Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation

Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration

CHS response is reduced significantly after depletion of Gr-1⁺ neutrophils in vivo.

<p>Naive C57BL/6 mice were treated with DNFB as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a> to generate DNFB-CHS model. 1 day prior to DNFB challenge, mice were i.p. injected with 100 μg anti-Gr-1 or 100 μg isotype antibodies (rat-IgG2b). Neutrophils (CD11b⁺ Gr-1⁺) in blood, spleen, and challenged skin site at 48 hours after DNFB challenge were analyzed by FACS (A). The ear thickness was measured at 0–120 hours after challenge (B). *: P<0.05. 5 mice for each group were tested. Results are representative of three independent experiments.</p

CHS response is dramatically reduced after neutralization of IL-17 in vivo.

<p>Naive C57BL/6 mice were treated with DNFB as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a> to generate DNFB-CHS model. 1 day prior to DNFB challenge, mice were i.p. injected with 100 μg anti-IL-17 or 100 μg isotype antibodies (rat-IgG1). Neutrophils (CD11b⁺ Gr-1⁺) in blood, spleen, and challenged skin site at 48 hours after DNFB challenge were analyzed by FACS (A). The ear thickness was measured at 0–120 hours after challenge (B). **: P<0.01. 5 mice for each group were tested. Results are representative of three independent experiments.</p

The recruitment of Gr-1⁺ CD11b⁺ neutrophils, not the sensitized CD4⁺ or CD8⁺ T cells and NK cells, to DNFB-challenged skin is significantly influenced in dermal γδ T cell-deficient chimeric mice.

<p>A, The DNFB-induced CHS model using chimeric mice was established as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g003" target="_blank">Fig 3</a>. 48 hours after DNFB challenge, right ears were collected and digested to prepare single cell suspensions. After washed with cold PBS, skin cells were stained with fluorescence-conjugated antibodies for flow cytometry. B, The frequencies of CD4⁺ or CD8⁺ T cells, NK cells, and Gr-1⁺ CD11b⁺ neutrophils in the challenged skin. C, The numbers of skin infiltrated CD4⁺ or CD8⁺ T cells, NK cells, and Gr-1⁺ CD11b⁺ neutrophils. **: P<0.01. Results are representative of two independent experiments.</p

The sensitized CD4⁺ or CD8⁺ T cells infiltrate into DNFB-treated skin normally after intravenous transfer while Gr-1⁺ CD11b⁺ neutrophils are greatly reduced in dermal γδ T cell-deficient chimeric mice.

<p>A, Wild type CD45.1⁺ C57BL/6 mice were sensitized with DNFB at first 2 days. 5 days later, dLNs were collected to prepare cell suspension. After washed with PBS, 2 x 10⁷ CD45.1⁺ sensitized leukocytes were intravenously transferred to CD45.2⁺ BM or BM+thymocytes chimeric mice. 1 day after transfer, the ears of chimeric mice were treated with 0.25% DNFB. 48 hours later, the treated ears were collected for flow cytometry analysis. B, The frequency of total or donor T cells. C and D are the numbers of total or donor T (CD4⁺ or CD8⁺) cells in the skin, respectively. E and F are the frequency and number of total or donor Gr-1⁺CD11b⁺ neutrophils in the skin, respectively. NS: no significant. **: P<0.01. Results are representative of two independent experiments.</p

CHS response is remarkably reduced in TCR δ^-/- mice.

<p>WT or TCR δ^-/- mice were sensitized with 0.25% DNFB on left ears for 2 consecutive days. 5 days later, the right ears were challenged with one dose of 0.25% DNFB. The ear thickness was measured at 0–120 hours after challenge (A and B). *: P<0.05. 5 mice for each group were tested. Results are representative of two independent experiments.</p

H&E staining of DNFB-challenged skin.

<p>Naive C57BL/6 mice were treated with DNFB as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a> to generate DNFB-CHS model. 1 day prior to DNFB challenge, mice were i.p. injected with 100 μg anti-Gr-1, 100 μg anti-IL-17, or 100 μg isotype antibodies. 48 hours after DNFB challenge, the challenged skin site was harvested for H&E staining. 5 mice for each group were tested. Results are representative of two independent experiments. The bar represents 100 μm.</p

CHS response is significantly reduced in dermal γδ T cell-deficient chimeric mice.

<p>A, Generation of chimeric mice. 5–10 x 10⁶ neonatal thymocytes from newborn C57BL/6 mice (day 0–1 after birth) were transferred to half of the irradiated mice. 24 hours later, 5 x 10⁶ bone marrow (BM) cells from adult naïve C57BL/6 mice were intravenously transferred to all irradiated mice. At least 12 weeks later, these BM or BM + thymocytes chimeric mice were sensitized with 0.25% DNFB on left ears at first two days. 5 days later, the right ears were challenged with one dose of 0.25% DNFB. The ear thickness was measured at 0–120 hours after challenge (B). *: P<0.05. 5 mice for each group were tested. Results are representative of two independent experiments.</p

Dermal γδ T cells rapidly increase in situ and produce large amounts of IL-17 after DNFB challenge.

<p>The DNFB-induced CHS model using wild type C57BL/6 mice was established as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a>. 24 hours after DNFB challenge, right ears were collected and the frequency and number of dermal γδ T cells were analyzed by flow cytometry (A: of total skin cells; and B: per ear). In parallel, parts of skin cells were incubated in RPMI 1640 media supplemented with 5% FCS, 1% P/S, 0.25% DNFB and Brefeldin A at 37°C. 6–7 hours later, IL-17 production of total dermal γδ T cells and the proportion of IL-17-producing Vγ4⁺ cells were analyzed by intracellular staining (C). D, Vγ4⁺ γδ T cells were depleted by i.v. injection of anti-Vγ4 antibody or isotype antibody for 3 consecutive days. DNFB-induced CHS model was created as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a>. Ear swelling was measured during 0–120 hours after challenge. E: Vγ4⁺ cells were depleted in LN and skin after 3 consecutive days of anti-Vγ4 injection. F: The increase of ear thickness after DNFB challenge. *: P<0.05. **: P<0.01. 5 mice for each group were tested. Results are representative of three independent experiments.</p