Killing human neuroblastoma or mouse NSC34 cells through the ADCC pathway.

<p>ADCC was performed using human neuroblastoma as target cells, PBMCs as effector cells, and pools of serum samples of ALS, healthy control, inflammatory bowel disease patients, and multiple sclerosis patients as IgG sources. The controls contain: neuroblastoma cells incubated with IgG pools from the different serum sources and co-cultures of neuroblastoma cells and PBMCs (<b>A</b>). FACS results from PBMCs pre-treated with anti-CD16 antibodies (<b>B</b>, left) and the heavy chain of ALS-IgG before and after PNGase-F treatment in SDS-PAGE and Western blot using ECL lectin (<b>B</b>, right); ADCC mediated by CD16-blocked effector cells or by ALS-IgG after PNGase-F treatment, as compared to the ADCC against neuroblastoma mediated by unblocked effector cells and untreated ALS-IgG (<b>C</b>); Killing of NSC34 cells by ADCC as described above was mediated by intact (untreated) ALS-IgG, PNGase-F treated ALS-IgG and by IgG from healthy controls (<b>D</b>). Neuroblastoma lysis by CD32- and CD64-positive THP1 cells was mediated by ALS-IgG, IgG of healthy controls, and in serum free of IgG (<b>E</b>). Spontaneous lysis was measured in neuroblastoma or NSC34 cultures. Triple staining of NeuN, Iba1, and ALS-IgG by anti-human IgGs conjugated to FITC demonstrates the localization of intact ALS-IgG in immune synapse (arrow) amongst microglia and neurons (<b>F</b>). Data represent the mean ± SD of triplicate measurements from triplicate independent experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, ** p<0.01, * p<0.05 and NS (Not significant) is represented versus the appropriate controls in each panel.</p

Coupling of IgG to target neuroblastoma and NSC34 cells and to CD16 on effector cells.

<p>FACS histograms presenting a shift in binding of serum pools of ALS patients to human neuroblastoma cells in comparison to binding of healthy control (CON), inflammatory bowel disease and multiple sclerosis serum pools to neuroblastoma cells (<b>A</b>); FACS histogram presenting shift in binding of purified IgG from serum pools of ALS patients to neuroblastoma cells relative to binding of purified IgG from healthy control (CON) (<b>B</b>); Dose-dependent coupling of purified ALS-IgG to human PANC1, HeLa, and neuroblastoma cells performed as described in A(<b>C</b>); Mean fluorescent intensity (MFI) calculated relative to control sample containing cells and serum that was free of IgG. Dose-dependent coupling of ALS-IgG to mouse NSC34 cells was performed as described above (<b>D</b>); Secretion of IFNγ by enriched human peripheral NK cells in response to interactions with pools of ALS, inflammatory bowel disease patients, patients of multiple sclerosis, and healthy control (CON) sera (<b>E</b>); Secretion of IL-2 by BW-CD16 transfectants or BW cells in response to interactions with pools of ALS and healthy control sera (<b>F</b>), and in response to interactions with ALS-IgG and ALS IgG-depleted sera (<b>G</b>). Comparing the specificity of dose-dependent coupling of PNGase F-treated or untreated IgG of ALS patients and of the IgG of healthy volunteers, to CD16 (<b>H</b>). Data represent the mean ± SD of triplicate measurements from independent duplicate experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, *** p<0.005, ** p<0.01 and * p<0.05, versus the appropriate controls in each panel.</p

The Fc-N²⁹⁷-glycan of ALS-IgG contains a galactosylated glycan with bisecting-GlcNAc and lacking a core fucose.

<p>ANOVA t-test analysis of glycan amounts in individual or in pooled serum samples of healthy controls and inflammatory bowel disease, multiple sclerosis and ALS patients The glycan amounts and structures were determined by NP-HPLC and MALDI-TOF MS methods (<b>A</b>), ANOVA t-test analysis of A2BG2 with respect to gender (<b>B</b>) and age (<b>C</b>). Data represent means ± SD of measurements of 19 ALS, 24 healthy controls, 22 inflammatory bowel disease patients, and 6 multiple sclerosis patients or 6 pools generated by mixing four individual serum samples from every group (in multiple sclerosis two pools were generated by mixing three individual serum samples). Statistical significance, *** p<0.005, ** p<0.01 and *, p<0.05, versus the appropriate control.</p

Statistic analyses comparing N²⁹⁷-glycans digested from serum IgG of 19 ALS patients and of 24 healthy candidates.

<p>Fold of change: (+) assigns that N²⁹⁷glycan amounts in ALS IgG are higher relative to N²⁹⁷glycans from healthy control IgG and (−) assigns the contrary.</p

Statistic analyses comparing N-glycans derived from 19 ALS patient sera and from 24 healthy candidate sera.

<p>Fold of change: (+) assigns that N-glycan amounts in ALS sera are higher relative to glycans from healthy candidate sera and (−) assigns the contrary.</p

ALS-IgG co-localized with CD16 and microglia cells in brain and spinal-cord tissues of G93A-SOD1 mice.

<p>Representative confocal microscopic images of brain cortex and spinal cord slices taken from 130-day old G93A-SOD1 mice and age-matched littermates stained for CD16, hIgG, Iba1, NeuN (neurons) and counterstained with nuclear DAPI. Localization of ALS-IgG before and after PNGase-F treatment and of healthy control-IgG in wild-type and mSOD1 brain tissues. The boxed area is a high magnification of CD16- and intact ALS-IgG-positive cell (<b>A</b>); Quantity of ALS-IgG accumulation before and after PNGase-F treatment and of IgG from healthy control in mSOD1 brain slices (<b>B</b>); Quantity of ALS-IgG before and after PNGase-F treatment and of IgG from healthy control co-localized with CD16 in wild-type and mSOD1 brain tissues (<b>C</b>); Co-localization of intact ALS-IgG with microglia was detected in mSOD1 brain tissue (<b>D</b>), and co-localization of intact ALS-IgG with NeuN in mSOD1 brain and spinal cord tissues (<b>E</b>). The measurements were performed on five fields from 3–4 sections per mouse. Error bars indicate means ± SD. Asterisks denote the significance of differences relative to deglycosylated ALS-IgG or control-IgG in mSOD1 sections or ALS-IgG in non-SOD1 littermates, *** p<0.005 represents a comparison using ANOVA t-test.</p