T-Cell Activation: Post-Infection Diagnostic Tool for COVID-19.

COVID-19 is caused by the SARS-CoV-2 virus and has spread globally in 2020. Cellular immunity may serve as an important functional marker of the disease, especially in the asymptomatic cases. Blood samples were collected from 46 convalescent donors with a history of COVID-19 and 38 control donors. Quantification of the T-cell response upon contact with SARS-CoV-2 proteins in vitro was based on IFN-γ. Significantly higher numbers of activated cells were measured in patients who underwent COVID-19. Anti-SARS-CoV-2 T cells were detected weeks after the active virus disappeared from the organism. Repeated sample collection after five months proved that the T-cell activation was weaker in time in 79 % of the patients. In the majority of cases, the CD4+ helper T-cell subpopulation was responsible for the immune reaction. Moreover, different viral proteins triggered activation in CD4+ helper and in CD8+ cytotoxic T cells. Together, these findings suggest that the T-cell activation level identifies the individuals who underwent COVID-19 and may become a diagnostic tool for the disease.

Mysterious role of H3K56ac in embryonic stem cells.

Posttranslational modifications of histones belong to epigenetic mechanisms that regulate gene expression by chromatin structure changes. Generally, histone acetylation reduces its positive charge and consequently weakens the stability of the nucleosome. Acetylation of lysine 56 on histone H3 is implicated in the processes associated with loosened chromatin structure. H3K56ac is a mark for histones with high nucleosome turnover in the nuclear processes such as gene transcription, DNA replication and reparation in yeasts. During evolution, the main H3K56ac regulatory pathway was lost and the level of H3K56ac remained very low in mammalian cells. Moreover, the function of this modification still remains unclear. In this minireview, we summarize the recent knowledge of the ambiguous role of H3K56ac in mammalian embryonic stem cells.

Generation of human induced pluripotent stem cells using genome integrating or non-integrating methods.

Preclinical studies have demonstrated the promising potential of human induced pluripotent stem cells (hiPSCs) for clinical application. To fulfil this goal, efficient and safe methods to generate them must be established. Various reprogramming techniques were presented during seven years of hiPSCs research. Genome non-integrating and completely xeno-free protocols from the first biopsy to stable hiPSC clones are highly preferable in terms of future clinical application. In this short communication, we summarize the reprogramming experiments performed in our laboratories. We successfully generated hiPSCs using STEMCCA lentivirus, Sendai virus or episomal vectors. Human neonatal fibroblasts and CD34(+) blood progenitors were used as cell sources and were maintained either on mouse embryonic feeder cells or in feeder-free conditions. The reprogramming efficiency was comparable for all three methods and both cell types, while the best results were obtained in feeder-free conditions.

Haematopoietic developmental potential of human pluripotent stem cell lines.

The generation of haematopoietic progenitors from human pluripotent stem cells (hPSCs) presents great promise for cell-replacement therapies. However, current protocols for haematopoietic differentiation of hPSCs suffer from low efficiency and functional defects in the derived cells. The technology is also limited by variable ability of hPSC lines to generate blood cells in vitro. To address this issue, methodologies for haematopoietic differentiation in feeder-free conditions were applied to available human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines in this study. It was found that these cell lines did not generate haematopoietic progenitors to such an extent as did H1 and H9 hESC lines that were used for this purpose in the vast majority of relevant studies. These results suggest that for clinical application of blood cells derived from hPSCs, possibly from autologous hiPSCs, it is necessary to overcome the variability in the haematopoietic developmental potential of individual hPSC lines.

Relative expression of γδ T-cell receptor gene families detected by RT-PCR and capillary electrophoresis.

γδ T cells are intensively studied because their function in infection, allergy, autoimmune disease, cancer and post-transplant period is not yet fully understood. PCR-based techniques were established to study the γ variable (Vγ) and δ variable (Vδ) gene families. PCR product evaluation is routinely carried out by Southern blot analysis or the third complementarity-determining region spectratyping, but a fast and simple assessment of Vγ and Vδ gene family expression is missing. The aim of our study was to test capillary electrophoresis as a potential method for evaluating the composition of the γδ T-cell population. This report provides optimized PCR conditions for γδ T-cell receptor amplification. Further, it describes the utilization of capillary electrophoresis in the Agilent 2100 Bioanalyzer to evaluate the relative expression of Vγ and Vδ gene families after their amplification. An application of the methodology to peripheral blood mononuclear cell samples from patients during haemato-oncological treatment is shown. The described methodology is fast and simple to operate and is therefore suitable as a first screening of the γδ T-cell population composition in tissues of interest.

Microarray analysis using a limited amount of cells.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.