In this study we show that highly activated virus-specific CD8 + T cells expressing CCR5 and CXCR3 are found in the spinal cord of HSV-2-infected mice. This was associated with elevated levels of cognate chemokines in the spinal cord during HSV-2 infection.
We confirm previous studies showing that there are few CD8 + T cells in the spinal cord of naïve mice (21). For the first time though, we can show that the majority of these CD8 + T cells have a limited expression of chemokine receptors. 20–30% of the cells expressed CCR5 and/or CXCR3 and few/none expressed any of the other chemokine-receptors investigated; CCR2, CCR4, CCR6, CCR7, CXCR2 or CXCR4. In accordance, also the chemokine levels in the spinal cord were low in the absence of infection, as previously documented both in humans and in mice (14, 19). Thus, a limited number of CD8 + T cells may patrol the healthy spinal cord in what appear to be a chemokine-independent fashion.
Neuroinflammation caused by HSV-2 infection led to a pronounced increase of CD8 + T cells in the spinal cord, as shown by us here and by others (14). The viable fraction of the spinal cord CD8 + T cells in HSV-2 infected mice were CD44+ (and CD62L-) effector/memory cells, similar to what has been shown in other inflammatory diseases of the CNS (22) and in cell adhesion and migration to the CNS in general (23). The majority of the cells expressed granzyme B, and a few percent of them had recently degranulated. As previously shown (14), CD8 + T cells that were specific for the HSV-2 peptide SSIEFARL predominated in the spinal cord of HSV-2-infected mice, in accordance with the notion that only antigen-specific effector cells translocate to the CNS. We conclude that HSV-2 induced neuroinflammation in mice is associated with the presence of highly activated virus-specific CD8 + T cells in the spinal cord.
The majority of the spinal cord CD8 + T cells expressed CCR5 and several CCR5 ligands were expressed in the HSV-2-infected spinal cord. Studies in gene-deficient mice show that albeit CCR5 deficiency is associated with exacerbated CNS disease, it does not affect CD8 + T cell numbers in the CNS, neither during HSV-2 infection nor during intracranial infection with coronavirus (24, 25). This is in contrast to studies on non-viral CNS inflammation where CCR5 deficiency leads to an attenuated influx of CD8 + T cells and other immune cells to the spinal cord (26, 27). Many ligands for CCR5 are however expressed in the HSV-2-infected spinal cord. Thapa et al have previously reported the presence of CCL3 and CCL5 (14, 15, 24), and in this study we show that also CCL4 and in particular CCL8 are upregulated. The primary binding partner of CCL8 is CCR5, which is expressed on activated T cells (28) as confirmed by us. The latter is particularly interesting as CCL8 is one of the most prominent chemokines in HSV-2 meningitis patients (19). We conclude that CCR5 is present on most CD8 + T cells in the spinal cord during viral neuroinflammation, with concurrent high levels of CCR5 ligands including in particular CCL8.
Almost 60% of the CD8 + T cells in the spinal cord of HSV-2 infected mice also expressed CXCR3, and the cognate ligand CXCL10 was markedly upregulated in the infected spinal cord. CXCR3 is an important chemokine receptor for T cell migration; however it is debated if CXCR3 has a protective or detrimental function. On one hand mice deficient in CXCR3 succumb faster to HSV-2 infection, on the other hand HSV-1-infected mice lacking CXCR3 are protected from fatal encephalitis (15, 29), indicating a mouse strain and tissue-specific role of CXCR3 (30). Lack of the CXCR3 ligands CXCL9 and CXCL10 on the other hand increase the susceptibility to HSV-2 infection in mice, and reduce T cell migration into the spinal cord despite high levels of CCL2, CCL3 and CCL5 (14), implying that CXCR3/CXCL10 represent a major pathway for spinal cord T cell recruitment. IFN-gamma, which we could show is expressed in the infected spinal cord, induces the expression of CXCL10. CXCL10 is also upregulated in other neuroinflammatory infections like Theiler's murine encephalomyelitis virus, lymphocytic choriomeningitis virus and tick-borne encephalitis (31–33). Most importantly, the enhanced levels of CXCL10 correlate with data from cerebrospinal fluid from humans with HSV-2 meningitis (19).
A moderate proportion of CD8 + T cells in the infected spinal cord also expressed CCR2 and CCR4. CCR2, usually considered a monocyte marker, is found on 2–15% of T cells in both healthy humans and mice, where it bind ligands CCL2, CCL7, CCL8 and CCL12 (28, 34, 35). These four chemokines were strongly upregulated in the spinal cord of mice with HSV-2-induced neuroinflammation, as well as in human HSV-2 meningitis patients (19). Continuing the chain of promiscuous chemokine receptor/ligand interactions CCL5, together with CCL2 and CCL3, also interacts with CCR4. This chemokine receptor is normally considered a classical TH2 and Treg marker (36, 37). In humans it is also expressed on a subset of cytokine-producing but non-cytotoxic memory CD8 + T cells (38), which resemble a CD8 + T cells subset that are involved in the inhibition of HSV reactivation from latency (38). In summary, all measured chemokines binding either CCR2 or CCR4 increased in the spinal cord of HSV-2-infected mice, possibly contributing to the recruitment of CCR2- and CCR4-expressing CD8 + T cells.
CXCR4 was found on a limited number of spinal cord CD8 + T cells but was abundantly expressed on T cells in the spleen, indicating an inverse relationship to CNS homing. CXCR4 interacts with CXCL12, which is a known retention signal for CD8 + T cells at the blood-brain barrier. In West Nile virus infection it has been reported that disturbing the CXCR4/CXCL12 interaction allows migration of CD8 + T cells into the brain parenchyma resulting in increased viral clearance and survival (39, 40). In HSV-2 infection, the CXCL12 levels remained low in spinal cord confirming that the CXCR4/CXCL12 pathway is not a major driving force for CD8 + T cell presence in the spinal cord.