Alteration of Cell Surface Sialylation Regulates Antigen-Induced Naive CD8  T Cell Responses 1 Bhanu P. Pappu 2 and Protul A. Shrikant 3 The strength of interactions with APC instructs naive T cells to undergo programmed expansion and differentiation, which is largely determined by the peptide affinity and dose as well as the duration of TCR ligation. Although, most ligands mediating these interactions are terminally sialylated, the impact of the T cell sialylation status on Ag-dependent response remains poorly un- derstood. In this study, by monitoring TCR transgenic CD8  T cells, OT-I, we show that biochemical desialylation of naive OT-I T cells increases their sensitivity for agonist as well as partial agonist peptides. Desialylation enhances early activation and shortens the duration of TCR stimulation required for proliferation and differentiation, without increasing apoptosis. Moreover, desialy- lation of naive OT-I T cells augments their response to tumor-presented Ag. These results provide direct evidence for a regulatory role for sialylation in Ag-dependent CD8  T cell responses and offer a new approach to sensitize or dampen Ag-specific CD8  T cell responses. The Journal of Immunology, 2004, 173: 275–284. M ost glycosylated proteins and lipids are terminally modified by sialylation (1, 2). The role for sialyl-gly- cans in the normal functioning of the immune system is amply demonstrated by loss of regulated homing (3), aberrant activation that leads to autoimmunity (4), and loss of T cell ho- meostasis in animals with genetic deficiencies for various glyco- syltransferase activities (5–7). Naive T cell activation is regulated by multiple factors including the concentration and affinity of Ag, duration of Ag stimulation, and the costimulatory signals and the cytokine milieu present at the time of Ag interrogation (8, 9). In contrast, Ag-experienced T cells have less stringent requirements for Ag dose, duration, and co- stimulatory context for their activation (10). This condition is thought to occur due to inherent differences in their protein and gene expression that endows the Ag-experienced T cells with en- hanced ability to integrate activation signals during their secondary exposure to Ag (11, 12). Conversely, the changes produced in the cell surface by activation (e.g., expression of adhesion molecules, cytokine receptors) and the reduced sialylation, as demonstrated by increased binding of the plant lectin peanut agglutinin (PNA), 4 may permit distinct qualitative and/or quantitative receptor ligand interactions that sensitize Ag-experienced T cell responses (7, 11, 13, 14). The notion that the sialyl-glycans can impart regulatory potential to T cells is supported by several reports in which it has been demonstrated that genetic disruption of the normal N-glyco- sylation pathway promotes aberrant naive T cell activation result- ing in autoimmunity, whereas overexpression of branched O-gly- cans decreases primary T cell responses to Ag (4, 15). This may occur due to the extent of sialyl-glycans on peripheral T cell surface that by virtue of their size and charge, may tune Ag recognition by regulating the thresholds for activation (16). Furthermore, several el- egant studies have recently presented compelling evidence indicating that the differential sialylation of CD8 coreceptor modulates the TCR affinity during thymocyte selection and contributes to their dampening of the dynamic range for peptide recognition upon matu- ration in the thymus and egress into the periphery (17–19), and it has been reported that sialylation regulates the dimerization and activity of CD45; a phosphatase that restricts TCR signaling, clearly implicating sialyl-glycans in the regulation of Ag-specific peripheral T cell re- sponses (20). Collectively, these findings have generated significant interest in better understanding the precise impact of T cell sialylation on Ag recognition in the hope that thresholds for Ag-dependent ac- tivation of T cells can be either augmented (tumor immunity) or at- tenuated (transplantation, autoimmunity) by altering sialyl-glycans on T cell surfaces. To directly test the impact of the extent of sialylation on the Ag-specific responses of CD8 + T cells, we have biochemically reduced cell surface sialylation on naive TCR transgenic CD8 + T cells (OT-I) in vitro and studied the effects of dose, affinity, and duration of stimulation by Ag presented on either professional APCs (dendritic cells (DC)) or tumor cells. Our results show that surface sialylation attenuates the dynamic range of naive CD8 + T cells for Ag recognition and desialylation promotes their capacity to integrate the instructive signals that program their proliferation and differentiation in an Ag-specific manner. Materials and Methods Animals and cells The OT-I TCR transgenic mice (21), originally a gift from Dr. F. Carbone (Monash Medical School, Victoria, Australia) were bred to B6.PL- Thy1 a /Cy mice (The Jackson Laboratory, Bar Harbor, ME) and generously provided by Dr. M. F. Mescher (University of Minnesota, Minneapolis, MN). They were backcrossed up to 10 generations for Rag2 deficiency; OT-I/Pl-Rag -/- referred throughout the report as OT-I mice. C57BL/6 mice were purchased from The Jackson Laboratory. All animals were maintained under specific pathogen-free conditions and the experiments performed were in compliance with the relevant laws and institutional guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the Roswell Park Cancer Institute. Adherence-depleted lymph node cells from OT-I mice were the source of CD8 + T cells for all Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263 Received for publication February 11, 2004. Accepted for publication April 26, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Roswell Park Alliance Foundation and an Institu- tional Award from the American Cancer Society. P.A.S was a special fellow of the Leukemia and Lymphoma Society of America. 2 Current address: Department of Microbiology and Immunology, State University of New York, Buffalo, NY 14214. 3 Address correspondence and reprint requests to Dr. Protul A. Shrikant, Assistant Member, Department of Immunology, Roswell Park Cancer Institute, 322 Compre- hensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: Protul.Shrikant@roswellpark.org 4 Abbreviations used in this paper: PNA, peanut lectin agglutinin; DC, dendritic cell. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00