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. 2011 Nov;134(3):257-69.
doi: 10.1111/j.1365-2567.2011.03484.x.

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Cornelia Gujer  1 , Christopher Sundling, Robert A Seder, Gunilla B Karlsson Hedestam, Karin Loré
Affiliations

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Cornelia Gujer et al. Immunology. 2011 Nov.

Abstract

Interferon-α (IFN-α) produced at high levels by human plasmacytoid dendritic cells (pDCs) can specifically regulate B-cell activation to Toll-like receptor (TLR) 7/8 stimulation. To explore the influence of IFN-α and pDCs on B-cell functions in vivo, studies in non-human primates that closely resemble humans in terms of TLR expression on different subsets of immune cells are valuable. Here, we performed a side-by side comparison of the response pattern between human and rhesus macaque B cells and pDCs in vitro to well-defined TLR ligands and tested whether IFN-α enhanced B-cell function comparably. We found that both human and rhesus B cells proliferated while pDCs from both species produced high levels of IFN-α in response to ligands targeting TLR7/8 and TLR9. Both human and rhesus B-cell proliferation to TLR7/8 ligand and CpG class C was significantly increased in the presence of IFN-α. Although both human and rhesus B cells produced IgM upon stimulation, only human B cells acquired high expression of CD27 associated with plasmablast formation. Instead, rhesus B-cell differentiation and IgM levels correlated to down-regulation of CD20. These data suggest that the response pattern of human and rhesus B cells and pDCs to TLR7/8 and TLR9 is similar, although some differences in the cell surface phenotype of the differentiating cells exist. A more thorough understanding of potential similarities and differences between human and rhesus cells and their response to potential vaccine components will provide important information for translating non-human primate studies into human trials.

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Figures

Figure 1
Figure 1
B cell and dendritic cell (DC) subsets can be identified using the same markers in humans and rhesus macaques. (a) B cells were characterized based on their expression of CD20. Memory and naive B cells were further distinguished by their expression of levels of CD27 (high for memory B cells, low for naive B cells) in both human and rhesus macaques. Graphs show individual data of the percentages of B cells out of total peripheral blood mononuclear cells (PBMCs) (b) and percentages of CD27+ memory B cells out of total B cells (c). (d) DCs in both human and rhesus macaques were identified using exclusion of lineage markers, positive expression of MHC class II (HLA-DR) and CD123 and CD11c expression for plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), respectively. Flow cytometry plots of representative stainings are shown. (e) Graph shows data obtained from individual donors/animals on the percentages of pDCs and MDCs out of total PBMCs.
Figure 2
Figure 2
Human and rhesus B cells proliferate in response to Toll-like receptor 7/8 (TLR7/8) and TLR9 but not TLR3 ligation. Total rhesus (a) and human (b) peripheral blood mononuclear cells (PBMCs) were cultured in the presence of indicated TLR ligands. Proliferation was measured by thymidine incorporation after 5 days (top panels). Values are indicated in counts per minute (c.p.m.). The proliferation was also analysed using carboxyfluorescein succinimidyl ester (CFSE) dilution after 6 days (lower panels) gating on B cells lacking CD3 and CD14 expression and expressing some degree of CD20 or CD19 in the rhesus and human cultures, respectively. Numbers indicate percentages of proliferating B cells.
Figure 3
Figure 3
Human and rhesus plasmacytoid dendritic cells (pDCs) produce interferon-α (IFN-α) in response to Toll-like receptor 7/8 (TLR7/8) and TLR9 ligation. Total human and rhesus peripheral blood mononuclear cells (PBMCs) were stimulated with CpG C or TLR7/8-L. (a) IFN-α expression in CD123+ pDCs was measured after 11 hr by intracellular staining and flow cytometry. One representative donor is shown. Data for separate individuals are shown to the right. (b) IFN-α levels in similar cultures were measured by ELISA after 24 hr of stimulation (n = 10 rhesus and n = 11 human). (c) IFN-α levels in response to CpG C and TLR7/8-L were compared between rhesus and human cultures using a Mann–Whitney U-test. Data represents mean ± SEM.
Figure 4
Figure 4
Interferon-α (IFN-α) enhances B-cell proliferation to Toll-like receptor 7/8 ligand (TLR7/8-L) and CpG C both in human and rhesus cultures. (a) Increasing concentrations of IFN-α were added to sorted human B cells with or without TLR7/8-L stimulation. Proliferation was measured by thymidine incorporation after 5 days (n = 5). (b) Sorted human and rhesus B cells were stimulated with TLR7/8-L or CpG C in presence or absence of IFN-α for 5 days and proliferation was analysed (n = 11). Data represent mean ± SEM.
Figure 5
Figure 5
Discrepancy of human and rhesus B-cell differentiation markers. (a) Sorted B cells were stimulated with Toll-like receptor 7/8 ligand (TLR7/8-L) or CpG C in the presence or absence of interferon-α (IFN-α) for 5 days. Differentiation to plasma cells was measured by flow cytometry using high expression of CD27 in human cells. CD20 expression is also depicted in the histograms and below, CD20 histograms are shown from CD27high versus the rest of the B cells. (b) The same staining was applied for rhesus cells. Graphs show data obtained from different individuals of (c) CD27high human B cells or (d) rhesus B cells with low expression of CD20.
Figure 6
Figure 6
IgM expression in human and rhesus B cells as a measure of differentiation. (a, b) Differentiation to plasma cells was measured using IgG and IgM stainings in both human and rhesus macaque cells, respectively. Compiled data of percentage of IgM-expressing B cells in human and rhesus are shown to the right. (c) Percentage of CD27high cells are compared with the percentage IgM in human B cells. (d) Percentage CD20− cells are compared with the percentage IgM in rhesus as well as human B cells. The correlation analysis shows compiled data from all the different stimulation conditions used.
Figure 7
Figure 7
The respective B-cell differentiation pattern in human and rhesus B cells correlate to IgM production. IgM levels were measured by ELISA in supernatants of B cells that were stimulated with Toll-like receptor 7/8 ligand (TLR7/8-L) or CpG C in the presence or absence of interferon-α (IFN-α) for 5 days. Levels of IgM secretion by stimulated human B cells (a) as well as rhesus B cells (b) are shown (n = 3 for human, n = 5 for rhesus). Data represents mean ± SEM. (c) Percentages of IgM+ B cells found by flow cytometry strongly correlate with the IgM levels measured by ELISA in both human and rhesus cultures. (d) Percentage of CD27high B cells in human and CD20low B cells in rhesus, respectively, correlate strongly to IgM levels found in the culture. The correlation analysis includes data points from all the different stimulation conditions.
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