Fig 2: 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 3: Loss of FAK kinase function reduces survival of MMTV-Wnt1-driven basal-like mammary tumors. a Representative images of Ctrl-Wnt1, cKD-Wnt1, and cKO-Wnt tumors immunostained for Ki67 and corresponding quantification of percentage of positive cells per field of view (n = 4 for each genotype). b Representative images of Ctrl-Wnt1, cKD-Wnt1, and cKO-Wnt tumors immunostained for cleaved caspase 3 and corresponding quantification of percentage of positive cells per field of view (Ctrl-Wnt1, cKD-Wnt n = 8; cKO-Wnt n = 7). c Representative images of Ctrl-Wnt1, cKD-Wnt1, and cKO-Wnt tumors immunostained for CD31 and corresponding quantification of percentage of CD31-positive area per field of view (n = 3). d Representative images of Ctrl-Wnt1, cKD-Wnt1, and cKO-Wnt tumors immunostained for CD8 and corresponding quantification of percentage of CD8-positive cells per field of view (Ctrl-Wnt1 n = 7; cKD-Wnt, cKO-Wnt n = 5). All statistical tests for c–f include ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05, **p = 0.01

Fig 4: HLA class I and PDL1 expression in serial tumor biopsies from DC vaccine treated patients. The pattern of HLA class I expression by melanoma cells is shown in matched pre- and post-therapy lesions for one representative patient (Pt#10). Scale bars, 250 μm (A). Differences in intratumoral PDL1 expression are illustrated in the graph as the percentage of PDL1 expressing cells on the total cell number (n = 14, *p = 0.0353) (B). A representative example of the staining is shown (Pt#13). Scale bars, 100 μm (C). PDL1 expression distribution in matched samples within the naïve/CT/RT cohort and the immuno_treated cohort is reported in right and left graphs (n = 9, **p = 0.0078 and n = 5, p = 0.6250), respectively. CR and PR Patients are displayed in green, SD in black, and PD in red. Open circles denote on-treatment samples, open triangles post-treatment ones (D). A positive correlation was found between CD8 and PDL1 expression (Spearman r = 0.4948, p = 0.0074) (E). Statistical comparisons are based on the non-parametric two-tailed Wilcoxon signed-rank test. Only values statistically significant are reported: *p < 0.05 and **p < 0.01.

Fig 5: Quantitative analysis of intratumoral CD8+ and CD4+ T cells of serial tumor biopsies from DC vaccine treated patients. The number of intratumoral CD8+ T cells per mm2 in matched pre- and post- treatment samples is plotted in graphs. Graph showing all patients (n = 16, p = 0.2144) (A). Right and left graphs showing naïve/CT/RT (n = 9, *p = 0.0195), and immuno_treated (n = 7, p = 0.2969), respectively (B). Whole slide images (WSI) of CD8 staining in the pre- and post-vaccine lesions of one representative patient (Pt#16). Scale bars 1 and 2 mm for the left and the right WSI panel, respectively. Higher magnification images for the mark-up CD8 stain in pre- and post- treatment samples are shown. Scale bars, 100 µm (C). The number of intratumoral CD4+ T cells per mm2 in matched pre- and post- treatment samples is plotted in graphs. Graph showing all patients (n = 9, p = 0.4258) (D). Right and Left graphs showing naïve/CT/RT (n = 5, p > 0.9999) and immune_treated (n = 4, p = 0.1250), respectively (E). CR and PR Patients are displayed in green, SD in black, and PD in red. Open circles denote on-treatment samples, open triangles post-treatment ones. Statistical comparisons are based on the non-parametric two-tailed Wilcoxon signed-rank test. Only values statistically significant are reported: *p < 0.05.