Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells

Abstract

Humoral immunity depends on intrinsic B cell developmental programs guided by systemic signals that convey physiologic needs. Aberrant cues or their improper interpretation can lead to immune insufficiency or a failure of tolerance and autoimmunity. The means by which such systemic signals are conveyed remain poorly understood. Hence, further insight is essential to understanding and treating autoimmune diseases and to the development of improved vaccines. ST6Gal-1 is a sialyltransferase that constructs the α2,6-sialyl linkage on cell surface and extracellular glycans. The requirement for functional ST6Gal-1 in the development of humoral immunity is well documented. Canonically, ST6Gal-1 resides within the intracellular ER-Golgi secretory apparatus and participates in cell-autonomous glycosylation. However, a significant pool of extracellular ST6Gal-1 exists in circulation. Here, we segregate the contributions of B cell intrinsic and extrinsic ST6Gal-1 to B cell development. We observed that B cell-intrinsic ST6Gal-1 is required for marginal zone B cell development, while B cell non-autonomous ST6Gal-1 modulates B cell development and survival at the early transitional stages of the marrow and spleen. Exposure to extracellular ST6Gal-1 ex vivo enhanced the formation of IgM-high B cells from immature precursors, and increased CD23 and IgM expression. Extrinsic sialylation by extracellular ST6Gal-1 augmented BAFF-mediated activation of the non-canonical NF-kB, p38 MAPK, and PI3K/AKT pathways, and accelerated tyrosine phosphorylation after B cell receptor stimulation. in vivo, systemic ST6Gal-1 did not influence homing of B cells to the spleen but was critical for their long-term survival and systemic IgG levels. Circulatory ST6Gal-1 levels respond to inflammation, infection, and malignancy in mammals, including humans. In turn, we have shown previously that systemic ST6Gal-1 regulates inflammatory cell production by modifying bone marrow myeloid progenitors. Our data here point to an additional role of systemic ST6Gal-1 in guiding B cell development, which supports the concept that circulating ST6Gal-1 is a conveyor of systemic cues to guide the development of multiple branches of immune cells.

Keywords: B cell; ST6Gal-1; glycosylation; humoral immunity; sialylation; sialyltransferase.

Figure 1

B cell development in St6gal1-KO mice. St6gal1-KO (KO) mice were backcrossed onto a C57BL/6J (WT) background for 15 generations to control for strain-specific differences. (A) Schematic of B cell development from the immature to mature stages in the bone marrow and spleen, as proposed by Carsetti and colleagues. (B) Frequency of B cells in the bone marrow (upper panel) and B cell subpopulations (lower panel) in WT and KO mice (n = 5). (C) Splenic mass and cell counts in WT and KO mice (upper panels). Frequencies of splenic B cell subpopulations in WT and KO mice (lower panel; n = 10). (D) Hematoxylin and eosin-stained spleens, with location of relevant anatomical compartments (WP, white pulp; RP, red pulp; MZ, marginal zone). Immunofluorescence microscopy of B220 (red) and marginal zone marker MARCO (green). (E) Mean fluorescence intensity of cell surface CD19, CD24, IgM, and CD23 in IgM-high bone marrow B cells, with FSC and SSC of gated cells shown (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), IgM-high, and mature (BMM), as well as splenic IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) (>94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin (n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin (n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets (n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations (n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.

Figure 3

Cell non-autonomous ST6Gal-1 influences sialylation and abundance of early transitional B cell populations. CD45.1+ whole bone marrow cells from wild-type or St6gal1-KO mice were adoptively transferred to irradiated CD45.2+ hosts. Mice were allowed to recover for 6 weeks before analysis of bone marrow and splenic B cells. (A) SNA reactivity of bone marrow and splenic B cell subsets of CD45.1+ donor cells. (B) Frequencies of CD45.1+ IM, IgM-high, BMM, IgD-/CD21-, IgD+/CD21+, MZ, and FO B cells as a fraction of total CD45.1+ B cells (n = 5). (C) Immunofluorescence microscopy staining anti-IgM (red) and anti-IgD (green) in chimeras. Splenic B cell populations indicated are identified accordingly - T1: IgM+/IgD-, extrafollicular; T2 and FO: IgM-variable/IgD+, follicular, MZ: IgM+/IgD-, marginal sinus. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Cell non-autonomous ST6Gal-1 influences serum IgG. Wild-type or St6gal1-KO mice reconstituted for 6 weeks with wild-type bone marrow were assayed for serum IgG (n = 7, 9). **** P < 0.0001.

Figure 5

Systemic ST6Gal-1 influences long-term survival, but not homing of splenic B cells. (A) CFSE-labeled splenocytes from day 6 wild-type mice were intravenously injected into μMT and μMT/ST6KO mice. 24 h later, spleens were analyzed. CFSE+ IgM+ cells were identified in spleens of recipient mice 24 hrs post-injection. The frequency of adoptively transferred B cells was equal between μMT and μMT/ST6 DKO mice (quantified in Supplementary Figure 5C). (B) CD3−/B220+ wild-type or St6gal1-KO splenic B cells were transferred intravenously into μMT or μMT/ST6KO mice, and recipients sacrificed after 28 days. Representative microscopic analysis of spleens for IgM expression is shown (n = 3).

Figure 6

Extrinsic sialylation promotes transitional B cell development and CD23 expression. Wild-type immature B cells were cultured for 40 h with or without recombinant ST6Gal-1. Phenotypically B220-low (IM), IgM-hi (T), and B220-high (M) B cells are designated. In all panels, absence or presence of recombinant ST6Gal-1 (rST6G) in culture is indicated with “−” or “+.” (A) Mean fluorescence intensity for SNA is shown (n = 7). (B) Relative size of B cell populations after culture period (n = 7). (C) Expression of cell surface IgM and CD23 in the transitional population before culture, and after culture with or without extrinsic sialylation (n = 3). Results are representative of 3 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 7

Extrinsic sialylation enhances BAFF and BCR-mediated pro-survival signaling. (A) Wild-type bone marrow immature B cells were extrinsically sialylated with recombinant enzyme (rST6G), then stimulated with recombinant BAFF for 15 or 30 min, before western blot analysis of cytosolic proteins. (B) The membrane portion of cell lysate was subjected to immunoprecipitation with SNA-agarose beads, and the enriched fraction probed for BAFFR by western blot. ~5% of input is shown, whereas ~20% of input is represented in the immunoprecipitation. (C) Bone marrow CD23-/B220+ B cells were purified by magnetic separation, extrinsically sialylated by recombinant ST6Gal-1, then stimulated with functional grade anti-IgM F(ab')2 for indicated times. Anti-pTyr (4G10) blot identifies total tyrosine phosphorylation events. (D) Immature B cells were cultured in presence of BAFF and ST6Gal-1 and stimulated with anti-IgM F(ab')2 antibody with or without IL-4 and anti-CD40 antibody. Survival was quantified after 18 h by DAPI uptake, and results are representative of 3 independent experiments (n = 3). *P < 0.05, **P < 0.01.