Imaging flow cytometry: A method for examining dynamic native FOXO1 localization in human lymphocytes.

While flow cytometry can reliably assess surface and intracellular marker expression within small cell populations, it does not provide any information on protein localization. Several key transcription factors (TF) downstream of lymphocyte surface receptors are regulated by nuclear versus cytoplasmic localization, and one such TF is Forkhead box O1 (FOXO1). FOXO1 integrates antigen-binding, co-receptor activation and metabolic signals in lymphocytes, leading to proliferation and differentiation. Importantly, the nuclear or cytoplasmic localization of FOXO1 is key for gene expression leading to different lymphocyte phenotypes. In effector lymphocytes (Teff), for example, lymphocyte receptor (TCR) signaling leads to an Akt-dependent phosphorylation of FOXO1. Phosphorylated FOXO1 is excluded from the nucleus, promoting proliferation and effector functions. In contrast, nuclear retention of FOXO1 is essential for early and late development of T and B cells and for the thymic development and stability of regulatory T cells. Given the critical role of FOXO1 localization as an indicator and determinant of function, quantification of FOXO1 cellular localization in human lymphocytes can help determine immune cell activation and activity in experimental and clinical scenarios. The standard method used to determine subcellular protein localization is the analysis of nuclear and cytoplasmic protein extracts by Western blotting (WB). However, available techniques, such as WB, are limited by a requirement for a large number of cells and inability to determine FOXO1 localization in individual cells or sub-populations. In contrast, a standardized method using an imaging flow cytometer (IFC) such as the Amnis ImagestreamX Mark II, would provide both qualitative, per-cell localization information, as well as quantitative data on gated sub-populations. To this end, we report the development and optimization of an IFC protocol to examine native FOXO1 localization in human lymphocytes. A human CD4+ lymphocyte line, HuT102, as well as primary human T cells, were assessed for dynamic FOXO1 localization after treatment with a lymphocyte receptor signaling mimic (PMA/Ionomycin). IFC nuclear translocation analysis permitted us to precisely quantify the alterations over time in nuclear and cytoplasmic localization of native FOXO1 on a per cell basis, including within specific, user-defined sub-populations of cells. For human lymphocytes, using IFC to assess and quantify dynamic FOXO1 localization allows the user to simultaneously study multiple lymphocyte subpopulations as well as to delineate differing effects of dynamic FOXO1 localization that may be lost when other available methods are used.

Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration.

Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαß rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTßR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.

Molecular characterization and evolution of self-incompatibility genes in Arabidopsis thaliana: the case of the Sc haplotype.

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.