DOI: 10.5061/dryad.sbcc2frgj
title: IL1b induction of endothelial surface IL15 by let7c3p downregulation

description: Expression of IL-15 on the surface of human graft endothelial cells (ECs)
 bound to the IL-15Rα subunit can increase the activation of CTLs,
 potentiating allograft rejection. Our previous work showed that surface
 expression of this protein complex could be induced by
 alloantibody-mediated complement activation through increased IL-1β
 synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of
 NF-κB. In this article, we report that cultured human ECs express eight
 differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter
 the expression level of any IL-15 transcript but induces surface
 expression independently of RNA polymerase II-mediated transcription while
 requiring new protein translation. Mechanistically, IL-1β causes an
 NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby
 relieving a block of translation of IL-15 surface protein. Let7c-3p
 anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence
 of complement activation or of IL-1, enabling IL-15 transpresentation to
 boost CD8 T cell activation. Because of the complexity we have uncovered
 in IL-15 regulation, we recommend caution in interpreting increased total
 IL-15 mRNA or protein levels as a surrogate for
 transpresentation. 
description: # IL1b induction of endothelial surface IL15 by let7c3p downregulation
 Dataset DOI:
 [10.5061/dryad.sbcc2frgj](https://doi.org/10.5061/dryad.sbcc2frgj) ##
 Description of the data and file structure qRT-PCR performed per
 manuscript methods description under cytokine treatments as described. IF
 performed as described; captured on EVOS FL2 auto microscope. Quantified
 in ImageJ, reported in .xls file. Western Blotting was performed as
 described in the manuscript methods. ### Files and variables #### File:
 DICER_knockdown_WB_-_B-actin.png **Description:** DICER siRNA treatment WB
 beta-actin loading control #### File: DICER_knockdown_WB_-_DICER.png
 **Description:**  DICER siRNA WB probe for DICER&gt; #### File:
 MFI_Data_values.xlsx **Description:**  MFI values for quantified
 immunofluorescence. Please reference the associated manuscript for
 treatment conditions. Values tabulated on per cell measured fluorescent
 intensity for each relevant fluorophore. Units: Fluorescent units
 Abbreviations: IL: Interleukin. DAPI: 4',6-diamidino-2-phenylindole,
 SELE: E-selectin. IFN: Interferon. DMSO: dimethyl sulfoxide. B11:
 Bay-117086. DRB: 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole. CHX:
 Cycloheximide. si(TARGET): siRNA against Target. let7c Inh: Let-7c
 anti-miR #### File: Raw_qRT_PCR_Ct.xlsx **Description:**  Cycle times for
 qRT-PCR experiments. Units: Cycle Abbreviations: IL: Interleukin. GAPDH:
 Glyceraldehyde-3-phosphate dehydrogenase. DAPI:
 4',6-diamidino-2-phenylindole, SELE: E-selectin. IFN: Interferon.
 DMSO: dimethyl sulfoxide. B11: Bay-117086. DRB:
 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole. CHX: Cycloheximide.
 si(TARGET): siRNA against Target. let7c Inh: Let-7c anti-miR. IP:
 Immunoprecipitation. #### File: p65_Knockdown_WB_-_B-actin.tif
 **Description:**  p65 knockdown beta actin loading control #### File:
 p65_Knockdown_WB_-_p65.tif **Description:** p65 knockdown p65 probe. ####
 File: FACS_Experiments.zip **Description:** pSTAT5A flow cytometry
 experiments containing FCS files. APC: CD8. PE: Phospho-STAT5A. #### File:
 IF_Images.zip **Description:** RAW images of immunofluorescence
 experiments. ## Code/software ImageJ, flow cytometry analysis software. No
 specialty software required.