Targeting the Microtubule-Network Rescues CTL Killing Efficiency in Dense 3D Matrices.

Renping Zhao,Aránzazu Del Campo,Bin Qu,Essak S Khan,Eva C Schwarz,Dalia Alansary,Wenjuan Yang,Kim S Friedmann,Markus Hoth,Xiangda Zhou

doi:10.3389/fimmu.2021.729820

Renping Zhao, Aránzazu Del Campo + Show 8 more

Open Access

https://doi.org/10.3389/fimmu.2021.729820

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Abstract

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors.

Highlights

Cytotoxic T lymphocytes (CTLs), which are activated CD8+ T cells, compose an essential arm of the immune system to fight aberrant cells like tumorigenic and pathogen-infected cells [1, 2]
In matrices with larger pore sizes (Figures 2J, K) the cytotoxic efficiency of CTLs was enhanced (Figure 2L). These results suggest that smaller pore size and higher stiffness in dense collagen matrices lead to impaired CTL killing efficiency as a result of hindered migration, whereby CTL migration persistence is mainly determined by matrix stiffness while the velocity is likely determined by the pore size of the matrix
We found that two physical properties of matrices are decisive for T cell migration: pore size and stiffness of the fibrils

Summary

Introduction

Cytotoxic T lymphocytes (CTLs), which are activated CD8+ T cells, compose an essential arm of the immune system to fight aberrant cells like tumorigenic and pathogen-infected cells [1, 2]. CTLs recognize their targets via engagement of T cell receptors (TCRs) with the cognate antigens presented on the surface of target cells [3,4,5]. Once the matching antigens are identified, activation of TCRs triggers the downstream signaling cascades to re-orientate the CTL killing machinery towards the contact site, termed immunological synapse (IS) [6,7,8]. The major killing mechanisms employed by CTLs are lytic granules (LGs) and Fas/FasL pathway [9]. In order to locate their targets, CTLs need to migrate through peripheral tissues across the three-dimensional (3D) extracellular matrix (ECM)

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