Pattern recognition receptor ligand-induced differentiation of human transitional B cells

Abstract

B cells represent a critical component of the adaptive immune response whose development and differentiation are determined by antigen-dependent and antigen-independent interactions. In this study, we explored the effects of IL-4 and pattern-recognition receptor (PRR) ligands on B cell development and differentiation by investigating their capacity to drive the in vitro maturation of human transitional B cells. In the presence of IL-4, ligands for TLR7/8, TLR9, and NOD1 were effective in driving the in vitro maturation of cord blood transitional B cells into mature, naïve B cells as measured by CD23 expression, ABCB1 transporter activation and upregulation of sIgM and sIgD. In addition, several stimulation conditions, including TLR9 ligand alone, favored an expansion of CD27+ IgM memory B cells. Transitional B cells stimulated with TLR7/8 ligand + IL-4 or TLR9 ligand, with or without IL-4, induced a significant subpopulation of CD23+CD27+ B cells expressing high levels of sIgM and sIgD, a minor B cell subpopulation found in human peripheral blood. These studies illustrate the heterogeneity of the B cell populations induced by cytokine and PRR ligand stimulation. A comparison of transitional and mature, naïve B cells transcriptomes to identify novel genes involved in B cell maturation revealed that mature, naïve B cells were less transcriptionally active than transitional B cells. Nevertheless, a subset of differentially expressed genes in mature, naïve B cells was identified including genes associated with the IL-4 signaling pathway, PI3K signaling in B lymphocytes, the NF-κB signaling pathway, and the TNFR superfamily. When transitional B cells were stimulated in vitro with IL-4 and PRR ligands, gene expression was found to be dependent on the nature of the stimulants, suggesting that exposure to these stimulants may alter the developmental fate of transitional B cells. The influence of IL-4 and PRR signaling on transitional B cell maturation illustrates the potential synergy that may be achieved when certain PRR ligands are incorporated as adjuvants in vaccine formulations and presented to developing B cells in the context of an inflammatory cytokine environment. These studies demonstrate the potential of the PRR ligands to drive transitional B cell differentiation in the periphery during infection or vaccination independently of antigen mediated BCR signaling.

Fig 1. Transitional B cells from human cord blood and peripheral blood mononuclear cells.

Transitional B cells were FACS-sorted based on expression of surface markers, CD19, CD24, CD38 and staining by Rhodamine 123. (A) Total cells were gated based on lymphocyte morphology, followed by gates on viable CD19⁺CD3^- cells, and finally Rhodamine 123 fluorescence. (B and C) Expression of sIgD, CD21, CD23, CD5, and sIgM are shown as histogram plots of median fluorescence intensity (MFI) for high (B) and low (C) Rhodamine 123 staining B cells. (D) Freshly isolated PBMCs were labeled with fluorochrome-conjugated antibodies against CD19, CD24, CD38, CD21, CD23, IgD, and IgM. Total cells were gated based on lymphocyte morphology, followed by viable CD19⁺ cells, and CD38^hi CD24^hi expression. Expression of CD23, sIgM, CD21, and sIgD by these CD38^hi CD24^hi transitional B cells are shown as MFI histogram plots (shaded histograms are isotype controls).

Fig 2. Transitional B cell differentiation into follicular-like or marginal zone-like phenotypes.

FACS-sorted transitional B cells were cultured with IL-4 and individual PRR ligands at 5x10⁵ cells/mL for 2 days. Expression of CD23 and R123 exclusion were evaluated as indicators of transitional B cell maturation. R123 negative, CD23^hi cells (upper boxes) correspond to mature, FO-like B cells. R123 negative, CD23^-, CD21⁺ (bottom boxes) correspond to either immature or MZ-like B cells. Purified transitional B cells were cultured with (A) IL-4 alone; (B) R848+IL-4; (C) iE-DAP+IL-4; (D) GLA+IL-4; (E) Poly (I:C)+IL-4; (F) TDB+IL-4; (G) CpG+IL-4; and (H) CpG alone. (I-J) Compiled data of five independent experiments measuring percentages of CD23^hi cells (I); CD23 MFI values (J), and percentage of CD23^-CD21⁺ cells (K). Statistical significance was determined using Student’s t test for % CD23^hi (I), CD23 MFI (J), % CD23^-CD21⁺ (K) values. * = p<0.05, ** = p<0.01.

Fig 3. R123 expression highlights three distinct populations of immature transitional, maturing transitional, and mature B cells.

(A) Baseline staining of unstimulated FACS-sorted transitional B cells with R123 (left panel), CD21 and CD23 (middle panel), and sIgD and sIgM (right panel). (B) Characteristics of transitional B cells cultured with IL-4: percentages of R123^neg, R123^lo, and R123^hi cells (panel 1); graphical distribution of R123^neg (orange), R123^lo (blue), and R123^hi (red) subpopulations (panel 2); CD21 and CD23 expression of R123 subpopulations (panel 3); and sIgD and sIgM expression of R123 subpopulations (panel 4). (C) Characteristics of transitional B cells cultured with IL-4+R848: percentages of R123^neg, R123^lo, and R123^hi cells (panel 1); graphical distribution of R123^neg, R123^lo, and R123^hi cells (panel 2); CD21 and CD23 expression of R123 subpopulations (panel 3); and sIgD and sIgM expression of R123 subpopulations (panel 4).(D) Model of human transitional B cell maturation stimulated by IL-4 alone or IL-4 + PRR ligand. The immature, transitional B cell possesses a characteristic phenotype as shown. Stimulation with IL-4 alone or IL-4 + PRR ligand results in the development of both CD23⁺ and CD23^- mature B cells that express ABCB1. The CD23⁺ population upregulates expression of CD23, sIgM, and sIgD prior to full induction of ABCB1. Expression of CD23 and ABCB1 by these B cells is markedly enhanced when they are stimulated with both IL-4 and certain PRR ligands, including R848 and CpG. The CD23^- population upregulates sIgM and sIgD expression and ABCB1 without inducing CD23. This population is more prominent in cells stimulated with IL-4 alone than in those stimulated with the IL-4+PRR ligand combination.

Fig 4. Induction of transitional B cell development into memory B cells.

FACS-sorted transitional B cells were cultured with IL4, R848+IL4, CpG, or CpG+IL4 for 2 days and evaluated for expression of CD23 and the B cell memory marker, CD27. (A) Follicular B cell maturation was determined by R123 extrusion and CD23 acquisition in cultures stimulated with IL-4 (panel 1), R848+IL-4 (panel 2), CpG (panel 3), and CpG+IL-4 (panel 4). Boxed area corresponds to R123^negCD23⁺ mature B cells. (B) CD27 and CD23 expression of cultures stimulated with IL-4 (panel 1), R848+IL-4 (panel 2), CpG (panel 3), and CpG+IL-4 (panel 4). (C) Pie-chart display of relative amounts of cells expressing the following surface phenotypes: CD23⁺CD27^-, purple; CD23⁺CD27⁺, blue; CD23^-CD27⁺, green; CD23^-CD27-, orange. Culture stimulation conditions are shown above each pie chart. (D-G) sIgM and sIgD expression of subpopulations of cells expressing different combinations of CD23 and CD27 in cultures stimulated with IL-4 (panel 1), R848+IL-4 (panel 2), CpG (panel 3), and CpG+IL-4 (panel 4).

Fig 5. Sorting strategies for human cord blood transitional B cells and human tonsil follicular B cells.

(A) Cord blood mononuclear cells were stained with R123 and chased for 3 hours. Following R123 pulse/chase, cells were stained with fluorochrome-conjugated antibodies against CD3, CD19, CD24, and CD38. Transitional B cells were sorted based on high R123 expression following the initial gating strategy. (B) Tonsil mononuclear cells were isolated from fresh tonsil tissue via mechanical homogenization and Ficoll density gradient centrifugation. Cell suspensions were stained with fluorochrome-conjugated antibodies against CD3, CD19, CD23, IgD, IgM. Follicular B cells were sorted as CD3-, CD19+, CD23+, IgD+, IgM+/-.

Fig 6. Transcriptome analysis of tonsil follicular B cells and cord blood transitional B cells.

(A) Mean differences plot showing the gene expression fold change for follicular and transitional B cells. Statistically significant differences in gene expression between tonsil follicular B cells and cord blood transitional B cells are in red. (B) RT-qPCR confirmation of select genes (FCER2, RUNX1, and ADAM28) identified from the AmpliSeq analysis in purified FO and Tr B cells. Gene expression fold change is expressed as the ratio of gene expression in follicular B cells/transitional B cells.

Fig 7. Pathway analysis of tonsil follicular B cells and cord blood transitional B cells.

Heatmap display of follicular B cells (black band) and transitional B cells (yellow band) from three individual samples for each cell type. Pathways analyzed were (A) IL-6 signaling, NF-kB signaling, PKA signaling; (B) BCR signaling, Cyclins & Cell Cycle Regulation, Th1/Th2 activation; (C) IL-4 signaling, Notch signaling, PI3K signaling in B lymphocytes. Specific genes of interest are boxed in red. Heat map keys are provided on the right of each figure.

Fig 8. TNFR superfamily transcriptome analysis of tonsil follicular B cells and cord blood transitional B cells.

Listed are genes upregulated in follicular B cells (top) and in transitional B cells (bottom). A heatmap display of follicular B cell (black band) and transitional B cell (yellow band) populations from three individuals for each cell type is shown on the right.

Fig 9. Gene expression analysis of transitional B cells stimulated with CpG+IL-4 or CpG alone.

Purified cord blood transitional B cells were cultured with various stimuli, cultured cells were isolated on day 2 and cDNA was prepared for real-time qPCR. (A) CD23 and CD27 expression of cells cultured with IL-4 (panel 1), R848+IL-4 (panel 2), CpG (panel 3), and CpG+IL-4 (panel 4). (B) Gene expression fold change was calculated using CpG-stimulated cultures as the reference condition as compared to cultures stimulated with IL4 (blue), R848+IL-4 (red), and CpG+IL-4 (green). Transcripts evaluated were CD27, PRDM1, FCER2(CD23), PTPN6, ADAM28, and TNFRSF17. (C) List of upregulated genes for tonsil follicular B cells as compared to cord blood transitional B cells from transcriptome analysis.