Biophysical Characterization of CD6-TCR/CD3 Interplay in T Cells.

Marjolein B M Meddens,J Joris Witsenburg,F Burcu Celikkol,Joost Te Riet,Svenja F B Mennens,Roland Brock,Alessandra Cambi,Johannes S Kanger,Ben Joosten,Carl G Figdor

doi:10.3389/fimmu.2018.02333

Abstract

Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors.

Highlights

T cells play an important role in the execution of the adaptive immune response by regulating the activity of innate and other adaptive immune cells or directly executing effector functions, such as killing by cytotoxic T cells
CD6 has been recognized as a T cell receptor (TCR) co-receptor, the nature of the interaction with the TCR/CD3 complex has not been resolved
Wildtype Jurkat T lymphoma cells were seeded on microprinted antibody spots (5 μm in diameter) that were composed of 100% αCD6 or different concentrations of αCD3 (110-100%; diluted with mouse IgG2A-isotype control antibody) surrounded by fibronectin

Summary

Introduction

T cells play an important role in the execution of the adaptive immune response by regulating the activity of innate and other adaptive immune cells or directly executing effector functions, such as killing by cytotoxic T cells. During the T cell-APC contact, TCR microclusters are laterally transported during local cell surface receptor rearrangement creating a typical bull’s eye-shaped pattern at the T cell-APC interface, termed “the immunological synapse” (IS) [3, 4]. This lateral TCR microcluster transport results in TCR accumulation in the center of the IS, forming the central supramolecular activation cluster or “cSMAC,” together with co-stimulatory molecules such as CD2, CD4/CD8 and CD28 [4,5,6]. LAT forms a signaling hub, the so-called LAT signalosome, which through various signaling molecules such as SLP-76 and GRB2, initiates downstream events, such as calcium fluxing, actin reorganization, integrin inside-out signaling and gene expression, leading to T cell activation and effector functions [12, 14]

Methods

Results

Conclusion