Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96

Abstract

The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

Figure 1

Determination of β-gal expression in R/gal and RS/gal transfectants. Serial dilutions of NP-40 lysates of R/gal (open triangles) and RS/gal (closed triangles) transfectants or untransfected RMA cells (closed circles) were incubated with the chromogenic substrate X-gal and enzyme activity was determined by measuring the absorbance at 620 nm.

Figure 2

Induction of β-gal–specific CTL responses using gp96 molecules isolated from P815, P13.1, R/gal, and RS/gal cells. (A) BALB/c mice were immunized with gp96 molecules isolated from P815 cells (open triangles), P13.1 cells (closed triangles), or with PBS (closed circles). Spleen cells were stimulated in vitro with the β-gal–derived peptide TPHPARIGL and the CTL activity was tested on P13.1 cells at the indicated E/T ratio or on P815 incubated with different concentrations of the β-gal peptide at an E/T ratio of 3:1. (B) BALB/c mice were immunized with gp96 molecules isolated from RS/gal cells (open triangles), R/gal cells (closed triangles), or PBS (closed circles). Spleen cells were stimulated and CTL activity was determined as described in A.

Figure 3

Induction of minor H-specific CTL using gp96 molecules isolated from R/gal and RS/gal cells in BALB/c mice. Mice were immunized with PBS (closed circles) or gp96 molecules isolated from RS/gal (open triangles), R/gal (closed triangles) to induce cross-priming against minor H antigens of the C57BL/6 background. Spleen cells were stimulated in vitro with B10.D2 stimulator cells and tested for their ability to lyse BALB/c (left) and B10.D2 (right) Con A blasts.

Figure 4

TAP-independent recognition of minor H antigens by BALB.B anti-C57BL/6 CTL. BALB.B mice were immunized with C57BL/6 spleen cells (closed symbols) or left untreated (open symbols) and stimulated in vitro with irradiated C57BL/6 cells. CTLs were tested for their ability to recognize RMA cells (triangles) or RMA-S cells (circles).

Figure 5

Induction of minor H-specific CTL in 129/Sv mice upon immunization with gp96 molecules isolated from RMA and RMA-S cells. Mice were immunized with gp96 molecules isolated from RMA-S cells (open triangles), RMA cells (closed triangles), C57BL/6 spleen cells (open circles) or with PBS (closed circles) to induce priming against minor H antigens of the C57BL/6 strain. Spleen cells were stimulated in vitro with C57BL/6 stimulator cells and tested for their ability to lyse 129/Sv and C57BL/6 Con A blasts (A) or RMA and RMA-S target cells (B).