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Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0625
Received 15 September 2006/ Returned for modification 8 October 2006/ Accepted 4 November 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Current concepts of CD8+ T-cell apoptosis include the extrinsic or death receptor-mediated pathway and the intrinsic or mitochondrial death pathway (57). The extrinsic pathway is thought to depend on extracellular receptor-ligand binding to directly activate a death signal, which has been described as mediated by members of the tumor necrosis factor receptor family. Some studies have implicated a role for Fas and tumor necrosis factor signaling (28). These ligand-receptor systems have also been found to induce nonapoptotic signals, such as costimulation, depending on culture conditions (54). Importantly, mice deficient in these receptors and their ligands exhibit normal CD8+ T-cell apoptotic contraction following immune stimulation in vivo (21, 30, 39, 45, 49, 50).
In contrast, the intrinsic pathway of CD8+ T-cell apoptosis is thought to depend upon signals arising in a cell-autonomous manner which tip the balance of intracellular pro- and antiapoptotic proteins that include the Bcl-2 family, which regulate mitochondrial membrane permeability (17, 33). Indeed, T cells in mice overexpressing Bcl-2 are more resistant to apoptotic death and display prolonged survival in vivo in response to immunization with the superantigen Staphylococcus aureus enterotoxin B (SEB) (21, 56). The proapoptotic Bcl-2 family member Bim also appears to be a key intracellular factor regulating the death of mature T cells. Interestingly, in contrast to Bcl-2 overexpression, Bim-deficient mice accumulate peripheral T and B lymphocytes in vivo, and their thymocytes are resistant to apoptotic stimuli in vitro (5). Peripheral CD4+ and CD8+ T cells lacking Bim display prolonged survival in response to SEB immunization as well as following infection with herpes simplex virus (21, 45).
Mediators of CD8+ T-cell apoptosis include, but are not limited to, perforin, gamma interferon, granzyme B, and reactive oxygen species. Nevertheless, perforin-deficient mice exhibit normal contraction of CD8+ T cells in response to an attenuated strain of Listeria monocytogenes, while chronic lymphocytic choriomeningitis virus (LCMV) infection in these mice leads to accumulation of virus-specific CD8+ T cells (2, 34, 63). Gamma interferon-deficient mice display delayed T-cell contraction after bacterial and viral infection, which could be due to hyperproliferation with failure to effectively clear pathogen (2, 4, 31, 44). Gamma interferon-dependent inflammation early in the immune response does appear to regulate the initial expansion phase that makes CD8+ T-cell contraction possible (3). Apoptosis may also be mediated by reactive oxygen species, as reduction using the superoxide dismutase mimetic (MnTBAP) protected T cells from death in vitro (20). In addition, the role of serine protease inhibitor 6 has been recently described in protecting against CD8+ T-cell death caused by granzyme B-mediated breakdown of intracellular cytotoxic granules (62).
It is evident that the apoptotic death of postactivated CD8+ T cells can be modulated by multiple factors, although the potential remains for the existence of an apoptotic signaling network that is enabled and perhaps activated by one type or a few types of cell surface molecules. In this case, the attributes of such a cell surface molecule or modification might include its induction or activation at the cell surface following immune stimulation and perhaps at later stages of activation. A second expectation would be that the function of such a molecule among naive CD8+ T cells should induce caspase-dependent apoptotic signals that result in a decrease in peripheral CD8+ T-cell homeostasis in the absence of immune stimulation.
A candidate cell surface alteration that fits this profile of an apoptotic regulator operating in post-immune CD8+ T-cell contraction has been identified as a change in protein O glycosylation involving the ST3Gal-I sialyltransferase (48). A significant reduction in the sialic acid linkage on core 1 O-glycans indicative of ST3Gal-I deficiency marks activated and effector CD8+ T cells that are destined either for apoptosis or differentiation into memory CD8+ T cells, the latter of which appear with increased levels of sialylated core 1 O-glycans due to ST3Gal-I activity (14, 48). The unsialylated core 1 O-glycan structure is detected by the peanut agglutinin (PNA) lectin, and ST3Gal-I deficiency is also permissive for an increase of 1B11 antibody-reactive core 2 O-glycans (Fig. 1A). When this postactivated cell surface O glycotype is produced on naive CD8+ T cells, apoptosis occurs by caspase-dependent mechanisms that vastly reduce the pool of peripheral naive CD8+ T cells (48).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/; strain B6.129-Bcl2l11tm1.1Ast/J) (5), and RAG-1 null (Rag/; strain B6.129S7-Rag1tm1Mom/J) (35) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). GM2/GD2 synthase null (Galgt1/) mice have been described previously (29) and were provided by the Consortium for Functional Glycomics, grant number GM2116. CD43 null (CD43/) mice have been described previously (7, 32) and were kindly provided by H. Ziltener (Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada). Mice lacking core 2 GlcNAcT-1 (C2GNT1/) and ST3Gal-I (ST3Gal-I /) have also been previously reported (12, 48). Animals were used between 8 to 12 weeks of age and in compliance with standards and procedures approved by the UCSD Institutional Animal Care and Use Committee.
Cell preparation.
Single-cell suspensions were prepared from isolated lymphoid tissues in phosphate-buffered saline (PBS) containing 2% heat-inactivated fetal bovine serum (FBS) after red blood cells lysis with ammonium chloride solution (BD PharM Lyse; BD Biosciences, San Jose, CA). For enrichment of CD8+ T cells, negative depletion using magnetic beads was performed. Briefly, mixed cell suspensions from lymph nodes (pooled from axillary, brachial, cervical, inguinal, and mesenteric lymph nodes) and spleen were incubated with biotinylated antibodies to CD4, B220, Mac-1, NK1.1, and Gr-1 (BD Biosciences) and washed with PBS, and labeled cells were depleted with Dynabeads M-280 streptavidin (Invitrogen Co., Carlsbad, CA) according to the manufacturer's recommendations. Resulting cell preparations had a purity of 
90% CD8+ T cells.
Antibodies, lectins, and flow cytometry.
Cells were labeled for flow cytometry using annexin V-allophycocyanin (APC) according to the manufacturer's recommendations (Caltag Laboratories, Burlingame, CA) and in combination with 7-amino-actinomycin D (7-AAD), phycoerythrin (PE)-1B11, PE-CD25 (3C7), PE-CD122 (5H4), PE-CD127 (SB/199), PE- or fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (RM4-5), PE- or APC-conjugated anti-CD8
(53-6.7) (BD Biosciences), or PNA-FITC (Vector Laboratories, Burlingame, CA). Mouse Vß T-cell receptor (TCR) repertoire analysis was performed with FITC-conjugated antibodies to TCR Vß 2, 3, 4, 5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a (BD Biosciences). All antibody incubations for flow cytometry were performed on ice for 10 min. In some experiments, cell surface analyses were supplemented by intracellular labeling with PE-conjugated antibodies to either human Bcl-2 (6C8) or Armenian hamster immunoglobulin G isotype control (Ha4/8) using Cytofix/Cytoperm buffer (BD Biosciences) to permeabilize and fix the cells. Data were acquired with a FACSCalibur flow cytometer and analyzed with CellQuest software (BD Biosciences). Polyclonal rabbit anti-Bim antibody (BD Biosciences) was used in Western blotting of total thymocyte lysates.
PCR. ST3Gal-ITg CD8+ T cells from the lymph node and spleen (purified by negative depletion) were cultured at 37°C with 5% CO2, and aliquots were removed at 0, 24, 48, and 72 h postactivation (for 24 h) with immobilized anti-CD3 (145-2C11, 1 µg/ml; BD Biosciences) in RPMI 1640 media containing 10% heat-inactivated FBS, 1x penicillin-streptomycin-L-glutamine, and 2-mercaptoethanol (Invitrogen). Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase I (DNA-free; Ambion, Austin, TX) to remove any genomic DNA contamination. Reverse transcription was performed using 100 ng total RNA with 40 U Moloney murine leukemia virus (M-MLV) reverse transcriptase containing either oligo(dT)15 or random hexamer primers (Promega, Madison, WI) in a final volume of 20 µl. Control reactions lacking reverse transcriptase were performed in parallel. Resulting cDNA from reverse transcription (1 µl) was amplified by PCR using primers specific for the ST3Gal-I transgene or 18s rRNA (QuantumRNA 18s internal standards; Ambion). PCR products were separated on 2% agarose gels, stained with ethidium bromide, and quantified by densitometry.
In vitro T-cell activation. For in vitro apoptosis analysis, cells from lymph nodes or spleen were prepared as described, resuspended in RPMI 1640 media containing 10% FBS, 1x penicillin-streptomycin-L-glutamine, and 2-mercaptoethanol, and stimulated with immobilized anti-CD3 (145-2C11, 1 µg/ml; BD Biosciences) or ionomycin (0.5 µM)-phorbol myristate acetate (PMA, 10 ng/ml) (Sigma-Aldrich Co., St. Louis, MO) for 24 h in culture at 37°C with 5% CO2. The cells were then removed from activation stimuli, resuspended in fresh media, and further incubated without stimulus for the next 48 h. At this time (72 h postactivation), cells were removed from culture, labeled, and analyzed by flow cytometry. Where indicated, interleukin-2 (50 U/ml; R & D Systems, Minneapolis, MN) was added to the cultures after removal of anti-CD3 at 24 h.
Neuraminidase and lectin application. Lymph node cells (5 x 106) from C57BL/6 mice were isolated as described and treated with or without 3 mU protease-free neuraminidase (sialidase) from Vibrio cholerae (EC 3.2.1.18; Roche Diagnostics, Indianapolis, IN) in RPMI 1640 media for 25 min at 37°C. After washing with PBS, cells were resuspended at 1 x 106 cells/ml in RPMI 1640 media containing PNA, Erythrina cristagalli agglutinin (ECA) (Vector Laboratories), or media alone. Where indicated, lectin addition was preceded by a 6-h incubation with immobilized anti-CD3 (145-2C11, 1 µg/ml) or accompanied by D-galactose (25 mM; Sigma-Aldrich). Using live (7-AAD–) CD8+ T cells, annexin V-APC detected phosphatidylserine externalization and caspase activity was assessed by 6-carboxyfluorescein (FAM)-VAD-FMK (Invitrogen) cleavage. DNA fragmentation was measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay (Promega), all according to the manufacturer's recommendations. After 24 h in culture at 37°C with 5% CO2, cells were counted using a hemacytometer and trypan blue exclusion to identify live cells. The proportion of CD8+ T cells in culture was determined by flow cytometry and used in calculating the total CD8+ T-cell count.
LCMV infection.
ST3Gal-ITg, ST3Gal-I/, and wild-type littermate mice were inoculated with an intraperitoneal injection of 2 x 105 PFU of LCMV-Armstrong in 0.2 ml sterile PBS. On days 0, 8, 15, and 30 postinoculation, mice were euthanized, the spleen and lymph nodes were harvested, and cell suspensions were prepared for counting and flow cytometry as described. LCMV-specific T cells were defined by labeling with PE-conjugated Db gp33-41 major histocompatibility complex (MHC) class I tetramers (Beckman Coulter, Fullerton, CA) and in combination with other markers as described above.
SEB immunization. Mice were injected intravenously on day 0 with 150 µg SEB from Staphylococcus aureus (Sigma-Aldrich) in 0.2 ml sterile PBS. On days 0, 2, and 10 postinjection, mice were euthanized, the spleen and lymph nodes were harvested, and cell suspensions were prepared for counting and flow cytometry, as well as used to enrich CD8+ T cells by negative depletion followed by culture in RPMI 1640 as described above. CD8+ T cells expressing the Vß8 TCR were identified using the PE-conjugated anti-Vß8 antibody (F23.1; BD Biosciences) and in combination with other antibody markers, as described.
Adoptive transfer of T cells.
Thymocytes (5 x 106) isolated from Bim/, ST3Gal-I/, and Bim//ST3Gal-I/ mice, enriched for double-negative and CD8 single-positive thymocytes (present in identical proportions) as described above, were suspended in 0.2 ml sterile PBS and adoptively transferred into RAG-1-deficient recipient mice (35) by intravenous injection. After 2 weeks, mice were euthanized, and lymphocyte suspensions were prepared from the peripheral blood, lymph nodes, or spleen. CD8+ T cells were identified by antibody labeling, and the percentage of annexin V+ cells was assessed by flow cytometry as described.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ CD8+ T cells and support the view that CD8+ T-cell apoptosis may normally occur among postactivated CD8+ T cells bearing altered O glycosylation. Constitutive ST3Gal-I transgene expression in T-cell ontogeny and peripheral homeostasis. The model of post-immune CD8+ T-cell apoptosis we previously reported predicts that the apoptosis of CD8+ T cells due to absence of sialic acid on core 1 O-glycan structures would be inhibited by constitutive expression of ST3Gal-I (48). We therefore generated transgenic mice using the human ST3Gal-I cDNA, which is over 90% identical to the mouse sequence, expressed in the T-cell lineage by the human CD2 promoter (Fig. 2A). ST3Gal-I transgenic (ST3Gal-ITg) mice were viable, fertile, and normal upon examination. Histologic examination of the thymus revealed an overall normal organization and appearance in sections stained with hematoxylin and eosin but a profound reduction in PNA ligands in both the cortex and medulla, indicative of high-level ST3Gal-I function in thymocytes (Fig. 2B). Elevated ST3Gal-I expression did not significantly affect thymocyte cellularity or ontogeny, judged by CD 4/CD8 subset frequencies (Fig. 2C). The CD8 single-positive (SP) thymocyte population, however, displayed specific alterations in TCR Vß repertoire expression (Fig. 2D). Notably, frequencies of Vß9 and Vß12 were reduced and induced, respectively. These findings are opposite of, and consistent with, those observed in ST3Gal-I-deficient CD8 SP thymocytes and likely reflect alterations proposed in some MHC class I interactions that are modified by CD8 O glycosylation (36). The transgenic increase in core 1 O-glycan sialylation was measured as a three- to fivefold decrease on average in PNA binding to ST3Gal-ITg thymic and peripheral CD8+ T cells among the spleen, lymph nodes, and blood (Fig. 2E and data not shown). However, this increase in ST3Gal-I function did not significantly alter peripheral lymphoid tissue cellularity or total CD8 T-cell numbers in the lymph nodes or spleen of littermate mice at 8 to 10 weeks of age or those aged for 7 to 12 months (Fig. 2F and data not shown).
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chain CD132 (data not shown) (see Fig. S1 in the supplemental material). No significant change was observed in ST3Gal-I transgene mRNA expression throughout the in vitro activation time course (Fig. 3C), suggesting that the appearance of unsialylated core 1 O-glycans on peripheral postactivated CD8+ T cells is due to a posttranscriptional mechanism inactivating ST3Gal-I function or removing the sialic acid linkage produced by ST3Gal-I.
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/ mice with LCMV and further measuring the level of annexin V binding, which has been used to identify virus-specific CD8+ T cells undergoing apoptosis (60).
We observed a close relationship between unsialylated core 1 O-glycans detected by PNA and annexin V binding on gp33-41 tetramer-positive cells after LCMV infection (Fig. 4A). Moreover, among ST3Gal-ITg tetramer-positive cells, only those that expressed unsialylated core 1 O-glycans were positive for annexin V binding, as was observed in vitro upon anti-CD3 activation (Fig. 3A). The induction of unsialylated core 1 O-glycans among gp33-41 tetramer-positive T cells peaked at day 8 in both ST3Gal-ITg and wild-type mice with a 6- to 10-fold increase in PNA ligands, respectively, that subsided by day 30, in agreement with previous findings (Fig. 4B) (14, 48). Both wild-type and ST3Gal-ITg mice showed similar expansion and contraction responses involving total CD8+ T cells and gp33-41 tetramer-positive CD8+ T cells in the lymph nodes (Fig. 4C) and the spleen (Fig. 4D). Notably, ST3Gal-I/ cells, which constitutively displayed unsialylated core 1 O-glycans, also maintained the highest percentage of annexin V+ cells throughout the infection time course, which corresponded with low numbers of viable cells in the lymph nodes and spleen at every time point tested. ST3Gal-I/ mice also displayed reduced numbers of both total and gp33-41 tetramer-positive CD8+ T cells at all time points examined, although distinct expansion and contraction phases involving these cells were observed. These observations are consistent with previous results implicating the normal response of ST3Gal-I-deficient naive CD8+ T cells to TCR stimuli in the presence of attenuated cytotoxic T-cell activity in vivo to MHC-mismatched tumor cell inoculation due to reduced CD8+ T cell numbers (48).
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/ mice, we found a pattern in the SEB response similar to that observed with LCMV challenge, specifically, all CD8+ T cells that became annexin V+ also expressed unsialylated core 1 O-glycans (Fig. 5A). In addition, the expansion and contraction of Vß8+ CD8+ T cells displayed similar kinetics among all genotypes (Fig. 5B). When compared to anti-CD3 stimulation or LCMV inoculation, many fewer cells were annexin V+ at the indicated times assayed, although those that were also expressed the highest levels of unsialylated core 1 O-glycans. Interestingly, the majority of of Vß8+ CD8+ T cells from both wild-type and ST3Gal-ITg mice became annexin V+ PNA+ during a subsequent 24- to 48-h culture period ex vivo (Fig. 5C). As with LCMV infection, the corresponding ST3Gal-I/ CD8+ T cells constitutively expressed unsialylated core 1 O-glycans, coinciding with a high percentage that were annexin V+ PNA+ throughout the time course. Decreased viable cell numbers upon in vitro culture were comparable among wild-type, ST3Gal-ITg, and ST3Gal-I/ Vß8+ CD8+ T cells (Fig. 5D). Identical to the in vitro findings, transgenic ST3Gal-I expression in the context of antigen stimulation in vivo maintained sialylated core 1 O-glycans among activated T cells, while apoptotic T cells were both PNA+ and annexin V+.
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The absence of core 2 GlcNAcT-1 failed to restore normal CD8+ T cell levels in mice also lacking ST3Gal-I. Identical results were observed among mice lacking either CD43 or Galgt1 in the context of ST3Gal-I deficiency (Fig. 6A). Restoration of normal CD8+ T-cell homeostasis was achieved, however, in ST3Gal-I-deficient mice bearing the human ST3Gal-I transgene. Levels of core 2 O glycosylation, unsialylated core 1 O-glycans, and annexin V binding were also measured in these resting and activated CD8+ T cells. Remarkably, transgenic ST3Gal-I expression blocked the induction of core 2 O-glycans that are produced in the medial Golgi apparatus following immune activation (Fig. 6B) (48). As increased PNA ligand formation and annexin V binding continues among CD8+ T cells in ST3Gal-ITg mice, we can conclude that core 2 O-glycan branching is not a determinant in the glycan structure contributing to peripheral homeostasis in postactivated CD8+ T cells. Identical results were obtained among ST3Gal-ITg CD8+ T cells during the in vivo immune response to LCMV (data not shown). CD43 deficiency also failed to diminish the induction of unsialylated core 1 O-glycans and annexin V binding, while the level of core 2 O-glycans apportioned to this glycoprotein appeared to reflect more than 60% of the level typically induced following activation. In addition, there was no contribution to CD8+ T-cell homeostasis evident among mice lacking glycolipids produced by Galgt1 or in collaboration with ST3Gal-I.
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/ CD8+ T cells (see Fig. S2A in the supplemental material). Bcl-2 transgene expression did not reduce the high level of PNA ligands or decrease the percentage of annexin V+ CD8+ T cells in Bcl-2Tg/ST3Gal-I/ mice, compared with ST3Gal-I deficiency alone (Fig. 7A). A significant decrease in peripheral CD8+ T cells was observed in ST3Gal-I-deficient mice regardless of the presence of the Bcl-2 transgene, while no differences were observed in the CD4+ T-cell population (Fig. 7B and C).
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/ Vß8+ CD8+ T cells, remaining indirectly proportional to apoptotic marker expression among Vß8+ CD8+ T cells from wild-type and ST3Gal-I-deficient mice (Fig. 7F and data not shown). ST3Gal-I deficiency attenuates the accumulation of CD8+ T cells in the absence of Bim. Unlike Bcl-2 transgenic mice, Bim-deficient mice accumulate lymphocytes even among experimentally and immunologically naive animals (5). The presence or absence of Bim was verified among all genotypes (see Fig. S2B in the supplemental material). We further examined mice deficient in both ST3Gal-I and Bim and compared results with littermates that were either wild type or deficient in either ST3Gal-I or Bim alone. Peripheral CD8+ T cells from Bim-deficient mice expressed low levels of annexin V binding, similar to Bcl-2 transgenic cells. In contrast to results obtained with the Bcl-2 transgene, Bim deficiency significantly reduced the frequency of annexin V+ CD8+ T cells in the absence of ST3Gal-I, compared with ST3Gal-I deficiency alone (Fig. 8A). This coincided with restoration of splenic CD8+ T cell numbers to wild-type levels, while lymph node CD8+ T-cell cellularity was increased by more than twofold to about 50% of normal (Fig. 8B). These findings were specific to the CD8+ T-cell population, as CD4+ T cells continued to accumulate excessively in the absence of Bim, regardless of ST3Gal-I function (Fig. 8C).
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Induction of apoptotic CD8+ T cells in dose-response O-glycan cross-linking, among cell compartments, and in the leukopenic state. The phenotypic modulation of CD8+ T-cell apoptosis in ST3Gal-I deficiency by Bcl-2 and Bim may be explained by the expression of an endogenous stimulus more abundant in the lymph nodes than in the spleen and absent from in vitro cell cultures. Such a stimulus may be a lectin, similar in specificity to PNA, that cross-links one or more key O-glycoproteins bearing unsialylated core 1 O-glycans and thereby induces apoptosis. Alternatively, Bim and Bcl-2 may reside in partial epistatic relationships with ST3Gal-I within apoptotic signaling pathways that contribute to the post-immune CD8+ T-cell contraction phase. To further discern among such possibilities, we explored the dose-response relationship of CD8+ T cells from Bcl-2 transgenic and Bim-deficient mice in the presence or absence of ST3Gal-I. No reduction was observed in the sensitivity to PNA-induced annexin V induction or cell death in vitro, implying that neither increasing Bcl-2 expression nor Bim deficiency alters the efficacy of apoptotic signaling invoked by cross-linking glycoproteins bearing unsialylated core 1 O-glycans (Fig. 9A). In addition, there remained significant differences in the frequency of apoptotic cells among different peripheral compartments of wild-type and ST3Gal-I-deficient mice, similar to those seen in the absence of Bim, with the highest frequencies of annexin V+ cells in the spleen and lymph nodes compared to thymus and blood (Fig. 9B).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relationship of CD8+ T-cell apoptosis to altered protein O glycosylation. Following immune activation in vitro or in vivo, both CD4+ and CD8+ T cells produce a measurable change in protein O glycoslyation detected by a 10- to 20-fold increase in PNA lectin binding to the intact cell surface. Nevertheless, induction of unsialylated core 1 O-glycans induces apoptosis only among the CD8+ T-cell population in mice lacking ST3Gal-I (48). We have now found that producing this O-glycan change by cell surface desialylation of wild-type CD8+ T cells sensitizes them to PNA-induced apoptosis, which is preceded by the induction of DNA fragmentation, caspase activation, and phosphatidylserine externalization. The specificity of this response to unsialylated core 1 O-glycans was evident as increased ECA lectin binding to other galactose-bearing glycan branch termini also occurred upon sialidase treatment but failed to alter cell viability, eliminating the possibility of a nonspecific effect of global desialylation.
The acquisition of the annexin V+ cell surface phenotype with induced levels of PNA+ unsialylated core 1 O-glycans tracked directly with frequencies of apoptotic death and reduced peripheral CD8+ T-cell numbers in all genotypes of CD8+ T cells studied. This O-glycan determinant of apoptotic fate is present 48 to 72 h following TCR stimulation but does not likely participate in immune activation, as constitutive transgenic expression of ST3Gal-I results in PNA– T cells that respond normally to immune stimuli. This O-glycan change, however, invariably precedes apoptosis in the absence of recent TCR stimulation, and when immune signals appear to be waning, as in the context of limiting IL-2 levels. Interestingly, this change in protein O glycosylation may require direct TCR stimulation, as ionomycin and PMA treatment, which activates T cells but bypasses direct TCR stimulation, fails to induce unsialylated core 1 O-glycans coincident with reduced apoptotic death.
The close relationship of annexin V binding on CD8+ T cells with the increased presence of unsialylated core 1 O-glycans was further evident upon in vivo immune activation by viral challenge (LCMV) and bacterial superantigen (SEB) immunization. In all studies, clonal expansion was followed by a measurable contraction phase, the latter being proportional to the frequency of PNA+ annexin V+ CD8+ T cells among the relevant responding populations. TCR stimulation by viral or bacterial antigens invariably elevated unsialylated core 1 O-glycan levels prior to increased annexin V binding, which together marked apoptotic cell frequencies that correlated directly with total viable peripheral cell numbers. Remarkably, transgenic ST3Gal-I expression again abolished the appearance of unsialylated core 1 O-glycans among activated CD8+ T cells but not among the apoptotic annexin V+ population that is increased in frequency during the contraction phase. As previously indicated in multiple genotypes and contexts, viable CD8+ T cells remaining following the contraction phase may be memory CD8+ T cells, which are more similar to resting CD8+ T cells in having increased levels of sialic acid on cell surface core 1 O-glycans (14, 48).
Structure and regulation of protein O glycosylation in CD8+ T-cell apoptosis. The appearance of unsialylated core 1 O-glycans following TCR stimulation is normally accompanied by an induction of core 2 O-glycan branching (47). We have now shown that core 2 O-glycan branching is not involved in CD8+ T-cell apoptosis as induced by the absence of ST3Gal-I. Core 2 GlcNAcT-1 deficiency did not affect CD8+ T-cell homeostasis or rescue peripheral CD8+ T-cell numbers in mice also deficient in ST3Gal-I. The frequency of CD8+ T cells that were PNA+ annexin V+ remained elevated, similar to ST3Gal-I deficiency alone. The normal induction of core 2 O-glycan branching upon T-cell activation may modulate the production of selectin ligands that regulate lymphocyte trafficking (55). This finding implicates the unsialylated core 1 O-glycan Galß1-3GalNAc-Ser/Thr as a glycan structure that controls apoptotic signaling in CD8+ T cells.
Efforts to block the loss of sialic acid on core 1 O-glycans following CD8+ T-cell activation involved the successful production of ST3Gal-I transgenic mice bearing constitutive ST3Gal-I function throughout T-cell ontogeny and immune stimulation. Human ST3Gal-I enzyme function in the mouse thymus was evident, along with the ability of the human transgene to rescue CD8+ T-cell numbers in the absence of endogenous mouse ST3Gal-I. The loss of PNA binding in cortical thymoyctes correlated with changes in the same TCR Vß chains affected in ST3Gal-I deficiency and, in an opposing manner, consistent with a role for ST3Gal-I activity in altering CD8-MHC interactions (36, 37). These relatively minor changes in the TCR Vß repertoire, however, did not alter mature T-cell activation responses (data not shown). Unexpectedly, elevated and constitutive ST3Gal-I expression blocked the appearance of 1B11 binding determinants upon CD8+ T-cell activation that require core 2 GlcNAcT-1, supporting the view that ST3Gal-I competes with core 2 GlcNAcT-1 for glycoprotein substrates in vivo (48, 51).
Remarkably, increased ST3Gal-I expression fails to inhibit the appearance of unsialylated core 1 O-glycans on postactivated CD8+ T cells. This finding implies that loss of ST3Gal-I function following CD8+ T-cell stimulation reflects a posttranscriptional regulatory mechanism. The ability of the transgene to compete with core 2 GlcNAcT activity in the medial Golgi apparatus and sialylate all core 1 O-glycans in resting CD8+ T cells reveals that unsialylated core 1 O-glycans likely result from an event that occurs in the late (trans) Golgi network or in post-Golgi processing. This is consistent with the absence of significant difference in endogenous ST3Gal-I mRNA levels among resting and activated wild-type CD8+ T cells (1). Elevated levels of unsialylated core 1 O-glycans may reflect increased sialidase activity upon T-cell activation (14, 26, 59); however, other studies have found that absence of specific sialidase function, or broad inhibition of sialidase activity, had no effect on the appearance of unsialylated core 1 O-glycans and instead implicated a de novo process by which newly synthesized glycoproteins lack sialic acids on core 1 O-glycans (1). Efforts to produce antibodies to investigate glycosyltransferase regulation have been mostly unsuccessful, but such reagents will be needed to more fully define the mechanisms controlling ST3Gal-I function.
Mechanism of apoptotic regulation by ST3Gal-I. It is clear that not all glycoproteins normally bearing unsialylated core 1 O-glycans participate in CD8+ T-cell apoptosis. We have found that the CD43 glycoprotein, which carries unsialylated core 1 O-glycans in addition to the majority of core 2 O-glycans induced following CD8+ T-cell activation, and has been implicated in apoptosis (6, 42, 48), does not participate in apoptosis among ST3Gal-I-deficient CD8+ T cells. In addition, the susceptibility of CD8+ T cells to PNA-induced apoptosis was not altered by the addition of IL-7 and did not coincide with altered expression levels of various cytokine receptors (data not shown). Among glycoprotein candidates other than CD43, both CD8 and CD45 are also modified with unsialylated core 1 O-glycans (1, 8, 36, 37, 48, 61). Recently, cross-linking of CD8 with antibodies to CD8 or MHC class I ligands has been found to induce apoptosis among immature CD4+ CD8+ thymocytes but not mature CD8+ T cells (16), and this does not appear to be altered by loss of ST3Gal-I (22). Interestingly, de novo synthesis of CD45 after CD8+ T-cell activation can account for the majority of increased PNA ligands (1), and CD45 has been found to participate in apoptosis (43, 46); however, our studies indicate that antibodies to CD45 are unable to induce or modulate PNA-induced apoptosis (data not shown) (48).
The identification of the relevant glycoprotein(s) and apoptotic pathway(s) involved will be necessary to resolve how core 1 O-glycan sialylation inhibits CD8+ T-cell apoptosis and may require a glycoproteomics approach that is increasingly able to characterize biologically relevant glycan structures on glycoproteins (9, 10, 37). The dysfunction of one or a few glycoproteins can be responsible for the emergence of major phenotypes among glycosyltransferase-deficient mice, and such studies have implied that protein and glycan determinants can collaborate in the production of endogenous ligands for mammalian lectins (18, 40, 41).
Here we have used an inside-out approach to investigate the apoptotic pathway in ST3Gal-I function by probing the effect of intracellular Bcl-2 levels and Bim deficiency. Remarkably, the ability of Bcl-2 to reduce the CD8+ T-cell contraction phase of an immune response to SEB in vivo was eliminated by ST3Gal-I deficiency, resulting in reduced numbers of peripheral CD8+ T cells which were mostly annexin V+. Bcl-2 transgene expression continued to increase cell viability among all cells in vitro, concurrent with reduced frequencies of CD8+ T cells that were PNA+ and annexin V+ at the cell surface. Transgenic Bcl-2 expression did not, however, interfere with the production of unsialylated core 1 O-glycans postactivation; therefore, the ability of Bcl-2 to inhibit the contraction phase in the presence of unsialylated core 1 O-glycans may reflect augmented downstream TCR and IL-2 receptor signaling, reducing the onset and rate of the contraction phase. Consistent with this interpretation, CD8+ T-cell accumulation fails to occur in Bcl-2 transgenic mice, wherein normal peripheral levels of CD8+ T cells exist.
In contrast, the intermediate phenotype observed in Bim- and ST3Gal-I-deficient mice includes a reduction in the frequency of apoptotic CD8+ T cells compared with ST3Gal-I deficiency alone and appears to reflect a balance between the hyperaccumulation of CD8+ T cells due to Bim deficiency and the ongoing effect of ST3Gal-I-deficient apoptosis. This is consistent with the finding that Bim deficiency failed to reduce the efficacy of PNA treatment as a means to induce apoptotic cell death in vitro in the absence of ST3Gal-I. A similar result was seen with Bcl-2 transgenic CD8+ T cells in vitro and may further reflect the reduction in antigen levels and immune signaling that occur upon isolated ex vivo T-cell culture. These findings suggest an endogenous apoptotic stimulus that is lacking in vitro and is mimicked by PNA binding.
We propose that an endogenous multivalent lectin induces CD8+ T-cell apoptosis during the contraction phase of the immune response by cross-linking one or more cell surface O glycoproteins that are modified by unsialylated core 1 O-glycans following TCR activation (Fig. 10A). In this model, CD8+ T-cell immune activation via TCR stimulation initiates formation of a default and extrinsic apoptotic pathway to cell death that operates when immune signaling wanes. In Bim deficiency, unlike with elevated Bcl-2 levels, the vast increase in CD8+ T-cell numbers saturates lectin binding sites and thereby reduces peripheral T-cell numbers to a normal homeostatic level (Fig. 10B). Sialylation by ST3Gal-I normally protects naive and memory CD8+ T cells from this apoptotic stimulus in compartments where the apoptotic lectin is present and capable of cross-linking unsialylated core 1 O-glycans. Reconstitution kinetics among lymphopenic RAG-1-deficient mice were consistent with the presence of an apoptosis-inducing lectin more highly expressed in lymph node compartments compared with the spleen and absent from blood circulation, a finding consistent with the presence of a CD8+ T-cell contraction phase following SEB immunization in lymph nodes, but not in the spleens or blood of Bim- and ST3Gal-I-deficient mice. Previous studies found that the contraction of Bim-deficient CD8+ T cells proceeded normally in the lymph nodes after herpes simplex virus infection and occurred to a greater degree in the lymph nodes than in the spleen in the case of SEB (21, 45). The identity this putative apoptosis-inducing lectin is not yet known, although candidates include the ß-galactosidase-binding mammalian galectins, several of which have been reported to induce apoptosis in T cells (13, 23, 25, 46, 58).
The homeostasis of CD8+ T cells may be critically dependent on the presence or absence of multiple factors. Nevertheless, we have found that a specific alteration in protein O glycosylation during late stages of TCR stimulation and manifested on the cell surface by a posttranscriptional mechanism regulating ST3Gal-I function can play a dominant role in the CD8+ T-cell contraction phase of the immune response by linking extracellular glycoprotein structure with molecular interactions that lead to apoptotic signal formation. Further resolving this process will elucidate a molecular pathway from the cell surface by which apoptotic signal formation controls peripheral CD8+ T-cell homeostasis. The ability to modulate CD8+ T-cell numbers by altering ST3Gal-I activity may be useful in reducing cytotoxic T-cell activity in tissue transplantation and attenuating pathogenesis in diseases such as glomerulonephritis and diabetes.
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ACKNOWLEDGMENTS |
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This research was supported by a grant from the National Institutes of Health (P01-HL57345). J.D.M. acknowledges support as an Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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Published ahead of print on 13 November 2006.
Supplemental material for this article may be found at http://mcb.asm.org/.
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, statistically significant difference from wild-type littermates (P < 0.05, paired t test).
