Is Suppressor Function in Mouse CD4 + CD25 + Tregs Due Wholly to GRAIL?

Abstract

The (E3) ubiquitin ligase, GRAIL (gene related to anergy in lymphocytes), is associated with CD4 + T cell anergy, a state of T cell nonresponsiveness to antigen. MacKenzie et al . found that amounts of GRAIL mRNA and protein were greater in mouse CD4 + CD25 + Tregs than in CD4 + CD25 – naïve cells. Transfer of naïve CD4 + T cells from TCR-DO11.10/Rag2 –/– (DO11) mice to BALB/c mice, followed by injection of the mice with OVA peptide, induced tolerized CD4 + CD25 + cells detected with the DO11-specific antibody KJ1.26. This is a routinely used method for studying tolerance, because the donor cells express a T cell receptor (TCR) that responds only to OVA peptide, and the donor mice cannot produce any mature T cells because of the lack of the Rag2 gene (recombinase activating gene 2). KJ1.26 + CD25 + cells had larger amounts of GRAIL mRNA and Foxp3 mRNA and protein than did the KJ1.26 + CD25 – subset, as measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. A retroviral system was used to generate cell lines producing large amounts of GRAIL (DO11 GRAIL) or GFP (DO11 GFP). Co-culture of naïve CD4 + CD25 – T cells with antigen-presenting cells, OVA peptide, and DO GFP cells resulted in proliferation of naïve cells. However, when DO11 GRAIL cells were used instead of DO11 GFP cells, proliferation of naïve CD4 + CD25 – T cells was suppressed. DO11 GRAIL cells produced more transforming growth factor-β (TGF-β), a cytokine associated with Treg function, than did DO11 cells. DO11 GRAIL cells also had larger amounts of the surface Treg markers, CD25, GITR, and CTLA4. Surprisingly, DO11 GRAIL cells did not produce the cytokine, IL-10, or the transcription factor, Foxp3, both of which are associated with Treg function. However, this study demonstrates the presence of GRAIL in CD4 + CD25 + Tregs and suggests a role for GRAIL in suppressor function. D. A. MacKenzie, J. Schartner, J. Lin, A. Timmel, M. Jennens-Clough, C. G. Fathman, C. M. Seroogy, GRAIL is up-regulated in CD4 + CD25 + T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype. J. Biol. Chem. 282 , 9696-9702 (2007). [Abstract] [Full Text]