Extended Data Fig. 7. RNA-seq analysis of VRC01-class bnAb precursors.
a) Bulk RNA-seq analysis showing the row-wise z score of differentially expressed genes in VRC01gHL B cells from vaccinated mice compared to naive cell (2-fold cutoff, adj p < 0.01). n = 3 mice for group 1-4, n = 4 mice for group 5. (b) Venn diagrams summarizing bulk RNA-seq analysis showing the number of differentially expressed genes in VRC01gHL cells from each group compared to naive cells (1.5-fold cut-off, adj p <0.05, DeSeq2 pairwise comparisons). (c) t-Distributed stochastic neighboring embedding (tSNE) two-dimensional plot of single-cell RNA-seq data shows different clustering patterns of VRC01gHL cells from different immunization groups. n = 42 cells (4 mice) for naïve, n = 55 cells (4 mice) for Ser4-eOD, n = 53 cells (4 mice) for pSer-eOD (Ag–), n = 58 cells for pSer-eOD (Ag+) (4 mice), n = 35 cells (2 mice) for 60mer. Pooled data from two adoptive transfer experiments. (d) Normalized expression of MHC II genes and March1 by VRC01gHL cells. Horizontal lines in violin plots indicate the first quartile, the median, and the third quartile. Each circle represents a cell. n = 37 cells (2 mice) for Ser-eOD + alum, n = 37 cells (2 mice) for pSer-eOD + alum (Ag–), n = 46 cells (2 mice) for pSer-eOD + alum (Ag+), n = 35 cells (2 mice) for eOD-60mer. (e–h) C57BL/6 mice adoptively transferred with 1x106 CTV+ VRC01gHL B cells were immunized by i.p. injection of 10 µg AF647-labelled Ser4-eOD-GT5 or pSer8-eOD-GT5 together with 1 mg alum. (e) Flow cytometry analysis showing changes in MHC II and CD83 expression patterns on VRC01gHL B cells. Number indicates percentage of CD83+ MHC IIhi VRC01gHL B cells. Two independent experiments were performed with similar results. (f) Bar graphs show percentage of CD83+ MHC IIhi VRC01gHL B cells in (E). Bars represent the mean. Pooled data from two experiments. n=7 for group 1 and 2, n=6 for group 3 and 4, n=3 for group 5. (g) Flow cytometry analysis showing changes in CD86 expression patterns on VRC01gHL B cells. Two independent experiments were performed with similar results. (h) Bar graphs show gMFI of CD86 on VRC01gHL cells in (G) normalized to unprimed group. Bars represent the mean. Data combined from two independent experiments. ***, p = 0.002; ****, p < 0.0001 by One-way ANOVA with Tukey’s post-test. (i) Day 28 sera from C57BL/6 mice and NLRP3 KO mice (n=5 animals/group) immunized with pSer8-eOD (red, black) or Ser4-eOD (grey) was analyzed by ELISA for eOD-specific IgG antibody responses. Statistical comparison by One-way ANOVA with Tukey’s post-test. (j) Day 14 sera from BL/6 mice (red) and IL18 KO mice (black) (n=5 animals/group) immunized with pSer8-eOD was analyzed for eOD-specific IgG antibody responses. p=0.69 by two-tailed Student’s t-test.