5, bottom panel; Supporting Information Fig. S1D). This result is consistent with the hypothesis that in the presence of polyclonal Treg cells, fewer cells leave the LN to enter the circulation, and fewer cells are therefore available to respond to antigen at a distant site. To begin to explore potential mechanisms by which Treg cells might inhibit T-cell trafficking from the site of immunization, we initially compared the phenotype of Teff

cells primed in the presence or absence of Treg cells. There were no differences between the two groups for a variety of markers tested. A summary of various markers, cytokines and chemokine/chemokine receptors that Maraviroc manufacturer were consistently found to be unaltered between the two groups can be found in Supporting Information Table 1. These results suggested to us that the presence of a higher number of Treg cells does not result in global and dramatic alterations to the immune response, but influences immunological

outcomes check details by targeting very specific pathways. To elucidate these pathways, we purified Teff cells from mice that had been immunized in the presence or absence of Treg cells and subjected mRNA from these cells to microarray analysis. Remarkably, very few genes were found to be up or downregulated by more than three-fold between the two groups (data not shown), further confirming the notion that Treg cells do not induce global changes. Notably, two of the genes that were found to be different between the two groups were involved in cell migration and trafficking. CXCR4 was found to be decreased over four-fold in the presence of Treg cells. We confirmed this observation both at the protein and at the mRNA level (Fig. 6). We also confirmed at the protein level decreased

expression of Syndecan-4, a molecule involved in cell motility 11. An additional molecule that has been well characterized as being important in the trafficking of T lymphocytes is the sphingosine 1-phosphate receptor before 1 (S1P1) 12. S1P1 levels are rapidly downregulated on T cells following entry into the LN. As T cells are primed and differentiate, they upregulate S1P1 allowing the cell to respond to high levels of S1P in the circulation and exit the LN in response to the concentration gradient 13, 14. We observed a dramatic decrease in S1P1 expression at the mRNA level in Teff cells that had been primed in the presence of Treg cells. This observation provides a potential mechanistic explanation for the retention of Teff cells in the LN. By altering the expression of S1P1 on Teff cells, Treg cells would affect the ability of these cells to migrate out of the LN and into the circulation. It remains to be determined whether Treg cell-mediated suppression of S1P1 upregulation on Teff cells is direct or indirect. Both polyclonal and antigen-specific Treg cells are capable of suppressing immune responses in vitro and in vivo.