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The human gut microbiota is a diverse and complex ecosystem that is involved in beneficial physiological functions as well as disease pathogenesis. Blastocystis is a common protistan parasite and is increasingly recognized as an important component of the gut microbiota. The correlations between Blastocystis and other communities of intestinal microbiota have been investigated, and, to a lesser extent, the role of this parasite in maintaining the host immunological homeostasis. Despite recent studies suggesting that Blastocystis decreases the abundance of beneficial bacteria, most reports indicate that Blastocystis is a common component of the healthy gut microbiome. This review covers recent finding on the potential interactions between Blastocystis and the gut microbiota communities and its roles in regulating host immune responses. [ABSTRACT FROM AUTHOR]

Development of thymocytes through the positive selection checkpoint requires the rearrangement and expression of a suitable T cell receptor (TCR) α-chain that can pair with the already-expressed β-chain to make a TCR that is selectable. That is, it must have sufficient affinity for self MHC-peptide to induce the signals required for differentiation, but not too strong so as to induce cell death. Because both alleles of the α-chain continue to rearrange until a positively-selectable heterodimer is formed, thymocytes and T cells can in principle express dual α-chains. However, cell-surface expression of two TCRs is comparatively rare in mature T cells because of post-transcriptional regulatory mechanisms termed “phenotypic allelic exclusion”. We produced mice transgenic for a rearranged β-chain and for two unrearranged α-chains on a genetic background where endogenous α-chains could not be rearranged. Both Vα3.2 and Vα2 containing α-chains were efficiently positively selected, to the extent that a population of dual α-chain-bearing cells was not distinguishable from single α-chain-expressors. Surprisingly, Vα3.2-expressing cells were much more frequent than the Vα2 transgene-expressing cells, even though this Vα3.2-Vβ5 combination can reconstitute a known selectable TCR. In accord with previous work on the Vα3 repertoire, T cells bearing Vα3.2 expressed from the rearranged minilocus were predominantly selected into the CD8+ T cell subpopulation. Because of the dominance of Vα3.2 expression over Vα2 expressed from the miniloci, the peripheral T cell population was predominantly CD8+ cells. [ABSTRACT FROM AUTHOR]

The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP4) is essential for thymocyte positive selection by regulating plasma-membrane association of the protein tyrosine kinase Itk downstream of the T cell receptor (TCR). IP4 can act as a soluble analog of the phosphoinositide 3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate (PIP3). PIP3 recruits signaling proteins such as Itk to cellular membranes by binding to PH and other domains. In thymocytes, low-dose IP4 binding to the Itk PH domain surprisingly promoted and high-dose IP4 inhibited PIP3 binding of Itk PH domains. However, the mechanisms that underlie the regulation of membrane recruitment of Itk by IP4 and PIP3 remain unclear. The distinct Itk PH domain ability to oligomerize is consistent with a cooperative-allosteric mode of IP4 action. However, other possibilities cannot be ruled out due to difficulties in quantitatively measuring the interactions between Itk, IP4 and PIP3, and in generating non-oligomerizing Itk PH domain mutants. This has hindered a full mechanistic understanding of how IP4 controls Itk function. By combining experimentally measured kinetics of PLCγ1 phosphorylation by Itk with in silico modeling of multiple Itk signaling circuits and a maximum entropy (MaxEnt) based computational approach, we show that those in silico models which are most robust against variations of protein and lipid expression levels and kinetic rates at the single cell level share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains at the plasma membrane. This identifies MaxEnt as an excellent tool for quantifying robustness for complex TCR signaling circuits and provides testable predictions to further elucidate a controversial mechanism of PIP3 signaling. [ABSTRACT FROM AUTHOR]

Thymocyte negative selection is a requirement for the development of self tolerance. Although it is possible to assay the induction of cell death in thymocytes in vitro using antibody cross-linking, this stimulus is much stronger than the normal range of T cell receptor ligands that could be encountered during normal development. Signaling in thymocytes is finely balanced between positive and negative selection stimuli, where a negative selecting ligand can be only marginally higher affinity than a positive selecting ligand. We have therefore developed an assay for the induction of negative selection that can distinguish such cases, and that is amenable to high-throughput analysis. The assay is based on the induction of activated caspase 3 in thymocytes expressing a defined T cell receptor, after stimulation with MHC-peptide tetramers in vitro for 24 hours or less. [ABSTRACT FROM AUTHOR]

The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP4) is essential for thymocyte positive selection by regulating plasma-membrane association of the protein tyrosine kinase Itk downstream of the T cell receptor (TCR). IP4 can act as a soluble analog of the phosphoinositide 3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate (PIP3). PIP3 recruits signaling proteins such as Itk to cellular membranes by binding to PH and other domains. In thymocytes, low-dose IP4 binding to the Itk PH domain surprisingly promoted and high-dose IP4 inhibited PIP3 binding of Itk PH domains. However, the mechanisms that underlie the regulation of membrane recruitment of Itk by IP4 and PIP3 remain unclear. The distinct Itk PH domain ability to oligomerize is consistent with a cooperative-allosteric mode of IP4 action. However, other possibilities cannot be ruled out due to difficulties in quantitatively measuring the interactions between Itk, IP4 and PIP3, and in generating non-oligomerizing Itk PH domain mutants. This has hindered a full mechanistic understanding of how IP4 controls Itk function. By combining experimentally measured kinetics of PLCγ1 phosphorylation by Itk with in silico modeling of multiple Itk signaling circuits and a maximum entropy (MaxEnt) based computational approach, we show that those in silico models which are most robust against variations of protein and lipid expression levels and kinetic rates at the single cell level share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains at the plasma membrane. This identifies MaxEnt as an excellent tool for quantifying robustness for complex TCR signaling circuits and provides testable predictions to further elucidate a controversial mechanism of PIP3 signaling.