Fig 2: Distribution analyses of categorized HLA-DR and marker of interest high-low Iba1-positive populations. One MOI was co-labelled with HLA-DR and pan myeloid cell marker, Iba1, in 10-µm thick normal human middle temporal gyrus sections. The point intensities (PI) of HLA-DR and MOI P2RY12 (A, H, and O), TMEM119 (B, I, and P), CD74 (C, J, and Q), CD206 (D, K, and R), CD32 (E, L, and S), CD163 (F, M, and T), or L-Ferritin (G, N, and U) measured on Iba1-positive cells were used to categorize each Iba1-positive cell as either HLA-DRhigh MOIhigh, HLA-DRhigh MOIlow, HLA-DRlow MOIhigh, or HLA-DRlow MOIlow. Chi-square analyses were used to determine whether HLA-DR expression and MOI expression are independent of one another (A–G); data are presented as pooled Iba1-positive cells from the GM of all 6 normal cases. MOIhigh cells were grouped based on their HLA-DR status, and the proportion of HLA-DRhigh or HLA-DRlow cells determined to be MOIhigh were compared with a student’s t-test (H–N); data are presented as mean ± SD (n = 6). HLA-DRhigh cells were subsequently grouped based on their MOI status, and the proportion of MOIhigh or MOIlow cells determined to be HLA-DRhigh were compared with a student’s t-test (O–U); data are presented as mean ± SD (n = 6). Significance of differences between high-low populations: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Microglial cell density, pTDP-43, and GFAP load are increased in ALS. Microglial cell density was quantified from round 1 immunolabelling (A and B) and pTDP-43 load and astrogliosis was quantified from round 2 (C–F). Examples of immunofluorescent images and binary masks are taken from stage 4 ALS case, MN13 (A, C, and E). Total microglia were identified by creating separate microglial marker binary masks from HLA-DR, CD68, CD74, and Iba1, which were then combined to create a microglial marker master mask (A). Each object within this mask was considered a single microglial cell. The microglial cell density in the motor cortex and hippocampus was quantified and compared between control and ALS cases (B). Aggregates of pTDP-43 were detected, and binary masks of immunoreactivity were generated (C). The integrated intensity of pTDP-43 aggregates identified was quantified and normalised to total tissue are to generate a load measure. The pTDP-43 load was compared between control and ALS motor cortex and hippocampus (D). The area of GFAP immunoreactivity identified was quantified and normalised to total tissue are to generate a measure of astrogliosis (E and F). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. Microglial densities and pathology loads were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: ****p ≤ 0.0001, **p ≤ 0.01, *p ≤ 0.05

Fig 4: Distribution of CD74+ ID3+ ILCFR in human tonsillar B cell follicles.a Histocytometry analysis showing the frequency of CD19− CD8− CD4− CD74+ ID3+ ILCFR in a tonsillar tissue section (tonsil #1) and b Overlay showing the distribution of CD74+ ID3+ ILCFR (red dots) with respect to CD19+ B cells (dark blue dots) and CD4+ T cells (yellow dots) in the same tonsil. c Bar graph summarizing the frequencies of intra- and extra- follicular ILCFR (CD19− CD8− CD4−CD74+ ID3+) as a frequency of total ILCFR in five tonsils. d Confocal images showing the tonsil area imaged, distribution of B cell follicles as denoted by CD19 (dark blue) and Ki67 (cyan) as well as ID3 positive cells (red). e Close up of a B cell follicle. Dotted lines demarcate the area of the follicle (LZ) as well as the dark zone (DZ) as defined by the density of Ki67 staining (cyan). B cells are shown in blue (CD19+), proliferating cells in cyan (Ki67+), ID3 in red, CD4 in yellow and CD8 in magenta. f Zoomed in details of the red rectangular enclosures shown in (e). The location of a CD74+ (green) ID3+ (red) cell is shown with respect to the positioning of CD19+ (blue) Ki67 (cyan) cells or CD8+ (magenta) and CD4+ (yellow) lymphoid cells. g Zoomed in close-ups confirming the positioning and phenotype of ILCFR (CD19-CD4− CD8− CD74+ ID3+). Images were acquired at ×40 (NA 1.3) with no zoom. Images shown are sequential digital magnifications of 150 um (d), 30 um (e), 10 um (f), and 2 um (g). A total of five biologically independent human tonsils were imaged.

Fig 5: Microglia express functional markers in the normal and ALS post-mortem human motor cortex and hippocampus. Two rounds of immunohistochemical labelling were used to visualise microglial, anatomical, and pathological markers in the motor cortex (A–H) and hippocampus (I–P) from human ALS (stage 4 shown) and neurologically normal control cases. Round 1 was comprised of microglial markers, including L-ferritin (green), HLA-DR (magenta), CD68 (red), CD74 (yellow), and Iba1 (cyan), with a Hoechst nuclear counterstain (blue) (A-B, E–F, I–J, M–N). Round 2 was comprised of pathological and anatomical markers, including pTDP-43 (green), NeuN (magenta), GFAP (red), and lectin (yellow) with a Hoechst nuclear counterstain (blue) (C–D, G–H, K–L, O–P). Images from rounds 1 and 2 were aligned using the Hoechst nuclear counterstain. Scale bars (A, C, E, I, K, M, O) = 100 µm and scale bars (B, D, F, H, J, L, N, P) = 20 µm