Fig 2: The bone marrow contains a major population of isotype-switched non-proliferating memory B cells.a Quantification of NP-specific IgG2a/b+ spleen, peripheral lymph nodes, blood and bone marrow (BM) memory B cells. Female C57BL/6 mice immunized with NP-KLH/LPS SC. Numbers of NP-binding IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) determined by flow-cytometry on d421 or d426 post immunization; pooled from two independent experiments. OVA ctrl: staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI- small lymphocytes (cf. Supplementary Fig. 5). Lines connect samples from one individual, paired one-sided t-test for spleen and BM samples. b Flow-cytometric quantification of Ki-67 expression in IgG2b+ Bsm (IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI- small lymphocytes) splenic naïve (IgM+IgD+IgG2b-CD19+CD38+CD138-GL7-CD11c- PI- small lymphocytes) and germinal center (GC) (CD19+CD38loGL7+CD11c-PI- lymphocytes) B cells. Frequencies of Ki-67+ cells within the subset, data in right graph from two independent experiments using pooled cells from 4 to 20 C57BL/6 mice, paired one-sided t-test. c Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in female C57BL/6J mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3× NP-CGG/IFA. Analysis performed after 7 days of CyP. IgG2b+ B cells quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI- small lymphocytes, CD19+ B cells as CD19+CD138-PI- lymphocytes (Welch’s test, one-sided). Representative data for one of two independent experiments (n = 5 per group). Boxplot indicates median, first and third quartiles, whiskers: 1.5 IQR. d IgG2b+ B memory cells (Ki-67- IgD- Blimp1-GFP-) are dispersed as single cells throughout the BM. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20 µm. Micrograph representative of five slides from two female C57BL/6 mice. e Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10 µm. f Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10 µm (gray) of a cell stained for the respective molecule. g Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b+ Bsm. Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI- small lymphocytes, histogram plots representative of three biological replicates from C57BL/6 females. Source data for Fig. 1a–c, f are provided as a Source Data file.

Fig 3: Reduced CD8 T cell response to ZIKVBR is associated with virus detection at later time points following infection.a, b Frequency (a) and number (b) of CD8aloCD11ahi CD8 T cells in the peripheral blood of mice at indicated dpi with ZIKVCDN or ZIKVBR. Data are representative of two independent experiments with n = 3 mice per group, sampled repeatedly at indicated time points. Data were analyzed by two-tailed, unpaired Student’s t-test at each time point. In a day 5 p = 0.0041, day 7 p = 0.0004, day 10 p = 0.0015, day 14 p = 0.0067, day 20 p = 0.0073, day 30 p = 0.0132, day 40 p = 0.0137, day 50 p = 0.0157. In b day 5 p = 0.0004, day 7 p = 0.0014, day 10 p = 0.0003, day 14 p = 0.0218, day 20 p = 0.0169, day 30 p = 0.0002, day 40 p = 0.0371, day 50 p = 0.0327. c, d Frequency (c) and number (d) of CD11a+CD49d+ CD4 T cells in the peripheral blood of mice at indicated dpi with ZIKVCDN or ZIKVBR. Data are representative of two independent experiments with n = 3 mice per group, sampled repeatedly at indicated time points. Data were analyzed by two-tailed, unpaired Student’s t-test at each time point. In d day 0 p = 0.0407. e Viral RNA was quantified in the spleen using RT-qPCR analysis. Data are presented as PFU equivalents per gram of tissue after comparison to a standard curve of Ct value versus log10(PFU) for each isolate. Mice were sacrificed 12 hpi and 3, 5, and 7 dpi with ZIKVCDN or ZIKVBR, or following mock-infection or injection with UV-ZIKV (12 hpi only). Data are representative of three independent experiments, with n = 3 mice per group at each time point. Data were analyzed by two-tailed, unpaired Student’s t-test at each time point, 12 hpi p = 0.001, 5 dpi p = 0.000079, 7 dpi p = 0.00014. f, g Viral burden in the spleen (f) and kidney (g) were quantified via plaque assay 7 dpi with each ZIKV isolate. N.D. indicates no data recorded above the LOD (dotted line). Data are pooled from two experiments with n = 5 mice per group. All data are shown as mean ± SEM and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Red squares = ZIKVCDN-infected mice, blue triangles = ZIKVBR-infected mice, black squares UV-ZIKV-injected mice. Source data are provided as a Source Data file.

Fig 4: Resistant Tac Mice Exhibit Increased Expansion of GC B Cells and Increased Titers of Parasite MSP1-Specific Antibodies Preceding Clearance of P. yoeliiC57BL/6N mice from Taconic Biosciences (Tac) and Charles River (CR) Laboratories were infected with P. yoelii parasitized red blood cells (pRBCs).(A) Percentage of RBCs infected with P. yoelii (% parasitemia) on the indicated day post-P. yoelii infection. Data (means ± SEs) are cumulative results from 3 experiments. Numbers within each day are the fold difference between the mean % parasitemia in Tac and CR mice.(B–I) Spleens were removed at days 8, 11, 14, 17, and 21. Representative fluorescence-activated cell sorting (FACS) plots and total numbers of antigen-specific CD4 cells (CD4+CD49d+CD11a+), T follicular helper cells (Tfh) (CD4+CD44+CXCR5+BCL6+), plasmablasts (CD19+B220+IgD-CD138+), and germinal center B (GCB) cells (CD19+CD95+GL7+) were determined. Total cell number data (means ± SEs) are cumulative results (n = 6–8 mice per time point) from 2 (day 0) or 3 (days 8, 11, 14, 17, 21) independent experiments and were analyzed by unpaired t test.(J) Serum was collected from Tac and CR mice 14 days post-P. yoelii infection. Sera was serially diluted and reacted against MSP119-coated plates. MSP1-specific antibodies were detected by ELISA. Data (means ± SDs) are representative of 3 experiments and were analyzed by unpaired t test.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.