Figure 6.

Early Tfh help optimizes somatic hypermutation during SARS-CoV-2 Spike protein vaccine boosting

(A) Schematic of experiment. Control (Cd4^Cre and Cxcr5^wt) or Tfh-DTR (Cd4^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with an adjuvanted SARS-CoV-2 Spike protein vaccine and given DT until day 11. The mice received a SARS-CoV-2 vaccine boost on day 30 and draining lymph nodes were harvested on day 38.

(B) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T (left), CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh (middle), and CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr (right) cells on day 38.

(C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells.

(D) Serological analysis of SARS-CoV-2 Spike or RBD-specific IgG.

(E) Percentage of SARS-CoV-2 Spike-specific clones out of the total IgG⁺ GC B cells from single-cell GC cultures (left). Number in the center of the circle plot indicates total number of IgG⁺ GC B cells analyzed.

(F) Affinities of Spike-specific antibodies from single GC B cell cultures.

(G) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells, including analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data.

(H) Analysis of clonal distribution from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. N75 index is represented on a per-mouse basis.

(I) Schematic of deletion during boosting experiment. Control (Cd4^Cre and Cxcr5^wt) or Tfh-DTR (Cd4^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with a SARS-CoV-2 Spike protein vaccine and were boosted on day 30, followed by DT treatment. Draining lymph nodes were harvested on day 38.

(J) Spike- and RBD-specific IgG in serum from mice treated as in (I).

(K) Single GC B cell cultures from GC B cells as in (I). Numbers indicate total numbers of IgG⁺ GC B cells analyzed. Data are from a single experiment and are representative of two independent repeats with n = 4–8 mice per group (A–C), are concatenated data from two independent experiments with n = 4–8 mice per group (D), are concatenated data from two independent experiments (E and F), are concatenated data from n = 3 mice per group from one representative experiment (G and H), or are concatenated data from two independent repeats with n = 4–6 mice per group (J and K). Student's two-tailed unpaired t test (A–D, J) or Mann-Whitney Test (G). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figure S5.