Is the TAM receptor Axl a receptor for lymphocytic choriomeningitis virus?

Abstract

A recent publication indicated that overexpression of Axl, a cellular receptor that negatively regulates Toll-like receptor signaling, enhanced the entry of viruses pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV) in vitro. In testing the biological relevance of these observations, we found differences in neither viral kinetics between LCMV infections of Axl(-/-) and wild-type mice nor T-cell responses prior to spontaneous viral clearance. Thus, Axl is not required for productive LCMV infection of mice.

Fig 1

LCMV clone 13 infection of adult immunocompetent mice. Axl^−/− and control C57BL/6J mice were infected with 2 × 10⁶ PFU of virus i.v. Data are representative of two independent experiments (n = 4). (A) Serum was taken at the indicated time points, and plaque formation on Vero cells was assessed. (B) Mice were weighed at the indicated time points, and each result presented is a percentage of the mouse's weight at t = 0. L.O.D., limit of detection.

Fig 2

Cytokine responses of T cells stimulated with immunodominant LCMV peptides. Mice were infected with 2 × 10⁶ PFU of LCMV clone 13 i.v. or with 2 × 10⁵ PFU of LCMV Armstrong i.p., and spleens were harvested at 8 days postinfection (**, P < 0.005; ***, P < 0.0005; n.s., not significant). (A, B) Splenocytes were incubated with H-2^b MHC class I-restricted epitopes NP_396-404 and GP_33-41, MHC class II-restricted epitope GP_61-80, 50 U/ml IL-2, and 1 mg/ml brefeldin A for 5 h at 37°C. Cells were stained with antibodies against murine Thy1.2, CD4, CD8α, IFN-γ, TNF-α, and IL-2, and the levels of each were assessed by flow cytometry. Representative graphs are shown for intracellular cytokine staining of CD3⁺ CD8⁺ (A, lower panels) and CD3⁺ CD4⁺ (B, right panels) cells. (C) Splenocytes were stained with fluorescently conjugated MHC tetramers (GP_276-286 and GP_33-41) and antibodies against murine CD3ε and CD8α. Representative graphs are shown for each condition (left panels).

Fig 3

Cytotoxic T lymphocyte activity of LCMV-infected H-2^b (C57BL/6J) mice. Mice were infected with 2 × 10⁶ PFU of LCMV clone 13 i.v. or with 2 × 10⁵ PFU of LCMV Armstrong i.p., and spleens were harvested at 8 days postinfection (n = 4). LCMV Armstrong and LCMV clone 13 share identical sequences for all known H-2^b epitopes. Splenocytes were incubated for 5 h at 37°C with ⁵¹Cr-labeled H-2b (MC57) cells infected with LCMV Armstrong 2 days prior. Gamma radiation in the supernatant, indicating cell lysis and release of ⁵¹Cr-integrated proteins, was measured. Levels of gamma radiation in supernatants of splenocytes incubated with uninfected H-2^b target cells were subtracted from those of splenocytes incubated with infected H-2^b target cells, and the results are expressed as percentages of the total lysis (top panels). Splenocytes were also incubated with MHC-mismatched H-2^d target cells (BALB CL7), and gamma radiation levels were measured (they are expressed as percentages of the total lysis [bottom panels]) and served as a negative control.