A-D Attenuation of T cell activation response by TGFβ1-LAP-loaded on anti-CD3/CD28/LAP beads. A) TGFβ1-LAP on anti-CD3/CD28/LAP activating beads, shown in ng/ml final concentration, holds back bead-induced proliferation of human T lymphocytes in dose-dependent manner, nearing a complete attenuation at highest doses. Proliferation was assayed by [3H]thymidine incorporation, depicted in counts per minute (cpm). Data present averages and SEMs of 3 independent experiments with hexuplicate cultures each. B) Inactive LAP and TGFβ1-LAP were loaded onto anti-CD3/CD28/LAP beads in parallel titrations. TGFβ1-LAP on activating beads recapitulated dose-dependent silencing of T cell proliferative response to TCR/CD28 activation. Loading of LAP elicited significant inhibition of proliferation only at highest doses (n = 4). **, *** depict respective significant difference of p <0.01, 0.005 to 100% proliferation response elicited by anti-CD3/CD28/LAP beads. C) Western blots show that bead-induced tyrosine phosphorylation of TCR-proximal signalling proteins PLCγ, ZAP-70, and LAT is attenuated by TGFβ1 presentation on anti-CD3/CD28/LAP beads. β-actin was probed as a loading control. Representative anti-phosphotyrosine blots of 3 independent experiments are shown. D) Gradual release of TGFβ1 cytokine by TGFβ1-LAP-loaded beads into the aqueous supernatant of ELISA wells.