A lymphocytic choriomeningitis virus glycoprotein variant that is retained in the endoplasmic reticulum efficiently cross-primes CD8(+) T cell responses

Abstract

Recent studies indicate that T cell cross-priming preferentially occurs against long-lived, stable proteins. We have studied cross-priming by using the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), a protein that normally is not MHC class I cross-presented. This study shows that a C-terminally truncated, noncleavable variant of LCMV-GP led to the accumulation of stable, soluble GP trimers in the endoplasmic reticulum (ER) of the antigen donor cell, and thereby converted LCMV-GP into a potent immunogen for cytotoxic T lymphocyte cross-priming. Immunization of mice with tumor cells expressing an ER-retained LCMV-GP variant cross-primed protective antiviral cytotoxic T lymphocyte responses in vivo at least 10,000-fold better than immunization with cells expressing the cross-presentation-"resistant" wild-type LCMV-GP. Thus the ER is a cellular compartment that can provide antigen for cross-presentation, and modifications affecting stability and subcellular localization of the antigen significantly increase its availability for MHC class I cross-presentation. These findings impinge on vaccine strategies.

Fig. 1.

C-terminally truncated LCMV-GP is retained within the ER. (A) Cartoon of full-length WT LCMV-GP. LCMV-GP is translated as a single precursor protein (GPc) into the lumen of the ER. The leader sequence (L) at the N terminus is indicated. After transport to the Golgi complex, GPc is cleaved by the protease S1P/SKI-1 into the peripheral subunit GP-1 and the transmembrane subunit GP-2. Several features within GP-2 are highlighted: an N-terminal fusion peptide (green), a central coiled-coil core (dark blue), and a helical region (light blue) before the transmembrane region (TM). (B) Truncated LCMV-GP fibritin fusion protein (GPER). The S1P/SKI-1 cleavage site is mutated to avoid proteolytic processing, and the C-terminal half of GP-2 is replaced by a trimerization-promoting fibritin sequence (F). (C) Native Western blot analysis of total cell lysate (CL) and of concentrated cell supernatant (SN) of MC57 cells stably expressing GPER. (D–O) Subcellular localization of WT LCMV-GP in stably transfected MC57 cells (MC-GP) and of GPER in stably transfected MC57 cells (MC-GPER). GP expression is detected with the GP-1-specific mAb KL25 (red). (D and E) MC-GP transfected with GFP-KDEL (green) being either permeabilized (D) or nonpermeabilized (E) before KL25 staining. (F and G) KL25 stained, permeabilized MC-GPER, either costained with the ER marker calnexin (green) (F) or transfected with GFP-KDEL (green) (G). (H–O) Triple staining of MC-GP (H–K) and MC-GPER (L–O) with KL25 (red), GFP-KDEL (green), and calnexin (blue). (Scale bars: 2 μm.) (P) HeLa cells transiently transfected with WT LCMV-GP (GP) or with truncated fibritin fusion protein (GPER) were radioactively labeled and chased for 3 h before lysis and immunoprecipitation with the GP-1-specific Ab KL25. Immunoprecipitates were treated with peptide-N-glycosidase F, treated with Endo H, or left untreated as indicated. Bands representing the glycosylated or deglycosylated precursor protein (gGPc or GPc), the glycosylated or deglycosylated GP-1 subunit (gGP-1 or GP-1), the glycosylated or deglycosylated GP-2 subunit (gGP-2 or GP-2), and the glycosylated or deglycosylated truncated GPER fusion protein (gGPER or GPER) are indicated by arrows. (Q) MC-GPER (black line), MC-GP (gray line), and parental MC57 cells (gray solid line) as assessed by FACS after intracellular KL25 staining.

Fig. 2.

ER-retained GPER is cross-presented in vivo. (A) Lysis of unpulsed (Left) and gp33 peptide-pulsed (Right) MC-GPER (circles) and MC-GP (triangles) cells by GP-specific CTLs. Open diamonds, parental MC57 cells. E:T ratio, ratio of effector to target cells. (B) LCMV-GP-specific tetramer-positive CD8⁺ T cells in blood of C57BL/6 mice on day 9 after priming with MC-GPER. (C and D) GP-specific CTL responses in CB6 F₁ and B/c × H8 F₁ mice after priming i.p. with 5 × 10⁶ MC-GPER. The frequency of specific IFNγ-producing CD8⁺ T cells in peritoneal exudate (C and D Lower) and spleen (D Upper) on days 7, 10, and 13 (d7, d10, and d13) after immunization was measured after in vitro restimulation for 6 h with the indicated peptides. (C) FACS plots of peritoneal exudate cells from individual mice on day 10. (D) Spleen cells (Upper) and peritoneal exudate cells (PEC) (Lower). Open circles, MC57-immunized CB6 F₁ mice; filled circles, MC-GPER-immunized CB6 F₁ mice; shaded circles, MC-GPER-immunized B/c × H8 F₁ mice. Mice infected with GP-recombinant vaccinia virus (VV-G2, grey triangles and diamonds) were used as positive controls. Symbols represent individual mice.

Fig. 3.

Cross-presentation of GPER does not result from increased antigen dose. CB6 F₁ and B/c × H8 F₁ mice were immunized with titrated numbers of MC-GPER or MC-GP, and IFNγ-producing CD8⁺ T cells in spleen (data not shown) and peritoneal exudate cells (data of individual mice are shown) on day 10 after immunization were analyzed after in vitro restimulation using the indicated peptides.

Fig. 4.

Protective CTL responses induced by GPER-expressing tumor cells. BALB/c mice were i.p. immunized with parental MC57 (MCwt), MC-GPER, GP-recombinant vaccinia virus (VV-G2), or were left naïve as controls, and challenged 15 or 41 days later with 200 pfu of LCMV-WE. Virus titers (days 15 and 41 are shown) and T cell responses (day 15 is shown) were assessed in spleens on day 4 after LCMV challenge. (A) gp283-specific CD8⁺ T cell responses on day 15 after immunization with MC-GPER. (B) Virus titers in mice that received the LCMV challenge either 15 days (Upper) or 41 days (Lower) after being immunized as indicated (open circles). CD4-depleted (open diamonds) animals were immunized with MC-GPER and challenged 15 days later with LCMV-WE. (C–E) Ex vivo cytotoxicity (C) and frequency of IFNγ-producing (D) or TNFα-producing (E) gp-283 and np118-specific CD8⁺ T cells in spleens on day 4 after virus challenge.

Fig. 5.

ER-retained GP, but not WT GP, is a potent immunogen for cross-priming of protective antiviral CTLs. BALB/c (H-2^d) mice were primed with titrated numbers of MC-GPER (filled circles) or MC-GP (open circles) and challenged with LCMV-WE on day 15 after immunization. Four days later, virus titers (A Upper), ex vivo cytotoxicity (A Lower), and IFNγ-producing (B Left) or TNFα-producing (B Right) virus-specific CD8⁺ T cells were assessed using spleen cells. Filled bars, MC-GPER; open bars, MC-GP. N.D., not determined. Symbols represent individual mice in A, the mean ± SEM of groups of 3–4 mice in B.