Calreticulin, a peptide-binding chaperone of the endoplasmic reticulum, elicits tumor- and peptide-specific immunity

Abstract

Calreticulin (CRT), a peptide-binding heat shock protein (HSP) of the endoplasmic reticulum (ER), has been shown previously to associate with peptides transported into the ER by transporter associated with antigen processing (Spee, P., and J. Neefjes. 1997. Eur. J. Immunol. 27: 2441-2449). Our studies show that CRT preparations purified from tumors elicit specific immunity to the tumor used as the source of CRT but not to an antigenically distinct tumor. The immunogenicity is attributed to the peptides associated with the CRT molecule and not to the CRT molecule per se. It is further shown that CRT molecules can be complexed in vitro to unglycosylated peptides and used to elicit peptide-specific CD8(+) T cell response in spite of exogenous administration. These characteristics of CRT closely resemble those of HSPs gp96, hsp90, and hsp70, although CRT has no apparent structural homologies to them.

Figure 1

Gp96 and CRT preparations. Gp96 and CRT purified from Meth A sarcoma and liver of BALB/cJ mice were resolved by SDS-PAGE, silver-stained, or immunoblotted using antibodies as described in Materials and Methods.

Figure 2

CRT preparation is free of other peptide-binding proteins of similar size. CRT preparations from Meth A sarcoma were resolved on SDS-PAGE and were immunoblotted with antibodies to Erp 57 or PDI (as described in Materials and Methods). The lane marked − was silver-stained directly and was not immunoblotted. Meth A cell lysate was used as source of protein for each immunoblot.

Figure 3

Immunogenicity of CRT and gp96 preparations from Meth A sarcoma. Mice were immunized with 20 μg of gp96 or 5.5 and 11 μg of CRT (molar equivalent of 10 and 20 μg of gp96) preparations from Meth A sarcoma or normal liver of BALB/cJ mice as indicated. Immunization was carried out in 200 μl vol subcutaneously twice at a weekly interval. Mice were challenged with 100,000 live Meth A cells intradermally 1 wk after the last immunization. Each line represents the kinetics of tumor growth in one mouse.

Figure 4

Interaction of CRT, gp96, and hsp70 with ATP or ADP. CRT, gp96, or hsp70 were purified from Meth A cells and were incubated with Tris-buffer containing 150 mM NaCl and 1 mM dithiothreitol at pH 8.0 or the above buffer containing 10 mM ATP or ADP and 2 mM Mg²⁺ and 0.5 mM Ca²⁺ at room temperature for 2 h. The mixtures were centrifuged through centricon 10 and the eluates were applied to a reverse phase column R2H. The x-axis represents elution time and the y-axis represents the absorbance at 214 nm. Specific peaks were obtained from hsp70 treated with ATP but not with ADP or with buffer alone. Peptides could not be eluted from gp96 or CRT after treatment with ATP or ADP. A, buffer; B, plus ATP; C, plus ADP.

Figure 5

CRT binds to peptides in vitro. CRT or gp96 (40 pmol) were incubated with iodinated synthetic peptide (2 nmol, NH₂-Ser-Leu-Ser-Asp -Leu-Arg-Gly -Tyr - Val-Tyr-Gln-Gly-Leu-Lys-Ser-Asn-Val-Ser-COOH) in phosphate buffer in 20 μl reaction volume at 25°C for 20 min, 37°C for 1 h, or 50°C for 10 min. After a brief centrifugation, the mixtures were incubated at 25°C for another 30 min. Samples were analyzed by SDS-PAGE and staining followed by autoradiography of the stained gel.

Figure 6

CRT-peptide complex reconstituted in vitro primes mice for antigen-specific CTL response. Mice were immunized with gp96 alone (50 μg), CRT alone (27.5 μg), peptide alone (50 μg), gp96–peptide complex (50 μg), or CRT–peptide complex (27.5 μg). After 10 d, spleens were removed and stimulated with VSV8 peptide (1 μM). The lymphocyte cultures were tested for cytotoxic activity on ⁵¹Cr-labeled EL4 cells or EL4 cells pulsed with the VSV8 peptide as targets. Open symbols, EL4; filled symbols, EL4 plus VSV8.