Exploring the Functional Role of Helios in Mucosal Associated Invariant T Cells using siRNA.

Mucosal associated invariant T (MAIT) cells are a fairly recently described population of innate-like T cells. In humans, MAIT cells represent an exceptionally abundant population in the blood, where they account for 1–10% of all T cells. However, compared to conventional T cells, which can display an almost unlimited T cell receptor (TCR) repertoire, the specificities of MAIT cell TCRs are limited. This evolutionarily conserved subset displays a semi-invariant TCR which recognises riboflavin metabolites, produced by a wide range of bacteria and fungi, and presented on the major histocompatibility complex (MHC)-class I related (MR1) molecule. The function and significance of MAIT cells in health and disease have only started to be unravelled, and it is becoming increasingly clear that MAIT cells are also modulated in non-microbial diseases. Interestingly, MAIT cells have been shown to exhibit a relatively high expression of the transcription factor, Helios, compared to most other T cell subsets. The function of this translational activator and repressor, encoded by the gene IKZF2, in the MAIT cell population remains obscure. This study focused on the functional role Helios may play in regulating the activation of MAIT cells. Thus, by using siRNA to silence Helios expression, and flow cytometry to analyse any potential alterations in MAIT cell activation markers, we aimed to be able to characterise the kinetics and functional role of Helios in peripheral MAIT cells of healthy individuals. Here, we clearly established a striking upregulation of Helios in MAIT cells following 24 hours of stimulation. Moreover, we were able to achieve a >50% knockdown of Helios at the protein level, in this subset of T cells. Nevertheless, no significant difference in any of the activation markers we investigated was present between the MAIT cells with reduced Helios expression and their controls. This could, however, of occurred as a result of the toxicity that the transfection had on the functionally of the T cells. From these results, it is difficult to conclude any clear role for Helios in the activation of MAIT cells, and consequently, further research needs to be performed before any clear conclusions can be drawn

Optimising protein detection with fixable custom oligo-labelled antibodies for single-cell multi-omics approaches

Background and Aim Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. Main Methods and Results We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. Conclusions and Implications Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.Peer reviewe

A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.Peer reviewe

Characterization of low-density granulocytes in COVID-19

Author summary The emergence of SARS-COV-2 and the ensuing COVID-19 disease has revealed an unprecedented need to understand the pathological mechanisms of acute respiratory infections in more detail. Granulocytes are highly abundant cells of the innate immunity, and thus first responders towards acute infections. However, their excessive activation can cause unwanted tissue damage and detrimental effects in humans. This study identifies a population of low-density granulocytes (LDGs) in COVID-19 patient samples, which has been poorly described in the context of acute infections so far. These cells were subclassified and found to be mainly of immature phenotypes. Further characterization revealed COVID-19 LDGs as a phenotypically diverse population with immunosuppressive characteristics, which seemed to be in line with an elevated recruitment and activation of granulocytes. Altogether, these findings suggest LDG may play a role in COVID-19 disease progression. Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.Peer reviewe

Systems-Level Immunomonitoring from Acute to Recovery Phase of Severe COVID-19

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Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

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Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

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Pinterest as a Tool: Applications in Academic Libraries and Higher Education

Pinterest, a pinboard-style social photo-sharing website, has become a popular site for many individuals who collect images that help them plan, organize, and explore any topic of interest. Launched in March 2010, Pinterest now has over 10 million users and is continuing to grow. Libraries and educators are starting to explore this new type of social media and how it can be used to connect with and inspire their patrons and students. This article will look at how the University of Regina Library is currently using Pinterest to engage and interact with the University community. This social tool has appealed not only to librarians but educators as well. Pinterest is starting to have an impact on the way educators teach and present information and ideas to their students. The popularity of Pinterest has even inspired other image-based social media sites such as Learnist. After developing a Pinterest account for the library, a list of best practices were created. The library looked at copyright considerations and developed a series of questions to help us determine whether to pin or repin an image. This article will look at how Pinterest can be used in libraries and higher education, and some of the copyright considerations involved in using this image-driven site