Taken together, these results indicate that induction of CD8+ T-cell responses at mucosal sites upon i.m. immunization is independent of a given vaccine platform. Antigen-experienced CD8+ T cells may traffic to the GT with

the help of specific sensors that remain to be identified, or alternatively this process may be random. To gain further insight into the vaccine-induced CD8+ T cells that homed to the GT, we conducted a detailed phenotypical analysis of Gag-specific CD8+ T cells induced by the different immunization protocols, comparing cells isolated from spleen, blood, ILN and the GT at different times after immunization. In some assays, we also tested cells isolated from NALT; Wnt inhibitor the latter were tested for comparison as a population of cells homing to a distinct mucosal site. Phenotypes of Gag-specific

CD8+ T cells isolated from systemic sites and the GT were phenotypically distinct, and this was especially pronounced at 1 year after the i.m./i.m. prime-boost vaccine Akt inhibitor regimen. The phenotypes suggest that most tet+CD8+ T cells present in the GT remain fully activated and would be expected to start target cell lysis immediately upon encounter of infected cells. We evaluated markers that are known to be upregulated on cells derived from the intestinal mucosa. Studies have demonstrated high levels of CD69 expression on intestinal CD8+ cells 22, 30, but expression of CD69 was not increased in the GT at any of the time points analyzed. Although α4β7 has been linked to the genital migration of subsets Obatoclax Mesylate (GX15-070) of CD4+ cells 31, and is a well-known marker for homing of T cells to the intestinal mucosa, our results do not suggest that α4β7 affects homing of CD8+ T cells to the GT. CD103 was slightly increased in tet+CD8+ T cells from the GT at early time points, and by 1 year after immunization became strongly upregulated. In the adoptive transfer experiment, CD103 was low on the Gag-specific CD8+ T cells isolated from the vaccinated donors and upon transfer

remained low on cells isolated from all compartments but the GT, where an increase was observed. Again, these data argue against the notion that CD103 supports mucosal homing but rather suggest that CD103 may contribute to the retention of CD8+ T cells within the GT. The adoptive transfer experiment also showed that Gag-specific CD8+ T cells from the spleen could readily migrate to the GT to a similar extend as observed in vaccinated mice. This argues against the need for a distinct differentiation pathway during activation to allow for migration of CD8+ T cells to the mucosa, as had been described for T-cell homing to GALT 32 or for CD4+ T cells of the female GT 33. On the other hand, the observation that at 2 wk upon i.m. immunization frequencies of Gag-specific CD8+ T cells were ∼10-fold higher in blood but only ∼2-fold higher in the GT than upon i.n.