CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαβ Complex.

Beatriz Garcillán,Raquel G Laborda,Marina S Mazariegos,Ana V Marin,Daniel Chacon-Arguedas,Maria L Toribio,Paula P Cárdenas,Rebeca F Megino,Patricia Fuentes,Anaïs Jiménez-Reinoso,Edgar Fernández-Malavé,Miguel Muñoz-Ruiz,José R Regueiro

doi:10.3389/fcell.2021.608490

Abstract

The human αβ T-cell receptor (TCR) is composed of a variable heterodimer (TCRαβ) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαβ assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

Highlights

T lymphocytes detect the presence of antigens through a cell surface complex termed the T-cell receptor (TCR)
CD3 heterodimers are formed through interactions between their extracellular Ig domains and associate with TCR chains through intramembrane contacts to form the hexameric TCR, which is exported to the Golgi apparatus where it interacts with the ζζ/CD2472 homodimer (Dietrich et al, 1999; Wucherpfennig et al, 2010)
Our results indicate that both CD3γ and CD3δ chains in both knocked down (KD) cell lines were EndoH sensitive and, endoplasmic reticulum (ER) resident, suggesting that TCR complexes in the absence of CD3γ or CD3δ are retained in the ER

Summary

Introduction

T lymphocytes detect the presence of antigens through a cell surface complex termed the T-cell receptor (TCR). This receptor is composed of a variable heterodimer (either TCRαβ or TCRγδ) responsible for ligand recognition and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472) that participate in the assembly and surface expression of the TCR complex and in the delivery of intracellular signals (Call et al, 2004). Hexameric TCR ensembles that reach the Golgi apparatus but fail to assemble with ζζ/CD2472 homodimers are sorted to the lysosomal pathway for degradation (Bonifacino et al, 1990; Wileman et al, 1990, 1993; Letourneur and Klausner, 1992; Lee, 1998; Delgado and Alarcon, 2005)

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