CC chemokines impart crucial chemotactic and nonchemotactic effects on T cells that are thought to play critical roles in immune responses (2
). Thus, given the costimulatory functions of CCR5 ligands (7
) and the recruitment of CCR5 at the immunological synapse (9
), we hypothesized that reduced CCR5 expression might result in impaired T cell activation. Consistent with this thesis, here we report that CCR5 is a determinant of IL-2 and CD25 expression in activated T lymphocytes and provide evidence that these effects are mediated via NFAT and affect IL-2-dependent biological processes such as STAT5 phosphorylation and T cell proliferation.
Taub et al. demonstrated that CC chemokines costimulate T cell proliferation through the release of IL-2 and the increased expression of CD25 (8
). The results of our study extend this observation by indicating that CCR5 is one of the receptors through which these effects might be mediated. Given the redundant nature of the chemokine system, it is possible that other CCRs are involved in T cell responses. However, our results obtained in three knockout models (i.e., CCR1−/−
, and CCR5−/−
) mice suggest that among these three CCRs, CCR5 might have a greater impact on IL-2 production and IL-2-dependent events than CCR1 or CCR2. There are abundant data demonstrating that IL-2 positively regulates the expression of CCR5 (18
). Here we report the converse: CCR5 favors IL-2 production and CD25 expression in activated T cells. This inference is supported not only by our findings in primary T cells from CCR5-deficient mice, but also from in vitro experimental approaches such as Ab-mediated blockade of CCR5. That maraviroc did not affect IL-2 production in vitro may be due to differences in the receptor configuration and the resulting functionality of Ab-bound vs inhibitor-bound forms of CCR5 (29
). Taken together, these observations support the existence of a positive bi-directional feedback between CCR5 expression and IL-2 production during T cell activation. This might ensure survival, expansion, and migration of Ag-specific memory as well as Th1 cells (i.e., CCR5-bearing cells) during the effector phase of adaptive immune responses.
An important but often underappreciated phenotypic difference between naive and memory T cells is the differential expression of CCR5 on these cell types, which is significantly greater in memory cells. We observed that memory T cells (high CCR5 expression) produce higher levels of IL-2 in response to TCR stimulation than naive T cells (low CCR5 expression) and that signaling via CCR5 results in NFAT transactivation during T cell activation. In this regard, our findings might account for the expanded functional capacity of memory cells in terms of IL-2 secretion and cell proliferation (61
), and they are also consistent with a recent report showing that accumulation of NFAT mediates IL-2 expression in memory, but not naive, CD4+
T cells (62
As compared with naive cells, memory T cells have less stringent activation requirements and are thus more permissive to activation (63
). Therefore, based on our findings, here we propose that CCR5 might serve as a costimulatory receptor for memory T cells during secondary immune responses. We acknowledge fully that there are many other differences between naive and memory T cells and, consequently, IL-2 production might be influenced by factors independent of CCR5 expression. Despite this caveat, our findings strongly suggest that in addition to conferring inflamed tissue homing capacity, the acquisition of memory/effector function (characterized by a shift from CCR7 to CCR5 surface expression) might confer a preferential advantage for enhanced secretion of IL-2 and proliferation. Similarly, our findings might help explain why Th1 cells, which akin to memory cells exhibit a phenotype characterized by high surface expression of CCR5, produce more IL-2 than do Th2 cells, which do not express high levels of this receptor (64
The activation of the NFAT pathway following stimulation of CCR5 is not unprecedented but is controversial. For example, Paya and colleagues showed that CCR5 stimulation alone is sufficient to induce expression of FasL by a NFAT-dependent pathway in primary CD4+
T cells (65
). In contrast, Cicala et al. (66
) found that treatment of PBMC with the CCR5 ligand CCL4 does not induce NFAT. However, in the latter study the authors also found that specific inhibitors of CCR5 prevented R5 gp120-mediated NFAT activation. The results of the present study provide additional support for the presence of NFAT-dependent pathways that are mediated via CCR5 in T lymphocytes.
The enhanced NFAT transactivation observed in the context of high CCR5 expression appears to be the result of a quantitative increase in NFAT levels. Specifically, we observed a significant increase in the levels of NFAT intranuclear translocation in CCR5-expressing T cells that also exhibited increased IL-2 production. This observation is consistent with a recent report suggesting that the translocation of NFAT into the nucleus in activated T cells serves as a switch of the cellular phenotype from an IL-2-nonproducing to an IL-2-producing cell (67
). Thus, the link between CCR5 and NFAT expression has biological relevance, as it suggests that the NFAT-influencing effects of CCR5 might regulate the number of Th cells actively producing IL-2 during an immune response (67
IL-2R is a heterotrimer composed of three protein subunits (α, β, γ) (45
). The β-chain exists in a complex with the γ-chain and binds to IL-2 with intermediate affinity (45
). This complex is converted into a high-affinity receptor by binding to the α-chain (CD25), whose expression is highly induced by T cell activation (45
). Here we demonstrate that CCR5 expression influences the up-regulation of CD25 during T cell activation, and that it may underlie the previously reported correlation between CCR5 and CD25 expression on activated T cells (60
). Although IL-2 may signal through its intermediate affinity receptor, the influence of CCR5 on CD25 expression is likely to be biologically relevant, as evidenced by the fact that activated CD25-null T cells are resistant to Fas-mediated activation-induced cell death, a mechanism of peripheral tolerance that is dependent on IL-2 expression (69
The observation that CCR5-CCR5 ligand interactions influence IL-2 production are consistent with a previous report showing that mice genetically inactivated for CCL5 exhibit not only impaired IL-2 production and T cell proliferation in vitro but also reduced delayed-type hypersensitivity responses in vivo (71
). Similarly, we previously found that both humans and mice deficient in CCR5 expression have impaired cell-mediated immunity, as evidenced by reduced delayed-type hypersensitivity skin test responses (27
). Since IL-2 is a critical component of anti-viral cell-mediated immunity, the findings reported herein provide additional support to our premise that by influencing cell-mediated immunity, CCR5 may affect anti-HIV responses (27
). Notably, the phenotype of low CCR5 expression–low IL-2 levels may provide a basis for the recent reports demonstrating increased susceptibility to infection with West Nile virus (28
) in individuals homozygous for the CCR5
mutation, and with the higher incidence of herpesvirus and influenza infections observed during clinical trials in individuals who received the CCR5 inhibitor maraviroc as compared with individuals from the placebo arm of the study (www.pfizerpro.com/product_info/selzentry_pi_warnings.jsp
). The observation of reduced T cell activation in the context of deficient CCR5 expression might also help explain the association between CCR5
mutation and lower susceptibility to inflammatory disorders, including rheumatoid arthritis, Kawasaki disease, and coronary artery disease (72
). On the other hand, our findings might account for the prolonged graft survival observed after blockade of CCR5 in vivo as well as in CCR5-deficient recipients (76
). Thus, the perturbations in IL-2 production and T cell proliferation resulting from deficient CCR5 expression might be therapeutically relevant in graft rejection and inflammatory disorders where down-regulation of T cell responses is highly desirable.
Our results have implications for the treatment of HIV infection with CCR5 antagonists. For example, administration of maraviroc has been shown to have beneficial effects on CD4+
T cell recovery in HIV+
individuals even in clinical settings where CCR5 may not play a major role as a coreceptor (80
). This observation suggests that in addition to their effects on viral entry, absence or blockade of CCR5 might provide an immunological benefit by regulating the degree of host immune activation. However, in our study we did not find that maraviroc influences IL-2 and CD25 levels, whereas a mAb that specifically binds to the second extracellular loop of CCR5 did. This is a surprising observation considering that both 2D7 Ab as well as maraviroc inhibit ligand-induced calcium flux. It is possible that 2D7 and maraviroc have differential effects on other secondary pathways downstream of CCR5 that may be required for optimal IL-2 expression. Such a scenario is plausible, as it has been previously suggested that distinct biological responses of CCR5 may be mediated through different sets of receptor conformations (81
). However, additional studies are required to confirm that maraviroc does not influence immune responses.
The results of our studies with 2D7 have translational value as in addition to small molecule CCR5 antagonists, CCR5 mAbs are also being developed for human use. The results of a phase I clinical trial with HGS004, a fully human IgG4 mAb, was recently reported (83
). HGS004 inhibits HIV envelope-dependent cell fusion and viral entry as well as chemokine binding to CCR5 in a manner that is akin to 2D7. Intriguingly, administration of low doses of this Ab was associated with a striking increase in circulating CD4+
T cell counts as early as day 1 after Ab administration without having any effects on HIV viral loads. Based on the studies reported herein, it will be important to evaluate whether these prominent effects on CD4 recovery yet minimal effects on viral replication that are associated with administration of low doses of CCR5 mAb are in part because this therapeutic agent affects the immune activation state as observed for 2D7 herein.
At least three additional lines of evidence further support a link between CCR5 and T cell activation in the context of HIV infection. The first is the fact that natural hosts of SIV/HIV infection are characterized by both low CCR5 expression (84
) as well as low levels of activation (85
), despite having high levels of viral replication. In light of these reports, our findings indicate that low CCR5 expression may be one of the driving forces that underlie the low T cell activation status observed in these animals. Supporting this hypothesis and consistent with the synchronized expression of CD25 and CCR5 in CD4+
PP T cells activated in vitro (60
), here we found a significantly reduced number of CD4+
T cells in PP from CCR5
-null mice. This circumstantial evidence suggests that the level of T cell activation in the GALT might be influenced by the level of CCR5 expression. Second, we have found that in HIV+
individuals the level of CCR5 expression is highly correlated with the degree of T cell activation (our unpublished data), which is consistent with the preferential expression of CCR5 in activated CD4+
T cells from these patients (88
). Finally, the fact that HIV R5 but not X4 strains induce increased T cell activation favoring HIV survival and spread (89
) further supports a role for CCR5-mediated T cell costimulation in HIV pathogenesis.
In summary, we herein demonstrate that CCR5 expression is a regulator of IL-2 and CD25 expression during T lymphocyte activation, an effect that might be mediated through NFAT transactivation/translocation. Furthermore, these effects of CCR5 levels have biological consequences, as they impact IL-2-dependent processes such as STAT5 phosphorylation and T cell proliferation. These findings may have important clinical implications, as they emphasize careful immunological evaluation of HIV+ patients receiving CCR5 antagonists such as mAbs and suggest that blockade of CCR5 might be a beneficial intervention in T cell-driven conditions such as autoimmunity, graft rejection, and possibly HIV.