Fig 2: Gating strategy for identification of B cell subsets and characterization of memory B cells (MBCs)(A) Identification of B cell subsets from PBMCs. Lymphocytes are first identified based on their size and granularity as indicated by the forward scatter (FSC) and sideward scatter (SSC) signals. Subsequently, doublets and dead cells are excluded from the analysis, as well as T cells, monocytes and NK cells, based on expression of CD3, CD14 and CD56, respectively. B cells are identified by expression of CD19 and/or CD20 and subsequently further divided into B cell subsets. Memory B cells (MBCs) are identified as IgD-CD20+ cells as opposed to IgD+ naïve B cells and IgD-CD20- antibody-secreting cells.(B) Identification of antigen-specific MBCs. After exclusion of streptavidin-binding MBCs, antigen-specific cells are identified as double positive for the respective antigen probes (S1, RBD, S2).(C) Identification of isotypes IgM, IgG and IgA within the MBC population.(D) MBC activation and proliferation status, depicted as overlaid contour plots and histograms of total IgG+ MBCs (red) and S1-specific IgG+ MBCs (purple). Resting, intermediate, atypical, and activated MBCs are identified by expression of CD21 and CD27 (left panel). CD71 serves as a marker of proliferation activity (right panel). Representative contour plots are shown for donor 1, day 19 after the second SARS-CoV-2 vaccination (A and B) and donor 2, day 8 after the third vaccination (C and D).

Fig 3: Longitudinal eOD-GT8 60-mer-Specific BGC and Non-BGC Cell Responses in Draining LNs after a Single IM or s.c. Immunization(A) Flow cytometry identification of total eOD-GT8 60-mer-specific LN B cells before and after immunization (top and bottom samples pairs are from individual RMs). Gated on CD20+ B cells. Full gating in Figure S2.(B) Frequency of total eOD-GT8 60-mer-specific B cells after IM or s.c. immunization (Mann-Whitney, one tailed).(C) Flow cytometry identification of eOD-GT8 60-mer-specific plasmablast (KI67+BCL6−) or GC (KI67+BCL6+) B cells in the LN.(D) Frequency of KI67+BCL6− plasmablasts among eOD-GT8 60-mer-specific B cells in the draining axillary LN over time.(E) Frequency of BGC cells among total eOD-GT8 60-mer-specific B cells in the draining axillary LN over time (Mann-Whitney, two tailed).(F) Representative flow cytometry gating of eOD-GT8 60-mer-specific B cells among BGC cells.(G) Frequency of eOD-GT8 60-mer-specific BGC cells in the draining axillary LN over time (Mann-Whitney, two tailed).(H) Total number of eOD-GT8 60-mer-specific BGC cells in the LN FNA sample of the draining axillary LN.(I) Total number of eOD-GT8 60-mer-specific B cells in the LN FNA sample of the draining axillary LN.*p < 0.05. Each point represents an individual LN FNA sample. n = 8, four LN FNAs per immunization condition at each time point.See also Figures S2 and S3.

Fig 4: T cell response post-MVA immunization.a Germinal center Tfh response (CXCR5++PD1++) (at MVA-1**p = 0.008, at MVA-2 *p = 0.031) and b germinal center B-cell response (BCL6 + Ki67+) (**p = 0.008) measured on CD4 + T cells and CD20 + B cells respectively in FNAs processed draining inguinal lymph node. The shaded area represents the range of germinal center responses at pre-vaccination time points from MVA-ID animals. Data were not available for the MVA-IM group. Left: representative flow-cytometer plots post-MVA immunization. c Circulating non-Tfh response (CXCR5-Ki67+) and Tfh response (CXCR5 + Ki67+) (***p = 0.0007) measured on blood CD4 + T cells (frozen PBMC) post-MVA-1 immunization. Left: representative flow-cytometer plots. d Th1(CXCR5- CXCR3+) /Tfh(CXCR5 + CXCR3-) in blood Ki67 + CD4 + T cells (frozen PBMC) (*p = 0.034). Left: Representative flow cytometer plots. Asterisks in all the above data, denote statistically significant differences measured by Mann–Whitney rank-sum (two-tailed). e Total SHIV BG505 (Gag + Env) specific IFN-γ + CD4 T cell responses and CD8 T cell responses measured in blood. Boxes in all the box plots extend from the 25th percentile to the 75th percentile of the dataset and the line inside the box denotes the median. The whiskers outside the box descend to the dataset’s minimum values and ascend to the maximum values. f Kaplan–Meier curves in animals with low and high frequencies of total SHIV BG505 (Gag + Env) specific IFN-γ + CD4 T cells (all vaccinated animals included and stratified as low and high, based on the median frequency as cut off) post-MVA-1 immunization compared with control animals. Statistical significance by log-rank test (*p = 0.028). g–i Spearman’s correlation (two-sided) between Day 7 MVA-1 induced IFN-γ + CD4 T cells and g MVA-1, Day 7 Th1/Tfh ratio, h Wk68 vaginal IgA (ng anti-SOSIP IgA per µg total IgA), i Wk70, serum ADP score. In all the correlation plots, p and r values were color-coded to represent the vaccine group (red: MVA-ID, Blue: MVA-IM, Black: both groups combined). These values were not shown for the group that didn’t have a statistically significant p value <0.05. DNA-1: first DNA; DNA-2: second DNA; MVA-1: first MVA; MVA-2: second MVA; Prt-1: first protein; Prt-2: second protein. Source data are provided as a Source Data file.

Fig 5: Flow cytometric analyses of human lung DC subsets. As depicted, human lung samples are pregated on single, live, CD45+ cells. The CD45+ population of the lung then is further gated on CD3− and CD16/CD19/CD20− cells (also called LIN−), before gating on all HLA‐DR+ cells. From here, monocytes are excluded by gating on CD88− cells only and CD14−CD88− and CD14+ CD88− are used for subsequent gating on the different DC subsets. Within the CD88− population, pDC can be identified as CD123+CD169− cells and pre‐DC as CD123int/+CD169+ cells. Subsequently, the CD123/CD169 double negative population is further gated on CD141+ cDC1 (optional also CADM1+) or CD1c+ cells, which contain a mixture of cDC2 (DC2 and DC3). These CD1c+ cDC2 can be subdivided using CD5 and CD14 and by gating on the CD5+CD14− DC2, CD5−CD14− DC3, and CD5−CD14+ DC3 subsets. Samples shown here were acquired on a CYTEK Aurora 5L.