ppGalNAcT-1 supports lymphocyte homing to specific lymph nodes. CMFDA-labeled lymphocytes (2.5 × 10⁷) obtained from wild-type (WT) mice were injected into the tail veins of recipients of the indicated genotypes. Lymphoid aggregates, as denoted in the legend to Fig. 4, were harvested 1 h (A) or 24 h (B) after injection. CMFDA⁺ T and B lymphocytes were quantified by flow cytometry. Data are means ± SEM for eight mice of each genotype. An unpaired t test indicated significance (***, P < 0.001; **, P < 0.01; *, P < 0.05).

Reduced IgG and plasma B-cell abundances during ppGalNAcT-1 deficiency. (A) Splenic GCs analyzed 14 days after immunization with DNP-KLH, indicating a reduced abundance of IgG as well as colocalization of the apoptosis marker activated caspase-3 with IgG-expressing B cells. Magnification, ×1,000. (B) Reduced frequency and number of viable IgG⁺ IgM⁻ B220⁺ B cells in the spleens of ppGalNAcT-1-deficient mice 8 days after immunization with DNP-KLH. (C) Reduced frequency and number of splenic plasma B cells (CD138⁺) in ppGalNAcT-1-deficient mice, measured 8 days after immunization with DNP-KLH. Experiments shown are representative of data obtained from analyses of three to six littermates of the indicated genotypes.

Loss of O-glycans produced by ppGalNAcT-1-deficient mice increases GC macrophage infiltration and B-cell apoptosis. (A) Abundances of tingible-body macrophages (CD68⁺) and markers of cell apoptosis, including activated caspase-3 and TUNEL, were increased in ppGalNAcT-1 deficiency among GC B cells analyzed in situ 14 days after immunization with DNP-KLH. Magnification, ×1,000. (B) CD68, activated caspase-3, and TUNEL measurements obtained by quantifying fluorescent signals. (C) Percentage of CD68⁺ macrophage colocalization with apoptotic cell markers, including activated caspase-3 and TUNEL. (D) Centroblast (CB) and centrocyte (CC) populations among splenic GC B cells (B220⁺ GL7⁺) measured 8 days after immunization with DNP-KLH by forward-scatter (FSC) flow cytometry. (E) Increased abundance of activated caspase-3 detected among permeabilized centroblasts and centrocytes from ppGalNAcT-1-deficient mice by flow cytometry. Analyses were accomplished 8 days after immunization with DNP-KLH. Experiments shown are representative of data obtained from three to six littermates of the indicated genotypes. WT, wild type.

Tissue-selective deficiency in L-selectin ligand and MECA-79 epitope expression among peripheral lymph nodes. (A) Histological analysis of inguinal (ILN) and mesenteric (MLN) lymph nodes incubated with CD4-FITC, CD8-FITC, and B220-Rho and visualized by fluorescence microscopy. Magnification, ×100. (B) Unaltered expression levels of L-selectin on CD19⁺ and CD3⁺ lymphocytes in spleen, blood, and inguinal and mesenteric lymph nodes, as indicated by flow cytometry. Dotted lines represent background cell fluorescence using isotype-specific control antibodies. (C) Frozen sections of inguinal and mesenteric lymph nodes stained with L-selectin-IgG chimera or MECA-79 antibody. Magnification, ×400. (D) Relative levels of L-selectin ligands and MECA-79 expression on HEVs were obtained by quantifying fluorescent signals from serial and parallel tissue sections of inguinal lymph nodes (ILN) and mesenteric lymph nodes (MLN), using deconvolution microscopy and MetaMorph software analysis (see Materials and Methods). WT, wild type.

Altered blood lymphocyte levels with reduced protein O glycosylation. (A) Leukocyte counts were obtained from blood of wild-type and ppGalNAcT-1-deficient mice. Cell numbers are expressed as means per μl of whole blood ± standard errors of the means (SEM) among 21 mice, including littermates of the indicated genotypes. WBC, white blood cell; Neu, neutrophil; Lym, lymphocyte; Mon, monocyte; Eos, eosinophil; Bas, basophil; RBC, red blood cell; Plt, platelet. An unpaired t test indicated significance (***, P < 0.001; *, P < 0.05). (B) Reduced protein O glycosylation on the cell surfaces of circulating leukocytes, detected by PNA lectin binding. Reduced PNA binding was observed among Gr1⁺ neutrophils and B220⁺ B cells. In contrast, close-to-wild-type levels of O-glycans detected by PNA binding levels were noted among unactivated circulating CD3⁺ T cells. WT, wild type.

Leukocytosis involving T and B cells is associated with a reduction in lymph node cellularity. (A) Circulating numbers of T cells (CD3⁺) and B cells (CD19⁺) were significantly increased in ppGalNAcT-1-deficient mice. (B) Leukocyte abundance was reduced among distinct peripheral lymphoid aggregates, including inguinal lymph nodes (ILN), axillary lymph nodes (ALN), cervical lymph nodes (CLN), and mesenteric lymph nodes (MLN). No significant change occurred among the Peyer's patch (PP), spleen (Spl), thymus (Thy), and bone marrow (BM) compartments. (C) B-lymphocyte (CD19⁺) numbers were significantly reduced among multiple lymphoid aggregates. (D) T-lymphocyte (CD3⁺) numbers were reduced most significantly among inguinal lymph nodes. Data are means ± SEM for 12 littermate pairs of the indicated genotypes. An unpaired t test indicated significance (***, P < 0.001; **, P < 0.01; *, P < 0.05). WT, wild type.

Diminished GC development and B-cell expansion upon immunization of ppGalNAcT-1-deficient mice. (A) Splenic follicles and GCs from mouse spleens prior to and 8 days subsequent to immunization with DNP-KLH were detected with hematoxylin and eosin (H.E.) and with GL7 antibody. WP and RP indicate white pulp and red pulp, respectively. Images were magnified 100×. Small arrows within white pulp denote GC B cells. The percentage of white pulp bearing GCs and the size of GCs were measured. (B) GC B cells in the spleen were further analyzed in situ 14 days after immunization with DNP-KLH. T- and B-cell zones remained intact, and follicular dendritic cells (FDCM1⁺) were present at normal levels. (C) GC markers include GL7 antibody binding among B220⁺ B cells, while PNA binding dependent upon O glycosylation was substantially decreased in ppGalNAcT-1-deficient mice. Magnification (B and C), ×400. (D) Reduced frequency of splenic GC B cells (B220⁺ GL7⁺) as well as reduced total splenic B220⁺ B-cell number among ppGalNAcT-1-deficient mice 8 days after immunization with DNP-KLH. Results shown are representative of data obtained from analyses of three to six littermates of the indicated genotypes. WT, wild type.

Reduced selectin ligand expression on ppGalNAcT-1-deficient neutrophils attenuates selectin-mediated rolling and inflammation in a model of acute peritonitis. (A) Reduced E- and P-selectin ligands among circulating Gr1⁺ cells, detected by selectin-IgM chimera binding and flow cytometry. Loss of selectin-IgM chimera binding in the presence of EDTA is also shown. (B) Expression levels of adhesion molecules (CD11a, CD11b, CD18, and CD62L) and selectin counterreceptors (CD24 and PSGL-1) on Gr1⁺ cells in blood, analyzed by flow cytometry. The results shown in panels A and B are representative of three experiments with separate littermates. (C) Numbers of rolling leukocytes on P-selectin and E-selectin were reduced in ppGalNAcT-1-deficient mice, analyzed using blood-perfused microflow chambers at 5.9 dynes/cm². Data presented are means ± SEM for at least six chambers, using cells from three separate littermates of the indicated genotypes. *, P < 0.05 compared to ppGalNAcT-1. FOV, field of view. (D) Peritoneal cells were collected, and Gr1⁺ neutrophils were counted prior to (0 h) or after (2 or 4 h) intraperitoneal injection of casein to initiate acute peritonitis. Data are presented as means ± SEM for eight mice representing littermates of the indicated genotypes (***, P < 0.001). WT, wild type.

Normal antigen receptor activation contrasts with attenuated antibody production due to loss of ppGalNAcT-1. (A) Serum Ig levels among 8-week-old naive mice (n = 16). Points represent measurements for individual animals. The median Ig levels are depicted as horizontal bars (means ± SEM are indicated). An unpaired t test indicated significance (***, P < 0.001; *, P < 0.05). (B) The expression levels of activation markers (CD44, B7.2, and I-A^b) and CD40 were analyzed by flow cytometry on CD19⁺ lymphocytes derived from the spleen (Spl), peripheral lymph nodes (PLN), and mesenteric lymph nodes (MLN). The results shown are representative of three separate experiments. Dotted lines represent the fluorescence of cells stained using an isotype-specific control antibody. (C) B lymphocytes were isolated and stimulated by antibody to IgM or LPS. The proliferation response was measured by [³H]thymidine incorporation. Data are presented as means ± SEM for three mice of the indicated genotypes. (D) Anti-DNP antibody levels produced in response to immunization with 10 μg of the T-cell-independent antigen DNP-Ficoll, measured at the indicated times. (E) Anti-DNP antibody levels measured before and subsequent to a secondary immunization (arrow) using 100 μg of the T-cell-dependent antigen DNP-KLH. Multiple dilutions of sera were assayed, with the results shown reflecting the linear range of responses (OD₄₀₅ measurements) (data not shown). Data are presented as means ± SEM for eight mice of the indicated genotypes. WT, wild type.

ppGalNAcT-1 mutagenesis and loss of enzyme activity by deletion of exon 3. (A) Construction of ppGalNAcT-1 targeting vector for subsequent Cre-loxP recombination. (B) Cre recombination results in the deletion of exon 3, producing the null allele (type 1), or the flanking of exon 3 by loxP sites to generate the floxed allele (type 2). Restriction enzyme sites for panels A and B are indicated as follows: A, ApaI; B, BamHI; R, EcoRV; X, XhoI; Sp, SpeI; N, NotI. (C) Genomic Southern blot analysis of targeted ES cell clones (6-6, 1-4, and 3-3), using the loxP probe. (D) Genomic Southern blotting of tail DNAs, using a genomic probe, indicated the presence of germ line type 1 and type 2 mutations in the gene encoding ppGalNAcT-1. (E) Enzyme activity was assayed with total protein extracts from various tissues by using the peptide substrate PRFQDSSSKAPPPLPSPSRLPG. O-glycosylated products were profiled by anion-exchange chromatography and evaluated by reverse-phase high-performance liquid chromatography. Data are represented as means ± standard deviations (SD) for three separate experiments. SG, salivary gland; Spl, spleen; Thy, thymus. (F) Polypeptide GalNAcT-1 cDNA structures sequenced from the kidney tissues of mice bearing either the wild-type or deleted (Δ) allele. (G) ppGalNAcT enzyme activity towards the EA2 peptide (PTTDSTTPAPTTK) in COS7 cell extracts following transfection of cDNAs expressed by the pIMKF3 vector. Data are represented as means ± SD for three separate experiments. WT, wild type.