The role of chemokines in regulating immune responses during chronic viral infections is poorly understood. Here we investigated the importance of RANTES in response to a chronic infection where CCR5 is not a viral co-receptor. RANTES was more highly expressed during chronic LCMV infection compared to acute infection. While the absence of RANTES did not impact T cell responses following acute LCMV infection, a different scenario emerged during chronic LCMV infection. During chronic infection, CD8 T cells become exhausted and their dysfunction was characterized by a loss of cytokine production, reduced cytotoxicity and increased inhibitory receptor expression, all of which can hinder the ability to control the infection 
. In the absence of RANTES, CD8 T cell exhaustion was more severe with reduced virus-specific CD8 T cell numbers, cytokine production and higher expression of inhibitory receptors. The cytotoxic potential of virus-specific CD8 T cells responding to clone 13 infection in RANTES−/−
mice was also reduced compared to WT controls. Consistent with the more severe exhaustion of the CD8 T cell response, mice lacking RANTES also had higher viral loads. Thus, the absence of RANTES resulted in the dysfunction of virus-specific CD8 T cells and poor viral control suggesting that RANTES has an important role in regulating and/or sustaining optimal immune responses during chronic viral infection.
There are a number of ways in which the absence of RANTES could result in the higher viral titers and reduced CD8 T cell function during clone 13 infection. First, slightly higher viral loads at the beginning of the response could lead to more severe CD8 T cell exhaustion. One possible mechanism for RANTES affecting viral load is via one of the main cell types infected by LCMV, macrophages. Macrophages play a key role in the immune defense against LCMV. Marginal zone macrophages and metallophilic macrophages may act as filters, controlling the spread of LCMV 
. The increased tropism of LCMV clone 13 for macrophages and DCs is thought to result in the ability of the virus to persist 
. The absence of RANTES could impact macrophage function or survival. For example, RANTES is essential to prevent apoptosis of macrophages infected with Sendai virus 
. Thus, it will be important to investigate the role of RANTES in regulating DC and macrophage differentiation during persisting infections. A second possibility is that RANTES regulates the homing dynamics of the T cells, preventing T cell migration to the peripheral tissues or microenvironments and therefore limiting the ability of these cells to control the infection. However, during chronic LCMV infection the LCMV-specific CD8 T cells were found in spleen, blood and liver showing that the virus-specific T cells could still migrate to peripheral tissues at least at the level of the whole tissue. This observation does not rule out potential differences in movement within tissue, however, and a more detailed analysis of the migration dynamics of exhausted CD8 T cells in the absence of RANTES could be important. Third, CD4 T cell help could be reduced/absent in mice lacking RANTES. At least with LCMV Armstrong infection, CD4 T cell help appears to be intact as CD8 T cell memory cells are fully functional upon secondary challenge. Moreover, LCMV-specific CD4 T cell expansion and cytokine production in RANTES−/−
mice were similar to WT mice in response to both LCMV Armstrong and LCMV clone 13. While the phenotype of the CD8 T cells in RANTES−/−
mice was similar to CD4-depleted mice, the viral titers in mice lacking CD4 T cells was much higher suggesting that the CD4-depleted phenotype is more severe. Given that CD4 T cells also produce RANTES, it is possible that CD4 T cells are an important source of RANTES during LCMV clone 13 infection but that remains to be determined. A fourth possibility is that RANTES directly affects T cell activation/differentiation leading to reduced effector functions and that loss of RANTES directly results in functional defects in T cells leading to higher viral loads. While RANTES has been shown to act as a costimulator of T cells 
T cells responded similarly to WT CD8 T cells in a competitive environment suggesting that the importance of RANTES during LCMV clone 13 infection was not due to direct costimulation of CD8 T cells, though other receptors capable of binding RANTES could have a role.
CD8 T cell activation/differentiation is clearly negatively impacted by the absence of RANTES since IFNγ production by CD8 T cells was reduced even at day 8 p.i. in RANTES−/−
mice and this reduced IFNγ production was even more dramatic at the chronic stage of disease. Given that IFNγ has been shown to regulate the ability to clear LCMV infection 
, this initial decrease in IFNγ at the early stage of infection could result in a reduced ability to control viral replication, leading to further CD8 T cell exhaustion. Our data supports a role for RANTES in allowing the efficient activation and differentiation of CD8 T cells that are required to help control clone 13 infection. Interestingly, RANTES was not required for memory CD8 T cells to clear clone 13 infection. This observation, along with the similar T cell response to acute LCMV infection supports a role for RANTES during a sustained infection and further supports the model that minor defects early in the response to a rapidly disseminating infection are magnified as the infection persists leading to more severe T cell dysfunction and pathogen persistence.
Transcription factors that regulate effector functions of CD8 T cells during LCMV infection include Tbet and eomesodermin 
. Tbet expression was reduced in the absence of RANTES during LCMV clone 13 infection. How the absence of RANTES regulates the expression of Tbet, however, is currently unclear. These findings do suggest that the CD8 T cells responding to clone 13 in RANTES−/−
mice have differential expression of transcription factors compared to those from WT mice and perhaps these differences in transcription factor regulation impact their effector functions. Determining whether this effect can be directly attributed to RANTES or is a byproduct of higher viral load requires further investigation.
Interestingly, while CD8 T cell numbers and function were clearly reduced in the absence of RANTES, the CD4 T cells were not as sensitive to the loss of RANTES. CD4 T cells were unaffected in terms of numbers and the ability to produce IFNγ. Thus, the absence of RANTES had differential effects on CD4 versus CD8 T cells. These observations are somewhat surprising given that both CD4 and CD8 T cells produce RANTES and express the main receptor, CCR5. Further, this observation suggests that at least some aspects of CD4 and CD8 T cell exhaustion are regulated differently during chronic LCMV infection. Perhaps the differential effects of RANTES on CD4 versus CD8 T cells could be due to differences in expression of the other receptors for RANTES.
The CCR5-Δ32 mutation is found at a high frequency in European populations and is thought to have arisen through selective pressure during Yersinia pestis or variola major infection 
. While absence of CCR5 can clearly be protective against HIV, CCR5 plays a role in protecting against WNV and tick-borne encephalitis. CCR5 may also play a protective role in the response against yellow fever virus; viscerotropic disease following yellow fever virus (YFV) vaccination in one subject was associated with the CCR5-Δ32 polymorphism as well as an additional mutation in the RANTES promoter 
. The dichotomy of protection versus susceptibility of various infections and the use of CCR5 inhibitors suggests the need for more research on subjects with the CCR5-Δ32 mutation in terms of susceptibility to infection with different pathogens.
Understanding the role of RANTES during chronic infection is highly relevant due to the interest in CCR5 inhibitors for the treatment of HIV. CCR5 inhibitors prevent the entry of the R5-tropic stains of HIV virus into the cell 
. While CCR5 inhibitors can be of tremendous benefit to those infected with the CCR5-tropic stain of HIV, our data suggests that blocking the RANTES pathway could negatively influence ongoing immune responses to other persisting infections. Many patients infected with HIV are also co-infected with other pathogens and the effect of the RANTES
CCR5 pathway on these co-infections is not well understood. As many as 30% of HIV-infected patients in western Europe and the USA are coinfected with hepatitis C virus (HCV) and complications from HCV coinfection have emerged as a significant cause of morbidity and mortality 
. Given the role of RANTES in regulating responses to the flaviviruses WNV and YFV, and that serum levels of CC-chemokines are increased in patients infected with chronic hepatitis 
, it will be interesting to determine whether RANTES also plays a role in regulating T cell responses to another member of the flavivirus family HCV.
Our data suggest that therapeutic interventions targeting the RANTES pathway could have negative effects on the ability to control some chronic infections and indicates the need for further research into any link between the CCR5-Δ32 mutation and persistent infections. These observations also suggest that blocking the RANTES
CCR5 receptor pathway could alter the development and or quality of antiviral immune responses to chronic viral infection and, therefore, CCR5 inhibitors that block only HIV binding but not the RANTES
CCR5 pathway may be more ideal.