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1.
Figure 4

Figure 4. A common set of 742 significant genes in subjects that had been previously vaccinated.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

Day 0 normalized gene expression values for the common set of 742 significant genes in S02, S03, and S04. These subjects reported influenza vaccination within the past three years (Red indicates increased gene expression level, blue indicates decreased levels). S02, which had the earliest peak response, had received all available influenza vaccines in the previous 3 years. Samples from S05 and S06, who reported no vaccination in the previous three years, had fewer significantly time-varying genes (S05, 174 genes) (S06, 2 genes) in common with the others (p-values in Supplementary Table S6).

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
2.
Figure 1

Figure 1. Hemagglutinin Inhibition Activity of Daily Serum Samples.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

Blood was drawn one day within a week prior to vaccination (pre-V), at vaccination (day 0), daily days1 to 10, and day 21 after vaccination. Serum samples showed heterogeneous humoral responses to the three virus types in the vaccine, A/California/7/2009 (A/California), A/Perth/16/2009 (A/Perth), and B/Brisbane/60/2008 (B/Brisbane). With immunity classified as an HAI titer > 1:40, S06 was not previously immune to any of the three viruses. S05, who had reported not being vaccinated in the previous three years, was immune to A/California only. S01, S07, and S10 were initially immune to all three viruses and the other subjects showed a mix of initial immunity. At day 21, all subjects except S08 and S12 qualified as immune. See also Supplementary Figure S1 and Supplementary Tables S1 and S2.

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
3.
Figure 3

Figure 3. Quantitative RT-PCR confirms patterns of expression of B cell Differentiation Genes.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

To confirm expression levels of B cell differentiation genes by qRT-PCR, we assayed RNA from eight subjects collected in two cohorts previous to the cohort used for RNA Seq data. These subjects had varied serum vaccine responses as assessed by (a) HAI titer, (b) vaccine-specific ELISA, and (c) flow cytometric analysis. Expansion of plasmablasts and plasma cell numbers were not clear in all subjects. T cell contamination was evident in two samples. (d) Increased expression of CD27, CD38, XBP1, CD59, PRDM1 and IRF4 and decreased expression of IRF8, BACH2, and PAX5 was expected at peak plasmablast/plasma cell expansion. While not all genes correlated with phenotype (correlations in Supplementary Table S5), expression levels changed as expected based on phenotype.

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
4.
Figure 7

Figure 7. PBMC Gene Expression Patterns Suggest Myeloid/Dendritic Cell Migration.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

PBMC were isolated in parallel with enriched B cell samples. (a) FPCA analysis of RNA Seq data showed a primary eigenfunction that explained over 70% of the variation across the timepoints for four subjects (S02, 81.44%) (S03, 86.68%) (S04, 72.8%) (S05, 57.15%) (S06, 85.32%). Second eigenfunctions explained most of the remaining variation in three subjects (S02, 13.11%) (S03, 7.24%) (S06, 10.2%). S05 had a third eigenfunction that explained 13.85% of variation. The first eigenfunction for S02, S03, and S04 peaked day 1. (b) The largest positive loadings on these functions were enriched for genes expressed by myeloid/DC lineages (gene symbols in red), (Supplementary Table S8). The first eigenfunction for S05 had few myeloid/DC genes. Second and third eigenfunctions, peaking day 1, both contained small numbers of myeloid/DC genes. The first eigenfunction of S06 peaking on day 1 was enriched for myeloid/DC genes, but expression changes were modest. Day 8 there was a sample processing error.

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
5.
Figure 6

Figure 6. Upstream analysis of time-varying B cell gene sets.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

Ingenuity Pathways Analysis of significant time-varying genes of subjects S02 (5256 genes), S03 (1309 genes), S04 (2147 genes) was performed. Upstream modifiers with a Z-score of 2.0 or higher, indicating probable activation, are shown. Molecule sets downstream of these modifiers were not mutually exclusive. All three subjects showed probable activation by upstream cytokines typical of the germinal center reaction, including IL2, IL4, IL5, IL6 and CD40LG across days of peak response. Upstream modifiers unique to each subject suggested immunological differences. Of interest, late in the peak response of S04, IFN-related signaling molecules were identified. This could be the result of a previous innate-type stimulus such as interaction with plasmacytoid dendritic cells or viral DNA-initiated toll-like receptor activation. Our method identified dynamic gene regulation patterns with common as well as subject-specific upstream regulatory elements underlying the larger post-vaccine response.

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
6.
Figure 5

Figure 5. Expression of the PCgs in Differentiating Plasma Cells.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

(a) We found strong correlations between PCgs expression and CD19+ CD27++ CD38++ CD138 plasmablasts and CD19loCD20loCD27++ CD38++ CD138+ plasma cell percentages (number of correlating genes in each panel). (b) Normal human IgM- B cells were stained with pacific blue succinimidyl ester (PBSE) to show proliferation and stimulated for 60 hr in vitro with CpG2009 ODN and cytokines IL-2, IL-10, IL-15, and BAFF (n = 6 subjects). The cells were FACS-sorted into three stages of differentiation (Undivided, CD27lo, and CD27hi). (c) Row-normalized RNA expression levels from gene array data previously published by our group23 were re-examined for the PCgs (genes ordered as Figure 4 and Supplementary Table S6, red = upregulation, blue = downregulation). Genes upregulated in the PCgs also tended to be upregulated in the most differentiated CD27hi cells, expressed at moderate levels in less differentiated CD27lo cells, and at lowest levels in undivided cells. The converse was true of PCgs downregulated genes. (d) Of 2033 genes significantly different between CD27lo and the more differentiated CD27hi plasmablasts, 366 genes were shared with the PCgs (full list, Supplementary Table S6). (e) The proportion of PCgs genes expressed increased as resting memory B differentiated through plasmablast states and then to fully differentiated CD138+ plasma cells.

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.
7.
Figure 2

Figure 2. Subject-specific B cell vaccine response characteristics.. From: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature.

(a) A single gene expression eigenfunction accounted for 90% of sample variation in each subject vaccinated in the previous three years (S02, S04, and S04). Multiple eigenfunctions were found in RNA Seq data of subjects not previously vaccinated (S05, S06) (full list of significant genes in Supplementary Table S3). (b) By HAI titer, S02, S03 and S04 had previous immunity to two HA antigens. S02 had increases in antibody levels at least 24 hours before S03 and S04 (p-values, Supplementary Table S2). (c) Anti-vaccine IgM, IgG, and IgA levels parallel changes in HAI titer (p-values Supplementary Table S2). (d) ELISPOT assays show functional vaccine-specific IgA, IgG, and IgM ASC increase as serum antibody levels increase. (e) Phenotypic changes of CD3CD19+CD20+CD27 naïve B cells, CD19+CD27+CD20+CD38+CD138 memory B cells, CD19+CD27++CD38++CD138 plasmablasts and CD19loCD20loCD27++CD38++CD138+ plasma cells. Plasmablast and plasma cell populations increase as ASC appear (p-values Supplementary Table S4). (f) Expression of genes (ratio to day 0) change as expected based on available literature in S02, S03 and, S04 (p-values, Table S3). CD5 and SERPINB9 levels decrease at peak response consistent with a fractional decrease in naïve and cytolytic B cells. Fewer genes changed significantly in S06 and S05, subjects reporting no vaccination in the previous 3 years. S02 showed peak response at day 5, earlier than S03 and S04. (N/D = no data).

Alicia D. Henn, et al. Sci Rep. 2013;3:2327.

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