An Immunodominant MHC Class II-Restricted Tumor Antigen Is Conformation Dependent and Binds to the Endoplasmic Reticulum Chaperone, Calreticulin

Abstract

There is accumulating evidence that CD4+ T cell responses are important in antitumor immunity. Accordingly, we generated CD4+ T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4+ hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4+ response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4+ epitope.

Folding of an MHC class II-restricted tumor antigen controls its antigenicity via MHC-guided processing

“…gp90 is synthesized in the endoplasmic reticulum in many mouse tumor lines as a precursor protein before cleavage in the Golgi apparatus to form the extracellular glycoprotein gp70 and transmembrane protein p15E (23)(24)(25). We have found that only gp90 synthesized by CT26 stimulates a panel of CD4 ϩ T cell hybridomas generated by the immunization of syngeneic BALB/c mice with CT26 (22). In contrast, gp90 from other mouse tumors is not stimulatory.…”

“…1d). We have shown previously that gp90 antigenicity correlates with CRT binding (22), so we next examined which of the conformers identified by nonreducing SDS/PAGE was complexed to CRT by coimmunoprecipitation. Only the floppy conformer, gp85, from CT26-P cells was coprecipitated with CRT (Fig.…”

“…In contrast, gp90 from other mouse tumors is not stimulatory. Furthermore, the antigenicity of gp90 correlates with binding to the lectin-like chaperone calreticulin (CRT) and is strictly dependent on its conformation, namely, denaturation and unfolding abrogate recognition of gp90 by all of the established tumor-specific T cell hybridomas (22). Here we investigate the molecular basis for the conformation-dependent and tumorspecific antigenicity.…”

“…Here we describe how the notion of MHC-guided processing is related to the immunodominance of a recently identified MHC class II-restricted tumor antigen, murine leukemia virus (MuLV) envelope protein gp70/90 from mouse colorectal tumor CT26 cells (22). gp90 is synthesized in the endoplasmic reticulum in many mouse tumor lines as a precursor protein before cleavage in the Golgi apparatus to form the extracellular glycoprotein gp70 and transmembrane protein p15E (23)(24)(25).…”

Age-dependent tolerance to an endogenous tumor-associated antigen

“…A CTL epitope p15E 604-611 /H-2K b was also identified from these cells [24] as well as from B16 tumor cells [25]. In CT26 tumor cells, a unique glycoslylated I-E d -restricted epitope contributes to the CD4+ antitumor response [26], and CD8+ T cells respond to the dominant antigen gp70 423-431 /H-2L d (the AH1 antigen) [18]. Gp70 mRNA and T cell responses to gp70-derived antigens are also detectable in other murine tumor cells and models including the 4T1 mammary carcinoma [27], A20 lymphoma [28], and B16 and S91 melanoma cells [18,29].…”