Anti-GITR Antibody Treatment Increases TCR Repertoire Diversity of Regulatory but not Effector T Cells Engaged in the Immune Response Against B16 Melanoma.

Crosslinking of glucocorticoid-induced TNF family-related receptor (GITR) with agonist antibodies restores cancer immunity by enhancing effector T cell (Teff) responses while interfering with intra-tumor regulatory T cell (Treg) stability and/or accumulation. However, how anti-GITR antibody infusion changes T cell receptor (TCR) repertoire of Teffs and Tregs engaged in anti-tumor immune response is unclear. Here, we used a transgenic mouse model (TCRmini) where T cells express naturally generated but limited TCR repertoire to trace the fate of individual T cells recognizing B16 melanoma in tumor-bearing mice, treated or non-treated with an anti-GITR monoclonal antibody DTA-1.

Assessment of caspase mediated degradation of linker for activation of T cells (LAT) at a single cell level.

Caspase/Granzyme B mediated protein degradation is involved in elimination of activated T cell receptor (TCR) signaling molecules during processes of thymocyte selection and maintenance of peripheral homeostasis of T cells. Key components of TCR signaling cassette including LAT undergo biological inactivation in response to pro-apoptotic or anergy inducing environmental stimuli. Although available Western immunoblotting-based techniques are appropriate for detection of protein degradation in bulk populations of target cells, quantitative assessment of this process at a single cell level requires a different approach.