Glycosyl-Phosphatidylinositol Reanchoring Unmasks Distinct Antigen-Presenting Pathways for CD1b and CD1c 1 David H. Geho,* John D. Fayen,* Robin M. Jackman, 2² D. Branch Moody, ² Steven A. Porcelli, ² and Mark L. Tykocinski 3 * ‡ Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phospha- tidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal se- quence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol- specific phospholipase C sensitivity of the CD1b  DAF and CD1c  DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c  DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b  DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c  DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b  DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, sug- gesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation. The Journal of Immunology, 2000, 165: 1272–1277. T he CD1 family of proteins is made up of nonpolymorphic, transmembrane-anchored Ag-presenting molecules that associate noncovalently with  2 -microglobulin (1). Two CD1 isoforms, CD1b and CD1c, present mycobacterial lipid and glycolipid Ags to specific T cell subsets, including CD4 - CD8 - and CD4 - CD8 + T cells (2– 6). Many of the Ags presented by CD1 molecules possess a structural motif comprised of a hydrophilic head group connected to two hydrophobic aliphatic tails (5, 7). A recent crystal structure of the mouse CD1d1 isoform revealed that the amino-terminal  1 and  2 domains form a hydrophobic groove (8). According to modeling studies, the other CD1 isoforms are predicted to share this putative Ag-binding structure (7). Presum- ably, the lipid tails of these aliphatic Ags are anchored in the hy- drophobic binding groove of CD1 proteins. TCR engagement by CD1 proteins is probably mediated by the amino-terminal  1 and  2 domains of CD1 (9). The process by which CD1 proteins engage and bind Ags may be distinct for each isoform. Ag presentation of lipid Ags by CD1b is disrupted by glutaraldehyde fixation of cell surfaces before ex- posure to Ags, suggesting that Ags must be internalized for pre- sentation to occur (10). Furthermore, inhibition of endosomal acid- ification by chloroquine treatment also blocks CD1b-mediated presentation of lipid Ags to T cells (3, 10). Taken together, this evidence suggests that Ag uptake and transport to acidic endoso- mal compartments are required for effective presentation by CD1b (5). Much less is known about the Ag-processing requirements of CD1c. Experimental evidence suggests that tyrosine-based motifs present in the cytoplasmic tails of many CD1 proteins direct their cellular distributions (11, 12). Detailed studies have reported the subcellular distribution of human CD1b, the majority of which is localized inside the cell and is preferentially distributed within late endosomes or lysosomes. At this intracellular site CD1b most likely encounters and binds its cognate Ags (11–14). Localization of CD1b to endosomal compartments is strongly dependent on the tyrosine-based motif in its short cytoplasmic tail. In contrast, pre- liminary observations indicate that a majority of CD1c, which also contains a tyrosine-based motif in its cytoplasmic tail, is present at the cell surface, with a substantially lower intracellular pool. While a portion of the intracellular CD1c is found in late endosomal compartments, it appears that a larger fraction probably enters early endosomes, from which it may be subsequently recycled back to the cell surface (R. Jackman, V. Briken, and S. Porcelli, unpublished observation). Consequently, although their cellular distributions partially overlap, it has been hypothesized that CD1b and CD1c may survey different intracellular compartments for nonpeptide Ags (12). Glycosyl-phosphatidylinositol (GPI) 4 reanchoring of Ag-pre- senting molecules has varying, context-dependent effects on their Ag-presenting functions (15–19). Whereas Ag-preloaded, exog- enously reincorporated, GPI-modified MHC class I proteins retain *Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; ² Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and ‡ Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 Received for publication June 8, 1999. Accepted for publication May 16, 2000. 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 National Institutes of Health Grants RO1CA74958 and RO1AI31044. 2 Current address: BankBoston Robertson Stephens, 555 California Avenue, San Francisco, CA 94104. 3 Address correspondence and reprint requests to Dr. Mark L. Tykocinski, Depart- ment of Pathology and Laboratory Medicine, University of Pennsylvania, 6 Gates Pavilion, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail address: mlt4@mail.med.upenn.edu 4 Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol; DAF, decay- accelerating factor; GMM, glucose monomycolate; MFI, mean fluorescence intensity; PI-PLC, phosphatidylinositol-specific phospholipase C; TCM, T cell medium. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00