Third-party CD4⁺ iNKT cells inhibit Tcon proliferation and induce a Th2 immune phenotype. Photon emission of Tcons from luc⁺ C57BL/6 donor mice was measured by BLI to assess the in vivo expansion capacity of alloreactive T cells in donor and third-party CD4⁺ iNKT-cell–treated BALB/c recipient mice. (A) Representative serial bioluminescence images. (B) Bioluminescence signal intensity time course. Error bars indicate SEM. Shown is 1 of 2 independent experiments with 5 mice per group. (C) To determine alterations in immune polarization in third-party CD4⁺ iNKT-cell–treated BALB/c recipient mice, murine sera, and Thy1.1⁺CD4⁺FoxP3⁻ T cells from spleens were analyzed at day +10 by multiplex assay and ICS, respectively. Error bars indicate SEM. Pooled data from 2 independent experiments with 6 mice per group are shown. *P ≤ .05; **P ≤ .01; ***P ≤ .001.