Reporter assays failed to provide evidence for a BACL receptor.

<p>(A) Bright expression of FLAG-tagged hBACL-CD3ζ or hKLRG2-CD3ζ chimera on stably transfected BWZ.36 cells detected by flow cytometry using anti-FLAG mAb M2 (solid lines). IgG1 isotype control stainings are filled. (B) Functional responsiveness of BACL-CD3ζ or KLRG2-CD3ζ expressing BWZ.36 reporter cells upon overnight stimulation with immobilized mAb M2, but not with control IgG1. (C) BWZ.36-hKLRG2-CD3ζ cells or mock-transfected BWZ.36 controls were cultured overnight with 293T cells transiently transfected with hBACL or mock-transfected 293T cells. (D, E) Mock-transfected BWZ.36 controls or BWZ.36-hBACL-CD3ζ reporter cells were cultured overnight with indicated NK cell lines (D) or freshly isolated human PBMC or NK cells (E). (C – E) Treatment of BWZ.36 cells with ionomycin and PMA (P/I) served as a positive control.</p

The <i>CLEC2L</i>-encoded CTLR is highly conserved and a member of the CLEC2 subfamily of CTLR.

<p>(A) Amino acid sequence alignment of the CTLDs of mouse Clec2l (mClec2l) and human CLEC2L (hCLEC2L) with NKC-encoded CLEC2 family members and other NKC-encoded CTLRs of human or mouse origin. Alignment starts with the first out of six highly conserved cysteines (Cys1 to Cys6) of the CTLD that are highlighted in black and numbered (bottom line) accordingly. The ‘WIGL’ motif of the hydrophobic core is indicated by asterisks (bottom line) and shaded, as are other conserved CTLD residues. Dots indicate sequence gaps. (B) Phylogenetic tree of the CTLD sequences shown in (A). Tree was generated with the PHYLIP program (<a href="http://evolution.genetics.washington.edu/phylip.html" target="_blank">http://evolution.genetics.washington.edu/phylip.html</a>).</p

<i>CLEC2L</i> encodes a disulfide-linked homodimeric CTLR readily expressed on the cell surface.

<p>(A) 293T cells transiently transfected with carboxyterminally FLAG-tagged mouse CLEC2L cDNA (solid line) or vector control (filled) were analysed by flow cytometry using anti-FLAG mAb M2. (B, C) Immunoblots of 293T cells transiently transfected with FLAG-tagged mouse CLEC2L cDNA. CLEC2L proteins in cellular lysates were detected with mAb M2. Lysates of mock-transfected 293T cells (mock) served as controls. (B) Lysates of 293T cells expressing mouse CLEC2L proteins were separated by reducing (red; left panel) or non-reducing SDS-PAGE (non-red; right panel). Lysates were treated with PNGase F for protein deglycosylation (reducing conditions) where indicated. (C) Lysates of 293T cells expressing wildtype mouse CLEC2L (wt) or mouse CLEC2L mutants were separated by reducing (left panel) or non-reducing SDS-PAGE (right panel). Stalk region of CLEC2L was mutated, with cysteines 93 and/or 96 substituted by alanines.</p

CLEC2L is predominantly expressed in the brain.

<p>(A) Quantitative RT-PCR of CLEC2L transcripts in various human tissues. Data are normalized to human TBP and arbitrarily set relative to CLEC2L transcript levels of kidney (*). (B) Quantitative RT-PCR of CLEC2L transcripts in various tissues of C57BL/6 mice. Data are normalized to 18S rRNA and arbitrarily set relative to CLEC2L transcript levels of kidney (*). (C) <i>In situ</i> hybridization of C57BL/6 mouse brain sections confirmed pronounced CLEC2L expression in the brain with particularly high levels in cerebellar Purkinje cells. <i>In situ</i> hybridization was performed using DIG-labeled sense (control) and anti-sense mouse CLEC2L probes. (D) Quantitative RT-PCR of CLEC2L transcripts in various brain regions isolated from C57BL/6 mice. Whole brain sample (**) was included for comparison. Data are normalized to 18S rRNA and arbitrarily set relative to CLEC2L transcript levels of kidney (*). (E) <i>In situ</i> hybridization of tissue sections of human cerebellum using DIG-labeled sense (control) and anti-sense human CLEC2L probes.</p

The <i>CLEC2L</i> gene and its products.

<p>(A) Map of the genomic region containing the <i>CLEC2L</i> gene in human and mouse. Boxes represent genes, arrows transcriptional orientation. The distance of the orphan genes <i>CLEC2L</i> and <i>KLRG2</i> is indicated. Adjacent genes are HIPK2 (homeodomain interacting protein kinase 2) and LUC7L2 (LUC7-like 2). (B) Schematic representation (true to scale) of the exon/intron structure of the human <i>CLEC2L</i> gene with the five exons numbered and the 3′ UTR sequence in light gray. (C) Schematic representation of protein domains encoded by the five exons. Cyt = cytoplasmic domain, TM = transmembrane domain, CTLD = C-type lectin-like domain. (D) Alignment of the human and mouse CLEC2L amino acid sequences. Dashes indicate identical amino acids, dots represent sequence gaps. The conserved cysteines of the CTLD are in black, the cysteines of the stalk are marked by arrows. The ‘WIGL’ motif is shaded and the predicted transmembrane region boxed.</p

Tumor-associated BACL expression does not affect tumor growth <i>in vivo</i>.

<p>(A) Ectopic expression of BACL or MICA by the respective RMA transfectants as revealed by flow cytometry with anti-BACL IgY or anti-MICA mAb AMO1 (solid lines). Stainings of mock-transfected RMA for control (filled). (B) 3x10⁵ RMA-BACL, RMA-MICA*07, or RMA-mock cells were subcutaneously inoculated in syngeneic C57BL/6 mice and tumor growth was monitored for the indicated period with a metric caliper. While differences in tumor size were significant (starting from day 9) between RMA-mock (n  = 4) and RMA-MICA*07 (positive control, n  = 3), there was no significant difference (n.s.) in tumor size between RMA-mock and RMA-BACL (n  = 4). Error bars represent SEM.</p

Brain-associated BACL molecules.

<p>(A, B) Depiction of BACL proteins from mouse and human brain. (A) BACL proteins immunoprecipitated from cellular lysates of mouse cerebrum and cerebellum, respectively, were subjected to reducing (right) or non-reducing SDS-PAGE (left) and detected by immunoblotting with anti-BACL IgY. No BACL was detected in lysates of spleens or in control immunoprecipitates (pre-immune IgY). (B) BACL proteins immunoprecipitated from cellular lysates of human cerebellum and cortex, respectively, were subjected to reducing (right) or non-reducing SDS-PAGE (left) and detected by immunoblotting with anti-BACL IgY. No BACL was detected in control immunoprecipitates (pre-immune IgY). (A, B) Cellular lysates were included as input controls (inp).</p

Patient characteristics.

<p>Patient characteristics.</p

MRI scans before (a-e) and under (f-j) therapy.

<p><b>a, f:</b> Reduced leptomeningeal enhancement (white arrows) after 8 weeks of therapy with bevacizumab and lomustine in patient 3. <b>b, g:</b> Regression of leptomeningeal contrast-enhancing nodule (white arrow) on the septum pellucidum on T1-weighted images after eight weeks of therapy with bevacizumab and temozolomide in patient 4. <b>c, h:</b> This regression (black arrow) in patient 3 was also visible on T2-weighted images, which makes pure pseudoresponse unlikely. <b>d, i:</b> Regression of leptomeningeal contrast-enhancing nodules (white arrowheads) on the surface of the medullar conus and the lumbar nerve roots on T1 weighted images (Th10-L2) in patient 9 before and after radiotherapy plus eight weeks of therapy with bevacizumab and lomustine. <b>e, j:</b> This regression of contrast-enhancement (white arrowheads) in patient 9 was also apparent in the thoracic spine (Th5-Th9) which was not treated with radiotherapy.</p

Treatment and outcome.

<p>Treatment and outcome.</p