Extended Data Fig. 2: Kinetic analysis of GC response in aged C57BL/6 mice after 1W1K-NP in alum immunization.
From: Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging
(a) Gating strategy for the analysis of GC B cells, Tfh and Tfr populations in the iLNs of adult and aged C57BL/6 mice immunized with 1W1K-NP in alum. (b) Single-cell sorting strategy of NP + IgG1+ GC B cells from iLNs of C57BL/6 mice 21 days after immunization with 1W1K-NP in alum for VH186.2 sequencing. (c) Representative flow cytometry plots identifying GC B cells (Ki67+Bcl6+B220+CD4−) in the inguinal lymph nodes (iLNs) of adult and aged C57BL/6 mice 10 days after subcutaneous immunization with 1W1K-NP in alum and their frequency (d) and total number (e) at the indicated timepoints post immunization. (f) Representative flow cytometry plots identifying Tfh cells (PD1+CXCR5+CD4+Foxp3−B220−) in the iLNs of adult and aged C57BL/6 mice 10 days post-immunization with 1W1K-NP in alum and their frequency (g) and total number (h) at the indicated timepoints post immunization. (i-k) Representative ELISpot well images (left) and quantification (right) of bone marrow NP23 (i) and NP2 (j) specific IgG1 antibody-secreting cells (ASCs) in C57BL/6 mice 21 days after immunization. (k) Affinity maturation of bone marrow ASCs from C57BL/6 mice as determined by the ratio of NP2/NP23-specific ASCs. For (i-k) the data is representative of two independent experiments (n = 12) where each symbol represents a mouse, and the bar height represents the median. The p-values were obtained by performing an unpaired, two-tailed Mann Whitney U test. (l-n) Adult and aged C57BL/6 serum antibody titers of NP20 (l) and NP7 (m) specific IgG1 at the indicated timepoints after immunization with NP-KLH in alum and antibody affinity maturation (n) as determined by the ratio of NP7/NP20 IgG1 titers. For (l-n) antibody titers shown are displayed as arbitrary units (AU). For (c-h, l-n) The data are representative of two independent experiments (n = 16) where each symbol represents the mean ± SD and p-values were generated by performing a two-way ANOVA with Sidak’s multiple comparisons test.
