Micron-scale exclusion of CD45 enhances activation of TCR zeta associated protein of 70 kDa tyrosine kinase. (P1192)

Abstract

Abstract CD45, a transmembrane protein tyrosine phosphatase, is critical for activation of T cells. CD45 is known to modulate T cell signaling by dephosphorylating the inhibitory Tyr 505 of Lck, but at the same time it has been argued that exclusion of CD45 from the vicinity of the TCR is necessary for activation of T cells. While many data support the notion that CD45 exclusion is necessary for triggering of TCR signaling, there is no evidence that CD45 exclusion is sufficient. In this work, we designed substrates to test if local exclusion of CD45 can trigger TCR without aid of TCR ligation. To test this hypothesis, mouse CD4 T cells were seeded onto surfaces micropatterned with an antibody targeting the extracellular domain of CD45. These surfaces were created by first defining 1- or 2-µm diameter features of PLL-g-PEG onto glass coverslips, followed by adsorption of anti-CD45 to the remaining regions. These substrates depleted CD45 from the PLL-g-PEG patterned regions and enhanced production of pZap70 over levels observed on uniformly coated anti-CD45 surfaces as determined by total internal reflection fluorescence. Surprisingly, the areas of higher pZap70 were not localized in the specific regions of CD45 exclusion, but were concentrated in the center of the interface, approximating early stages of signaling in a T cell immunological synapse. These results demonstrate that local control over CD45 distribution can alter T cell signaling, independent of TCR-MHC recognition.