Activation of Murine CD4+ and CD8+ T Lymphocytes Leads to Dramatic Remodeling of N-Linked

This information is current as Elena M. Comelli, Mark Sutton-Smith, Qi Yan, Margarida of September 29, 2021. Amado, Maria Panico, Tim Gilmartin, Thomas Whisenant, Caroline M. Lanigan, Steven R. Head, David Goldberg, Howard R. Morris, Anne Dell and James C. Paulson J Immunol 2006; 177:2431-2440; ;

doi: 10.4049/jimmunol.177.4.2431 Downloaded from http://www.jimmunol.org/content/177/4/2431

Supplementary http://www.jimmunol.org/content/suppl/2006/08/08/177.4.2431.DC1 Material http://www.jimmunol.org/ References This article cites 68 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/177/4/2431.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Murine CD4؉ and CD8؉ T Lymphocytes Leads to Dramatic Remodeling of N-Linked Glycans1

Elena M. Comelli,2* Mark Sutton-Smith,‡ Qi Yan,* Margarida Amado,* Maria Panico,‡ Tim Gilmartin,† Thomas Whisenant,† Caroline M. Lanigan,† Steven R. Head,† David Goldberg,§ Howard R. Morris,3‡ Anne Dell,‡ and James C. Paulson4*

Differentiation and activation of lymphocytes are documented to result in changes in associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGc␣2-6Gal sequence, and a corresponding increase in glycans carrying the Gal␣1-3Gal sequence. This change was Downloaded from accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galac- tosyltransferase, ␣1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding that recognize terminal sequences of N-linked glycans. The Journal of Immunology, 2006, 177: 2431–2440. http://www.jimmunol.org/

ifferentiation and activation of lymphocytes are accom- ST3Gal Inull mice that are constitutively PNAhigh undergo rapid panied by programmed remodeling of cell surface gly- apoptosis in the periphery, reducing the CD8 population to 10% of D cans of with biologically important con- wild type (2). Yet, conversion from PNAlow to PNAhigh is a natural sequences. Notably, the conversion of the peanut agglutinin consequence of activation of wild-type CD8 cells resulting from (PNA)5 high (PNAhigh) phenotype of immature CD4/CD8 medul- down-regulation of ST3Gal I (2, 8, 9). In contrast, ST3Gal I is lary thymocytes to the PNAlow phenotype of the mature single- differentially regulated in activated and polarized Th1 and Th2 positive CD8 and CD4 thymocytes results from conversion of the CD4 cells leading to the PNAhigh and PNAlow phenotype, respec- ␤ ␣ PNA ligand, the O-linked glycan Gal 1-3GalNAc Thr/Ser, to its tively (10). The additional increased expression of fucosyl- and by guest on September 29, 2021 sialylated form, NeuAc␣2–3Gal␤1-3GalNAc␣Thr/Ser, that is no sialyltransferases in Th1 cells promotes synthesis of the selectin longer recognized by PNA due to increased expression of a sia- ligand sialyl-Lewis X (NeuAc␣2-3Gal␤1-4[Fuca1-3]GlcNAc), lyltransferase, ST3Gal I (1–4). In CD8 T cells, this glycosylation while Th2 helper cells lack sialyl-Lewis X sequences because a change reduces the affinity of CD8 for MHC class I, suggesting key fucosyltransferase, Fuc-T VII, is not expressed. Such differ- that sialylation of CD8 O-glycans modulates CD8 function during ential glycosylation accounts for the selectin-mediated recruitment selection and maturation of CD8 T cells (5–7). Naive CD8 cells of of CD4 Th1 cells to sites of inflammation (11–17). Insights into the importance of N-linked glycan structures in *Departments of Molecular Biology and Molecular and Experimental Medicine and lymphocyte biology have also been obtained by ablation or over- †DNA Microarray Core Facility, The Scripps Research Institute, La Jolla, CA 92037; expression of key glycosyltransferases. For example, ablation of ‡ Division of Molecular Biosciences, Imperial College, London, United Kingdom; and the GlcNAc transferase Mgat5 leads to increased TCR signaling §Scripps-Palo Alto Research Center Institute for Advanced Biomedical Sciences, Palo Alto, CA 94304 and autoimmune disease, and promotes Th2 over Th1 responses, a ␤ Received for publication February 17, 2006. Accepted for publication April 26, 2006. result of the loss of N-linked glycans with a GlcNAc 1-6Man The costs of publication of this article were defrayed in part by the payment of page branch that interacts with galectins and reduces TCR signaling by charges. This article must therefore be hereby marked advertisement in accordance restricting TCR clustering (18). A sialyltransferase that elaborates with 18 U.S.C. Section 1734 solely to indicate this fact. the terminal sequence NeuAc␣2-6Gal␤1-4GlcNAc on N-linked 1 This work was supported by National Institutes of Health Grants AI50143 (to J.C.P.) and O-linked glycans has been shown to block the binding of ga- and GM074128 (to D.G.) and by research grants from the Biotechnology and Bio- logical Sciences Research Council and the Wellcome Trust (to H.R.M. and A.D.). lectin-1 that induces T cell death by clustering of CD45 and re- Microarray analysis was performed by the Microarray Core of the Consortium for duction of its phosphatase activity (19). The same sequence has Functional (GM62116). A.D. is a Biotechnology and Biological Sciences been documented to be the glycan ligand for CD22 (Siglec-2), a Research Council Professorial Fellow. regulator of B cell signaling, that has been proposed to mediate 2 Current address: Nestle´Research Centre, Lausanne, Switzerland. adhesion of B cells to T cells (20–25). The expression of the en- 3 Current address: M-SCAN Mass Spectrometry Research and Training Centre, Sil- wood Park, Ascot SL5 7PZ, U.K. zyme responsible for the synthesis of the sequence, ST6Gal I, was 4 Address correspondence and reprint requests to Dr. James C. Paulson, Department noted to be down-regulated in a microarray analysis of Ag-acti- of Molecular Biology and Molecular and Experimental Medicine, The Scripps Re- vated CD8 cells, but the consequences on the structures of CD8 search Institute, 10550 North Torrey Pines Road, MEM-L71, La Jolla, CA 92037. cell glycans were not investigated (26). E-mail address: [email protected] Despite the importance of glycosylation in lymphocyte function, 5 Abbreviations used in this paper: PNA, peanut agglutinin; MS, mass spectrometry; SNA, Sambucus nigra agglutinin; GS, Griffonia simplicifolia; RMA, robust multiar- changes in glycosylation are largely studied through indirect ray analysis. means such as probing for the presence or absence of specific

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2432 GLYCOSYLATION CHANGES IN ACTIVATED LYMPHOCYTES glycan sequences with plant or animal lectins or - Briefly, cells were resuspended in PBS containing 5 mg/ml BSA and 2 specific Abs. Although this approach has provided biologically mM EDTA and incubated with anti-CD4, anti-CD8, or anti-CD19 mi- important information, it is limited to examining aspects of struc- crobeads for 15 min at 4°C for purification of CD4, CD8 T cells, or B cells, respectively. After washing, cells were resuspended in the same ture relevant to the specificity of the probes used, and does not buffer and applied to the column. Purity of cell preparations was always address the extent to which changes occur. Indeed, there have been Ն90%, as judged by flow cytometry. Purified cells were either washed few investigations of glycosylation changes in lymphocytes in with PBS twice and immediately frozen and stored at Ϫ80°C until use which direct analysis of glycan structure has been conducted. An or immediately used for flow cytometry analysis. exception is a rigorous analysis conducted nearly 20 years ago Flow cytometry investigating the O-linked glycans of human leukosialin before ϫ and after activation of human T cells (27). This study revealed that Purified CD4, CD8 T cells and B cells were incubated in aliquots of 5 105 cells in 100 ␮l of PBS containing 10 mg/ml BSA with or without the predominant disialylated “core 1” glycans of naive T cells GS-I-B4-FITC lectin from Griffonia simplicifolia (GS; EY Laboratories) at (NeuAc␣2-3Gal␤1-3(NeuAc␣2-6)GalNAc␣Thr/Ser) were re- a concentration of 20 ␮g/ml or Sambucus nigra agglutinin (SNA) lectin placed by larger branched “core 2” structures (NeuAc␣2-3Gal␤1- (Vector Laboratories) at a concentration of 40 ␮g/ml, for 30 min on ice. 3[NeuAc␣2-3Gal␤1-4GlcNAc␤1-6] GalNAc␣Thr/Ser) in acti- Flow cytometry data were acquired on viability-gated cells using a vated cells (27), now known to be the nonfucosylated precursor of FACSCalibur flow cytometer and analyzed with the CellQuest software system (BD Biosciences). the O-linked sialyl-Lewis X ligand of P-selectin (28). The change was concluded to result from altered expression of two glycosyl- Analysis of glycan transferase gene expression ␣ transferases that form the NeuAc 2-6GalNAc linkage and Glc- Analysis of gene expression was conducted using a custom gene mi- NAc␤1-6GalNAc linkages, enzymes that compete to form the al- croarray (GLYCOv1 chip) produced by Affymetrix for the Consortium Downloaded from ternative branch points in these structures, respectively. for Functional Glycomics (͗www.functionalglycomics.org͘), and con- In principle, such analysis can now be conducted by high taining probe sets for 122 glycosyl- and sulfotransferases (29). Three throughput methods for assessment of glycan structure and expres- independent experiments were performed for each activation condition of each lymphocyte type (CD4, CD8, and B lymphocytes), resulting in sion of the glycosyltransferase genes responsible for their synthe- triplicate RNA samples per condition. RNA was extracted using the sis (29). To this end, we have investigated changes in glycosyla- Qiagen RNeasy mini kit according to the manufacturer’s protocol. After tion of N-linked glycans of resting and activated murine T and B treatment with DNase I amplification grade (Invitrogen Life Technol- http://www.jimmunol.org/ lymphocytes using MALDI-TOF mass spectrometry (MS) profil- ogies), total RNA was purified with RNeasy columns using the cleanup protocol (Qiagen) and was quality checked with an Agilent Bioanalyzer ing for assessment of glycan structure, and a custom gene microar- (Agilent Technologies). For glycosyltransferase expression analysis, 5 ray based on Affymetrix technology, developed and annotated by ␮g of total RNA was amplified and biotin-labeled using the Bioarray the Consortium for Functional Glycomics (͗www.functionalgly High Yield RNA Transcript Labeling kit (Enzo Life Sciences) and then comics.org͘) for evaluating changes in expression of glycosyl- hybridized to the GLYCOv1 chip. One chip per biological replicate was transferase genes. A dramatic change was observed in the terminal run. Hybridization and scanning of the chip were performed according to the Affymetrix recommended protocols. glycosylation pattern of N-linked glycans of CD4 and CD8 T cells. The MAS 5.0 algorithm (Affymetrix) was used to determine present (P) and In freshly isolated cells, the predominant complex type N-glycans absent (A) absolute calls for each sample. All marginal (M) calls were inter- contained terminal sialic acid, in the NeuGc␣2-6Gal sequence that preted as absent. Genes were finally considered present if they had been as- by guest on September 29, 2021 forms the ligand of murine CD22 and is implicated in regulation of signed a present call in at least two of the three biological replicate samples (Group Call). Expression signals were then generated using the Robust Mul- galectin-mediated cell death. In contrast, the activated cells exhib- tiarrayAnalysisalgorithm(Ref.30and͗http://stat-www.berkeley.edu/ϳbolstad/ ited a dramatic decrease in sialylated glycans, and a corresponding RMAExpress/RMAExpress.html͘). Unsupervised hierarchical clustering by increase in glycans with the Gal␣1-3Gal terminal sequence. Gene sample was performed with BRB ArrayTools using the default settings for microarray analysis revealed that the changes in N-glycan struc- correlation and linkage (͗http://linus.nci.nih.gov/BRB-ArrayTools.html͘). tures in activated T cells corresponded to changes in the expression Statistical comparisons were made for each gene to determine whether the signals in the fresh and activated samples were statistically significant. of a sialyltransferase and galactosyltransferase responsible for the Accordingly, the robust multiarray analysis (RMA) signals of the triplicate synthesis of these terminal structures. The approach of using fresh samples of each lymphocyte type were compared with the signals of MALDI-TOF MS for profiling glycan structure coupled with gene the triplicate samples of each activation condition corresponding to that microarray analysis to survey glycosyltransferase expression may lymphocyte type. The CD4 and CD8 T cell experiments, which involved three different activation conditions, warranted the use of the Dunnett’s prove to be a powerful combination for investigating glycosylation test, a modified t test, which controls for the “experiment wise” or “per changes in differentiating or activated cells. experiment” error rate (31, 32). It does not produce a p value, rather the computed qЈ value is compared against a table of Dunnett’s critical values Materials and Methods determined for the appropriate ␣ value as follows: Preparation and culture of splenocytes ៮ A Ϫ ៮ x c xi qЈ ϭ t ϭ C57BL/6 male mice (6–10 wk old) were obtained from The Scripps Re- dunnett ͱ2MSE search Institute Custom Breeding core and used to prepare splenocytes, as n previously described (9). In this report, fresh cells refer to cells not sub- jected to cell culture, and resting and activated cells are those subjected to ͸͑ Ϫ ៮͒2 ͸ͩ xi x ͪ cell culture in the absence or presence of activating agents, respectively. ͸ 2 Ϫ 8 si n 1 For T cell activation experiments, splenocytes were plated (10 cells/plate) where: MSE ϭ ϭ (1) with immobilized anti-CD3 (BD Pharmingen) and cultured for 72 h in k k RPMI 1640 medium supplemented with either 4 ng/ml IL-2 and 5 ng/ml IL-12, or 4 ng/ml IL-2 and 10 ng/ml IL-4, or 4 ng/ml IL-2 (R&D Systems). k is the number of groups, and n is the sample size. ␣ ϭ For B cell activation, splenocytes were cultured for 72 h in RPMI 1640 A two-tailed Dunnett’s test was applied. The critical values for ϭ ϭ ␯ ϭ Ϫ ϭ Ϫ medium supplemented with 10 ␮g/ml anti-IgM (Jackson ImmunoResearch 0.05, k 4 groups, degrees of freedom (error) e (n k) (12 ϭ Laboratories) and 10 ng/ml IL-4. 4) 8, were: ␣ 0.05 qЈ ϭ tdunnett(2) ϭ qЈͩ ,ve,kͪ ϭ qЈͩ ,8,4ͪ ϭ 2.88 (2) Lymphocyte purification 2 2 CD4 and CD8 T cells and B cells were purified from either fresh, resting, For the B cell experiments, the two groups were fresh and activated, and a or activated splenocytes using the MidiMACS system (Miltenyi Biotec) Student t test, assuming the variances were equal, was applied (the result by positive selection, according to the manufacturer’s protocols. of an Fmax test for variance on all probe sets was that the variance was The Journal of Immunology 2433 equal for all but three cases, and in all three cases the variance was very ditions for T cells were chosen to determine whether early glyco- small, with the control sample variance effectively 0): sylation changes were influenced by cytokines that promote CD4ϩ ␣ 0.05 T cell polarization to Th1 (IL-2/IL-12) and Th2 (IL-2/IL-4) sub- tcritical(2) ϭ tͩ ,vͪ ϭ tͩ ,4ͪ ϭ 2.776 (3) 2 2 sets. Lymphocytes were purified by positive selection using either Characterization of B and T lymphocytes N-glycans anti-CD4-, anti-CD8-, or anti-CD19-coated magnetic microbeads for CD4 T cells, CD8 T cells, or B cells, respectively, yielding cell Approximately 25 million fresh and 15 million activated B and T lympho- cytes, respectively, were used for each analysis. Cell preparations were populations 90–98% pure (Fig. 1). The remaining cells were dis- homogenized, detergent extracted, reduced/carboxy methylated, dialyzed, carded. Activated cells are compared with fresh lymphocytes be- and digested with trypsin as previously described (33). N-glycans were cause lymphocytes cultured in the absence of cytokines were sub- released from each proteolyzed extract by peptide:N-glycanase (PNGase F; ject to extensive cell death. Three independent experiments were Roche) treatment and were subsequently permethylated using the sodium done for each activation condition for mRNA extraction and mi- hydroxide procedure, as described elsewhere (33). MALDI-TOF MS, elec- trospray MS/MS, and methylation analyses were then performed following croarray analysis, and at least two experiments for each activation previously published strategies (34). condition were subjected for evaluation of N-glycan structure by MALDI-TOF MS analysis. Results Strategy for analysis of changes in N-linked glycosylation Analysis of N-glycans from fresh and activated lymphocytes The overall strategy for analysis of changes in N-linked glycosyl- Portions of representative MALDI-TOF MS profiles showing the ation was to activate freshly isolated mixed splenocytes in vitro for mass region with complex N-glycans are shown in Fig. 2 for fresh 72 h, purify the cell type of interest, and either subject the purified and activated T and B lymphocytes. Striking differences are ob- Downloaded from cells to glycan analysis, or extract mRNA for analysis on the gly- served upon comparison of fresh and activated CD8 and CD4 T cogene microarray. Each preparation of lymphocytes involved cells as illustrated by the major peaks annotated in symbol format. pooling splenocytes from 10 to 30 mice, and subjecting them to The predominant peaks in the fresh cells are disialylated bianten- activation conditions selected for T cell activation (anti-CD3 with nary and monosialylated triantennary glycans. In contrast, the pre- IL-2/IL-12 or IL-2/IL-4) or B cell activation (anti-IgM with IL-2/ dominant peaks in the cells activated with IL-2/IL-12 are di- and IL-4) as described in Materials and Methods. The activation con- tribranched structures disubstituted with dihexose termini. Similar http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Purification of B cells and T cells from mixed splenocytes before and after activation. CD8 T cells, CD4 T cells, and B cells were purified from mixed splenocytes before or after activation using the MACS system with anti-CD8-, anti-CD4-, or anti-CD19-coated magnetic beads, respectively. Phenotypic analysis of purified fresh and activated cell populations was performed by flow cytometry and showed Ն90% purity of all cell preparations, as indicated. Purified cells were used either for RNA preparation for microarray analysis, or were processed to extract glycans for MALDI profiling and methylation analysis. 2434 GLYCOSYLATION CHANGES IN ACTIVATED LYMPHOCYTES

profiles were observed for CD8 and CD4 T cells activated with IL-2/IL-4 (data not shown). As an example of the dramatic change between fresh and activated cells, note that the predominant peak representing the disialylated biantennary glycan at mass 3027 in fresh CD8 and CD4 cells is largely absent in the profiles of the corresponding activated cells. In contrast, the predominant dihex- ose-terminated biantennary glycan at mass 2653 in activated CD8 and CD4 cells is a minor peak in the profiles from fresh cells. More detailed annotations of the MS spectra, using the cartoonist algo- rithm (35), are provided in Fig. S1 (supplemental data).6 Further characterization of the major species by MS/MS anal- ysis confirmed that the termini of the complex glycans were NeuGc-Hexose-HexNAc in the fresh cells and Hexose-Hexose- HexNAc in the activated cells (data not shown). Methylation anal- ysis revealed that there was a significant change in the substitution of galactose at the 6 and 3 positions for N-glycans of fresh and activated (IL-2/IL-12) CD8 T cells (Table I). Although the meth- ylation analysis comprises results from all glycans including high

mannose glycans, galactose occurs only as a penultimate or ter- Downloaded from minal residue, so the substitution pattern of the galactose residues provides direct information about the terminal substitutions on complex N-linked glycans. Substitution of galactose at the 6 po- sition went from 42 to 9% of the total galactose in fresh and ac- tivated cells, respectively, while substitution at the 3 position went

from 11 to 53%. Taken together with the MALDI-TOF data show- http://www.jimmunol.org/ ing loss of sialic acids and an increase in terminal hexose, the data are consistent with the predominant terminal sequences on fresh and activated CD8 T cells as sequences previously reported on N-linked glycans of murine lymphocytes, NeuGc␣2-6Gal␤1-4Gl- cNAc (36, 37), and Gal␣1-3Gal␤1-4GlcNAc (38), respectively. In marked contrast to the dramatic changes in the major com- plex type N-glycans of fresh and activated T cells, there was little difference in the major N-linked glycans of fresh and activated B cells. In both fresh and activated B cells, the predominant struc- by guest on September 29, 2021 tures were biantennary capped with one or two NeuGc residues.

Altered expression of terminal Gal␣1-3Gal and NeuGc␣2-6Gal linkages in activated T cells To confirm that the Gal␣1-3Gal sequence did indeed increase fol- lowing activation of CD8 and CD4 T cells, FACS analysis was

performed using the GS-I-B4 lectin from G. simplicifolia, which exhibits high specificity for this sequence. As shown in Fig. 3,

there was a 10- to 30-fold increase in binding of the GS-I-B4 lectin in activated T cells relative to resting T cells. This observation is consistent with the dramatic increase in the Gal␣1-3Gal sequence detected in analysis of the N-linked glycans (Fig. 2 and Table I). Conversely, there was an activation-dependent decrease in the Sia␣2-6Gal sequence in CD8 cells, as detected by the SNA lectin specific for this sequence. Although a significant change in SNA binding was not detected in CD4 cells, it is possible that the Sia␣2- 6Gal sequence is also present on glycoproteins with O-linked gly- FIGURE 2. MALDI-MS profiles of N-glycans of fresh and activated cans, and/or glycolipids, which are turning over slower than gly- T and B cells. Shown are comparisons of MALDI-MS profiles of N- coproteins that comprise the majority of N-linked glycans profiled linked glycans from fresh (A, C, and E) and activated (B, D, and F)T in Fig. 2. No change in binding of either the GS-I-B or SNA and B cells. CD8 T cells (A and B) and, CD4 T cells (C and D), were 4 lectins to activated B cells relative to resting B cells was observed. activated with anti-CD3 and IL-2/IL-12, B cells (E and F) were acti- vated with anti-IgM and IL-4. Major peaks representing complex type Taken together, these phenotypic results are consistent with the ␣ N-glycans of either the fresh or activated cells are annotated with struc- increase in the Gal 1-3Gal linkage and a decrease in the tures in symbol form. Dramatic changes are evident in the profiles of NeuGc␣2-6Gal sequences detected on activated T cells by direct both the activated T cell populations, while little change is evident analysis of N-linked glycans. following activation of B cells. More detailed annotations are given in supplemental data (Fig. S1).

6 The online version of this article contains supplemental material. The Journal of Immunology 2435

Table I. Methylation analysis of N-linked glycans of fresh and activated (IL-2/IL-12) CD8 T cells

Relative Abundance

Elution Time (Min) Characteristic Fragment Ions Assignment Fresh IL-12

18.73 102, 118, 129, 145, 161, 162, 205 Terminal galactose 0.09 0.12 19.92 101, 118, 129, 234 3-Galactose 0.02 0.17 20.43 118, 129, 145, 189, 233 6-Galactose 0.08 0.03 18.47 102, 118, 129, 145, 161, 162, 205 Terminal mannose 0.77 0.55 19.62 129, 130, 161, 190, 234 2-Mannose 1.62 0.42 20.02 118, 129, 145, 189, 233 6-Mannose 0.04 0.02 20.81 129, 130, 190, 233 2,4-Mannose 0.02 0.03 21.19 129, 130, 189, 190 2,6-Mannose 0.01 0.04 21.36 118, 129, 189, 234 3,6-Mannose 0.27 0.42 16.95 102, 118, 131, 175 Terminal fucose 0.07 0.06 22.28 117, 143, 145, 159, 203, 205 Terminal GlcNAc 0.03 0.02 23.18 117, 159, 233 4-GlcNAc 1.00 1.00 24.02 117, 159, 346 3,4-GlcNAc Ͻ0.01 0.01 24.48 117, 159, 261 4,6-GlcNAc 0.05 0.09 Downloaded from

Microarray analysis of the expression of cytokine and glycan fold, 11 could be considered not meaningful (e.g., statistically sig- transferase genes nificant differences between samples with all absent calls). How- Ͼ For gene expression analysis, mRNAs were extracted from puri- ever, for the 29 with differences 1.5-fold, only one would be fied cell populations of fresh and activated lymphocytes and sub- considered not meaningful. Of these, only 13 of the genes code for enzymes that are potentially relevant to the biosynthesis of N- jected to analysis on a custom Affymetrix-based DNA microarray http://www.jimmunol.org/ containing murine and human glycosyltransferase, sulfotrans- linked glycans, and the remainder are primarily relevant to the ferase, and cytokine genes, made available by the Consortium for synthesis of other glycan classes including 5 for O-linked glycans, Functional Glycomics. Three independent RNA preparations were 4 for proteoglycans, 2 for glycolipids, 2 for glycophosphatidyl- made for each experimental condition comprising fresh and acti- inositol anchors, and 2 with undefined activity. vated CD4 and CD8 T cells (anti-CD3 with IL-2, IL2/IL-12, or Of the two genes coding for enzymes involved in terminal gly- IL-2/IL-4) and B cells (anti-IgM with IL-2/IL-4) for a total of 30 cosylation of O-linked glycans (Table II), one is ST3Gal I, which separate RNA preparations. Labeled samples were prepared for each RNA and then hybridized to microarrays yielding three sets of data for fresh cells and each activation condition. Present and by guest on September 29, 2021 absent calls were assigned by the MAS 5.0 algorithm (Affymetrix), and a gene was deemed present if at least two of the three triplicate samples gave a present call (present call). Expression signals were generated using the RMA algorithm expressed in log 2 (30), and differences between expression in the fresh and activated cells were assessed for statistical significance and converted to “fold differences” for presentation. The GLYCOv1 chip also contained cytokine genes which al- lowed further confirmation of the programmed activation resulting from cytokines present during activation. The changes observed were consistent with the early (72 h) programmed changes in gene expression following in vitro activation of CD4 cells with IL-12 or IL-4 (39, 40). In particular, increased expression of the IL-4 and IL-13 genes was observed only in CD4 cells cultured with IL-4, consistent with a CD4 Th2 response. In contrast, expression of these genes was not detected in CD4 cells cultured with IL-12, but expression of the IFN-␥ gene is highest in these cells, consistent with the early stages of conversion to the Th1 subtype. Samples were compared for expression of 122 glycosyltrans- ferase and sulfotransferase genes by unsupervised hierarchical cluster analysis as shown in Fig. 4. Expressions of genes in fresh FIGURE 3. FACS analysis of cell surface Gal␣1-3Gal (A) and Sia␣2- B and activated B cells and fresh CD4 and CD8 T cells are more 6Gal (B) sequence in resting and activated lymphocytes. Splenocytes iso- closely related to each other than activated CD4 and CD8 T cells, lated from C57BL/6 mice were cultured with (thick line) or without (thin which cluster separately. Of the 122 glycan transferase genes eval- line) activator as described in Materials and Methods and the medium was supplemented with IL-2/IL-12 for the preparation of activated T cells. Af- uated, 52 genes exhibited one or more statistically significant dif- ter 72 h in culture, resting or activated splenocytes were harvested and ferences between activated cells and the corresponding fresh cells CD4, CD8 T cells, and B cells were purified as described in Materials and (see Materials and Methods for statistical analysis and Table S1 Methods. Purified lymphocyte cell populations were evaluated for expres- available as supplemental data for a complete listing of results for sion of the terminal Gal␣1-3Gal and Sia␣2-6Gal sequences by FACS anal- all genes). Of these, 23 showed differences of Ͻ1.5-fold, and 29 ysis using GS-I-B 4 and SNA lectins, respectively, as described in Mate- showed differences Ͼ1.5-fold. Of the 23 with differences Ͻ1.5- rials and Methods. 2436 GLYCOSYLATION CHANGES IN ACTIVATED LYMPHOCYTES

noted by Kaufmann et al. (41) to be induced rapidly following activation of T cells and diminishing within 24 h.

Glycan transferase changes relevant to N-linked glycans Of the results summarized in Table II, 13 genes relevant to the synthesis of N-linked glycans exhibited significant changes in gene expression. Seven of these are involved in the synthesis of ele- ments of N-linked glycan structure common to all cells because they either 1) participate in the synthesis of the high mannose lipid-linked intermediate (mannosyltransferases), 2) are subunits of oligosaccharyltransferase that transfers the glycan to asparagine (DAD1, ribophorin I and II), or 3) form the ubiquitous Gal␤1– 4GlcNAc sequence that extends the branches of most complex type N-linked glycans (b4GalT1). Consistent with the constitutive FIGURE 4. Unsupervised hierarchical clustering analysis of glycosyl- expression of N-linked glycans in all cells, these genes are ex- transferases expression in fresh and activated lymphocytes. The dendro- pressed in all resting and activated cell populations. The six genes gram has been constructed using centered correlation and average linkage. involved in the synthesis and transfer of the lipid-linked interme- The three biological replicates are shown for each sample, fresh (F) and

diate in general showed increased expression in activated T cells, Downloaded from differently activated (aIgM ϭ anti-IgM; 2 ϭ IL-2; 4 ϭ IL-4; 12 ϭ IL-12) with 25 of 42 comparisons showing significant 1.5- to 2.9-fold as explained in Materials and Methods. increases in signal, consistent with the increased need for N-linked glycans in proliferating cells. forms the O-linked glycan sequence NeuAc(Gc)␣2-3Gal␤1-3Gal- The remaining six genes are involved in the synthesis of termi- NAc and regulates the expression of PNA receptors in developing nal sequences in N-linked (and O-linked) glycans. In contrast to thymocytes (1, 2) and in resting and activated peripheral CD8 T the genes involved in the synthesis of the core N-glycan sequences, http://www.jimmunol.org/ cells (9) and CD4 T cells (10). Although there is only a decrease these genes were differentially regulated, showing both positive of 1.3- to 1.5-fold in the signal between fresh and activated T cells and negative changes in expression (Table II and Fig. 5). Of these, (and B cells), it is statistically significant, and appears to be suf- the ␤-galactoside ␣2-6 sialyltransferase (ST6Gal I) and ␤-galac- ficient to cause hyposialylation of O-glycans resulting in the PNA- toside ␣1-3 galactosyltransferase (␣1-3GalT) are responsible for high phenotype resulting from the increased expression of the the synthesis of the terminal sequences NeuGc␣2-6Gal␤1-4Glc- Gal␤1-3GalNAc ligand recognized by PNA (9, 10). The increase NAc (42) and Gal␣1-3Gal␤1-4GlcNAc (43), respectively, that ac- in the other enzyme, ST6GalNAc IV transferase, was previously count for the major changes in terminal glycosylation of N-linked by guest on September 29, 2021 Table II. Expression fold changes in glycosyltransferase expression in resting and activated lymphocytesa

CD4 Activation Condition CD8 Activation Condition

CD4 Present IL-2- IL-2- CD8 Present IL-2- IL-2- B Cell Present B Cell Common Calls (Fresh: IL-2 IL-12 IL-4Calls (Fresh: IL-2 IL-12 IL-4 Cells Fresh: Anti-IgM Fold GenBank Subcategory Name Activated) Fold change (Active-Fresh)Activated) Fold change (Active-Fresh) Activated Change Number

N-linked glycan core synthesis N-glycan-transferase DAD1b P:PPP 1.612 1.533 1.579 P:PPP 1.057 Ϫ1.078 1.073 P:P 1.083 U83628 N-glycan-transferase Ribophorin I P:PPP 1.391 1.359 1.549 P:PPP 1.370 1.469 1.395 P:P Ϫ1.115 BC016080 N-glycan-transferase Ribophorin II P:PPP 1.659 1.578 1.803 P:PPP 1.400 1.279 1.294 P:P 1.025 NM_019642 Mannosyltransferase ALG13 P:PPP 1.572 1.350 1.890 P:PPP 1.547 1.533 2.027 P:P 1.356 AA215144 Mannosyltransferase DPM1 P:PPP 2.663 2.407 2.894 P:PPP 2.056 1.878 1.763 P:P 1.052 AB004789 Mannosyltransferase DPM2 P:PPP 2.191 2.126 2.231 P:PPP 1.851 1.963 2.089 P:P 1.334 NM_010073 Galactosyltransferase b4GalT1 P:PPP 1.001 Ϫ1.019 Ϫ1.122 P:PPP 1.344 ؊1.458 ؊1.399 P:P 1.686 J03880 Terminal Glycosyltransferases -N- and O-linked glycans Galactosyltransferase ␣1,3GalT P:PPP 1.920 2.067 2.085 P:PPP 2.079 2.063 1.859 P:P ؊2.765 AF297615 Sialyltransferase ST6Gal I-Long t P:PPP ؊3.061 ؊3.622 ؊3.820 P:AAA ؊5.939 ؊5.689 ؊5.742 P:P 1.293 BB768706 Sialyltransferase ST3Gal IV A:PPP 1.468 1.656 1.742 A:PPP 1.688 1.432 1.594 A:A Ϫ1.027 BC011121 Sialyltransferase ST3Gal VI P:AAA ؊1.549 ؊1.593 ؊1.789 P:AAA ؊3.067 ؊2.884 ؊3.251 P:P Ϫ2.564 NM_018784 Sialyltransferase ST8Sia IV P:PPP ؊4.597 ؊3.685 ؊4.902 P:PPA ؊6.684 ؊5.615 ؊7.057 P:P Ϫ3.135 X86000 Sulfotransferase GlcNAc6ST-1 A:PPP 1.544 2.075 1.687 A:APA 1.499 1.742 1.460 A:A 1.042 AB011451 Terminal glycosyltransferases - O-linked glycans Sialyltransferase ST3Gal I P:PPP ؊1.338 ؊1.298 ؊1.336 P:PPP ؊1.605 Ϫ1.381 Ϫ1.585 P:P Ϫ1.486 X73523 Sialyltransferase ST6GalNAc IV P:PPP 1.594 1.765 1.857 P:PPP 1.370 1.739 1.612 P:P 1.268 Y15779

a Mixed splenocytes pooled from 10–30 mice were activated using either anti-CD3 (for T cells) or anti-IgM (for B cells) in the presence of cytokines (IL-2, IL-2/IL-12, or IL-2/IL-4) as described in Materials and Methods. Following purification of the desired cell type, RNA was purified and gene expression was analyzed on a single GLYCOv1 microarray. Each experiment was done in triplicate, and the data for all three experiments were analyzed together to assess statistically significant changes in gene expression. Genes were scored as being present (detected) or absent (undetected) using the Affymetrix MAS 5.0 algorithm, and are either listed in the table as present (P) or absent (A) if they received that score in at least two of the three experiments. These are summarized in the present call column for fresh cells followed by the corresponding call for each of the related activation conditions (e.g. P:AAA indicates the gene was present in fresh cells and absent in each of the three activation conditions). The expression values (levels) were calculated using the Robust Multi-array Averaging (RMA) algorithm generated by the software package RMA express, which computes a log-2 expression value for each gene (Materials and Methods). Using the RMA value calculated for fresh cells as a reference, the change in RMA value was determined for each activation condition, and is expressed as a “fold change‘, with positive values representing an increase, and negative values representing a decrease. Values for “absent” calls are in italics and values for “present” are in normal font. Values in bold indicate statistically significant differences from the values in the corresponding fresh cells. A complete list of results for all 122 glycosyltransferase and sulfotransferase genes is provided in supplementary material (table 1S). b Defender against cell death 1. The Journal of Immunology 2437

decrease in RMA signal, the opposite from T cells. These results are consistent with the observed retention of N-linked glycans with the terminal NeuGc␣2-6Gal sequence in both resting and activated B cells (Fig. 2). The changes in expression of the other four genes do not appear relevant to the major changes seen in the N-glycan MALDI-TOF MS profiles documented in Fig. 2. The ST8Sia IV enzyme exhibits a dramatic decrease following activation of all three lymphocyte types. ST8Sia IV is a polysialyltransferase that transfers one or ␣ ␣ more sialic acid residues in the [NeuAc(Gc) 2-8]nNeuAc(Gc) 2- 3Gal sequence on N-linked glycans of a limited number of glyco- proteins including neural cell adhesion molecule (45). To date, such sialylated structures have not been reported on lymphocytes, FIGURE 5. Changes in the expression of glycosyltransferases that form and while they may be present in small amounts, they do not com- terminal sequences in N-linked glycans following activation of lympho- prise an observable component of the N-glycans analyzed in this cytes. Shown are results for genes of terminal glycosyltransferase acting on report. Two enzymes that synthesize the sequence NeuAc(Gc)␣2- N-linked (or N- and O-linked) glycans that exhibited at least a 2-fold 3Gal␤1-4GlcNAc on N-linked and O-linked glycans, ST3Gal IV change in expression levels in fresh and activated CD8 T cells with IL-2/ and ST3Gal VI (46–48), were differentially expressed in T cells.

IL-12. Each enzyme is identified by its common name and the symbol ST3Gal IV went from absent to present, and ST3Gal VI went from Downloaded from structure of the product formed as terminal sequences found on N- and present to absent (Table II). The increase in expression of ST3Gal O-linked glycans, with the sugar transferred by the enzyme identified by an IV has been previously reported in activated CD4 cells following ء asterisk ( ). Each enzyme exhibits a unique pattern of expression relative 6–10 days of activation (11) and is believed to play a critical role to the others with respect to positive or negative regulation upon activation, in the formation of the sialyl-Lewis X linkage on O-linked glycans and differential expression in T and B cells. Comparisons with other en- zymes and activation conditions are summarized in Table II. that serve as ligands for selectins (11, 49). However, the dramatic

reduction in sialylated N-glycans in activated CD4 and CD8 T http://www.jimmunol.org/ cells (Fig. 2) indicates that ST3Gal IV is not a major contributor to glycans observed following activation of CD8 and CD4 T cells. terminal glycosylation of N-glycans. Although a decrease in the The 5- to 6-fold decrease in ST6Gal I, is consistent with the loss expression of the ST3Gal VI enzyme is consistent with loss of of the NeuGc␣2-6Gal sequence observed as the major structure in sialylated glycans (Fig. 2), it does not account for the net loss in fresh T cells. The decrease in ST6Gal I expression was also ob- 6-substituted galactose and increase in 3-substituted galactose. Fi- served by quantitative RT-PCR, which showed a 6- to 10-fold nally, the GlcNAc6ST-1 is a sulfotransferase that forms the Gal␤1- reduction (data not shown). Conversely, there was a 2-fold in- 4(6S)GlcNAc sequence. Although sulfated structures are not cap- crease in the ␣1-3GalT, consistent with the formation of the tured in the MALDI-TOF profiling protocols used for analysis of Gal␣1-3Gal terminal sequence observed as the predominant struc- N-glycans in this report and could have been missed, the product of by guest on September 29, 2021 ture in activated cells. In preliminary experiments conducted with this enzyme has been shown to be expressed primarily on high en- a spotted cDNA microarray (44), changes in the expression of dothelial venules of lymph nodes and not on lymphocytes (50, 51) ST6Gal I were evident within 24 h and increased at 48 and 72 h, while the increase in ␣1-3GalT was evident by 48 h (data not shown). As diagrammed in Fig. 6, these two enzymes compete for Discussion a common substrate, Gal␤1-4GlcNAc, on growing N-linked gly- Comparison of the N-glycan structures following activation of T can chains. Thus, the effects of the decrease in expression of and B lymphocytes has revealed a heretofore unrecognized change ST6Gal I and increase in expression of ␣1-3GalT are amplified in terminal glycosylation of N-linked glycans of activated murine through their competition in the biosynthetic pathway. T lymphocytes involving the loss of charged sialylated glycans Conversely, in B cells, the ST6Gal I gene showed slightly in- terminated with the NeuGc␣2-6Gal sequence, and an increase in creased expression (2), and the ␣1-3GalT gene showed a 3-fold uncharged glycans terminated with the Gal␣1-3Gal sequence. This

FIGURE 6. ST6Gal I and ␣1-3 GalT compete for a common substrate in terminal glycosylation of N-linked glycans. The ␤-galactoside ␣2-6 sialyltransferase (ST6Gal I) and ␤-galactoside ␣1-3 galactosyltransferase (␣1-3GalT) use the same acceptor substrate sequence on growing N-linked glycan chains, resulting in the forma- tion of a terminal Sia␣2-6Gal␤1-4GlcNAc and Gal␣1- 3Gal␤1-4GlcNAc sequences, respectively. The two en- zymes effectively compete for the same substrate because neither can use the product of the other as a substrate. Changing the relative amounts of these two enzymes should, in principle, result in a corresponding change in the terminal sequences they produce on com- plex type N-glycans. 2438 GLYCOSYLATION CHANGES IN ACTIVATED LYMPHOCYTES change corresponds to changes in expression of the two glycosyl- Gal␣1-3Gal sequence is not expressed. It will be of interest to transferases responsible for their synthesis, a decreased expression assess the glycosylation changes in human lymphocytes to see of the sialyltransferase ST6Gal I, and increased expression of the whether the same loss of sialylated glycans occurs, and if so, what galactosyltransferase ␣1-3GalT, respectively. In contrast to the if any sequence replaces the Gal␣1-3Gal sequence in murine cells. change observed in T cells, B cells maintain their sialylated N- With the increasing attention of the roles of glycan-binding pro- linked glycans following activation, consistent with increased ex- teins in immune function, the paucity of information on glycan pression of ST6Gal I and decreased expression of ␣1-3GalT. structures on leukocytes has become increasingly evident, and is a Reports from other laboratories corroborate aspects of these limiting factor in elucidating the biology they mediate (60–64). findings for regulation of the ST6Gal I gene in activated lympho- The approach used in this report has general use for detecting cytes in vivo. In particular, using an in vivo transgenic lympho- changes in glycosylation of closely related leukocyte cell popula- cytic choriomeningitis virus model for Ag activation of CD8 cells, tions (e.g., pre-/postdifferentiation or activation, immature/mature, Kaech et al. (26) found ST6Gal I to be one of the genes that was etc.) and establishing the biosynthetic basis for those changes. down-regulated in CD8 effector cells 8 days after transfection. With present technology, the MALDI-TOF MS and supporting Moreover, effector cells exhibited reduced binding of the SNA that MS/MS and methylation analysis experiments provide meaningful recognizes the Sia␣2-6Gal sequence (Ref. 52, and L. Baum, T. data on the most abundant glycans. However, some subsets of Onami, and R. Ahmed, unpublished observations). This study re- glycans, for example sulfated structures, would be missed using capitulates in vivo the results obtained with CD8 cells in this re- protocols described in this report. Perhaps even more important, port. Although only modest reduction of SNA binding was ob- literally hundreds of minor glycans can be detected by MS anal- served comparing native and activated CD4 cells in this study, this ysis, but the subsequent annotation and verification of structure is Downloaded from may be due to the fact that the residual sialylated glycans at the end prohibitive. However, progress for developing automatic methods of 72 h are sufficient to support significant lectin binding to other of annotation with sophisticated algorithms is being made (65– glycan classes (e.g., glycolipids). In murine B cells, increased ex- 67), and technology advances in sample preparation and analysis pression of the ST6Gal I gene has been observed, mediated by a B will rapidly evolve. cell-specific promoter (53). Although peak expression occurs 10 Combining glycan structure analysis and global expression anal-

days after activation, significant increases in expression were ob- ysis using oligonucleotide microarrays may provide increasingly http://www.jimmunol.org/ served within 72 h as observed in this report. (Probe sets for one rich information about the regulation of glycosylation of pure cell of two alternatively spliced forms of ST6Gal I are shown in Table populations responding to environmental signals such as those II. The probe set corresponding to the “long form” is derived from studied here. It is estimated that there may be a total of ϳ250 the 3Ј end of a 4.3-kb mRNA, while the probe set for the “short glycan transferase genes in the murine (and human) genomes. Al- form” is at the 3Ј end of a ϳ2-kb mRNA resulting from an alter- though the microarray used for this study (GLYCOv1 chip) con- native polyA addition site 2 kb from the 3Ј end of the 4.3-kb tained 122 unique murine glycosyltransferase genes, an updated transcript. Although 4.3-kb message is the predominant mRNA for version, GLYCOv3, contains ϳ200 murine glycan transferase this gene in most human tissues (48), significant up-regulation of genes, providing more complete coverage of the biosynthetic path- the “short form” was seen in B cells (see supplementary material)). ways. An inherent problem with microarray data is that some by guest on September 29, 2021 The loss of the NeuGc␣2-6Gal sequence has functional impli- probe sets “do not work”. Two genes in particular, FucT-VII and cations relevant to the interaction of T cells and B cells with gly- core 2 GlcNAc transferase, known to be involved in Sialyl-Lewis can-binding proteins. The B cell membrane marker CD22 is well- X selectin ligand formation on O-linked glycans of CD4 Th1 T known as a regulator of BCR signaling, and is a member of the cells (11–16, 68), were not detected (see supplemental data). How- Siglec family (Siglec-2) with an extracellular N-terminal Ig do- ever, our recent experience is that redesign of the probe sets can main that recognizes the NeuGc␣2-6Gal sequence as a preferred largely remedy this problem and should lead to increasingly pow- ligand on murine cells (54, 55). CD22 interacts with erful data sets. Even with a complete set of genes, changes de- ligands on the same cell, in cis, and on adjacent cells, in trans. tected in gene expression may not be reflected in major changes in Interaction of CD22 with B cell ligands in cis has been suggested glycan structure (e.g., changes in ST3Gal VI and ST8Sia IV in this to modulate the activity of CD22 as a negative regulator of B cell report), because not all enzymes will be rate limiting, and some signaling (23, 25, 56, 57). However, CD22 was initially described may be expressed at levels below the threshold needed to compete as a T cell adhesion protein and has been demonstrated to interact with other enzymes in the pathway. However, recent efforts to with trans ligands on adjacent cells (23, 58, 59). Thus, differential develop algorithms that take global microarray data to predict gly- regulation of the NeuGc␣2-6Gal sequence may modulate CD22 can structure show promise (69), and could evolve, through infor- function through its complex interaction with cis and trans ligands, mation gleaned from data sets of expression data from cells with particularly in B-T cell interactions (e.g., B cell Ag presentation to known glycan profiles, to recognize significant from insignificant T cells). The ST6Gal I transferase expression has also been pro- changes in gene expression. Current efforts to combined advances posed to modulate binding of galectin-1 to CD45 on T cells lead- in glycan structure analysis and technologies for functional gene ing to cell death (19). In this case, sialylation of CD45 glycans expression analysis are likely to converge to provide a systems blocks the terminal Gal␤1–4GlcNAc-binding sites used as a li- biology approach to analysis of glycan structure and function that gand by galectin-1. Thus, reduced ST6Gal I expression in acti- will facilitate future investigations into roles of glycan-binding vated T cells could lead to increased binding of galectin-1. In this proteins in immune cell function (70). regard, following activation of CD4 and CD8 T cells, we observed significant enhancement of the binding of Erythrina cristagalli ag- glutinin, which binds the Gal␤1–4GlcNAc sequence (data not Acknowledgments shown). We thank Sarah Lustig for technical help and Anna Tran-Crie for assis- tance with preparation of the manuscript. Concomitant with a loss of the NeuGc␣2-6Gal sequence, N- linked glycans of activated T cells acquire the Gal␣1-3Gal se- quence, the product of the galactosyltransferase, ␣1-3GalT. In hu- Disclosures mans, this enzyme is catalytically inactive, and consequently the The authors have no financial conflict of interest. The Journal of Immunology 2439

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