T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue

Abstract

Much of our understanding of central nervous system (CNS) immunity has been gained from models involving pathological inflammation. Attenuated rabies viruses (RABV) are unique tools to study CNS immunity in the absence of conventional inflammatory mechanisms, as they spread from the site of inoculation to the CNS trans-axonally, thereby bypassing the blood-brain barrier (BBB), and are cleared without neutrophil or monocyte infiltration. To better understand the role of CD4 T cell subsets in the clearance of the virus from CNS tissues we examined the development of antiviral immunity in wild type (WT) and T-bet knock-out mice (T-bet−/−), which lack Th1 cells. Early control of RABV replication in the CNS tissues of WT mice is associated with the production of IFNγ, with antiviral effects likely mediated through the enhanced expression of type I interferons. Interestingly, IFN-α and −γ are overexpressed in the infected T-bet−/− by comparison with WT CNS tissues and the initial control of RABV infection is similar. Ultimately, attenuated RABV are cleared from the CNS tissues of WT mice by antibody locally produced by the activities of infiltrating T and B cells. While T and B cell infiltration into the CNS of infected T-bet−/− mice is comparable, their activities are not, the consequence being delayed, low level antibody production and prolonged RABV replication. More importantly, neither T-bet−/− mice immunized with an attenuated virus, nor WT mice with Th2 RABV-specific immunity induced by immunization with inactivated virus, are protected, in the long term, against challenge with a pathogenic RABV.

Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors

“…Quantitative real-time PCR (qPCR) was performed using iQ Supermix or iQ SYBRR Green Supermix (Bio-Rad Laboratories, Hercules, CA), for specific mRNAs with a Bio-Rad iCycler (BioRad, Hercules, CA). The Primer/probe sets (IDT, Coralville, IA) used for L13, SPBN-GAS, CD4, CD8, κ-L chain, IFN-β and IFN-γ were described previously (31) with additional sets listed in Table 1. All probes are dual labeled 5′-6-FAM and 3′-BHQ-1.…”

“…Serum samples were collected 0, 8 and 12 days after infection and incubated on UV-inactivated RABV ERA-coated plates. Antibody titers were determined using secondary antibodies specific for IgG as well as IgG1 and IgG2a as described previously (31). A cell-based ELISA was used to detect the level and isotype of GL261-specific antibodies as described previously (18).…”

“…Immunofluorescent staining was performed on sections fixed in cold methanol for 10 min at -20°C, rinsed in PBS and incubated with primary antibodies, diluted in PBS containing 2% BSA, 5% goat serum, and 0.25% Triton X-100, overnight at 4°C. Antibodies for RABV nucleoprotein, NeuN, and CD4 have been described previously (31) and additional reagents are listed in Table 2. Slides were then incubated with fluorescence-conjugated secondary antibodies and mounted with Vectashield® Hard Set™ mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing DAPI.…”

“…To determine if type 1 immune mechanisms induced by virus infection may impact glioma growth in the absence of tumor cell lysis, we have used the attenuated, neurotropic rabies virus (RABV) SPBN-GAS. This RABV strain contains multiple attenuating mutations in its glycoprotein that prohibit its reversion to pathogenicity, ensuring its safety even in immunocompromised animals (30, 31). Such viruses spread to the brain via axons, bypassing the BBB, then replicate in neurons and astrocytes causing minimal cell death (32).…”

“…In normal mice this results in the production of high levels of type 1 cytokines and virus neutralizing antibody in the CNS tissues and, ultimately, clearance of the virus without histological or clinical evidence of pathology. Type 1 immune mechanisms are central to virus clearance from the CNS as mice lacking Tbet, the transcription factor associated with the development of Th1 cells, have a severe deficit in this process despite developing a strong RABV-specific Th2 response (31, 34–36). To determine if the induction of a type 1 response in CNS tissues would alter glioma growth, we infected congenic C57BL/6 and Tbet -/- mice bearing intracranial GL261 tumors with SPBN-GAS, monitoring the immune responses to tumor and viral antigens, cytokine production in the TME, and tumor growth.…”