Mechanically active integrins target lytic secretion at the immune synapse to facilitate cellular cytotoxicity.

Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the αLß2 isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.

High-fat diet disrupts REG3γ and gut microbial rhythms promoting metabolic dysfunction.

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota's capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.

Prostaglandin E2: a putative potency indicator of the immunosuppressive activity of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal cells in the marrow. MSCs have been shown to be immunosuppressive and have become an attractive therapeutic option for the modulation of undesired immune responses. Currently, ex vivo expanded human (h)MSCs are being utilized in clinical trials both in the USA and in Europe to treat a variety of immune disorders. hMSCs need to be harvested, isolated and expanded in culture. This necessary expansion may also result in decrease or loss of the immunomodulatory potential of hMSCs. Ideally, the intrinsic immunomodulatory activity (potency) of an hMSC preparation should be assessed prior to its administration. The goal of the experiments described here was to develop a simple potency assay for the immunomodulatory properties of hMSCs. The immunosuppressive activity of hMSCs conditioned media was tested in enzyme-linked immunosorbent spot assays (ELISpot) and the immunosuppressive activity of the conditioned media was correlated with the concentration of several cytokines present in these conditioned media. The concentration of prostaglandin E(2) in the media correlated with their immunosuppressive activity. The concentration of the other cytokines measured did not correlate with the immunosuppressive activity of the media. The dose-response effect could be replicated by adding PGE(2) to ELISpot assays. Furthermore, the immunosuppressive activity of the conditioned media was inhibitable by a neutralizing anti-PGE(2) antibody. These data suggest that measurement of PGE(2) in media conditioned by hMSCs exposed to inflammatory stimuli could be used as a surrogate measure of their immunosuppressive capacity. These findings need to be confirmed in vitro using different assays of immune function and validated in vivo to determine the level of correlation of these data with efficacy in pre-clinical models of immune disorders.

Fibroblast Growth Factor-2 Enhances Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells without Diminishing Their Immunosuppressive Potential.

Allogeneic hematopoietic stem cell transplantation is the main curative therapy for many hematologic malignancies. Its potential relies on graft-versus-tumor effects which associate with graft-versus-host disease. Mesenchymal stromal cells (MSCs) possess immunomodulatory properties that make them attractive therapeutic alternatives. We evaluated the in vitro immunosuppressive activity of medium conditioned by human MSCs from 5 donors expanded 13 passages with or without FGF-2. FGF-2 supplementation increased expansion 3,500- and 240,000-fold by passages 7 and 13, respectively. There were no differences in immunosuppressive activity between media conditioned by passage-matched cells expanded under different conditions, but media conditioned by FGF-treated MSCs were superior to population doubling-matched controls. The immunosuppressive activity was maintained in three of the preparations but decreased with expansion in two. The proliferation induced by FGF-2 did not result in loss of immunosuppressive activity. However, because the immunosuppressive activity was not consistently preserved, caution must be exercised to ensure that the activity of the cells is sufficient after extensive expansion.