C, Deb

Abstract Neurovirulent picornaviral infections are a significant health risk in developing nations. Unfortunately, the mechanisms of picornaviral clearance from the central nervous system (CNS) are less understood and therapeutic approaches are limited. We have established a mouse model of neurovirulent picornavirus infection using the Theiler’s murine encephalomyelitis virus (TMEV) which replicates during the first 12 days postinfection (dpi) primarily in the hippocampus but is cleared from the brain by 21 dpi. Intracerebral infection of TMEV triggers the early infiltration of natural killer cells (NKCs) followed by CD8+ T cells (CTLs) into the brain. We observed that both brain-infiltrating NKCs and CTLs express the NKG2D activating receptor on their surface and 50% of the the CTLs specific to the VP2121–130 viral capsid peptide also expressed NKG2D. Concomitantly, brains of infected mice expressed NKG2D ligands such as RAE-1, H-60 and MULT-1 and microglia were the cellular locus of these ligands. Importantly, systemic treatment with the NKG2D function blocking antibody CX5 resulted in a significant increase in viral load in the brain. These results suggest that activated and infected microglia stimulate NKCs and CTLs via NKG2D in a manner that restricts viral replication. NKG2D may thus serve as a potential therapeutic target to enhance viral clearance responses during CNS infection. This work was supported by a grant from the National MS Society and by Donald and Frances Herdrich.

Natural killer cells (NKC) and cytotoxic T lymphocytes (CTL) express NKG2D, a lectin‐like molecule that serves as a direct stimulatory receptor in the innate immune system and as a co‐stimulatory receptor in the adaptive system. In mice, the ligands for NKG2D are stress‐inducible molecules such as Rae‐1, H‐60 and MULT‐1. These MHC class I‐like molecules are generally absent in normal tissues but are upregulated by inflammation and virus infection. We have found that acute intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) elicits robust infiltration of NKG2D‐positive CTL and NKC into the brain coincident with the zenith and resolution of viral load. Likewise, using quantitative RT‐PCR we found that MULT‐1 was upregulated coincident with the peak of viral load while Rae‐1 (only epsilon and delta tested) and H‐60 remained undetectable. Therefore, we hypothesized that CTL and NKC recognition of MULT‐1 on neurons is required for efficient viral clearance. However, in vivo immunodepletion of NKC and CTL beginning one day prior to infection and continuing throughout the first 3 weeks postinfection did not prevent efficient viral clearance despite greater than 90% reduction in NKG2D‐positive cells in the brain immune infiltrate as assessed by flow cytometry. Thus, the role of MULT‐1 expression following TMEV infection remains unclear and experiments are ongoing to determine the cellular locus of MULT‐1 expression and recognition. This work was supported by grant RG3636 from the National Multiple Sclerosis Society and by Donald and Frances Herdrich.

Stress‐dependent regulation of the immune response is a general repair and maintenance system. Members of the MHC class I superfamily serve as indicators of cellular stress. Nonclassical class I molecules such as H60, MULT‐1 and the RAE‐1 family are generally absent in normal tissues. We found that intracerebral infection with TMEV upregulated expression of these stress‐response molecules in the brain at timepoints that coincided with the control of viral load. Moreover, we found that about 50% of the brain‐ infiltrating cytotoxic T cells (CTLs) specific for the TMEV peptide VP2121–130 were positive for the NKG2D activating receptor at 7 days postinfection. NKG2D binds to RAE‐1, H60, and MULT‐1 and costimulates CTLs. When we blocked NKG2D using the CX5 function blocking antibody we found a significant increase in viral load in the brain. Therefore, we hypothesize that cellular stress associated with viral infection induces NKG2D ligand expression on target cells that in turn costimulates NKG2D‐bearing VP2‐specific CTLs to promote viral clearance. Our study may ultimately impact the health of people at risk from encephalitis and neurologic complications associated with picornaviral infections and may provide new ways to promote viral clearance in individuals suffering from persistent virus infections of the brain.