The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

Abstract

By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.

Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation

“…CD4 + T cells were isolated from at least 50 different healthy donors to perform the experiments. CRISPR/Cas9-edited Jurkat cells deficient for NSM2 (ΔNSM) ( Börtlein et al, 2018 ) cells were cultured in RPMI/10%FBS.…”

“…Knockdown efficiencies were analyzed by RT-PCR as previously described ( Börtlein et al, 2018 ). Total RNA from 2 × 10 6 NSM2 and control siRNA-nucleofected T cells was isolated 5 days post-transfection using TRIzol Reagent (Life Technologies) after the manufacturer’s protocol.…”

“…Jurkat cell culture was supplemented with C16 ceramide as described previously ( Collenburg et al, 2016 ; Börtlein et al, 2018 ). Shortly, a total of 5 × 10 7 Jurkat-CTRL or Jurkat-ΔNSM cells were extensively washed and resuspended in RPMI/2% FBS containing 25 μM C16-ceramide (Avanti Polar Lipids), incubated overnight at 37°C, and washed three times with PBS before performing flow cytometry or mitochondria isolation.…”

“…When compared to those measured in brain or liver, expression levels of NSM2 in T-cells are rather low ( Hofmann et al, 2000 ). Nevertheless, NSM2 activity proved to have a substantial impact on T-cell cytoskeleton dynamics, morphological polarization, and migration toward chemotactic signals, and, most importantly, for the optimal performance of TCR signaling ( Gassert et al, 2009 ; Collenburg et al, 2017 ; Börtlein et al, 2018 ). Our more recent studies identified the TCR/NSM2/PKCζ pathway as crucial for TCR signal amplification and sustainment especially at low doses of stimulation ( Börtlein et al, 2018 ).…”

“…Nevertheless, NSM2 activity proved to have a substantial impact on T-cell cytoskeleton dynamics, morphological polarization, and migration toward chemotactic signals, and, most importantly, for the optimal performance of TCR signaling ( Gassert et al, 2009 ; Collenburg et al, 2017 ; Börtlein et al, 2018 ). Our more recent studies identified the TCR/NSM2/PKCζ pathway as crucial for TCR signal amplification and sustainment especially at low doses of stimulation ( Börtlein et al, 2018 ). At a cellular level, NSM2-driven ceramide production essentially regulated PKCζ - dependent microtubule-organizing center (MTOC) dynamics as required for recycling and sustained supply of TCR signaling components to the plasma membrane at the immune synapse.…”