Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy

Abstract

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Figure 1.

Ionizing radiation increases the expression of peptide– MHC class I complexes at the cell surface. (A) Cells were exposed to different doses of irradiation as indicated, and levels of peptide–MHC class I complexes at the cell surface were measured 18 h after γ-irradiation by flow cytometry. Fluorescence in the absence of the first antibody (W6/32) is indicated (background). (Inset) The mean fluorescence intensity (MFI) plotted for MHC class I as well as the transferrin receptor (TfR) after an 18 h culture after various doses of γ-irradiation (representative experiment). (B) Time course of radiation-induced MHC class I up-regulation. MelJuSo cells were exposed to various doses of γ-radiation and cultured for the indicated times before analysis of surface MHC class I expression by FACS. The MFI was determined and related to MHC class I expression in control MelJuSo cells, and the ratio was plotted. Marked long-term increases in MHC class I expression are observed at higher doses of radiation. (C) An HLA-A2 transgenic mouse was locally exposed to γ-irradiation at a dose of 25 Gy. 24 h later, the mouse was killed and sections of a kidney exposed to γ-irradiation and a kidney outside the radiation field were stained with a rabbit anti–MHC class I H chain serum followed by a secondary antibody coupled to Cy5. Background fluorescence was detected with the second antibody only. Images were made under identical settings.

Figure 2.

Ionizing radiation increases intracellular peptide levels in a dose-dependent manner. (A) TAP activity (which correlates with the amount of intracellular peptides) was measured through its lateral mobility in the ER membrane using FRAP. Lactacystin was used to deplete cells for peptides by inhibiting the proteasome. ATP depletion and proteasome inhibition inactivate TAP. Cycloheximide was used to inhibit protein synthesis. TAP activity was measured 1 h after a dose of 4 Gy. (n = 9; mean ± SD). (B) TAP activity measured 1–2 h after different exposures to γ-irradiation as indicated (n = 9; mean ± SD). (C) Control cells and cells exposed to 25 Gy radiation were microinjected with peptide substrates of different length (as indicated) and the half-life was determined (n = 8; mean ± SD). (D) (Left) MelJuSo cells are exposed to various doses of γ-radiation followed by a culture for 1 or 6 h, as indicated. In the last 30 min, cells were cultured in the presence or absence of the proteasome inhibitor MG132 to visualize the polyubiquitinated species produced but not converted by the proteasome. N-ethylmaleimide was added before lysis to inhibit deubiquitinating enzymes, and cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene flouride membranes for detection of ubiquitin with antibody FK2. The position of the marker proteins is indicated and the polyubiquitinated protein fraction quantified indicated by a bar (marked with an asterisk) on the right site of the gel. (Right) Quantification of the effects of radiation on polyubiquitination. The luminescence for the polyubiquitin pool (area indicated by an asterisk) of the Western blot was quantified, corrected for protein input, and related to the signal in the control cells. A marked increase in the polyubiquitin pool is observed after radiation when proteasomes are inhibited by MG132. (E) TAP activity measured over time after γ-irradiation at time point t = 0. The top line indicates TAP activity in nonirradiated cells; the bottom line indicates TAP activity in peptide saturated cells (each data point n > 3; mean ± SD).

Figure 3.

The mTOR pathway, ionizing radiation, and antigen presentation by MHC class I. (A) MelJuSo cells stably expressing GFP under the control of the CMV promoter were exposed to 25 Gy γ-irradiation in the presence or absence of the mTOR inhibitor rapamycin. GFP expression was determined by flow cytometry at 0, 4, or 24 h after γ-irradiation (n = 6; mean ± SD). (B) MelJuSo cells expressing TAP1-GFP were exposed to 25 Gy radiation and cultured for 24 h before analysis. Rapamycin was either present during the 24 h culture period or only during the last 3 h. Alternatively, translation was inhibited by cycloheximide added during the last hour before analysis. The intracellular peptide pool was detected by FRAP. (C) MelJuSo cells were exposed to 25 Gy radiation in the presence or absence of the mTOR inhibitor rapamycin before labeling the cells with the MHC class I antibody W6/32 for flow cytometric analysis. (n = 4; mean ± SD). (D) MelJuSo cells stably expressing HLA-A2/GFP under control of the CMV promoter were radiated at the doses indicated and cultured for another 6 h in the presence or absence of rapamycin, as indicated. Cells were labeled with ³⁵S-methionine/cystein 60 min before lysis, and endogenous and GFP-tagged MHC class I molecules, MHC class II molecules, and the free MHC class I H-chains were isolated, as indicated. The position of the molecules as well as the marker proteins on the 10% gel is indicated. NRS, normal rabbit serum control immunoprecipitation. (Right) Quantitation of the SDS-PAGE signals related to that from nonradiated control cells after subtraction of the same M_r area in the NRS lane.

Figure 4.

Ionizing radiation alters the MHC class I–associated peptide profile and immunological responses. (A) Mass spectrometry profiles of double-charged peptides eluted from corresponding rpHPLC fractions from nonirradiated and irradiated cells as indicated. The peptides marked by an asterisk are observed in both profiles, whereas the arrow indicates peptide CGI-51 that is observed only in the peptide fraction after irradiation. (B) Peptide sequences from proteins induced by γ-radiation as determined by MS and the corresponding proteins. Note that all peptides contain the anchor residues for HLA-A1 (in bold). (C) Identification of CTLs recognizing HLA-A1 tetramers containing irradiation-induced peptides. Flow cytometric analysis of human blood mononuclear cells gated on CD5⁺ and CD4⁻/CD19⁻ TCRγδ⁻ staining (leftmost panel) to determine the CTL population.

Figure 5.

Irradiation of tumor cells boosts the efficacy of CTL in vitro and in vivo. MC38 cells were treated with (A) buffer, (B) 20 Gy radiation, (C) rapamycin, or (D) radiation and rapamycin and recultured. After 24 h, the MHC levels were analyzed by flow cytometry. (A–D) The dashed line depicts isotype control antibody; the solid line depicts H2-K^b. Inset numbers depict the percentage of positive cells (mean fluorescent intensity). (E) ¹¹¹In-labeled MC38 cells treated with buffer (□), 20 Gy radiation (•), rapamycin (◯), or the combination of radiation and rapamycin (▴). After 24 h, the cells were cultured with different numbers of gp70-specific CTL for 18 h. (F) Temporal effects of H-2K^b expression after radiation. MC38 cells were exposed to various doses of radiation followed by culture for the times indicated. The surface expression of H2-K^b was subsequently determined by FACS analyses and the MFI plotted after being related to the MFI of H2-K^b expression of control MC38 cells. (G) C57BL/6 mice were injected with 3 × 10⁵ MC38 tumor cells s.c., and the volume of the tumor (in mm) was measured daily and plotted. (First panel) Mice receiving no additional treatment. (Second panel) Tumors in mice were subjected to external-beam irradiation (10 Gy) in situ on day 9 of tumor transplant (▴). (Third panel) Mice were adoptively transferred (IV) with 3 × 10⁶ gp70-specific CTL at day 10 (▵). (Fourth panel) Tumors in mice were subjected to external-beam irradiation (10 Gy) in situ at day 9 (▴) followed by adoptive transfer of gp70-specific CTL at day 10 (▵). The number of mice in each arm of the experiment and the number of mice without any detectable tumor is indicated in the figure.

Figure 6.

A model summarizing the three effects of ionizing radiation on MHC class I antigen presentation. The early effects are caused by the degradation of proteins that may be triggered or damaged by irradiation. Later effects are caused by activation of the mTOR pathway, which results in increased protein translation of proteins, and an increased generation of peptides from (the RDPs of) these new proteins. The increased peptide pool will enhance MHC class I assembly because peptides are the limiting factor. In addition, unique proteins will be expressed/up-regulated in response to ionizing radiation, resulting in novel peptides presented by MHC class I molecules.