ζ-Chain expression serves as a biomarker for MSC-dependent T-cell immunosuppression. (A) Expansion of Gr-1⁺Mac-1⁺ cells in the spleens of control and TLRL-treated mice was determined by FACS analysis. The results are presented as the mean value of 3 independent experiments, and standard deviations are shown. *P < .001 (Student t test). (B) Normal splenic T cells were isolated and coincubated with Gr1⁺Mac-1⁺-enriched (95% purity) cells from LPS-treated mice at different ratios. After 16 hours, T cells were stained for ζ-chain and analyzed by FACS, and expression was compared with that of the originally separated T cells (cultured without MSCs, “1:0”). The histogram shows one experiment representative of 2 performed. (C) Paraffin-embedded sections of spleens from control (i,iii) or LPS-treated (ii,iv,v,vi) mice were stained with anti-CD3 or anti–Gr-1 antibodies. Photomicrographs of representative sections (I-IV;100×) and magnification of the boxed region (v,vi; 200×) are shown. (D) Kinetics of ζ-chain expression in the course of emergence and disappearance of the immunosuppressive environment. (E) Equal numbers of splenic T cells from control and LPS-treated (1 to 3 treatments; LPS I1-I3) mice (bottom panel) were lysed on days +2 and +8 and subjected to Western blot analysis. Expression of the ζ-chain and CD3ε was assessed by immunoblotting (IB) using specific antibodies. (F) Representative density plots of Gr-1⁺Mac-1⁺ cell distribution within the spleens of control and LPS-treated mice. (G) Splenocytes from control and LPS-treated mice from days +2 and +8 were activated with anti-CD3 and anti-CD28 antibodies; cell proliferation was then measured by BrdU incorporation in Thy-1.2 + cells using FACS analysis. Representative density plots of gated T cells are shown.