Abstract
How T cell receptors (TCRs) are triggered to start signaling is still not fully understood. It has been proposed that segregation of the large membrane tyrosine phosphatase CD45 from engaged TCRs initiates signaling by favoring phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3 molecules. However, whether CD45 segregation is important to initiate triggering is still uncertain. We examined CD45 segregation from TCRs engaged to anti-CD3 scFv with high or low affinity and with defined molecular lengths on glass-supported lipid bilayers using total internal reflection microscopy. Both short and elongated high-affinity anti-CD3 scFv effectively induced similar calcium mobilization, Zap70 phosphorylation, and cytokine secretion in Jurkat T cells but CD45 segregated from activated TCR microclusters significantly less for elongated versus short anti-CD3 ligands. In addition, at early times, triggering cells with both high and low affinity elongated anti-CD3 scFv resulted in similar degrees of CD3 co-localization with CD45, but only the high-affinity scFv induced T cell activation. The lack of correlation between CD45 segregation and early markers of T cell activation suggests that segregation of CD45 from engaged TCRs is not mandatory for initial triggering of TCR signaling by elongated high-affinity ligands.
Highlights
T cells represent major components of the cellular arm of the adaptive immune response
OKT3-1 (n = 26 cells, representative of three independent experiments). (D) A representative cell was imaged for CD3 and CD45 by total internal reflection fluorescence (TIRF) microscopy at 1, 2, 3, 5, and 10 min after contact with OKT3MA-BGP on a planar lipid bilayer. (E) Co-localization percentage between CD3 and CD45 measured by intensity of overlapped pixels for whole Jurkat cell activated by OKT3MA-BGP (n = 22 cells, representative of three independent experiments). (F) Co-localization percentage between CD3 and CD45 measured by intensity of the overlapped pixels for individual microclusters obtained from Jurkat cells activated by OKT3MA-BGP (n = 22 cells, representative of three independent experiments)
Our results suggest that segregation of CD45 from engaged T cell receptor (TCR) microclusters may not be absolutely required for initial triggering of TCR signaling
Summary
T cells represent major components of the cellular arm of the adaptive immune response. T cell receptor (TCR) triggering describes the initial signaling of the TCR upon binding to peptide–MHC (pMHC) complexes [1, 2]. Many models have been proposed to explain how binding of TCRs to pMHC molecules initiates signaling and activation of T cells [3], including increased proximity between CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) and Src family kinases caused by surface aggregation of engaged TCRs [4] or recruitment of CD4 and CD8-associated Lck [5], induced conformational changes that expose CD3 cytoplasmic domains to phosphorylation [6, 7], while other models postulate transfer of ligated TCRs to Lck-rich lipid rafts [8]. Despite numerous studies attempting to describe TCR triggering, the actual molecular mechanism remains controversial [3, 9, 10].
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