The Royal Society of Chemistry

Chemical Communications: Issue 94, 2021

DOI: 10.1039/D1CC05194F

Surface Presentation of Noncanonical Wnt5a Motif to Cytotoxic CD8+ T-Cell Promotes Its Mechanotransduction and Activation

ABSTRACT

  • This project utilized force analyzation devices and algorithms for the tracking and subsequent computational analysis of force generation during the activation of CD8+ cytotoxic T-Cells. Stronger immune responses exhibited positive correlation with higher levels of total cellular forces during CD8+ T-Cell activation. The potential signaling pathways to augment the mechanotransduction and immune responses remain largely unexplored. Here, we explored the effects of noncanonical Wnt activation on T-Cell mechano-transduction to find an effective molecular pathway to elevate the cytotoxicity of CD8+ T-Cells and increase the efficiency of immunotherapy by conjugating the designated activating antibodies and noncanonical WNT- activating ligand, Foxy5 peptides, on the PDMS micropillar force measure device. Results showed that Foxy5-mediated noncanonical Wnt activation effectively increased the force feedback in activated CD8+ T-Cells. This research furthers the field of mechanoimmunology by providing a novel signaling pathway to modulate the CD8+ T-Cell activation.

ILLUSTRATED METHODOLOGY

  • Step 1.1: Pillar Fabrication

    Make negative molds from silicon micro-pillar array detectors (mPADs) so they may be used to produce more 6um and 8um mPADs.

  • Step 1.2: Pillar Fabrication

    Attach the negative molds to glass slides using PDMS to form positive molds of micro-pillars.

  • Step 1.3: Pillar Fabrication

    Remove negative molds and dry the new mPADS made from PDMS.

  • Step 2: Antibody Protein Coating

    Conjugate the designated activating antibodies and noncanonical WNT-activating ligand, Foxy5 peptides, onto the PDMS micropillar force measurement devices.

    Transfer CD8+ T-Cell cultures to assorted PDMS micropillar force measurement devices to initiate antibody-to-cell interactions.

  • Step 3.1: Dynamic Cell Force Analysis

    Following mechanotransduction and monitored activation of CD8+ cells, photograph pillars under microscope. Images of pillar displacement are used to develop all.json files for Cellogram analysis.

  • Step 3.2: Dynamic Cell Force Analysis

    Run all.json files though Cellogram to calculate displacement metrics for each micro-pillar array.

  • Step 3.3: Dynamic Cell Force Analysis

    Run Cellogram results through MATLAB to perform computational analysis on displacement metrics. Comparisons against micro-pillar locations prior to activation allow for direct quantification of force magnitude and direction during T-Cell mechanotransduction.

  • Step 4.1: Cell Fixation and Staining

    Fix cells with Triton-X. Stain cells with DAPI to visualize nuclei under fluorescent microscopy.

  • Step 4.2: Cell Fixation and Staining

    Stain cells with Vinculin and Alexa Fluor-488 to visualize actin-integrin binding sites under fluorescent microscopy.

  • Step 4.3: Cell Fixation and Staining

    Stain cells with Phalloidin and Alexa Fluor-555 to visualize actin under fluorescent microscopy.

  • Step 4.4: Cell Fixation and Staining

    Stain cells with FAK (Focal Adhesion Kinase) and Alexa Fluor-647 to visualize integrin using fluorescent microscopy.

  • Step 4.5: Cell Fixation and Staining

    Overlay fluorescent images to produce final visualization of CD8+ T-Cells after activation and mechanotransduction.

SUMMARY OF FINDINGS

Micro-pillar displacement metrics and fluorescent imaging were both critical to our investigation into how foxy5 peptides modulate cytotoxic CD8+ T-cell activation and mechanotransduction. The metrics quantified the direction and magnitude of forces involved, while the staining and imaging techniques provided a visualization of the cells after activation. Together, these methods offered a comprehensive approach to understanding the underlying mechanisms of T-cell activation and the cytotoxic immune response. By elucidating these mechanisms at the cellular level, this research can contribute to the development of novel strategies for enhancing immune function against infection and disease.

Force direction dynamics data shows clear spreading movement (positive) after Foxy5 activation, and minimal spreading movement after Src (control) activation. These contrast to squeezing movement (negative).

Force dynamics data shows clear differences in the strengths of forces produced by T-Cells when activated by Foxy5 versus Scr (control).