| 1. |
- Assarsson, Erika
(författare)
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Acquisition and function of NK cell-associated molecules on T cells
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- One challenge for multicellular organisms, including humans, is to cope with a broad variety of microorganisms and their rapid replication and alterations. To do this, both fast and specific defense mechanisms are needed that can control threatening infections. Our immune system consists of two major parts: innate immunity, which is rapid and rather non-specific, and adaptive immunity, which is highly specific but requires more time for its development. Both innate (e.g. phagocytic cells and NK cells) and adaptive immune cells (B cells and T cells) are regulated by cellular interactions as well as by soluble factors. Each cell expresses a number of receptors, which can facilitate adhesion with other cells and/or mediate downstream signalling events that determine the outcome of immune reactions. In this thesis, I have focused on the expression of different NK cell-associated molecules on CD8+ T cells and the functional consequences of this. NKT cells have been classically defined as T cells expressing NK 1.1. These NKT cells are restricted to the MHC class I-like molecule CD1d and express an invariant T cell receptor, Jalpha281-Valpha 14. My initial finding was that IL-2-activated spleen cell cultures derived from mice deficient in classical NKT cells contained NK 1.1+ T cells (the classical definition of NKT cells). We found that these cells were derived from conventional IL-2 receptor-beta+CD8+ T cells that had acquired NK1.1 upon activation. Subsequently, we observed that similar cells also appeared in the lungs of influenza A virus-infected mice. During the peak of infection, up to 10% of the CD8+ T cells in the lungs coexpressed NK 1.1. Around one third of the NK1.1+CD8+ T cells were specific for a single influenza A virus-derived epitope (ASNENMDAM) as determined by tetramer stainings and antigen-induced IFNgamma production. Interestingly, these NK1.1+CD8+ T cells exhibited a memory phenotype and coexpressed other NK cell-associated molecules including inhibitory Ly49 receptors and 2B4. This showed that all NK1.1+ T cells do not belong to a certain lineage and that the expression of NK cell-associated molecules may represent a state of activation for T cells. We found that, similar to NK 1.1, the NK cell receptor 2B4 was expressed on a subset of memory- like CD8+ T cells and induced upon IL-2 stimulation and during influenza A virus infection. Therefore, we decided to address the function of 2B4 when expressed on CD8+ T cells. To our surprise, we found that 2B4 acted as a ligand, rather than a receptor, for CD48 expressed on neighboring T cells and augmented both antigen-driven and IL-2-induced proliferation. These results prompted us to study whether other 2B4-expressing cells could costimulate T cells through 2B4/CD48 interactions. It was observed that NK cells as well as 2B4-expressing tumor cells enhanced both activation and proliferation of the T cells in response to IL-2 or CD3-crosslinking. NK cells can regulate adaptive immune responses by shaping the cytokine milieu during infection or autoimmune conditions. These data suggest that in addition to cytokine production, direct physical interactions between NK cells and T cells influence both activation and proliferation of T cells. We speculate that 2B4/CD48 interactions between immune cells may facilitate maintenance and reactivation of CD8+ memory T cells and regulate adaptive immune responses.
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| 2. |
- Assarsson, Erika, et al.
(författare)
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Severe defect in thymic development in an insertional mutant mouse model.
- 2007
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Ingår i: Journal of immunology (Baltimore, Md. : 1950). - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 178:8, s. 5018-5027
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Tidskriftsartikel (refereegranskat)abstract
- Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.
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| 3. |
- Berggrund, Malin, et al.
(författare)
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Identification of candidate plasma protein biomarkers for cervical cancer using the multiplex proximity extension assay
- 2019
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 18:4, s. 735-743
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Tidskriftsartikel (refereegranskat)abstract
- Human papillomavirus (HPV) is recommended as the primary test in cervical cancer screening, with co-testing by cytology for HPV-positive women to identify cervical lesions. Cytology has low sensitivity and there is a need to identify biomarkers that could identify dysplasia that are likely to progress to cancer. We searched for plasma proteins that could identify women with cervical cancer using the multiplex proximity extension assay (PEA). The abundance of 100 proteins were measured in plasma collected at the time of diagnosis of patients with invasive cervical cancer and in population controls using the Olink Multiplex panels CVD II, INF I, and ONC II. Eighty proteins showed increased levels in cases compared to controls. We identified a signature of 11 proteins (PTX3, ITGB1BP2, AXIN1, STAMPB, SRC, SIRT2, 4E-BP1, PAPPA, HB-EGF, NEMO and IL27) that distinguished cases and controls with a sensitivity of 0.96 at a specificity of 1.0. This signature was evaluated in a prospective replication cohort with samples collected before, at or after diagnosis and achieved a sensitivity of 0.78 and a specificity 0.56 separating samples collected at the time of diagnosis of invasive cancer from samples collected prior to diagnosis. No difference in abundance was seen between samples collected prior to diagnosis or after treatment as compared to population controls, indicating that this protein signature is mainly informative close to time of diagnosis. Further studies are needed to determine the optimal window in time prior to diagnosis for these biomarker candidates.
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| 4. |
- Darmanis, Spyros, et al.
(författare)
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Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 14:2, s. 380-389
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Tidskriftsartikel (refereegranskat)abstract
- Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.
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| 5. |
- Lundberg, Martin, et al.
(författare)
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Multiplexed Homogeneous Proximity Ligation Assays for High-throughput Protein Biomarker Research in Serological Material
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 10:4, s. M110.004978-
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Tidskriftsartikel (refereegranskat)abstract
- A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pM sensitivity each consuming only 1 mu l of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.
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| 6. |
- Siegbahn, Agneta, 1947-, et al.
(författare)
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Development and validation of a quantitative Proximity Extension Assay instrument with 21 proteins associated with cardiovascular risk (CVD-21)
- 2023
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:11
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Tidskriftsartikel (refereegranskat)abstract
- Background Treatment of cardiovascular diseases (CVD) is a substantial burden to healthcare systems worldwide. New tools are needed to improve precision of treatment by optimizing the balance between efficacy, safety, and cost. We developed a high-throughput multi-marker decision support instrument which simultaneously quantifies proteins associated with CVD. Methods and findings Candidate proteins independently associated with different clinical outcomes were selected from clinical studies by the screening of 368 circulating biomarkers. We then custom-designed a quantitative PEA-panel with 21 proteins (CVD-21) by including recombinant antigens as calibrator samples for normalization and absolute quantification of the proteins. The utility of the CVD-21 tool was evaluated in plasma samples from a case-control cohort of 4224 patients with chronic coronary syndrome (CCS) using multivariable Cox regression analyses and machine learning techniques. The assays in the CVD-21 tool gave good precision and high sensitivity with lower level of determination (LOD) between 0.03-0.7 pg/ml for five of the biomarkers. The dynamic range for the assays was sufficient to accurately quantify the biomarkers in the validation study except for troponin I, which in the modeling was replaced by high-sensitive cardiac troponin T (hs-TnT). We created seven different multimarker models, including a reference model with NT-proBNP, hs-TnT, GDF-15, IL-6, and cystatin C and one model with only clinical variables, for the comparison of the discriminative value of the CVD-21 tool. All models with biomarkers including hs-TnT provided similar discrimination for all outcomes, e.g. c-index between 0.68-0.86 and outperformed models using only clinical variables. Most important prognostic biomarkers were MMP-12, U-PAR, REN, VEGF-D, FGF-23, TFF3, ADM, and SCF. Conclusions The CVD-21 tool is the very first instrument which with PEA simultaneously quantifies 21 proteins with associations to different CVD. Novel pathophysiologic and prognostic information beyond that of established biomarkers were identified by a number of proteins.
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