Figure 4. Frequencies of CSP-specific memory B cell subsets are greater in the DFD regimen than in the STD regimen at later time points.

PBMC were cultured for 5 days in the presence of PF-CSP and PF-16 antigens and analyzed for frequencies of B cell maturation subsets: switched memory (SM: CD20⁺CD10^–CD27⁺IgD^–IgG⁺), switched activated memory (sAM: CD20⁺CD10^–CD21^lowCD27⁺IgD^–IgG⁺) and Ki67 expression on total memory (Mem: CD20⁺CD10^–CD27⁺) B cells in DFD (n = 29) and STD (n = 14) regimens at T5, T6 and T7. (A–C) The scatter plots show PF-CSP-specific SM (A); sAM (B) and Ki67⁺(C) memory B cells. (D–F) PF-16-specific SM (D); sAM (E) and Ki67⁺(F) memory B cells. Data for the protected group are represented as dark blue open circles for DFD (P DFD, n = 25) and as light blue open circles for STD regimen (P STD, n = 10) and non-protected as red open circles (NP, n = 10) for both regimens. Statistical analysis was performed using the generalized linear mixed-effects model via Penalized Quasi-Likelihood to accommodate repeated measurements over time. Statistical significance is shown as: *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4—figure supplement 1. Gating strategy for the B cell subsets.

Live (Aqua^–) cells were gated from the singlets followed by gating for the CD3^–CD20⁺B cells from live lymphocytes. Mature B cells were identified as CD10^–CD20⁺ B cells. From the mature B cells, total memory B cells were identified as CD20⁺CD27⁺ cells. On the basis of the expression of CD21, CD27 and IgD, B cell maturation subsets were identified as naïve (CD21^hiCD27^–IgD⁺) B cells, resting memory (RM: CD21^hiCD27⁺) activated memory B cells (AM: CD21^lowCD27⁺) and atypical memory B cells (aMBC: CD21^lowCD27^–). On the basis of the IgD and IgG expression, unswitch and switch memory B cells were identified as IgD⁺IgG^– (UM: unswitch) and IgD^–IgG⁺ (SM: switch). Switch memory B cells in total memory, RM and AM were designated as SM, sRM and sAM, respectively, while unswitch memory B cells were designated as UM, uRM, uAM for total memory, RM and AM, respectively. All B cell subsets were gated for the expression of the proliferation marker Ki67.

Figure 4—figure supplement 2. CD80 expression on the total B cell, RM and AM subsets.

CD80 expression was analyzed in ex vivo B cell subsets at all the timepoints by flow cytometry. Data were compared longitudinally and also between the P (green line; n = 35) and NP (red line; n = 10) groups at each timepoint. (A) Representative flow cytometry dot plot for CD80 staining of B cells. (B, D, F) Line graphs with error bars, indicating mean ± standard error of mean (SEM), for frequencies of CD80 expression on total (B), RM (D) and AM B cells (F). (C, E, G) Scatter plots comparing CD80 expression between the DFD and STD regimens at T5, T6 and T7 for total (C), RM (E) and AM (G) B cells. Data for the protected group are represented by dark blue open circles for DFD (P DFD) and by light blue open circles for the STD regimen (P STD), whereas data for the non-protected group are shown as red open circles (NP) for both regimens. Statistical analysis was performed using the generalized linear mixed-effects model via Penalized Quasi-Likelihood to accommodate repeated measurements over time. Differences between timepoints in P and NP and between P and NP at each timepoint are not statistically significant.

Figure 4—figure supplement 3. Higher atypical memory B cells (aMBC) at T5 and T6 in the DFD group.

PBMC were cultured for 5 days in the presence of PF-CSP and PF-16 antigens, and atypical memory B cells (aMBC) were identified as CD3^–CD20⁺CD21^lo/negCD10^–CD27^– by flow cytometry. (A, B) Scatter plots showing aMBC specific to PF-CSP (A) and PF-16 (B). Data from the protected group are indicated as dark blue open circles for the DFD regimen (P DFD) and as light blue open circles for the STD regimen (P STD), whereas data from the non-protected group are represented as red open circles (NP) for both regimens. Statistical analysis was performed using the generalized linear mixed-effects model via Penalized Quasi-Likelihood package to accommodate repeated measurements over time. Statistical significance shown as: *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4—figure supplement 4. CSP-specific memory B cell subsets in P and NP subjects.

PBMC were cultured for 5 days in the presence of PF-CSP and PF-16 antigens, and B cell maturation subsets and Ki67 expression on B cell subsets were analyzed by flow cytometry. Line graphs with error bars indicate mean cell frequencies ± standard errors of the mean (SEM) for B cell subsets in the P (green; n = 35) and NP (red, n = 10) subjects for (A) SM, (B) sAM and (C) Ki67⁺ mem B cells. (D–F) Frequencies of PF-16-specific (D) SM, (E) sAM and (F) Ki67⁺ mem B cells. Statistical analysis was performed using the generalized linear mixed-effects model via Penalized Quasi-Likelihood to accommodate repeated measurements over time. Statistical significance is shown as: *, p<0.05; **, p<0.01; ***, p<0.001.