Cutting edge: FcR-like 5 on innate B cells is targeted by a poxvirus MHC class I-like immunoevasin

Abstract

Under selective pressure from host immunity, viruses have retained genes encoding immunoevasins, molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in antiorthopoxvirus immunity. In this study, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine FcR-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.

Figure 1

OMCP binds to B cells and monocytes/macrophages independently of NKG2D. A-C, C57BL/6 splenocytes (A), peritoneal cells (B), or human PBMCs (C) were stained with the indicated leukocyte subset markers. The gated cells were additionally stained with control DIII tetramers (lines 1) or OMCP tetramers (lines 2). OMCP tetramer staining following preincubation with 5 μg/mL isotype control mAb (lines 3), 5 μg/mL anti-NKG2D (lines 4), 2 μM DIII (control recombinant protein) (lines 5), or 2 μM recombinant soluble MULT1 (lines 6) is also shown. NK1.1⁺ cells were excluded from the CD11b and B220 gates (A – upper row). Results are representative of at least three independent experiments.

Figure 2

OMCP binds specifically to mouse FCRL5. A, Untransduced or library-transduced C1498 cells were stained with control (left) or OMCP (right) tetramers after the indicated round of sorting. Note that quadrants are not drawn equivalently on each population because cells were stained 2-3 days after sorting and therefore were stained on different days. However, control tetramer staining is shown for comparison on each day. B, Ba/F3 cells transduced with C57BL/6 or BALB/c FCRL5 were stained with nothing (upper row), control tetramers (middle row), or OMCP tetramers (lower row). Transductants are EGFP⁺; untransduced, EGFP^- Ba/F3 cells were included in each stain as an internal negative control. C, Ba/F3-FCRL5 (B6 allele) transductants or WEHI231 cells were stained with control tetramers (lines 1) or OMCP tetramers (lines 2). OMCP tetramer binding following preincubation of cells with 50 μg/mL isotype control mAb (lines 3) or anti-FCRL5 mAb (lines 4) is also shown. D, Plate-bound OMCP or RAE1δ were incubated with the indicated concentrations of FCRL1-Fc or FCRL5-Fc. Bound Fc fusion proteins were detected with anti-human IgG Fc-HRP. E-F, Splenic (E) or peritoneal (F) cells were stained with control tetramers (lines 1) or OMCP tetramers (lines 2). OMCP tetramer staining following cellular preincubation with 50 μg/mL isotype control mAb (lines 3) or anti-FCRL5 mAb (lines 4) are also shown. Results are representative of three independent experiments.

Figure 3

The three N-terminal Ig domains of FCRL5 are necessary and sufficient for OMCP binding. 293T cells transfected with Thy1.1 or the indicated FCRL5-Thy1.1 fusion construct were stained with negative control DIII tetramers (lines 1), OMCP tetramers (lines 2), isotype control mAb (lines 3), or anti-Thy1.1 mAb (lines 4). Results are representative of three independent experiments.