focused to a few individual clonotypes bearing ‘public’ TCRs that have shared sequences between individuals and are often high affinity. This makes CMV an excellent model for studying TCR-pMHC interactions. Several attempts to improve functionality of natural TCRs by engineering for higher affinity have resulted loss of specificity or reduced activation. Methods for direct selection of TCRs with improved activity are currently being developed, however they are very limited, whereas there are a variety of methods for affinity maturation of TCRs. A firm understanding of how TCR-pMHC binding affinity relates to activation would greatly benefit all T cell therapies. This work attempts to provide a better understanding of the relationship between TCR-pMHC affinity and T-cell activity by characterizing TCRs engineered in multiple platforms to obtain a range of affinities while maintaining antigen specificity. Additionally, we observe the relationship between affinity and activity of in vivo derived TCRs against CMV to help aid adoptive cell therapies, as well as the development of a vaccine to elicit potent T-cell responses.
Development of a model system to characterize antigen-specific T cell receptors based on TCR affinity and T cell function
(2021) Xia, Amanda; Jiang, Ning
Modern T cell cancer immunotherapies are based on the ability of cytotoxic T cells to recognize and attack infected cells. This process is activated by the T cell receptor (TCR) recognizing a peptide-bound major histocompatibility complex (pMHC) on the surface of target cells. It is of particular interest to target neoantigens, antigens derived from tumor-specific DNA mutations, to create a tumor-specific immune response. One method of broadening the T cell response is to genetically engineer the patient’s T cells to express neoantigen-specific TCRs isolated from healthy donors. It is important to understand how the affinity of the TCR-pMHC interaction relates to T cell function in the context of T cells expressing foreign TCR. Here, I created a model system to study human TCRs specific for cytomegalovirus (CMV) pp65, a commonly recognized CMV antigen. By transducing primary T cells with pp65-specific TCRs of known TCR affinity, I investigated how differences in TCR affinity related to T cell function. My model system demonstrated that transduced T cells could maintain high TCR expression over a sustained period and that the affinity of the TCR expressed positively correlated with cytokine production. This sets the foundation of a promising approach to study neoantigen-specific TCRs for the development of melanoma immunotherapy.
Environmental factors that impact neurological autoimmunity
(2024-05) Bazzi, Sam ; Melamed, Esther; Todd Triplett; R. Dayne Mayfield; Andrew Gaudet; Robert Messing
Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) whose etiology is poorly understood. Although genetic factors are known to contribute to disease risk, environmental factors are thought to play an even larger role in MS etiology. Recent work in the MS field has started to explore how genetic and environmental risk factors may interact to influence the onset and severity of MS. The goal of my PhD was to examine how three environmental factors, alcohol consumption, viral infection, and heat exposure, may function to modulate MS disease states either on their own or through their interaction with genetic factors. In my work on alcohol and MS, I explored the concentration-dependent effects of alcohol on oligodendrocytes, the myelinating cells of the CNS that are destroyed in MS. After conducting 3’-Tag RNA sequencing, I found that each dose of alcohol elicited unique changes to the transcriptome that spanned a variety of biological functions including inflammation, cell cycle regulation, and protein translation. I continued my work on alcohol by utilizing an animal model of binge alcohol consumption, the high drinking in the dark (HDID) mouse line. We found that HDID animals, which were selectively bred to consume large amounts of alcohol, had exhibited de novo genetic mutations that were highly enriched in immune pathways. I then conducted immunological assays that found disruptions to the innate and adaptive immune system in HDID mouse lines. During the COVID-19 pandemic, my work expanded to include a major new question that faced MS patients and their treating clinicians: how will immunosuppressive therapies commonly used in MS impact MS patients’ ability to fight off infection or react to vaccination? We followed MS patients on B cell-depleting therapies longitudinally over one year to assess spike antibody titers, lymphocyte composition, and T cell reactivity to SARS-CoV-2 antigens. We found that despite B cell depletion, MS patients mounted a similar T cell response to SARS-CoV-2 antigens. We also found that failure to establish humoral immunity did not result in severe disease. This work has identified cell signatures that may be utilized by clinicians to advise MS patients on the timing of vaccination against COVID-19. Finally, I explored the heat-induced worsening of MS symptoms, a syndrome known as Uhthoff’s phenomenon, and how it may be better defined clinically and potentially mediated through MS lesion location. Uhthoff’s phenomenon is not a well-characterized syndrome in terms of prevalence or specific symptomology, so we utilized survey measures to determine which specific symptoms are worsened during Uhthoff’s phenomenon. We then conducted MRI analysis of brain and spinal cord imaging to quantify CNS lesion location and quantity. We identified a set of symptoms that are worsened with heat in our MS patient cohort with Uhthoff’s phenomenon. We also found that MS patients with Uhthoff’s phenomenon were more likely to have a higher number of supratentorial lesions. My findings across these three projects have led to novel and impactful results that have furthered our understanding of how environmental factors may contribute to MS etiology and severity. My experiments on oligodendrocytes, HDID mice, and Uhthoff’s phenomenon have inspired further studies that are currently underway in our lab that may lead to a further understanding of how alcohol, viruses, and heat exposure impact MS.
High throughput methods for measuring TCR-pMHC affinity and specificity
(2017-11-09) Zhang, Shuqi (Ph. D. in chemical engineering); Georgiou, George; Jiang, Ning Jenny; Maynard, Jennifer; Alper, Hal; Davis, Mark
T cells are a crucial component of the human immune response. I developed two technology platforms that enable high-throughput measurement of TCR-pMHC affinity, sequence, and specificity from human T cells ex-vivo. I use these technologies to define the importance of TCR affinity, changes in the T cell repertoire with age, and properties of the cross-reactive T cell repertoire for cancer neoantigens.
In vitro generation of hematopoietic progenitors and functional T cells from pluripotent stem cells
(2010-08) Lin, Jian, 1980-; Roy, Krishnendu; Tucker, Philip W.; Stevens, Scott W.; Tian, Ming; Whiteley, Marvin
The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies. The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture.
Ligand-immobilized biomaterial surfaces for Notch signaling and T cell differentiation
(2012-12) Kim, Myunghee Michelle; Roy, Krishnendu; Maynard, Jennifer A; Schmidt, Christine E; Suggs, Laura J; Tucker, Haley
In vitro T cell differentiation from hematopoietic progenitor cells is a potential alternative source of T cells for adoptive therapy in treatment of cancers as well as T cell deficiencies. Presentation of Notch ligands immobilized on a surface is necessary in designing a stroma-free in vitro T cell differentiation system. Current methods for in vitro T cell differentiation have advanced greatly in the recent years, allowing development of functional T cells in vitro. However, these are limited to 2D coculture with stromal cells or culture on hard plastic surfaces with immobilized ligands, and have yet to report quantitative effects of variables such as substrate stiffness. This dissertation discusses the fabrication of 2D and 3D systems of various properties for presentation of Notch ligands for development of an efficient culture system, at the same time offering insight into the science of cell signaling and cell-material interactions. Magnetic microbeads, liposomes, as well as 2D and 3D soft hydrogel surfaces were fabricated to present Notch ligands at varying ligand densities and to study their quantitative effect on Notch signaling and T cell differentiation. The x findings of this dissertation demonstrate that substrate material plays a role in Notch signaling in combination with ligand density, and may affect downstream events of T lineage commitment. Insights gained from this research provide a new direction in the importance of culture substrate in ligand-presenting systems and allow development of new systems to support efficient generation of T cells in vitro.
Role of TCR affinity in function, proliferation, memory generation, and exhaustion during human viral infection
(2019-02-01) Williams, Chad Manjeot; Jiang, Ning Jenny; Brock, Amy; Maynard, Jennifer; Ehrlich, Lauren; Kim, Hyun Jung
TCR affinity is a strong predictor of T cell function, proliferation, exhaustion, and generation of immune memory. 2D TCR affinity quantification is derived from direct antigen interaction on the T cell itself, making it a more physiologically relevant form of TCR affinity information. 2D TCR affinity demonstrated a 1000-fold affinity range within in vitro cultured Naïve Hepatitis C Virus (HCV) specific CD8⁺ T cells from HCV negative individuals. 2D affinity predicted T cell function better than other affinity measurement techniques, and identified TCRs capable of a CD8 independent functional response. High affinity HCV TCRs were more sensitive to low concentrations of antigen presentation giving these TCRs a greater capacity for immune surveillance during infection. However, the interrogation of the role TCR affinity plays in the immune response of CD8⁺ T cells during an ongoing viral infection has yet to be realized. With the advent of high throughput TCR affinity quantification techniques like in situ TCR affinity and sequence test (iTAST), it is now possible for rapid ex vivo isolation and TCR affinity quantification of antigen specific T cells responding to an ongoing viral infection. iTAST was used to isolate naïve, effector, and memory subsets of Cytomegalovirus (CMV) specific CD8⁺ T cells in the blood and spleen of CMV infected individuals. 2D TCR affinity of naïve cells had 1000-fold affinity range, yet effector and memory subsets were highly enriched for cells expressing medium affinity TCRs in comparison to either high or low affinity counterparts. This was corroborated by TCR sequencing analysis of the same populations for which 2D TCR affinity distributions were characterized. Surprisingly, the functional response of medium affinity TCRs, despite more reliance on CD8, was similar to that of TCRs with an order of magnitude higher TCR affinity, and seemed to be linked to intrinsic differences in inhibitory marker regulation and expression of PD-1, Tim-3, and LAG-3. Additionally, medium affinity CMV TCRs gave CD8⁺ T cells a proliferative advantage during in vitro culture conditions. In summary, TCR affinity influences CD8⁺ T cell function, proliferation, and exhaustion thus helping maintain immunological memory within an ongoing human viral infection
The roles of dynein and dynein accessory proteins in T cell effector functions
(2013-05) Christian, Laura Manno; Poenie, Martin F.
T cell effector functions depend on focused secretion. This is accomplished by secretory vesicle (SV) clustering around the microtubule organizing center (MTOC) and MTOC translocation to the specialized site of cell-cell contact - the immunological synapse (IS). The dynein molecular motor has been implicated in both processes. To investigate the roles of dynein and dynein-associated proteins we used Jurkat cells expressing fluorescent CTLA-4 for SV tracking and molecular traps targeting dynein subunits to show that dynein intermediate chain (DIC) and the light chain LC8 are needed for both SV clustering and MTOC translocation. We also found that immunostaining with different anti-DIC antibodies labeled different pools of dynein at the IS in activated Jurkat cells. To discern how dynein separately accomplishes both MTOC and SV activities we cloned DIC cDNAs from Jurkat cell mRNA and obtained two isoforms, DIC2B and DIC2C. However, both isoforms were concentrated around the MTOC and formed a ring-like structure at the IS. We also saw little difference in dynein-binding proteins that co-immunoprecipitated with each isoform. We then investigated the roles of the dynactin component p150Glued and Lis1 protein in MTOC translocation and SV clustering. Surprisingly, p150Glued was concentrated around the MTOC but was not present at the IS. SVs marked by CTLA-4 showed clustering defects while MTOC translocation was not significantly affected in p150Glued siRNA knockdown cells. On the other hand, Lis1 immunostaining labeled a ring at the IS where it mimicked the distribution of the dynein ring thought to be involved in MTOC translocation. MTOC translocation was potently blocked in Lis1 siRNA knockdown cells but dynein recruitment was only slightly disrupted and there was no visible effect on actin localization at the IS. Overexpression of Lis1 or expression of Lis1 deletion mutants interfered with MTOC translocation and interfered with dynein recruitment, while actin was still localized at the IS. However, studies of calcium flux in response to T cell receptor (TcR) stimulation showed that these mutant-expressing cells had deficiencies in cell signaling from the TcR. These results suggest that MTOC translocation and SV clustering are mediated by dynein but likely involve different dynein-binding proteins.
The role of DISC1 in the organization of the T-cell immunological synapse
(2019-09-24) Maskalenko, Nicholas Alexander; Poenie, Martin F.; De Lozanne, Arturo; Chan, Clarence S; Jiang, Ning; Tucker, Haley O; Ehrlich, Lauren I
When T-cells contact an antigenic target or antigen-presenting cell, signaling through the T-cell receptor triggers formation of a specialized junction known as the immunological synapse (IS). In a previous study, DISC1 (Disrupted in Schizophrenia 1) was identified as a component of the IS in T-cells, where it forms a complex with dynein, Nde1, and Lis1. Here we show that there are two isoforms of DISC1 expressed in T-cells, one (DISC1 Lv) that colocalizes with mitochondria and the other (DISC1 L) that accumulates at the IS. The accumulation of DISC1 at the IS is also observed in OT-1 CTLs and NK cells. Additionally, we show that disrupting DISC1 using CRISPR/Cas9 technology leads to many changes at the synapse. Each isoform upon reintroduction restores specific changes. The translocation of mitochondria was dependent on the Lv isoform. Loss of the L isoform results in a defect in MTOC translocation, failure of Nde1 to move from the center of the IS to the pSMAC, and greatly reduced actin accumulation at the IS. This last effect depends on the association of DISC1 with Girdin, such that loss of expression of either protein gives the same phenotype. Girdin is involved in cytoskeletal remodeling in other cells but its function in T cells has not been previously described. The results of this work contribute to the understanding of MTOC translocation and mitochondrial movements, but they also generate new questions about the role and regulation of actin at the synapse. The loss of actin in Girdin or DISC1 deletions does not have a severe effect on MTOC translocation as is seen in cells treated with actin inhibitors. However, these treatments lead to a complete loss of Nde1, Lis1, and dynein from the synapse, suggesting that the dynein complex is ultimately linked to actin. These results point to DISC1 and Girdin functioning in a novel actin signaling pathway working at the IS