Extended Data Fig. 7: Spatial imaging and transcriptomics identifies factors that underlie Th2 and TFH cell localization.
a-d, The medLN were collected at indicated time points for immunofluorescence staining of B220 (blue), YFP (yellow) and CD45.1 (red). Scale bar, 500 µm. Zoom-in images show separately acquired regions of interest of the T cell zone based on CD4 and B220 staining. (d) Blimp-1-YFP + 1DER CD4 + T cells in T cell zone and B cell zone were quantified after 0d (n = 6), 3d (n = 9), 5d (n = 10) and 10d (n = 8). e-f, The medLN were collected for cross-section and immunofluorescence staining of B220 (blue), YFP (yellow), GATA3 (green) and CD45.1 (red) 3d after HDM immunization (left). Scale bar, 500 µm. Zoom-in images show separately acquired regions of interest of the T-B border. Quantification of the images showing the location of GATA3 + YFP + 1DER cells in T cell center or T-B border within the medLN (f, n = 8). g, genes in significant latent factor Z13 with high weighted expression in Th2 or TFH regions. h-m, Naïve CD45.1/2 + 1DER T cells were adoptive transferred into CD45.2+ recipients followed by HDM immunization for 3d (n = 2) or 5d (n = 2). medLN were collected for Curio Biosciences Slide Seeker protocol (Slide-seq). Serial sections were collected and stained for immunofluorescence of B220 (blue), CD4 (green), CD45.1 (red) and CD11c (magenta) (h, k, left). RCTD deconvolution was performed to assign regions with 7 cell types (i, l). RCTD, robust cell-type decomposition. Isolated regions mapped with Th2 transcriptomes or B cell transcriptomes were visualized by spatial location (j, m). n-o, Pathways analysis of genes identified in standalone latent factors enriched via SLIDE analysis performed on Slide-seq datasets at d3 (n) and d5 (o), the latent factors were identified for d3 (delta=0.085, lambda=1, spec=0.5, FDR = 0.1) and for d5 (delta=0.085, lambda=1, spec=0.1, FDR = 0.1). Each point represents one individual sample. Data are shown as means ± SD above and presented two (f) or three (d) independent experiments. Ordinary two-way ANOVA test (d), two-tailed unpaired Mann-Whitney t test (f). d: 0d versus 3d (T cell area)=0.0001, 0d versus 5d (T cell area)=0.0023; f: * P = 0.0281.
