Technical Reference #1821

1821.

Mechanism of T Cell Tolerance Induced by Myeloid-Derived Suppressor Cells Srinivas Nagaraj; Adam G. Schrum; Hyun-Il Cho; Esteban Celis; and Dmitry I. Gabrilovich, University of South Florida, Journal of Immunology, 184(1821), (2010)
Link To Paper

Abstract:
Ag-specific T cell tolerance plays a critical role in tumor escape. Recent studies implicated myeloid-derived suppressor cells (MDSCs) in the induction of CD8+ T cell tolerance in tumor-bearing hosts. However the mechanism of this phenomenon remained unclear. We have found that incubation of Ag-specific CD8+ T cells with peptide-loaded MDSCs did not induce signaling downstream of TCR. However it prevented subsequent signaling from peptide-loaded dendritic cells. Using double TCR transgenic CD8+ T cells we have demonstrated that MDSC induced tolerance to only the peptide which was presented by MDSCs. T cell response to the peptide specific to the other TCR was not affected. Incubation of MDSCs with Ag-specific CD8+ T cells caused nitration of the molecules on the surface of CD8+ T cells localized to the site of physical interaction between MDSC and T cells which involves preferentially only TCR specific for the peptide presented by MDSCs. Postincubation with MDSCs only nitrotyrosine-positive CD8+ T cells demonstrated profound nonresponsiveness to the specific peptide whereas nitrotyrosine-negative CD8+ T cells responded normally to that stimulation. MDSCs caused dissociation between TCR and CD3ζ molecules disrupting TCR complexes on T cells. Thus these data describe a novel mechanism of Ag-specific CD8+ T cell tolerance in cancer.

Materials & Methods:
Confocal microscopy Splenocytes from double TCR (DT) mice were cultured with specific peptide in the presence of MDSCs (at a 3:1 ratio) on a poly-d-lysine–coated glass-bottom culture dish (MatTek Ashland MA). The cells were labeled with anti-Va2 TCR APC anti-Vb 8.1 TCR Alexa 555 and anti-NT Alexa 488. Cells were viewed with a DMI6000 inverted Leica TCS AOBS SP5 tandem-scanning confocal microscope with a 340/1.30 NA oil immersion objective (Leica Microsystems Deerfield IL). Tunable 488 Argon and 546 and 633 laser lines were applied to excite the samples using AOBS line switching to minimize crosstalk between fluorochromes. Images and Zstacks were produced with three cooled photomultiplier detectors and the LAS AF version 1.5.1.889 software suite (Leica Microsystems).

Microscopic Technique
Confocal Microscopy