Fig 2: The majority of interleukin (IL)-31 expression in polymorphic light eruption (PLE) is by CD11b+ cells. Quantitative analysis of immunohistochemical staining showing the number of (a) interleukin (IL)31+ cells; (b) IL-31 receptor alpha complex (IL31RA)+ cells; and (c) oncostatin M receptor (OSMR)+ cells in different diseases. Visual analysis (a–c) was performed by counting positively stained cells in five of the most densely infiltrated microscopic fields randomly selected from the same section, at a magnification of × 40. (d) Representative images of double staining showing high numbers of CD11b+ cells expressing IL31 in PLE. (e–g) Quantitative analysis showing the number of (e) IL-31-expressing CD11b+, (f) IL31 expressing CD11b+ Siglec8+ (eosinophils); and (g) CD11b+ CD68+ cells (macrophages). Automated analysis for immunofluorescence (e–g) was performed using TissueQuest image analysis software v6·0 (TissueGnostics GmbH, Vienna, Austria). Briefly, nuclei were assessed by 4,6-diamidino-2-phenylindole mean intensity and area measurement, while cell surface and intracellular markers were detected inside and around nuclear staining, respectively. Number of positive cells/mm² was calculated and used for statistical analysis. Data are presented as mean ± SD. P values were determined by Mann–Whitney test. (f) All P values except one [atopic dermatitis (AD) vs PLE] remained significant after Bonferroni correction, setting significance to P = 0·0083; ns, not significant. Scale bar, 50 µm.

Fig 3: Assessment of eosinophils in melanoma patients upon the ICI therapy by flow cytometry. Peripheral blood samples from 17 patients (9 responders and 8 non-responders) were taken prior to the treatment (pre) and after the first infusion (post). (a) Representative dot plots identifying CD66b+CCR3+Siglec8+ eosinophils. (b) The frequency of eosinophils in melanoma patients is presented as the percentage of these cells within live CD66b+ granulocytes. (c) The percentage of responders or non-responders displaying changes in eosinophil frequencies after the therapy. Responders were characterized by CR, PR, and SD. *P < .05.

Fig 4: Siglec-8 IF antibody validation and accessory analyses. AIF staining for Siglec-5 (top) and Siglec-8 (bottom) in late-onset AD patient tissue (LOAD; Case ID: 03–06). Show are merge (Left; Red = Iba1, Green = Siglec, Blue = 488 background autofluorescence), background-corrected merge (Middle; Red = Iba1, Green == Siglec / 488 background), and background-corrected Siglec alone (Right). Scale bars = 50 µm.BAntibody validation of ab198690 on BV-2 with induced Siglec-8 (48 hours dox). Top: Siglec-8; Bottom: Siglec-8 2×YF. Blue = Hoechst 33342, Red = ab198690. Scale bar = 20 m.C–ERepresentative IF images for Iba1, Siglec-8, MHC-II, and A (Methoxy X04) in tissue from patients. Shown are: (C) white matter (WM) region of a LOAD patient (Case ID: 01–43); (D) grey matter (GM) region from a LOAD patient (Case ID: 03–06); and (E) GM region from an early-onset AD patient (Case ID: 00–22).F–GPercent of total Siglec-8 mask (F) area and (G) signal intensity that overlaps with Iba1, MHC, and the union (?) of Iba1 and MHC. ° indicates mask were dilated in by a given number of pixels prior to Boolean overlap calculation. 1 voxel ~ 2.4 µm. Values are median stack values from n = 3 selected 20× wide-field images.HPercent area coverage of Siglec-8 localized to Iba1 or MHC in grey matter (GM) and white matter (WM) across all analyzed images.IPercent area coverage of Siglec-8 localized to Iba1 compared to total Aß volume in each field of view across all analyzed images. Linear regression R2 value is shown for images with =10 µm2 total amyloid volume.

Fig 5: Siglec-F and Siglec-8 are upregulated in microglia across models of neurodegeneration AImmunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 µm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60× super-resolution max z-stack projections taken from coronal slices.B, CPercent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *P < 5e-2, **P < 1e-2, ***P < 1e-3, ****P < 1e-4; ns: not significant, using unpaired Student’s t-test, two-sided.D, EIF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aß from methoxy X04 (D) or D54D2 (E). Scale bars = 50 µm.FPercent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6-month WT (non-Tg) and 5XFAD mice. Box indicates quartiles, and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *P < 5e-2, **P < 1e-2, ***P < 1e-3, ****P < 1e-4; ns: not significant, using unpaired Student’s t-test, two-sided.GPercent area coverage of Siglec-F localized to Iba1 compared to total Aß plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with = 10 µm2 total amyloid volume.HCumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aß plaques in 6-month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing = 1 plaque of size = 10 µm2. n = 55 images (4 animals).I, JRelative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex postmortem patient tissue. Linear regression R2 values are shown on plots.KIF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20× wide-field; Case ID: 01-43). Scale bars = 50 µm. Images are 60× super-resolution max z-stack projections.L, MPercent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K), each point represents one WM or GM region average from each individual. In (L), values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ? = intersection, ? = union, NOT = inverse mask. Box indicates quartiles, and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *P < 5e-2, **P < 1e-2, ***P < 1e-3, ****P < 1e-4; ns: not significant, using unpaired Student’s t-test, two-sided.