Abstract
Purpose: :
Previous reports showed that matrix metalloproteinase 9 (MMP-9) expression is uniquely upregulated in CD8+ Treg cells and not other lymphocytes. We postulated that MMP-9 in the CD8+ Treg cell may be involved in trafficking of Treg cells to the peripheral tissues or contributes to CD8+ Treg suppressor function. Here we tested these postulates by studying the upregulation of MMP-9 expression and activity in ACAID CD8+ Treg cells and its ability to contribute to CD8+ Treg cells trafficking or suppression.
Methods: :
Induction of ACAID: Antigen was inoculated into the anterior chamber (a.c.) of OT-1 mice. A week later experimental mice were deliberately immunized (s.c.) with antigen and CFA. The following week, the spleen was extirpated , and CD8+ T cells were enriched and analyzed for Foxp3 and MMP-9 expression by flow cytometry. In Vitro ACAID: PEC were collected from wild type mice, treated with TGF-β2 and ovalbumin and used as tolerogenic APC (tol APC) with spleen cells from OT-1 mice. (Control,used OVA-pulsed APC). CD8+ T cells from 7 day cultures were then examined for Foxp3 and MMP-9 expression. Zymography: We examined the conditioned media from 48h cultures of ACAID CD8+ Treg cells for MMP-9 activity (zymography). Local adoptive transfer (LAT) assay. To test if MMP9 were needed for Treg suppression of T effector function, in vitro generated CD8+ Treg cells were incubated with a MMP-9 inhibitor prior to being co-transferred with sensitized cells and antigen directly into the ear pinnae of the mice.
Results: :
CD8+ T cells from both in vivo and in vitro ACAID modelsupregulated MMP-9 and Foxp3. A direct role for MMP-9 in the suppressor function of CD8 Treg cells was verified by the LAT assay since in vitro generated CD8+ Treg cells preincubated with MMP-9 inhibitor failed to suppress the DTH response.
Conclusions: :
Overall these data indicate that MMP-9 expression and activity is upregulated in FoxP3 +CD8 Treg cells and plays a direct role in their efferent suppressor function during ACAID.