Fig. 4: Antigen archiving is enhanced by sequential immunizations.

A Experimental design. Created in BioRender. Tamburini, B. (2025) https://BioRender.com/lp928uf. B Mean day 21 Ag-score is shown for LECs from mice that received a single immunization (day 0 or day 21) or dual immunization (day 0 and day 21). C UMAP projections show day 21 (BC2) Ag-scores for LEC subsets for single and dual immunizations. D Prior immunization enhances antigen archiving. Ag-score is shown for single and dual immunizations for the 21 day timepoint for each LEC subset. Other timepoints are shown in grey. P-values were calculated using a one-sided Wilcoxon rank sum test with Benjamini-Hochberg correction. The number of cells used for each comparison is shown below each boxplot. The center line, box limits, whiskers, and points represent the median, interquartile range (IQR), the range within 1.5×IQR from the box limits, and points outside this range (outliers), respectively. E Mean day 0 (barcode 1(BC1)) Ag-score is shown as described in (B). F UMAP projections show day 0 Ag-scores for LEC subsets as described in (C). G Successive immunization enhances retention of previously archived antigen. Ag-score is shown for the day 0 timepoint as described in (D). Values are shown for one biological replicate, a second biological replicate is shown in Figure S6. Each “biological replicate” contains cells from 18 mouse LNs pooled. P-values were calculated using a one-sided Wilcoxon rank sum test with Benjamini-Hochberg correction. H Day 0 (BC1) and day 21 (BC2) Ag-score is compared for LEC subsets for mice that received dual immunization. The Spearman correlation between BC1 and BC2 is shown for each subset. Values are shown for a single biological replicate, a second biological replicate is shown in Figure S6. Each “biological replicate” contains cells from 18 mouse LNs pooled. I The fraction of cells that archived antigens from one immunization (Ag-high), both immunizations (double-high), or were Ag-low but predicted to be Ag-high (Ag-competent) is shown for single (day 14:d14-PI; day 21:Group A; day 0:Group B) and dual immunized mice (Group C). J The expression of Ag-high modules described in Fig. 2 is shown for LECs that archived antigens from both immunizations (double-high), from only one immunization (Ag-high), or were Ag-low but predicted to be Ag-high (Ag-competent). Module scores are shown for the LEC subset matching the identified gene module. The Spearman correlation between the ordered antigen classes and Ag-high module scores is shown for each subset. One-sided p-values testing for an increase in module score were calculated and adjusted using the Benjamini-Hochberg procedure. Boxplots were drawn as described in (D). K Ag-low module scores are shown for LEC subset as described in (J). The Spearman correlation between the ordered antigen classes and Ag-low module scores is shown for each subset. One-sided p-values testing for a decrease in module score were calculated and adjusted using the Benjamini-Hochberg procedure.