Detection of full length agrin and CAF22 in mouse kidney.

<p>Kidneys from wildtype and NT^-/- mice were stained with antibodies against full length agrin (red) and CAF (green). Cell nuclei were marked with DAPI (blue). (<b>A,B,C</b>) Localization of full length agrin and CAF22 in the renal cortex (A,C) and at the transition between outer and inner medulla in wildtype kidney. (<b>D</b>) No staining related to CAF22 was detected in kidneys from NT^-/- mice. Original magnification 400x. (<b>E,F</b>) Higher magnification of proximal tubules in wildtype kidney show a punctuate subapical staining for CAF22 (arrows). (<b>G</b>) No subapical staining in proximal tubules of kidneys from NT^-/- mice.</p

mRNA expression of agrin and neurotrypsin.

<p><b>(A,B)</b> mRNA was extracted from various mouse organs and relative mRNA abundance of agrin and neurotrypsin assessed by real-time RT-PCR (n = 4 animals). <b>(C,D)</b> Mouse nephron segments were isolated by hand-dissection and relative mRNA abundance of agrin and neurotrypsin measured by real-time RT-PCR (n = 4 preparations per nephron segment).</p

Detection of human recombinant CAF22 in kidney.

<p>NT^-/- mice were pretreated with leupeptin and injected 60 min later with saline or human recombinant CAF22. Mice were fixed by perfusion 20 or 60 min after saline or CAF22 injection and tissue sections stained for CAF22 (green), actin (red), cathepsin B (red) or LAMP1 (red). Cell nuclei were marked with DAPI (blue). Original magnification 400–1000 x.</p

Fiber type distribution of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Consecutive muscle cross sections of <i>soleus</i> stained with myosine heavy chain (MHC) specific antibodies or cytochrome C oxidase (COX) staining as indicated. Control and treated SARCO mice show clearly separated type I and II fibers. SARCO mice have a significantly increased amount of hybrid fibers (indicated with asterisks). Cytochrome C staining of <i>soleus</i> sections shows a massive reduction of reactivity in SARCO mice which is reverted back to WT levels in treated SARCO mice. <b>B:</b> Quantitative analysis of muscle fibers of <i>soleus.</i> Bars represent mean values ± standard deviation. Control and treated SARCO mice have almost no hybrid fibers and are indistinguishable from each other. Compared to treated ones, SARCO mice have significantly increased hybrid fibers. Type I fibers are significantly decreased. The total fiber number in SARCO mice is also significantly decreased compared to treated animals. Number of animals: wt  =  3; SARCO treated  =  5, SARCO  =  3. * p<0.05; ** p<0.01. Scale bar: 100 µm.</p

NMJ morphology of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Confocal images of the NMJs in <i>soleus</i> muscle of P30 Control or SARCO mice, treated or not treated with NT-1654 as indicated. The postsynaptic AChRs were stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was stained with anti-neurofilament and synaptophysin antibodies (NF-Syn; green). The postsynapses were highly fragmented, partially or completely lost in <i>soleus</i> muscle of SARCO mice. The postsynapses of NT-1654 treated SARCO mice resembled those in Control mice, showing pretzel like structures and much less fragmentation. Scale bar: 50 µm. <b>B:</b> The postsynapses in Control mice showed fragmentation class 0 (no fragmentation, for illustration see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088739#pone.0088739.s003" target="_blank">figure S3</a>). The postsynapses of NT-1654 treated SARCO mice were mostly classified into class 1, whereas the postsynapse of SARCO mice showed class 1-4 with a peak at class 3. <b>C:</b> Many NMJs of SARCO mice have terminal nerve sprouting, which was dramatically reduced in the NT1654 treated SARCO mice. Data present mean ± standard deviation, n  =  2 mice, 100 NMJs were counted in each mouse.</p

NMJ recovery after sciatic nerve crush.

<p><b>A:</b> Confocal images of the neuromuscular junctions (NMJs) in <i>soleus</i> muscle of Thy1-YFP mice after 14 days sciatic nerve crushing, treated with NT-1654 or PBS (Control) as indicated. The postsynaptic AChR was stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was visualized by the transgenic expression of YFP in motor neurons (green). The NMJs in <i>soleus</i> muscle are fully re-innervated after 14 days sciatic nerve crushing. The NMJs of NT-1654 treated mice showed significantly less nerve sprouting than those treated with PBS (Control). The number of nerve sprouting in each NMJ was counted and shown in <b>B.</b> NMJs in the NT-1654 treated mice have significantly fewer events of nerve sprouting and significantly fewer number of nerve sprouts than those of Control mice. Data present mean ± standard error, *: p<0.05 (two ways t-test), 100 NMJs were counted in each mouse, n  =  3 mice, scale bar: 50 µm.</p

DataSheet_1_Siglec-7 represents a glyco-immune checkpoint for non-exhausted effector memory CD8+ T cells with high functional and metabolic capacities.pdf

While inhibitory Siglec receptors are known to regulate myeloid cells, less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state, and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells, which were found in patients’ peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer.</p