FcRL6, a new ITIM-bearing receptor on cytolytic cells, is broadly expressed by lymphocytes following HIV-1 infection by Timothy J. Wilson, Rachel M. Presti, Ilaria Tassi, Edgar T. Overton, Marina Cella, and Marco Colonna Blood Volume 109(9):3786-3793 May 1, 2007 ©2007 by American Society of Hematology

Structural features of human and mouse FcRL6. Structural features of human and mouse FcRL6. (A) Human FcRL6 is a 434–amino acid, type I transmembrane protein of the immunoglobulin superfamily. It contains a leader peptide, encoded by 2 exons, followed by 3 immunoglobulin domains. The protein also contains a cytoplasmic domain with a number of putative signaling motifs. The orthologous gene in mice encodes a type I transmembrane protein highly homologous to the rat protein Gp42. It contains only 2 immunoglobulin domains and lacks a cytoplasmic tail. Patterns within the ovals indicate the sequence homology within the immunoglobulin domains of FcRL6, Gp42, and FcγRI. FcγRI signals through a homodimer of the gamma chain of Fc receptors, which contains a cytoplasmic ITAM. The cDNA and protein sequences for human and mouse FcRL6 are published under GenBank accession numbers AY513661 and EF032497, respectively. (B) The cytoplasmic tail of FcRL6 encodes a series of putative signaling motifs. Most proximal to the membrane is a proline-rich sequence containing a potential SH3-binding PxxP motif (box) followed by 2 YxxV motifs, the second of which is a canonical ITIM (underlined). The last exon encodes a novel cysteine-rich domain containing 2 CxEVxC motifs (shaded) along with several acidic residues that may cooperate for metal binding. Timothy J. Wilson et al. Blood 2007;109:3786-3793 ©2007 by American Society of Hematology

Expression of FcRL6 on PBMCs Expression of FcRL6 on PBMCs. (A) FcRL6 is expressed on CD56dim NK cells, a subset of CD56+ CD3+ NKT cells, and a subset of CD8+ T cells. Expression of FcRL6 on PBMCs. (A) FcRL6 is expressed on CD56dim NK cells, a subset of CD56+ CD3+ NKT cells, and a subset of CD8+ T cells. In healthy individuals, FcRL6 is typically not expressed by CD4+ T cells, B cells, or monocytes. The lack of FcRL6 staining on CD14+ monocytes is indicated on the histogram (black line) overlaying the isotype control (gray line). (B) CD8+ T cells, purified by immunomagnetic isolation, were examined by 4-color flow cytometry to determine FcRL6 expression among CD8+ T-cell subsets. Expression of FcRL6 is found among CD28+ CD45RA− 2B4+ effector-memory cells and CD28− CD45RA+ 2B4+ effector cells. FcRL6 is not expressed by CD28+ CD45RA+ 2B4− naive cells, or by CD28+ CD45RA− 2B4− central memory cells. Timothy J. Wilson et al. Blood 2007;109:3786-3793 ©2007 by American Society of Hematology

FcRL6 can be down-regulated on NK cells by activating cytokines. FcRL6 can be down-regulated on NK cells by activating cytokines. Activation of NK cells with IL-2 or IL-15 leads to up-regulation of the surface marker CD56 and down-regulation of FcRL6. The differentiation of resting NK cells from CD56dim “cytotoxic” to CD56bright “regulatory” NK-cell phenotypes with IL-12 stimulation also leads to down-regulation of FcRL6. Timothy J. Wilson et al. Blood 2007;109:3786-3793 ©2007 by American Society of Hematology

Recruitment of SHP-2 by FcRL6 is dependent on tyrosine 371. Recruitment of SHP-2 by FcRL6 is dependent on tyrosine 371. (A) IP/Western analysis of pervanadate-treated primary NK cells shows phosphotyrosines in samples immunoprecipitated with anti-FcRL6 antibody, but not anti-CD56 control. (B) Phosphotyrosine-specific antibodies detected a strong 66-kDa band in immunoprecipitates from pervanadate-treated (PV), but not untreated (UT), NK92 cells stably expressing wild-type FcRL6. In addition, SHP-2 is recruited by FcRL6 in pervanadate-treated cells. Mutation of tyrosine 371, but not tyrosine 356, to phenylalanine leads to an abrogation of FcRL6 phosphorylation and a failure to recruit SHP-2. ERK immunoblot is shown as loading control. This result has been repeated twice. (C) Recruitment of SHP-2 by phosphorylated NKG2A in pervanadate-treated NK92 cells is shown as positive control. NKG2A recruits more SHP-2 than FcRL6, suggesting low stoichiometry of interaction between FcRL6 and SHP-2. Samples immunoprecipitated from untreated or pervanadate-treated NK92 cells with anti-NKG2A or a control antibody were immunoblotted with phosphotyrosine- and SHP-2–specific antibodies. ERK immunoblot is shown as loading control. Timothy J. Wilson et al. Blood 2007;109:3786-3793 ©2007 by American Society of Hematology

FcRL6 expression is dramatically up-regulated on lymphocytes in HIV-1–infected patients. FcRL6 expression is dramatically up-regulated on lymphocytes in HIV-1–infected patients. (A) Two-color FACS analysis of blood from HIV-1+ patients and healthy controls shows relative increases in FcRL6+ lymphocytes in HIV-1+ individuals. FACS plots showing increases in CD8+ FcRL6+ cells and CD4+ FcRL6+ cells are representative of the median expression levels for healthy and infected individuals. Up-regulation of FcRL6 on B cells in HIV-1+ is also seen, though less frequently than for T cells. Cells are gated by FSC/SSC to include lymphocytes. (B) Three-color FACS analysis of CD8+ T cells in HIV-1+ individuals. CD8+ FcRL6+ T cells (upper plot) express 2B4 (lower histogram) and therefore correspond to the effector/effector-memory population. (C) The distribution of FcRL6+ cells among CD4+ and CD8+ T cells is shown. Dramatic and statistically significant increases in FcRL6+ can be seen in both major populations of αβ-T cells. Elevation in the number of FcRL6+ CD8+ or CD4+ T cells does not correlate significantly with viral titer or CD4 count (not shown). Horizontal lines indicate mean values. Timothy J. Wilson et al. Blood 2007;109:3786-3793 ©2007 by American Society of Hematology