Eupalinilide E Inhibits Erythropoiesis and Promotes the Expansion of Hematopoietic Progenitor Cells

Hematopoietic stem cells (HSCs) are the progenitor cells that give rise to all blood cells. The ability to control HSC differentiation has the potential to improve the success of bone marrow transplants and the production of functional blood cells <i>ex vivo</i>. Here we performed an unbiased screen using primary human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) to identify natural products that selectively control their differentiation. We identified a plant-derived natural product, eupalinilide E, that promotes the <i>ex vivo</i> expansion of HSPCs and hinders the <i>in vitro</i> development of erythrocytes. This activity was additive with aryl hydrocarbon receptor (AhR) antagonists, which are also known to expand HSCs and currently in clinical development. These findings reveal a new activity for eupalinilide E, and suggest that it may be a useful tool to probe the mechanisms of hematopoiesis and improve the <i>ex vivo</i> production of progenitors for therapeutic purposes

Mst1 Directs Myosin IIa Partitioning of Low and Higher Affinity Integrins during T Cell Migration

<div><p>Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of <i>Mst1</i>-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. <i>Mst1</i> deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.</p></div

Mst1 is dispensable for integrin-dependent T cell polarization by required for CCL19-induced migration.

<p><b>A</b>) Wt and <i>Mst1^h/h</i> CD4 T cells were seeded into slide chambers pre-coated with 100 ng/mL ICAM-1-Fc prior to stimulation with CCL19. Polarization of CD44 to the uropod in comparison to LFA-1 expression was visualized by confocal microscopy. <b>B</b>) Computational scoring of CD44 and LFA-1 clustering during live imaging of wt and <i>Mst1^h/h</i> CD4 T cells on ICAM-1 coated chamberslides stimulated for 30 minutes with 100 ng/mL CCL19 in presence of 0.08 ng/mL Alexa647-anti-CD11a/LFA-1 (M17/4) and Alexa488-anti-CD44. For each time point, 99–166 individual cells were analyzed for receptor clustering. Student's t-test was performed to compare clustering efficiency for Mst1^wt and Mst1^h/h T cells. <b>C</b>) Transmigration of purified wt and <i>Mst1^h/h</i> CD4 T cells in response to 100 ng/mL CCL19 through 3 μm or 5 μm pores pre-coated with BSA or ICAM-1 Fc. Data is displayed as mean ± SEM of triplicate samples in a single experiment representative of 3–5 independent experiments. Student's t-test was performed to compare migration efficiency for Mst1^wt and Mst1^h/h T cells, * p<0.002, ** p<0.0001. <b>D</b>) CCR7 expression was determined by flow cytometry.</p

<i>WeeT</i> mice have reduced peripheral CD4 and CD8 T cells due to <i>Mst1</i> deficiency.

<p><b>A</b>) Representation of CD4 and CD8 T cells, CD11b⁺ and B cells in the peripheral blood of <i>Mst1^wt</i> and <i>Mst1^h/h</i> mice. <b>B</b>) Inheritance of homozygous C57BL/6 (B), 129Sv/ImJ (C) or heterozygous (H) SNPs in F2 mice generated by crossing <i>Mst1^h/h</i> mice from the original C57BL/6 background to 129Sv/ImJ. Genetic mapping of T-lymphopenic (<i>WeeT</i>) and normal mice isolated a 4.5 Mb region on chromosome 2 harboring the causative mutation. <b>C</b>) <i>Mst1^h/h</i> mice harbor an A to C transversion in exon 5 of the <i>Mst1</i> gene, resulting in change of Leu₁₅₇ within the Mst1 kinase C-lobe to Arg (L₁₅₇R). <b>D</b>) Similar abundance of <i>Mst1</i> transcripts in wt and <i>Mst1^h/h</i> T cells. <b>E</b>) <i>Mst1^h/h</i> T cells have reduced Mst1 protein levels in the presence or absence of proteosome (MG132) or caspase-3 inhibitors (Z-DEVD).</p

Mst1 is required for integrin-independent T cell polarization.

<p><b>A</b>) Wt and <i>Mst1^h/h</i> CD4 T cells were stimulated with 100 ng/mL CCL19 in PBS for 30 minutes. Polarization of CD44 to the uropod and LFA-1 distribution were visualized by confocal microscopy. <b>B</b>) Computational scoring of CD44 and LFA-1 clustering on wt and <i>Mst1^h/h</i> CD4 T stimulated with 100 ng/mL CCL19 in PBS prior to fixation and staining for LFA-1 and CD44 expression. Student's t-test was performed to compare clustering efficiency for Mst1^wt and Mst1^h/h T cells.</p

Mst1 regulates Myosin IIa localization and is required for partitioning of low and higher affinity LFA-1 molecules.

<p><b>A</b>) Wt and <i>Mst1^h/h</i> CD4 T cells expressing Myosin IIa-GFP were seeded into slide chambers pre-coated with 1 μg/mL ICAM-1-Fc and stimulated with CCL19 prior to fixation and staining of F-actin with Rhodamine-phalloidin. Three-dimensional image reconstructions from z-stacks of confocal micrographs are displayed. <b>B</b>) Wt and <i>Mst1^h/h</i> CD4 T cells expressing Myosin IIa-GFP were visualized by live TIRF microscopy. Arrows indicate bipolar morphology. Data are representative of 2 individual experiments with 150 cells per genotype. <b>C, D</b>) Wt and <i>Mst1^h/h</i> CD4 T cells were stimulated as above and stained with 2D7 (anti-low affinity CD11a/LFA-1, green) and M17/4 (anti-CD11a/LFA-1, red). <b>E</b>) Wt CD4 T cells stimulated as above with or without Blebbistatin treatment were stained with 2D7 and visualized by immunofluorescence. <b>F</b>) Quantification of cells with 2D7 localization at the trailing edge of untreated or Blebbistatin-treated Wt and Mst1^h/h CD4 T cells (data are representative of 2–3 individual experiments, n = 13–23).</p

Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen

Itpkb is required for T cell development.

<p><i>Itpkb</i>^<i>+/+</i> and <i>Itpkb</i>^<i>fl/fl</i> mice were treated with tamoxifen for 5 days followed by 2 days of rest. Thymocytes and splenocytes from tamoxifen-treated mice were compared to WT and <i>Itpkb</i>^<i>-/-</i> mice via flow cytometry. (A) Gating schemes used for thymocyte analysis with antibodies to CD4, CD8, CD3, and TCRb; numbers in the plots indicate the percentages of each gated population. (B) Total numbers of each thymocyte subset. (C) Gating scheme for analysis of the splenocytes stained with antibodies to CD4, CD8, CD3, and B220; numbers in the plots indicate the percentages of each gated population. (D) Total numbers of the indicated splenocyte populations. DP: double positive. Data from one representative experiment is shown. *, P < 0.05; **, P < 0.01.</p

Itpkb inhibitors block rat antigen-induced arthritis.

<p>(A) Lewis rats were immunized intra-dermally on Day -21 and -14 with methylated BSA (mBSA), followed by daily oral dosing of GNF362, or dexamethasone (Dex) as a positive control. On Day 0, the rats received an intra-articular injection of mBSA into the right knee joint. (B) Serum was sampled on Day +7, and IgG antibody titers to mBSA were determined by ELISA. The antibody titers were calculated by determining the average dilution at which half-maximal absorbance is detected after subtraction of background. Fold reduction in antibody titer over vehicle is shown in the table. Data shown is one representative experiment. (C) The diameters of the right and left knees were measured on Days 0, 2, 4, and 7, and the ratio of right over left knee diameters (R/L) is shown. (D) Histological analysis of the knee joint was performed blindly and scored on a 5-point scale at the termination of the study. Data shown is one representative experiment. *, P < 0.05; **, P < 0.01.</p

Itpkb is required for T cell function by negatively regulating SOC channels.

<p>WT and <i>Itpkb</i>^<i>fl/fl</i> mice were immunized with either the T-independent antigen, TNP-Ficoll, or the T-dependent antigen DNP-KLH. ELISA of TNP-specific IgG3 (A) or DNP-specific IgG1 (B) antibody responses on day 12 following immunization. Data from one representative experiment is shown (**, P < 0.01). <i>In vitro</i>, Itpkb-deficient mature lymphocytes are diminished in their proliferative capacity as measured by thymidine incorporation of purified CD4^<i>+</i> cells following stimulation with various concentrations of anti-CD3 plus anti-CD28 (C). The data from one representative experiment are shown with values representing the mean counts per minute (cpm) ± SEM. *P < 0.05. Analysis of Ca^<i>2+</i> responses using the Ca^<i>2+</i> sensitive dyes Fluo-4 and Fura Red were evaluated after cross-linking the antigen receptor either in the presence or absence of exogenous Ca^<i>2+</i>. Splenocytes gated on CD4 were treated with anti-CD3-biotin, followed by cross-linking with streptavidin (S.A.) in the presence of exogenous calcium (top panel), or in the absence of exogenous calcium, followed by calcium re-addition to examine SOC channel function (bottom panel). Ionomycin (Iono.) stimulation was used at the end of each run to control for equivalent dye loading. Data is shown as the mean fluorescent ratio of Fluo-3 and Fura-Red. Representative data of three independent experiments are shown.</p