Calreticulin Is Expressed on the Cell Surface of Activated Human Peripheral Blood T Lymphocytes in Association with Major Histocompatibility Complex Class I Molecules

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of β2-free MHC class I molecules. Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of β2-free MHC class I molecules. Calreticulin is a highly conserved and widely tissue distributed calcium-binding protein with a C-terminal KDEL endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; MHC I, major histocompatibility complex class I; β2m, β 2 microglobulin; PBL, partially purified human peripheral blood T lymphocyte; Ab, antibody; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis 1The abbreviations used are: ER, endoplasmic reticulum; MHC I, major histocompatibility complex class I; β2m, β 2 microglobulin; PBL, partially purified human peripheral blood T lymphocyte; Ab, antibody; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis retrieval sequence (1McCauliffe D.P. Lux F.A. Lieu T.S. Sanz I. Hanke J. Newkirk M.M. Bachinski L.L. Itoh Y. Siciliano M.J. Reichlin M. Sontheimer R.D. Capra J.D. J. Clin. Invest. 1990; 85: 1379-1391Crossref PubMed Scopus (140) Google Scholar, 2Sönnichsen B. Füllekrug J. Nguyen Van P. Diekmann W. Robinson D.G. Mieskes G. J. Cell Sci. 1994; 107: 2705-2717Crossref PubMed Google Scholar, 3Nash P.D. Opas M. Michalak M. Mol. Cell. Biochem. 1994; 135: 71-78Crossref PubMed Scopus (118) Google Scholar). However, calreticulin has also been found outside the ER, such as within the secretory granules of cytotoxic lymphocytes, the cell surface of melanoma cells and virus-infected fibroblasts, and the cytosol and nucleus of several cell types (Ref. 4Dupuis M. Schaerer E. Krause K.H. Tschopp J. J. Exp. Med. 1993; 177: 1-7Crossref PubMed Scopus (108) Google Scholar, 5Zhu J. Newkirk M.M. Clin. Invest. Med. 1994; 17: 196-205PubMed Google Scholar, 6White T.K. Zhu Q. Tanzer M.L. J. Biol. Chem. 1995; 270: 15926-15929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Rojiani M.V. Finlay B.B. Gray V. Dedhar S. Biochemistry. 1991; 30: 9859-9866Crossref PubMed Scopus (184) Google Scholar, 8Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (128) Google Scholar, reviewed in Ref.9Krause K.H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Given its lectin-like properties, calreticulin is considered to be an ER chaperone involved in the assembly and folding of nascent glycoproteins (10Peterson J.R. Ora A. Nguyen Van P. Helenius A. Mol. Biol. Cell. 1995; 6: 1173-1184Crossref PubMed Scopus (264) Google Scholar, 11Nauseef W.M. McCormick S.J. Clark R.A. J. Biol. Chem. 1995; 270: 4741-4747Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 12McDonnell M. Jones G.E. White K. Tanzer M.L. J. Biol. Chem. 1996; 271: 7891-7894Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Oliver J.D. Hresko R.C. Mueckler M. High S. J. Biol. Chem. 1996; 271: 13691-13696Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Mature MHC class I molecules are composites of a 44–49-kDa polymorphic heavy chain and a 12-kDa light chain (β2m) complexed with a cytosolic-processed peptide that are expressed on the plasma membrane of almost every nucleated cell (14Germain R.N. Margulies D.H. Annu. Rev. Immunol. 1993; 11: 403-450Crossref PubMed Scopus (952) Google Scholar). A number of ER resident molecular chaperones, such as calnexin, BiP, and transporter-associated protein, are involved in the assembly of the different composites (15Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar, 16Ortmann B. Androlewicz M. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar, 17Suh W. Cohen-Doyle M.F. Froh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (272) Google Scholar, 18Noesser E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (135) Google Scholar). Recently, calreticulin has also been shown to function as a chaperone in the assembly and folding of MHC class I molecules in the ER (19Sadasivan B. Lehner J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 20van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (75) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar, 22Solheim J.C. Carreno B.M. Hansen T.H. J. Immunol. 1997; 158: 541-543PubMed Google Scholar). Contrary to calnexin, calreticulin binds to MHC class I-β2m dimers and to transporter-associated protein via another chaperone, tapasin (19Sadasivan B. Lehner J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). After peptide loading and deglucosylation of N-linked glycans, calreticulin dissociates from the MHC class I-β2m dimers, thus allowing the final transport of mature MHC class I molecules to the cell surface (20van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (75) Google Scholar).Recent studies on the biosynthesis of TcR·CD3 complexes suggest that some chaperones such as calnexin can escape the ER retention mechanisms and be expressed on the cell surface of immature thymocytes in association with TcRαβ chains (23Wiest D.L. Burgess W.H. McKean D. Kearse K.P. Singer A. EMBO J. 1995; 14: 3425-3433Crossref PubMed Scopus (100) Google Scholar, 24Wiest D.L. Bhandoola A. Punt J. Kreibich G. McKean D. Singer A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1884-1889Crossref PubMed Scopus (86) Google Scholar, 25Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4183Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Zhang J. Salojin K. Gao J.X. Cameron M. Geisler C. Delovitch T.L. J. Immunol. 1998; 161: 2930-2937PubMed Google Scholar). On the contrary, studies on the biosynthesis of MHC class I molecules have never reported associations of ER chaperones with MHC class I molecules outside the ER and Golgi compartments (19Sadasivan B. Lehner J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 20van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (75) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar, 22Solheim J.C. Carreno B.M. Hansen T.H. J. Immunol. 1997; 158: 541-543PubMed Google Scholar, 28Solheim J.C. Harris M.R. Kindle C.S. Hansen T.H. J. Immunol. 1997; 158: 2236-2241PubMed Google Scholar, 29Zhang Q. Salter R.D. J. Immunol. 1998; 160: 831-837PubMed Google Scholar, 30Harris M.R. Yu Y.Y.L. Kindle C.S. Hansen T.H. Solheim J.C. J. Immunol. 1998; 160: 5404-5409PubMed Google Scholar). Given that calreticulin displays multiple functions and has long been associated with several chronic diseases (31Sontheimer R.D. Nguyen T.Q. Cheng S.T. Lieu T.S. Capra D. J. Invest. Med. 1995; 43: 362-370PubMed Google Scholar, 32Coppolino M.G. Dedhar S. Int. J. Biochem. Cell Biol. 1998; 30: 553-558Crossref PubMed Scopus (98) Google Scholar), we undertook a study of calreticulin expression on human peripheral blood T lymphocytes to gain new insights into possible biological functions of this molecular chaperone. In the present study, we report the finding that calreticulin is expressed at the cell surface of activated human peripheral blood T lymphocytes, where it is physically associated with a pool of unfolded MHC class I molecules.RESULTSActivated human PBLs were metabolically labeled with [35S]methionine for 15 min and chased in methionine-supplemented media at different times. Brij 96 detergent lysates were immunoprecipitated with anti-calreticulin Abs, and the immunoprecipitates were analyzed by SDS/PAGE. Numerous ER proteins co-precipitated with the 60-kDa calreticulin at time 0 after the 15-min pulse (Fig. 1). Among those, several have been previously identified as transporter-associated protein (70–72 kDa), tapasin (48 kDa), and MHC class I (46 kDa) (21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). Additional bands corresponding to unidentified proteins of ∼97, 50, 42, and 20 kDa were also visible. Interestingly, most of the pulse-labeled proteins, including calreticulin, rapidly disappeared 1 h after the initial pulse, with the marked exception of the 46-kDa band. Thus, 4 h after the pulse, although barely detectable labeled calreticulin was observed, a strong 46-kDa labeled protein was still co-precipitated by anti-calreticulin Abs (Fig. 1). Four h after the initial pulse, all proteins associated with calreticulin that are targeted to the cell surface would have reached the plasma membrane unless they were endocytosed and/or degraded. Among these, the survival of surface MHC class I molecules in activated human T cells is known to be increased (39Monos D.S. Cooper H.L. J. Immunol. 1983; 131: 341-346PubMed Google Scholar). Therefore, we suspected that newly synthesized "unlabeled" calreticulin was present in the cell surface of activated T cells in association with the mature 46-kDa MHC class I heavy chain.To address this question, resting and activated human PBLs were cell surface-labeled with biotin, lysed in Brij 96, and immunoprecipitated with the PA3–900 and W6/32 Abs. PA3–900 immunoprecipitated the 60-kDa calreticulin from lysates of biotinylated resting and activated PBLs (Fig. 2 A). The amount of precipitated calreticulin was, however, approximately 3-fold higher in activated PBLs. To note, a protein of 46 kDa similar to the one immunoprecipitated by W6/32 Abs, which recognize mature β2m-associated MHC I, was again co-precipitated with calreticulin in activated but not resting PBLs (Fig. 2 A). These results provided evidence that the ER resident chaperone calreticulin was expressed in the cell surface of human PBLs, that its expression increased after T cell activation, and that it was associated with a 46-kDa protein corresponding to the mobility of the MHC I heavy chain. The use of more stringent conditions during the cell lysis procedure (i.e. 2% Triton X-100), although decreasing markedly the amount of the co-precipitated 46-kDa protein, did not completely disrupt the association (Fig. 2 B). The observed difference between detergents did not reflect less protein extraction by Triton X-100, as both detergents extracted similar amounts of calreticulin (PA3–900 Ab) and MHC I heavy chain (W6/32 Ab). These results suggest that this association might be of physiological importance and rules out the possibility that the co-precipitation of the 46-kDa protein with the PA3–900 Ab is the consequence of an artifact resulting from cross-reactivity. It should be emphasized that there was some variation in the amount of the 46-kDa protein co-precipitated with calreticulin between the individuals studied.Calreticulin or calreticulin-like proteins have been described in almost every compartment of the cell. Recent reports have shown that human T cells express receptors for the C1q protein and that one of those receptors (cC1q-R) shows homology with calreticulin (40Chen A. Gaddipati S. Hong Y. Volkman D.J. Peerschke E.I.B. Ghrbrehiwet B. J. Immunol. 1994; 153: 1430-1440PubMed Google Scholar, 41Stuart G.R. Lynch N.J. Lu J. Geick A. Moffatt B.E. Sim R.B. Schwaeble W.J. Immunopharmacology. 1997; 38: 73-80Crossref PubMed Scopus (82) Google Scholar). To rule out the possibility that our PA3–900 antibody was recognizing the cC1q receptor, we performed depletion experiments. As shown in Fig.3, depletion of T cell surface calreticulin by four rounds of immunoprecipitation with the PA3–900 Ab did not abolish immunoprecipitation of the cC1q receptor. These data provide evidence that the calreticulin expressed in the surface of activated human T lymphocytes is apparently different from the C1q receptor. Next, we wanted to rule out the possibility that the calreticulin we were detecting on the cell surface of human PBLs could be result of intracellular biotinylation because of background labeling during the handling of the cells. Using activated human PBLs, immunoprecipitation of Brij 96 cell lysates from cell surface-biotinylated cells allowed the detection of cell surface-expressed molecules, such as calreticulin, but not of intracellular molecules such as the protein-tyrosine kinase FYN and the protein-tyrosine phosphatase SHP-1 (Fig.4, left panel). On the contrary, immunoprecipitation of biotinylated Brij 96 cell lysates allowed the detection of both sets of molecules, together with a number of co-precipitated proteins (Fig. 4, right panel). Interestingly, antibodies against the intracellular tyrosine kinase FYN were able to co-precipitate a strong band of similar mobility to the MHC class I heavy chain (Fig. 4, right panel).Figure 3The anti-calreticulin Ab PA3–900 does not recognize the C1q receptor. Five-day-activated human PBLs (20 × 106 cells) were surface-biotinylated and solubilized as indicated in the legend of Fig. 2. The cell lysate was subjected to four rounds of immunoprecipitation with PA3–900 and two additional rounds with beads, followed by immunoprecipitation with anti-C1q-R serum. Precipitates were boiled in 1% SDS and separated on a 12% SDS/PAGE under nondenaturing conditions. Proteins were transferred to nitrocellulose filters, and biotinylated proteins were visualized by ECL. Calreticulin (CRT) is indicated by an arrow. An asterisk indicates the C1q receptor. Molecular mass markers in kDa are indicated on the right.View Large Image Figure ViewerDownload (PPT)Figure 4Cell surface biotinylation detects cell surface calreticulin. Five-day-activated human PBLs were cell surface-biotinylated and lysed in Brij 96 (left) or lysed in Brij 96 (right), and cell lysates were biotinylated as indicated under "Experimental Procedures." Cell lysates were immunoprecipitated with antibodies against FYN, SHP-1, and calreticulin (CRT). Precipitates were boiled in 1% SDS and separated on 10% SDS/PAGE under nondenaturing conditions. Proteins were transferred to nitrocellulose filters, and biotinylated proteins were visualized by enhanced chemoluminiscence (ECL). Calreticulin, MHC I heavy chain, FYN, and SHP-1 are indicated by arrows. Molecular mass markers in kDa are indicated on the left.View Large Image Figure ViewerDownload (PPT)To further characterize calreticulin expression on human PBLs, immunofluorescence microscopy and flow cytometry were performed. For that purpose, cytospins of resting and activated human PBLs were stained with PA3–900 followed by a F(ab′)2 fraction of swine anti-rabbit fluorescein isothiocyanate conjugated Ab to avoid nonspecific binding to Fc receptors in activated T cells. The results demonstrated a faint cell surface expression of calreticulin in resting PBLs that markedly increased in activated T cells (Fig.5 A), which is in agreement with the immunoprecipitation results (Fig. 2). Permeabilized PBLs, either resting or activated, showed a conspicuous cytoplasmic staining (data not shown). To determine whether calreticulin was differentially expressed by the two major peripheral blood T cell subsets, two color flow cytometry studies were performed. The results showed that both CD8+ and CD4+ activated T cells expressed calreticulin at similar levels (Fig. 5 B).Figure 5Immunofluorescence identification of cell surface calreticulin. A, cytospins of fixed resting and 5 day-activated human PBLs were incubated with PA3–900 followed by a F(ab′)2 fraction of swine anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody, and calreticulin staining was visualized under green fluorescence. a and b, resting PBLs. c and d, activated PBLs. Ina and c, the PA3–900 Ab was omitted.B, five-day-activated human PBLs were double-stained for calreticulin (rabbit PA3–900 plus swine anti-rabbit-fluorescein isothiocyanate) and CD4-Cy5PE or CD8-PE. Ten-thousand viable cells were acquired in a FACScan and analyzed using the Lysys II software. Histograms show surface calreticulin expression on gated CD4+ and CD8+ activated PBLs. Dotted lines represent background staining.View Large Image Figure ViewerDownload (PPT)Finally, we wanted to confirm the identity of the 46-kDa protein co-precipitated with calreticulin. Because the W6/32 Ab was unable to co-precipitate a protein of similar mobility with calreticulin, we decided to include in our studies the conformation-independent Ab HC-10, which detects β2m-free MHC class I molecules. As shown in Fig. 6 A, calreticulin Abs were able again to co-precipitate low but significant amounts of a protein with 46 kDa. Re-precipitation of the primary immunoprecipitates with HC-10 demonstrated that the 46-kDa protein co-precipitating with calreticulin was indeed the MHC class I heavy chain (Fig.6 B). Immunodetection using the HC-10 Ab further confirmed that the 46-kDa band co-precipitating with calreticulin corresponds to the MHC class I heavy chain (data not shown). On the other hand, HC-10 but not W6/32 Abs were able to co-precipitate a protein of the same mobility as calreticulin from the surface of activated human PBLs (Fig.6 A). Re-precipitation of the primary immunoprecipitates with the PA3–900 Ab confirmed that calreticulin could be re-precipitated from HC-10 but not W6/32 primary immunoprecipitates (Fig.6 C), therefore demonstrating that calreticulin is associated with a pool of unfolded β2m-free MHC class I molecules on the cell surface of activated human PBLs.Figure 6Calreticulin (CRT) associates with unfolded MHC class I molecules on the surface of activated human PBLs. Five-day-activated human PBLs were surface-biotinylated and solubilized as indicated in the legend to Fig.2. Lysates corresponding to 107 cells were immunoprecipitated with beads (None), with W6/32 Abs (that recognize β2m-associated MHC class I molecules), with HC-10 Abs (that recognize β2m-free unfolded MHC class I molecules), and with PA3–900 Abs. Primary immunoprecipitates were boiled in 1% SDS, 20% of the sample separated on 7.5% SDS/PAGE under nondenaturing conditions, and proteins were transferred to nitrocellulose filters. Biotinylated proteins were visualized by ECL (A). The remaining immunoprecipitates were denatured in 2% SDS and re-precipitated with either HC-10 (B) or PA3–900 (C). Re-precipitates were boiled in 1% SDS, and aliquots were separated in 7.5% SDS/PAGE under nondenaturing conditions. Proteins were transferred to nitrocellulose filters, and biotinylated proteins were visualized by ECL+. Calreticulin and MHC class I heavy chain are indicated by arrows.View Large Image Figure ViewerDownload (PPT)DISCUSSIONRecent studies on calreticulin are disclosing the key role played by this molecular chaperone in the folding and assembly of MHC class I molecules in the ER (19Sadasivan B. Lehner J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 20van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (75) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar, 22Solheim J.C. Carreno B.M. Hansen T.H. J. Immunol. 1997; 158: 541-543PubMed Google Scholar). However, a number of studies indicate that calreticulin is involved in a number of biochemical and physiological functions unrelated with its ER chaperone role. These studies have shown that calreticulin can be found in different locations within the cell, from the nucleus to the plasma membrane of several cell lines (reviewed in Ref. 31Sontheimer R.D. Nguyen T.Q. Cheng S.T. Lieu T.S. Capra D. J. Invest. Med. 1995; 43: 362-370PubMed Google Scholar). How calreticulin can escape the ER retention mechanisms and move into the cytoplasm, nucleus, or plasma membrane is an enigma that challenges current biology. In addition, calreticulin is thought to be involved in a number of diseases, all sharing an immunological basis (32Coppolino M.G. Dedhar S. Int. J. Biochem. Cell Biol. 1998; 30: 553-558Crossref PubMed Scopus (98) Google Scholar, 42McCauliffe D.P. Sontheimer R.D. J. Invest. Dermatol. 1993; 100: 73-79Abstract Full Text PDF PubMed Scopus (0) Google Scholar). All these findings have led to the suggestion that calreticulin may represent a family of highly homologous proteins.The present study addressed the characterization of calreticulin expression in human peripheral blood T lymphocytes. Our interest in studying calreticulin relies 1) on our previous work demonstrating that T lymphocytes and classical MHC class I molecules play a regulatory role in tissue damage under excessive iron accumulation caused by a primary defect in an MHC class I like molecule (reviewed in Ref. 43de Sousa M. Porto G. J. Hepatol. 1998; 28: 1-7Abstract Full Text PDF PubMed Google Scholar) and 2) that calreticulin has also been described as an iron-binding protein (44Conrad M.E. Umbreit J.N. Moore E.G. Gastroenterology. 1993; 104: 1700-1704Abstract Full Text PDF PubMed Scopus (45) Google Scholar). The few studies of calreticulin in human T lymphocytes have shown that the levels of calreticulin mRNA and protein increase after mitogenic stimulation and that calreticulin is a constituent of lytic granules in lymphokine-activated killer cells (4Dupuis M. Schaerer E. Krause K.H. Tschopp J. J. Exp. Med. 1993; 177: 1-7Crossref PubMed Scopus (108) Google Scholar,45Burns K. Helgason C.D. Bleackley R.C. Michalak M. J. Biol. Chem. 1992; 267: 19039-19042Abstract Full Text PDF PubMed Google Scholar). The present results extend these studies by showing that calreticulin is expressed on the plasma membrane of peripheral blood human T lymphocytes and that cell surface expression increases after T cell activation.Previous studies have shown that calreticulin or calreticulin-like molecules are expressed on the plasma membrane of transformed cell lines of different origin. These studies raised the question of how calreticulin can be expressed at the cell surface because it lacks a transmembrane domain. A likely explanation is that cell surface calreticulin is anchored to other(s) surface molecules (46Zhu Q. Zelinka P. White T. Tanzer M.L. Biochem. Biophys. Res. Commun. 1997; 232: 354-358Crossref PubMed Scopus (55) Google Scholar, 47Sim R.B. Moestrup S.K. Stuart G.R. Lynch N.J. Lu J. Schwaeble W.J. Malhotra R. Immunobiology. 1998; 199: 208-224Crossref PubMed Scopus (80) Google Scholar). By cell surface biotinylation and immunoprecipitation, we have provided evidence that calreticulin co-precipitates with several T cell surface molecules, and we have identified MHC class I molecules as one of these T cell surface molecules. Escape of ER resident molecular chaperones to the plasma membrane has previously been reported in several cell types including thymic lymphoma cells (24Wiest D.L. Bhandoola A. Punt J. Kreibich G. McKean D. Singer A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1884-1889Crossref PubMed Scopus (86) Google Scholar, 27Zhang J. Salojin K. Gao J.X. Cameron M. Geisler C. Delovitch T.L. J. Immunol. 1998; 161: 2930-2937PubMed Google Scholar, 31Sontheimer R.D. Nguyen T.Q. Cheng S.T. Lieu T.S. Capra D. J. Invest. Med. 1995; 43: 362-370PubMed Google Scholar, 42McCauliffe D.P. Sontheimer R.D. J. Invest. Dermatol. 1993; 100: 73-79Abstract Full Text PDF PubMed Scopus (0) Google Scholar). It has been argued that this may result from changes in the relative availability of KDEL receptors present in post-ER compartments and known to be involved in the retrograde transport of proteins containing this C-terminal retrieval sequence (48Townsley F.M. Wilson D.W. Pelham H.R. EMBO J. 1993; 12: 2821-2829Crossref PubMed Scopus (110) Google Scholar, 49Griffiths G. Ericsson M. Krinjnse-Locker J. Nilsson T. Goud B. Soling H.D. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1994; 127: 1557-1574Crossref PubMed Scopus (167) Google Scholar, 50Llewellyn D.H. Roderick L. Rose S. Biochem. Biophys. Res. Commun. 1997; 240: 36-40Crossref PubMed Scopus (24) Google Scholar). Because we have detected calreticulin on the surface of normal human T lymphocytes, it is not unlikely to anticipate that other KDEL-containing ER chaperones such as BiP and GRP94 could also be expressed at the T cell surface as well.The association of calreticulin with MHC class I molecules on the surface of activated human T cells is intriguing considering the properties displayed by these polymorphic molecules. T cell surface MHC class I molecules are subjected to spontaneous cycles of endocytosis/re-expression via coated pits (39Monos D.S. Cooper H.L. J. Immunol. 1983; 131: 341-346PubMed Google Scholar, 51Machy P. Barbet J. Leserman L.D. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4148-4152Crossref PubMed Scopus (89) Google Scholar, 52Tse D.B. Pernis B. J. Exp. Med. 1984; 159: 193-207Crossref PubMed Scopus (89) Google Scholar, 53Machy P. Truneh A. Gennaro D. Hoffstein S. Nature. 1987; 328: 724-726Crossref PubMed Scopus (63) Google Scholar). Moreover, endocytosed β2m-associated MHC class I molecules are re-expressed at the cell surface as β2m-free unfolded MHC class I molecules (54DeMaria S. Schwab R. Bushkin Y. Cell Immunol. 1992; 142: 103-109Crossref PubMed Scopus (50) Google Scholar, 55Pickl W.F. Holter W. Stöckl J. Majdic O. Knapp W. Immunology. 1996; 88: 104-109Crossref PubMed Scopus (24) Google Scholar). On the other hand, recent studies have shown that calreticulin preferentially associates with unfolded MHC class I molecules in the ER and that the oligosaccharide moiety in the α1 domain and a residue within the α3 domain are critical for interaction with calreticulin (29Zhang Q. Salter R.D. J. Immunol. 1998; 160: 831-837PubMed Google Scholar, 30Harris M.R. Yu Y.Y.L. Kindle C.S. Hansen T.H. Solheim J.C. J. Immunol. 1998; 160: 5404-5409PubMed Google Scholar). Considering that the calreticulin-MHC I association was detected i