n.-primed mice. Figure 3B shows data for CD8+
T cells tested 4 and 10 wk after i.m. priming, at 4 wk after a booster immunization of i.m.-primed mice given i.vag. or i.m., and at 1 year after Bcl-2 inhibitor an i.m/i.m. prime-boost regimen. In all experiments, tet−CD8+ T cells from immune mice were also analyzed and their phenotypes mirrored those of naïve mice (data not shown). Four weeks after i.n. immunization with AdC6gag, CD44 was upregulated on Gag-specific CD8+ T cells from spleens, blood, ILN and NALT (Fig. 3A). This increase was less pronounced on tet+CD8+ cells from the GT, presumably reflecting that most T cells from the GT were already antigen-experienced. Most of the tet+CD8+ T cells from the GT expressed comparable levels of CD62L although a small population was CD62Lhi. It should be pointed out that expression of CD62L was also Y-27632 mouse low on most of the genital CD8+ T cells from naïve mice. Expression of α4β7 was low on most cells except for a small population of tet+CD8+ T cells present in spleen and blood. The booster immunization did
not have a pronounced effect on the expression of CD44, CD62L or CD27. α4β7 expression was again increased on some of the tet+CD8+ T cells from spleens and ILN. At 4 wk after i.m. immunization, CD44 expression was upregulated on tet+CD8+ T cells from spleens, ILN and GT (Fig. 3B). We detected a downregulation of CD62L expression on tet+CD8+ T cells from spleens, blood and the GT but not on those from ILN. CD27 expression was decreased on a subpopulation of tet+CD8+ T cells from blood, spleens and GT. At 4 wk after i.n. or i.vag. boost, expression levels of CD44, CD62L, CD27 and α4β7 mirrored those seen at 10 wk after priming, and there were no striking differences among groups that received an AdC6gag i.m. prime followed by a heterologous boost through the i.m. or i.vag. routes. At 1 year after the i.m. prime-boost vaccine regimen, expression of CD44 on tet+CD8+ T cells isolated from the different compartments (NALT was not tested in this experiment) overlapped with those seen on part of CD8+ T cells of
age-matched naïve mice. This may reflect an increase of CD44 expression on the control CD8+ T cells due to immunosenescence 15. Gag-specific CD8+ T cells isolated from the ILN and GT showed an increase in CD62L expression, which was unexpected for the latter compartment. In Inositol monophosphatase 1 blood and spleen, expression of CD62L and CD27 was similar or only slightly increased above those seen on unprimed CD8+ T cells, suggesting that the Gag-specific CD8+ T cells had differentiated into resting memory cells. Additional markers were analyzed on Gag-specific CD8+ T cells isolated from different compartments after an i.m./i.m. heterologous prime-boost regimen (Fig. 4). For the two early time points, i.e. 4 wk after priming or boosting, cells isolated from the vaginal mucosa were treated and analyzed separately from OUC. CD44, CD62L and CD27 were tested and found to mirror those shown in Fig.