r chambers. To determine whether T-cell migration was G protein-mediated, the cells were pre-incubated with 100 ng/ml pertussis toxin for 1 h at 37uC, washed, and then loaded in the chemotaxis chamber. In order to determine whether the migration of cells in response to R5 gp120 gradients was CCR5 dependent, cells were pre-incubated with the CCR5 antagonist, TAK-779, at a concentration of 40 nM. Statistical analysis All statistical analyses were performed in collaboration with Dr. Gebremichael at the Department of Biostatistics and Computational Biology at the Harvard School of Public Health. The Increased levels of Activation-Induced Cell Death occur during early infection but are not associated with HIV-1 gp120 in lymph nodes R-547 Previous in vitro studies have demonstrated that gp120 can enhance T cell sensitivity to AICD and therefore we hypothesized April 2011 | Volume 6 | Issue 4 | e18465 R5-SHIV Causes Multiple Defects in T Cell Function that the relative depletion of anti-gp120 responses might be due to enhanced activation of LN-derived T cells. We examined the ability of T cells to undergo AICD after 15963531 polyclonal stimulation and compared the results between T cells derived from either PB or LN from naive RM or RM during early infection at 5 and 12 weeks post-inoculation. Upon examination of CD4 and CD8 T cells derived from naive animals, we found that PB-derived T cells demonstrated induction of AICD and stimulationincreased apoptosis, as expected. Interestingly, the basal levels of CD4 T cell apoptosis at 5 and 12 weeks were higher in infected RM as compared to PB derived T cells from naive animals. In contrast to naive animals, PB-derived CD4 T cells from R5-SHIV-infected animals could not be further stimulated via anti-CD3 at either time point. A similar pattern was observed with respect to CD8 T cells: PB-derived cells had higher basal apoptosis at 5 weeks post-inoculation compared to PB-derived T cells from naive animals. Moreover, LN-derived CD8 T cells did not demonstrate enhanced levels of apoptosis upon stimulation with CD3 at either 5 or 12 weeks postinoculation. Increased levels of AICD in LN were not correlated with HIV gp120 levels in these tissues. These results suggest that AICD may not play a major role in April 2011 | Volume 6 | Issue 4 | e18465 R5-SHIV Causes Multiple Defects in T Cell Function immune dysregulation observed in LN of RM during early infection. Enhanced expression of PD-1 in LN derived T cells from RM during early infection Although, we observed no differences in apoptosis that were associated with reduced antigen-specific LN responses we proposed that T cell anergy may in part account for the observed immune dysregulation. Previous studies have demonstrated that during chronic HIV infection, levels of PD-1 are upregulated and correlated with anergic T cell responses to HIV antigens. Furthermore, the blockade of PD-1 restores T cell function in animal models. To further elucidate immune dysregulation in LN during early infection, we examined the level of PD-1 on T cells derived from PB and LN of naive and infected RM at 5 and 12 weeks post-R5-SHIV inoculation. We observed no major differences with respect to the proportion of T cells expressing PD-1 in any of the groups. However, when we examined 10188977 the amount of receptor on a per-cell basis, we observed higher levels of PD-1 on LN-derived CD4 T cells from R5-SHIV-infected RM at 5 weeks compared to cells from naive RM as measured by mean fluorescence intenr chambers. To determine whether T-cell migration was G protein-mediated, the cells were pre-incubated with 100 ng/ml pertussis toxin for 1 h at 37uC, washed, and then loaded in the chemotaxis chamber. In order to determine whether the migration of cells in response to R5 gp120 gradients was CCR5 dependent, cells were pre-incubated with the CCR5 antagonist, TAK-779, at a concentration of 40 nM. Statistical analysis All statistical analyses were performed in collaboration with Dr. Gebremichael at the Department of Biostatistics and Computational Biology at the Harvard School of Public Health. The Increased levels of Activation-Induced Cell Death occur during early infection but are not associated with HIV-1 gp120 in lymph nodes Previous in vitro studies have demonstrated that gp120 can enhance T cell sensitivity to AICD and therefore we hypothesized April 2011 | Volume 6 | Issue 4 | e18465 R5-SHIV Causes Multiple Defects in T Cell Function that the relative depletion of anti-gp120 responses might be due to enhanced activation of LN-derived T cells. We examined the ability of T cells to undergo AICD after polyclonal stimulation and compared the results between T cells derived from either PB or LN from naive RM or RM during early infection at 5 and 12 weeks post-inoculation. Upon examination of CD4 and CD8 T cells derived from naive animals, we found that PB-derived T cells demonstrated induction of AICD and stimulationincreased apoptosis, as expected. Interestingly, the basal levels of CD4 T cell apoptosis at 5 and 12 weeks were higher in infected RM as compared to PB derived T cells from naive animals. In contrast to naive animals, PB-derived CD4 T cells from R5-SHIV-infected animals could not be further stimulated via anti-CD3 at either time point. A similar pattern was observed with respect to CD8 T cells: PB-derived cells had higher basal apoptosis at 5 weeks post-inoculation compared to PB-derived T cells from naive animals. Moreover, LN-derived CD8 T cells did not demonstrate enhanced levels of apoptosis upon stimulation with CD3 at either 5 or 12 weeks postinoculation. Increased levels of AICD in LN were not correlated with HIV gp120 levels in these tissues. These results suggest that AICD may not play a major role in April 2011 | Volume 6 | Issue 4 | e18465 R5-SHIV Causes Multiple Defects in T Cell Function immune dysregulation observed in LN of RM during early infection. Enhanced expression of PD-1 in LN derived T cells from RM during early infection Although, we observed no differences in apoptosis that were associated with reduced antigen-specific LN responses we proposed that T cell anergy may in part account for the observed immune dysregulation. Previous studies have demonstrated that during chronic HIV infection, levels of PD-1 are upregulated and correlated with anergic T cell responses to HIV antigens. Furthermore, the blockade of PD-1 restores T cell function in animal models. To further elucidate immune dysregulation in LN during early infection, we examined the level of PD-1 on T cells derived from PB and LN of naive and infected RM at 5 and 12 weeks post-R5-SHIV inoculation. We observed no major differences with respect to the proportion of T cells expressing PD-1 in any of the groups. However, when we examined the amount of receptor on a per-cell basis, we observed higher levels of PD-1 on LN-derived CD4 T cells from R5-SHIV-infected RM at 5 weeks compared to cells from naive RM as measured by mean fluorescence inten