Acute HIV infection is associated with massive viral replication and loss of memory CD4 T cells in both mucosal and peripheral tissues, key aspects of viral pathogenesis that are well modeled in experimental infection of macaques with SIV. Beginning from a known time of inoculation, there is a lag, with limited viral replication for the first few days, after which infection amplifies explosively within a period of 2−3 days to reach peak by day 10 pi; this process appears to follow the same kinetics in the mucosa and periphery(1
). Given the highly activated mucosal tissue microenvironment, it has been generally believed that mucosal CD4 T cells were likely to be preferentially infected as compared to CD4 T cells elsewhere. However, the similarity with which infection proceeds in the mucosa and periphery suggests other factors likely play a role in this process.
Early viral infection is associated with an acute phase type response that likely induces a cascade of events leading to acute immune activation. Our results show that in experimental SIV infection, acute immune activation occurs very early, and prior to the amplification of viral infection within the memory CD4 T cell compartment. Interestingly however, the activation seen during early stages of infection was restricted to memory CD8 T cells without any apparent activation of CD4 T cells, observations in line with previous studies(5
). The selective activation of memory CD8 T cells but not memory CD4 T cells suggests that specific cytokines such as IL-15 play a role in this process. We found a significant correlation between the activation of CD8 T cells and plasma IL-15 levels.
It was surprising that increased levels of plasma IL-15 was not associated with increased proliferation or activation of memory CD4 T cells, but significantly correlated with the extent of viral infection in these cells. This apparent paradox was resolved by our findings that few memory CD4 T cells express IL-2Rβ (), and those that express them do so at significantly lower levels as compared to CD8 T cells (). Since IL-2Rβ signaling is critical for the activation and proliferation of T cells, these data indicate that the relative absence of increased activation and proliferation of memory CD4 T cells during the first 10 days of infection was likely due to the low levels of IL-2Rβ expression.
Burkett et al(32
) found that expression of IL-15Rα on T cells was dispensable for the generation of memory CD8 T cells. In murine studies, Kamimura et al(33
) showed that CD8 memory cells proliferated even after the depletion of IL-2 in vivo
. These memory CD8 T cells expressed IL-2Rα, and blockade of IL-2Rα signaling completely abolished the division of memory CD8 T cells. On the other hand, our results suggest that the low levels of IL-2Rα expression on CD4 T cells likely allows IL-15 to interact with these cells. However, the relative lack of any change in Ki-67 or HLA-DR expression on CD4 T cells during the first 10 days after infection indicates that IL-15 interaction with IL-2Rα on CD4 T cells was likely not strong enough to induce activation and proliferation but was sufficient to make the CD4 T cells more susceptible to SIV infection. Previous studies(34
) have shown that IL-15 can enhance HIV replication in the absence of proliferation.
Kaur et al(5
) reported little change in Ki-67+
T cells during the first 4 weeks of acute SIV infection. On the other hand, Picker et al(11
) reported an increased proliferation of effector memory CD4 T cells after administration of IL-15 protein to rhesus macaques. We did not specifically evaluate effector memory CD4 T cells. However, it is highly likely that the high dose of exogenous IL-15 (10 ug/Kg body weight/twice a week) used by Picker et al may have contributed to the proliferation of effector memory CD4 T cells by inducing the secretion of other factors, whereas the highest level of plasma IL-15 we observed was ~25 − 30 pg / ml at day 10 pi. Importantly, our findings indicate that increased activation or proliferation of memory CD4 T cells was not the primary cause for the increase in the level of infection in these cells at day 10 pi as compared to day 7 pi.
Though the low levels of IL-2Rβ explained the absence of any significant level of activation of memory CD4 T cells, it did not explain the highly significant correlation between plasma IL-15 levels and viral infection in memory CD4 T cells. We hypothesized that IL-15 interaction with the low levels of IL-2Rβ was sufficient to upregulate the expression of the receptors for SIV namely, CD4 and CCR5. In fact, we found a significant increase in the density of CD4 expression on memory CD4 T cells between day 7 and 10 pi that coincided with the increased level of SIV infection in these cells. SIV-gp120 binds to CD4 on CD4 T cells along with CCR5, and higher densities of CD4 expression on memory CD4 T cells significantly correlated (r = 0.78, p= 0.0002) with viral infection in these cells, suggesting that SIV may have bound to memory CD4 T cells more efficiently between day 7 and 10 pi leading to a higher a higher frequency of these cells being infected by day 10 pi.
Previous studies have shown that HIV binds weakly to cells expressing low levels of CD4(36
), and that the low level of CD4 expression on macrophages played an important role in restricting the entry of T tropic SIV strains such as SIVmac239(37
); this restriction could not be overcome by the overexpression of CCR5 on these cells. Kozak et al found that CD4 rather than CCR5 or CXCR4 expression determines the kinetics and pathways for gp120 binding, endocytosis, and proteolysis on cells that contain sufficient coreceptor for efficient infection(38
). Platt et al showed that cells with large amounts of CD4 and low traces of CCR5 are sufficient for maximum susceptibility to HIV-1 strains(39
). Finally, Pesenti et al demonstrated that susceptibility of macrophages to HIV-1 is significantly increased when they express high levels of CD4 as compared to macrophages that expressed low levels of CD4, whereas there was no significant difference in susceptibility based on CCR5 expression(40
These studies support our observations that the low level of infection in memory CD4 T cells coincided with the lower expression of CD4 on these cells, whereas an increased level of expression was associated with near total infection of most memory CD4 T cells. It is highly unlikely that the increased viral replication is the cause for the increased density of CD4 expression. If this were true, then at day 14 pi when the plasma viral loads are 2 logs higher than at day 7 pi, all of the memory CD4 T cells present should have been infected and carry viral DNA. This is however not the case as the frequency of CD4 memory T cells in both peripheral and mucosal tissues () that carry viral DNA at day 14 pi are less than 20% whereas most of the memory CD4 T cells at day 10 pi are infected and carry viral DNA. Likewise, the plasma viral loads are quite high at day 7 pi yet the level of infection in memory CD4 T cells is much lower than what is seen at day 10 pi.
In vitro culture experiments using highly purified CD4 T cells in the presence or absence of recombinant IL-15 confirmed our hypothesis that IL-15 plays a major role in early infection. IL-15 treatment upregulated the level of CD4 expression on highly purified CD4 T cells, an effect that was accompanied by a higher frequency of sorted CD4 T cells being infected with SIV (). The use of a defined system comprised of purified populations of CD4 T cells, and purified recombinant IL-15 minimized potential bystander effects from other cells.
We found no significant change in the level of plasma IL-7 levels during the early phase of infection (Suppl. Fig. 1d
). IL-7 is a cytokine produced by non-lymphoid cells, and plays a role in the homeostatic expansion of both naïve and memory CD4 and CD8 T cells(21
). Though we cannot completely rule out the potential role of IL-7 in acute immune activation we observed due to the small sample size, the relative lack of proliferation and activation of naïve CD4 and naïve CD8 T cells (Suppl. Fig. 2a & b
), and the minimal changes in proliferation and activation of memory CD4 T cells () all argue against a prominent role for IL-7 in immune activation seen during the first 10 days of acute SIV infection. The exact role of IL-7 may need to be evaluated using a larger group of animals to better clarify its contribution to early infection.
In conclusion, our data shows that IL-15 plays an important role in acute immune activation and increasing the susceptibility of memory CD4 T cells to SIV infection by upregulating the expression of CD4, the primary receptor for SIV, on memory CD4 T cells. Though our studies focused on peripheral memory CD4 T cells, the similarities in the kinetics of infection between peripheral and mucosal CD4 T cells suggest that IL-15 likely plays a role in increasing the susceptibility of CD4 T cells in the mucosa to SIV infection. However, additional studies need to be performed using a larger cohort of animals to better understand the role IL-15 plays in immune activation and immunopathogenesis of SIV infection in mucosal tissues.