The CD4 and CD3δε Cytosolic Juxtamembrane Regions Are Proximal within a Compact TCR–CD3–pMHC–CD4 Macrocomplex

Abstract

T-cell receptors (TCRs) relay information about peptides embedded within major histocompatibility complex molecules (pMHC) to the immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated CD3γε, CD3δε, and CD3ζζ signaling modules. CD4 then recruits the Src kinase, Lck, to the TCR-CD3 complex to phosphorylate the ITAMs, initiate intracellular signaling, and drive CD4+ T-cell fate decisions. While the six ITAMs of CD3ζζ are key determinants of T cell development, activation, and the execution of effector functions, multiple models predict that CD4 recruits Lck proximal to the four ITAMs of the CD3 heterodimers. We tested these models by placing FRET probes at the cytosolic juxtamembrane regions of CD4 and the CD3 subunits to evaluate their relationship upon pMHC engagement in mouse cell lines. The data are consistent with a compact assembly in which CD4 is proximal to CD3δε, CD3ζζ resides behind the TCR, and CD3γε is offset from CD3δε. These results advance our understanding of the architecture of the TCR-CD3-pMHC-CD4 macro-complex and point to regions of high CD4-Lck-ITAM concentrations therein. The findings thus have implications for TCR signaling, as phosphorylation of the CD3 ITAMs by CD4-associated Lck is important for CD4+ T-cell fate decisions.

Reciprocal TCR-CD3 and CD4 Engagement of a Nucleating pMHCII Stabilizes a Functional Receptor Macrocomplex

“…But three pieces of evidence raise questions about how, upon TCR-pMHCII engagement, CD4 positions Lck and the ITAMs in a sufficient local concentration for a sufficient duration for phosphorylation to occur; particularly for the weak interactions that drive positive selection and peripheral homeostasis ( Glassman et al, 2016 ; Kao and Allen, 2005 ; Stepanek et al, 2014 ; Wang et al, 2001b ; Zuñiga-Pflücker et al, 1989 ). First, crystallography data suggest that the TCR-CD3 complex, pMHCII, and CD4 adopt a V-like arch that could place the CD3 ITAMs, and, in particular, the six ITAMs of ζζ, ~100 Å from a CD4-associated Lck ( Wang et al, 2001a ; Yin et al, 2012 ).…”

“…Here, we tested predictions made by a V-like macrocomplex versus a more compact architecture in which CD4 docks along a composite surface formed by the TCR-CD3-pMHCII axis ( Figure S1A ) ( Glassman et al, 2016 ; Kuhns and Badgandi, 2012 ; Wang et al, 2001a ; Yin et al, 2012 ). In the latter model, TCR-CD3 dwell time on pMHCII would determine the duration with which the CD4 docking site is formed and thus influence CD4 dwell time within a mature macrocomplex.…”

A biomimetic five-module chimeric antigen receptor (5MCAR) designed to target and eliminate antigen-specific T cells

“…We also performed a flow cytometry-based fluorophore-linked immunosorbant assay (FFLISA) of detergent lysates from TCR + and 5M CAR + 58α − β − cells to confirm that TCR-CD3 and CRM pMHCII -CD3 complexes assemble analogously 14 . Latex beads coated with anti-CD3ε monoclonal antibodies (mAbs) were incubated with lysates and then stained with anti-CD3 ζ antibodies for analysis by flow cytometry.…”

“…6.0 µm streptavidin-coated polystyrene microspheres (Polysciences) were further coated with biotinylated anti-mouse CD3ε, washed, and incubated with 0.5 ml of DDM (1% n -dodecyl-b-D-maltoside) lysates from 5×10 6 5M CAR 58α − β − cells at 4°C for 1 hour. After washing, beads were probed with PE conjugated anti-mouse CD3 ζ (clone G3, Santa Cruz Biotechnology) and analyzed by flow cytometry 14 .…”

“…CD4 and CD8 are coreceptors that represent the fifth module on CD4 + or CD8 + T cells; they bind MHCII or MHCI, respectively, and associate non-covalently with the Src kinase, p56 Lck (Lck), that phosphorylates CD3 ITAMs. The coreceptors sequester Lck away from TCR-CD3 complexes until either CD4 or CD8 and the TCR both bind pMHC, at which point Lck is positioned proximal to the CD3 ITAMs to initiate signaling 14–17 . These 5-module pMHC-receptors can signal in response to a single agonist pMHC, direct CTL killing against just three pMHC, and direct distinct T cell responses according to the quantity and quality of the pMHC 1,2,11,18–23 .…”