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The Journal of Immunology, 2004, 173: 2462-2469.
Copyright © 2004 by The American Association of Immunologists

Ionizing Radiation Affects Human MART-1 Melanoma Antigen Processing and Presentation by Dendritic Cells1

Yu-Pei Liao*, Chun-Chieh Wang*,¶, Lisa H. Butterfield{ddagger}, James S. Economou{dagger}, Antoni Ribas{dagger}, Wilson S. Meng§, Keisuke S. Iwamoto* and William H. McBride2,*

Departments of * Radiation Oncology and {dagger} Surgery, Division of Surgical Oncology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; {ddagger} Department of Medicine, University of Pittsburgh, Cancer Institute, Pittsburgh, PA 15213; § Department of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282; and Department of Radiation Oncology, Chang Gung Memorial Hospital, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-127–35 that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-127–35 also protected mice against growth of B16-A2/Kb tumors in HLA-A2.1/Kb transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Exposure to ionizing radiation often leads to immunosuppression, although enhancement of immunity has been reported under certain circumstances, in particular with lower doses. Radiation-induced immunosuppression is most often attributed to lymphocyte killing, while immunoenhancement has been ascribed to elimination of a radiosensitive population of suppressor T cells (1). In this study, we report that ionizing radiation affects the function of dendritic cells (DCs),3 which are powerful, relatively radioresistant APCs (2, 3) that initiate naive T cell-mediated immune responses.

DCs are required to undergo phenotypic and functional changes following Ag capture that culminate in their transition to mature APCs before they can generate immunity (4, 5, 6). DC maturation is associated with loss of endocytic/phagocytic receptors and gain in expression of MHC class I and II, CD11a, CD40, CD54, CD58, CD80, CD83, and CD86 molecules (7, 8). The gain in costimulatory molecules promotes their ability to activate Ag-specific T cells and diminishes their ability to induce tolerance to self Ags (4, 9). Danger signals, including those generated by pathogens and proinflammatory cytokines, promote the maturation process (10, 11, 12). Because radiation induces the expression of a number of potential danger signals in tissues (13), its impact on DC function is of interest with respect to the generation of antitumor immunity and autoimmunity, both of which could affect the outcome of radiation therapy for cancer.

Peptides can be loaded onto MHC class I molecules by different mechanisms. In general, antigenic peptides from endogenously synthesized protein result from proteasomal degradation and are loaded onto MHC class I molecules in the endoplasmic reticulum (14, 15, 16). In contrast, exogenous peptides can bind to MHC class I molecules directly or are cross-presented (17, 18, 19). We have recently shown that radiation inhibits proteasome function in a number of different cell types (20, 21), leading to the possibility that Ag processing might be differentially affected depending on the pathway that is used.

We chose to use human melanoma Ag recognized by T cells (hMART-1) as a tumor Ag because we have previously studied its presentation when processed by the endogenous pathway and when loaded an exogenous peptide in both murine and human DC systems (22, 23, 24, 25, 26). hMART-1, a human melanocyte lineage-specific protein, is expressed by 75–100% of melanomas depending on their clinical stages (27). It is frequently recognized by CTLs from HLA-A2.1 patients (28, 29). This is a common HLA haplotype (30) for which immunodominant epitopes have been defined (29). Several groups (31, 32) have used hMART-1 as a target for immunotherapy of melanoma, including DC-based strategies (33, 34).

In this study, we report the effects of radiation on DC processing and presentation by the endogenous pathway and an exogenous loading of hMART-1 peptide. For the former, replication-deficient adenovirus was used to express the entire hMART-1 (AdVMART1) Ag. For the latter, the MART-127–35 peptide (the HLA-A2.1-restricted immunodominant epitope) was chosen. The results suggest that radiation modulates antitumor immunity by affecting DC function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice (H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME). HLA-A2.1/Kb transgenic mice ({alpha}1 and {alpha}2 HLA-A2.1 domains/{alpha}3 Kb domain) on a C57BL/6 background were kindly provided by Dr. L. Sherman (35) (The Scripps Research Institute, La Jolla, CA). Mice were bred and maintained in a strict defined-flora, pathogen-free environment in the American Association of Laboratory Animal Care-accredited Animal Facilities of Department of Radiation Oncology, University of California (Los Angeles, CA). Experiments used 6- to 8-wk-old female mice, and all local and national guidelines for the care of animals were adhered to.

Radiation

Cells were irradiated using a MARK-1–30 irradiator (Cs137 source; J. L. Shepherd & Associates, San Fernando, CA) at a dose rate of ~4.5 Gy/min.

Cell lines

The B16 melanoma and EL4 lymphoma cell lines, both of C57BL/6 origin, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The 293 human embryonic renal cells were used for amplification of adenoviral seed stocks. M202 is a MART-1-positive human melanoma cell line (23). Cell lines were maintained in vitro in DMEM (Mediatech, Herndon, VA) with 10% FBS (Sigma-Aldrich, St. Louis, MO) and 1% antibiotics-antimycotic solution: 10,000 IU penicillin, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B (Mediatech). An EL4 transfectant carrying the MART-1 cDNA and neomycin resistance gene, EL4(MART-1), was generated, as described previously (25), and maintained under constant G418 selection (0.5 mg/ml) in RPMI 1640 medium (Mediatech) with 10% FBS.

Generation of bone marrow-derived DCs

DCs were generated from murine bone marrow cells, as described by Inaba et al. (36), with modifications (24). Bone marrow cells were obtained from femurs and tibias (day 0) and cultured overnight in RPMI 1640 with 2% FBS and 1% antibiotics. Nonadherent cells were resuspended in RPMI 1640 supplemented with 2 ng/ml murine GM-CSF and 10 ng/ml murine IL-4 (BioSource International, Camarillo, CA), 10% FBS, 1% antibiotics, and 50 µM 2-ME (Sigma-Aldrich) at 1–2 x 106 cells/ml in a flask or 24-well plate. On day 4, 80–90% of the medium was removed, and the cells were fed fresh medium containing cytokines. After 8 days in culture, loosely adherent cells were harvested and used for experiments.

AdV transduction of DCs

The E1-deleted replication-defective adenoviral vector containing hMART-1 (AdVMART1) has been described previously (24). It was amplified in 293 cells, purified by centrifugation for 2 h at 24,000 rpm in a 30–60% sucrose gradient. DCs were transduced with AdVMART1 (multiplicity of infection = 100) at 37°C for 2 h in a total volume of 1 ml of RPMI 1640 medium with 2% FBS. Infection was terminated by adding 9 ml of RPMI 1640 with 10% FBS. Transduced DCs produce full-length hMART-1 protein and express MART-1 peptide on the cell surface (23). AdVMART1/DCs were kept at 37°C for an additional 20 min (24) and washed three times, and 5 x 105 cells were injected s.c. into each mouse in a volume of 100 µl of PBS. For assessment of the ability of DCs to stimulatelymphocyte responses, mice were immunized twice (1 wk apart). For AdVMART1/DC in vivo protection studies, they were immunized once.

RNA analysis of MART-1 expression

Total RNA was extracted from cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) and treated with DNase I (amplification grade; Invitrogen Life Technologies), according to manufacturers’ instructions. Each RNA sample was reverse transcribed using Qiagen OneStep RT-PCR kit (Qiagen, Valencia, CA) with primer pairs for MART-1: 5'-TTG GCA GAC GTC TAG ACT CGC TGG CTC TTA AGG T-3' and 5'-CCT CTT TCT CTC TAG ACC TGT GCC CTG ACC CTA CAA-3' (24).

Flow cytometric analyses

Cells were stained with relevant FITC- and PE-labeled Abs and analyzed using a FACSCalibur flow cytometry system (BD Biosciences, Mountain View, CA). The following mAbs were used: FITC anti-H-2Kb (mouse MHC class I), FITC anti-HLA-A2 (human MHC class I), FITC anti-I-Ab (mouse MHC class II), PE anti-CD80 (B7-1), FITC anti-CD40, and PE anti-CD86 (B7-2) (all from BD Pharmingen, San Diego, CA). FITC anti-mouse CD83 was purchased from BioCarta (San Diego, CA).

IFN-{gamma} and CD40L treatment

Bone marrow-derived DCs were generated, as described above, but on day 6 cells were irradiated in the flask and on day 8, 0.1 µg/ml mouse CD40L (R&D Systems, Minneapolis, MN) and 0.1 µg/ml IFN-{gamma} (BioSource International) were added with fresh RPMI 1640 medium with 10% FBS and 1% antibiotics. After 48 h, DCs were harvested, and cell surface molecule expression was analyzed by flow cytometry.

Peptide synthesis and pulsing of DCs

hMART-127–35 (AAGIGILTV), human {alpha}-feto-protein485–493 (CIRHEMTPV), and human tyrosinase1–9 (MLLAVLYCL) epitopes were synthesized at the University of California Peptide Synthesis Facility using standard F-moc strategies. DCs were pulsed with 25 µg/ml peptide in 1 ml of serum-free RPMI 1640 at 37°C. After 4-h incubation, cells were washed, and 5 x 105 DCs in 100 µl of PBS were injected s.c. into each animal. Mice were immunized twice (1 wk apart) for in vivo protection studies, but once for assessment of lymphocyte responses.

Generation of a B16-A2/Kb cell line

A B16-A2/Kb cell line was developed by dual transfection of B16 cells with the plasmids pSV2-neo (ATCC) and pA2Kb (pSV2 backbone with {alpha}1 and {alpha}2 domains of HLA-A2.1 and the {alpha}3 transmembrane and cytoplasmic region of Kb; a generous gift from Dr. L. Sherman) (37). Transfection was performed using Tfx-20 reagents (Promega, Madison, WI), according to the manufacturer’s instructions. Briefly, B16 cells were cultured in a 24-well plate. On the next day, DNA (0.5 µg of pSV2-neo and 0.5 µg of pSV2-A2Kb per well) was diluted in serum-free DMEM, mixed with Tfx-20 reagent (2:1 reagent-to-DNA ratios), added to the plate after removal of the medium, and incubated at 37°C for 1 h. Transfection was terminated by adding DMEM with 10% FBS to the cells. Positive clones were selected in G418 and screened for HLA-A2 expression by flow cytometry using fluorescent Abs (FITC anti-HLA-A2; BD Pharmingen). The B16-A2/Kb cell line was maintained in the presence of 0.5 mg/ml G418.

Measurement of immune function by ELISPOT

ELISPOT was used to assess MART-1-specific splenic lymphocyte responsesin immunized mice. Splenocytes were harvested on day 10–14 after immunization and depleted of RBC by treatment with ammonium chloride buffer (0.83% (w/v) NH4Cl, 0.14% (w/v) KHCO3, 0.002% Na2 EDTA, pH 7.3). MultiScreen-HA plates (Millipore, Bedford, MA) were coated with anti-IFN-{gamma} or anti-IL-4 Ab (BD Pharmingen). For the AdVMART1/DC study, cells were first restimulated with irradiated (50 Gy) EL4 or EL4(MART-1) (25:1 responder-to-stimulator ratios) in the presence of 10 U/ml hIL-2 (BioSource International) in a flask for 48 h. After washing and blocking plates with 10% FBS/PBS, restimulated splenocytes were added and incubated for an additional 24 h. For the MART-127–35-pulsed DC study, lymphocytes were added to the Ab-coated plate directly with 30 µg/ml peptides and 10 U/ml hIL-2 and incubated for 48 h. After incubation, plates were washed and incubated at 4°C with biotinylated anti-IFN-{gamma} or anti-IL-4 Ab (BD Pharmingen). HRP avidin D (Vector Laboratories, Burlingame, CA) diluted 1/2000 in blocking buffer (10% FBS/PBS) was added, and the plates were incubated at room temperature for 45 min. Spots were developed by adding 150 µl/well substrate buffer containing 0.4 mg/ml 3-amino-9-ethyl-carbazole (AEC tablets; Sigma-Aldrich) in 0.05 M sodium acetate buffer (pH 5.0) and 0.012% hydrogen peroxide (Fisher Scientific, Pittsburgh, PA) and counted using an ImmunoSpot Image Analyzer (Cellular Technology, Cleveland, OH).

In vivo protection studies

A total of 5 x 105 AdVMART1/DCs was administered s.c. in the left inner thigh of C57BL/6 mice. B16 tumor challenge (1 x 106 cells s.c. in 0.1 ml into the right thigh) was performed 10–14 days later using a single-cell suspension prepared from tumor growing in syngeneic mice, as described (24), with the following modification. Viable cells were isolated from minced tumors by treatment with 4.8 mg/ml Dispase II solution (Roche, Mannheim, Germany) for 40 min with stirring. After washing three times, cells were resuspended in PBS for injection.

A total of 5 x 105 MART-127–35 peptide-pulsed DCs was injected s.c. in the left inner thigh of HLA-A2.1/Kb transgenic mice twice with a 1-wk interval. B16-A2/Kb tumor cells (5 x 105), isolated from progressively growing tumors, as described earlier (24), were injected s.c. in the right thigh 10 days later.

Tumor sizes were measured three times per week using calipers and presented as mean tumor volume (mm3) using the following formula: 4{pi}/3 x (width/2)2 x (length/2).

Proteasome function assay

DCs were treated with 0 or 10 Gy irradiation and/or with 100 nM PS-341 for 3 h (a kind gift from Millenium Pharmaceuticals, Cambridge, MA). Proteasomes were extracted after treatment, as described previously (21). Chymotrypsin-like activity was measured using 100 µM SucLLVY-7-amido-4-methylcoumarin (Sigma-Aldrich) as fluorogenic substrate. Fluorescence (excitation, 380 nm; emission, 460 nm) was monitored every minute for 30 min using a TECAN Spectrafluor reader (Salzburg, Austria). Protein concentrations were determined by Bio-Rad protein assay (BioRad, Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of irradiation on the ability of DCs to process and present hMART-1 after AdVMART1 transduction

To determine whether irradiation affected presentation of Ag processed through the endogenous pathway, DCs (day 8) were irradiated or left nonirradiated, transduced immediately afterward with AdVMART1, and tested for their ability to generate MART-1-specific T cell responses following injection into syngeneic mice. Spleens were harvested 10–14 days after two immunizations 1 wk apart, and T cell responses were assessed by ELISPOT after 48 h of in vitro restimulation (Fig. 1).



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  		FIGURE 1. Irradiation of DCs inhibits their ability to generate MART-1-specific lymphocyte responses. C57BL/6 mice were immunized s.c. twice, 1 wk apart, with 5 x 105 DC treated with 0 or 10 Gy irradiation before AdVMART1 transduction, or were left unimmunized. Splenocytes were harvested 10–14 days after the last immunization and were restimulated in vitro for 48 h with EL4 ({square}), or EL4(MART-1) (), or without restimulation (control; {blacksquare}). ELISPOT assays for activated splenocytes producing IFN-{gamma} (A) and IL-4 (B) were performed. Nonspecific background values are common in mice injected with DC (26 ). The results are the mean ± 1 SEM of triplicate data of one representative of three independent experiments (*, p < 0.05 and **, p < 0.01; t test).

 
The number of responsive T cells in the spleens of unimmunized mice was very low, while lymphocytes from mice immunized with AdVMART1/DC responded specifically to in vitro MART-1 restimulation. In three independent experiments, irradiation was shown to abrogate the ability of AdVMART1/DC to generate MART-1-specific T cell responses (p < 0.01; t test). This was true for both IFN-{gamma} (Fig. 1A)- and IL-4 (Fig. 1B)-producing lymphocyte responses. The number of lymphocytes activated after immunization of mice with irradiated AdVMART1/DC was no greater than if mice were injected with untransduced DC (data not shown) or if there were no in vitro MART-1 stimulation.

Loss of Ag-presenting function by DC following irradiation was not due to reduced efficiency of AdVMART1 transduction, as assessed by RNA analysis for MART-1 (Fig. 2) or by immunohistochemistry (data not shown). It was also not due to radiation cytotoxicity. The viability of irradiated and nonirradiated DC was identical on injection, being in excess of 80% in all experiments, as assessed by trypan blue exclusion. Furthermore, culturing DCs for up to 48 h in vitro showed no difference in viability between irradiated and nonirradiated cells, presumably because cell proliferation was minimal, and therefore radiation damage was not expressed.



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  		FIGURE 2. Analysis of MART-1 mRNA expression in radiation-treated DCs. Irradiated (10 Gy) or nonirradiated DCs were transduced with AdVMART1 at a multiplicity of infection of 100. RT-PCR of MART-1 mRNA was assayed 24 h after transduction. Irradiation had no effect on MART-1 expression (~400 bp). M202, expressing MART-1, was used as a positive control (Ctrl+). The negative control (Ctrl–) was DC without AdVMART1 transduction. M, 100-bp size marker.

 
Irradiation affects protective antitumor immunity induced by AdVMART1/DC vaccination

Because irradiation of DC abrogated their ability to generate MART-specific immunity, we investigated whether this would translate into loss of protection against tumor growth. We have previously shown that AdVMART1/DC immunization of mice prevented or slowed growth of B16 melanoma cells (25) due to the fact that B16 melanoma cells express mMART, which has 68.8% homology with hMART (38). Injection of AdVMART1/DC completely protected mice against B16 challenge (Fig. 3). Irradiated (10 Gy) AdVMART1/DC had lost this protective ability. Tumors grew at a similar rate as in control unimmunized or in mice injected with untransduced DCs.



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  		FIGURE 3. Irradiation of DCs abrogates AdVMART1-induced protection of mice against growth of B16 melanoma cells. C57BL/6 mice were injected once with 5 x 105 irradiated (10 Gy) or nonirradiated DCs transduced with AdVMART1. A total of 106 B16 cells was injected 10–14 days after DC immunization. Tumor size was measured three times per week. Results are the mean volume ± 1 SEM of five mice in each group.

 
Effect of irradiation on expression of DC surface costimulatory molecules

A possible explanation for the loss of DC function following irradiation would be decreased expression of MHC and/or costimulatory molecules. To test this hypothesis, DCs were irradiated (day 8) and analyzed by flow cytometry for MHC class I, MHC class II, CD80, and CD86 expression. In a series of six experiments, MHC class I expression was not changed 24 h after irradiation. CD86 and MHC class II expression was marginally reduced by 2 or 10 Gy irradiation whether mean fluorescence (Fig. 4A) or percentage of positive cells (Fig. 4B) was the endpoint. Similar results were found when we analyzed expression 48 h after irradiation (data not shown). In addition, there was no significant change in expression of any of the molecules immediately after irradiation (data not shown).



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  		FIGURE 4. Flow cytometric analyses of DC costimulatory molecules. DCs were harvested (day 8), irradiated, and incubated for 24 h. Flow cytometric analysis of MHC I (H-2Kb), MHC II (I-Ab), CD80, and CD86 molecules is shown as mean florescence (A) and percentage of positive cells (B). In another experiment, DCs were irradiated on day 6, harvested on day 8, and treated with 0.1 µg/ml CD40L and 0.1 µg/ml IFN-{gamma} for 48 h. Flow cytometric analyses of mean fluorescence (C) and percentage of positive stained cells (D) are shown using FITC-conjugated anti-MHC I and II, CD40 and CD83 Abs, and PE-conjugated anti-CD80 and anti-CD86 Abs.

 
Another possible mechanism for radiation-induced loss in DC function would be failure of DCs to mature after irradiation. To test this hypothesis, DCs were cultured from bone marrow cells in the presence of GM-CSF and IL-4, but on day 6, cells were treated with 10 Gy or left unirradiated. We chose day 6 because it has been shown that a small portion of mature DC appears on day 8 (39) and to be better able to assess any effect of irradiation on the maturation process. Maturation was induced by addition of 0.1 µg/ml CD40L and 0.1 µg/ml IFN-{gamma} on day 8. Expression of costimulatory molecules was assessed 48 h later as mean fluorescence (Fig. 4C) and percentage of positivity (Fig. 4D). In a series of four experiments, CD40L and IFN-{gamma} treatment of unirradiated DCs induced expression of MHC class I, MHC class II, and CD86. Irradiated DCs had marginally decreased expression of MHC class II, CD83, and CD86 in the absence of maturation signals, in agreement with the results in Fig. 4, A and B, but irradiation did not blunt the response to IFN-{gamma} and CD40L.

It can be concluded that although radiation does affect expression of costimulatory molecules with and without the presence of maturation signals, its influence is unlikely to be sufficient to account for the loss in Ag presentation that we observed.

Effect of irradiation on the ability of DCs to present MART-127–35 following exogenous peptide pulsing

We examined whether presentation of exogenously loaded Ag would also be affected by DC irradiation. Irradiated and nonirradiated HLA-A2.1/Kb DCs were pulsed with MART-127–35 peptide. We have shown previously that this strategy could be used to generate tumor-specific MHC class I-restricted T cell responses in both mice and humans (40, 41). In contrast to the irradiation effects on the Ag presentation ability of AdVMART1/DC, in three independent experiments, ELISPOT assays of spleen cells from immunized HLA-A2.1/Kb transgenic mice showed that irradiation of DCs resulted in a significant increase in MART-127–35-specific IFN-{gamma}-producing T cell responses over nonirradiated DCs (p < 0.001; t test) (Fig. 5). In this experiment, there was a high background nonspecific response in irradiated DC-injected group, which is not uncommon in DC-injected mice; however, the difference between the MART-1-specific and nonspecific response was significant in each experiment (p < 0.05; t test).



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  		FIGURE 5. Irradiation enhances the ability of MART-127–35-pulsed DCs to generate MART-specific lymphocyte responses. HLA-A2/Kb mice were unimmunized or immunized (s.c.) with 5 x 105 irradiated (10 Gy) or nonirradiated DCs that had been pulsed with MART-127–35. Splenocytes were harvested after 10–14 days and restimulated in vitro with MART-127–35 (), or nonspecific {alpha}-feto-protein485–493 peptide ({square}), or left without restimulation (control; {blacksquare}). ELISPOT assays were performed to assess the frequency of splenocytes producing IFN-{gamma} at 48 h. MART-1-specific responses from mice immunized with irradiated DC were increased significantly compared with the nonirradiated DC group (p < 0.001; t test). In each DC-injected group, the number of MART-127–35-specific IFN-{gamma} spots was higher than that of nonspecific stimulation (*, p < 0.05 and **, p < 0.01; t test). Results are the mean spots ± 1 SEM of triplicate data of one representative of three independent experiments.

 
Generation of a B16-A2/Kb cell line

The effect of irradiation on the ability of peptide-pulsed DCs to present the HLA-A2.1-restricted epitope MART-127–35 and to generate protective antitumor immunity was next evaluated. To do this, we first transfected the poorly immunogenic B16 murine melanoma tumor with the gene for the chimeric MHC class I HLA-A2 molecule (A2/Kb). Flow cytometric evaluation identified stable transfectants of B16 expressing A2/Kb (referred to as B16-A2/Kb). Greater than 90% of cells were A2/Kb positive as compared with untransfected cells (data not shown). A2/Kb expression remained constant for at least 1 mo without G418 selection, although as a precaution cells were normally maintained in G418. Compared with the growth of B16 in C57BL/6 mice, B16-A2/Kb tumors in HLA-A2/Kb mice were slower to develop and take was more variable, but growth was progressive and relatively fast in those mice that did develop tumors (data not shown).

Irradiation enhances the ability of MART-127–35 peptide-pulsed DCs to protect mice against challenge with B16-A2/Kb tumors

Mice were immunized twice with irradiated or nonirradiated DCs pulsed with MART-127–35 peptide. Ten days after the last immunization, animals were challenged s.c. with viable B16-A2/Kb tumor cells. Mice injected with irradiated (10 Gy), peptide-pulsed DCs showed greater tumor growth delay than comparable mice injected with MART-127–35-pulsed DCs or unimmunized mice (Fig. 6). This in vivo protection study together with the previous data on the immune responses of mice injected with irradiated and MART-1-pulsed DCs (Fig. 5) confirm irradiation enhances the ability of peptide-pulsed DCs to stimulate immunity.



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  		FIGURE 6. Irradiation of DCs before pulsing with MART-127–35 enhances their ability to generate protective immunity against B16-A2/Kb tumor challenge. HLA-A2/Kb mice were immunized twice at a weekly interval with or without 10 Gy irradiated MART-127–35-pulsed DCs. Ten days after the last immunization, mice were challenged with viable B16-A2/Kb tumor cells. The percentage of mice that were tumor free with time is presented as a Kaplan-Meier plot (p < 0.0001 between the groups; log rank test).

 
Irradiation affects DC proteasome activities

The proteasome plays an important role in protein degradation and the generation of immunogenic peptides for MHC class I presentation. Because we have shown that irradiation inhibits proteasome function in tumor cells (21), we hypothesized this might also occur after irradiation of DC, and this might account for the loss of the ability of irradiated AdVMART1/DCs to present Ag. To investigate this hypothesis, we extracted proteasomes from DCs after treatment with 10 Gy radiation and/or 100 nM reversible proteasome inhibitor, PS-341 (3 h). Proteasomal activities were analyzed by fluorogenic assay. In repeated experiments, proteasome activity was down-regulated in DCs treated with irradiation and/or 100 nM PS-341 (Fig. 7). Radiation-induced inhibition was not complete, but varied ~50–60% of normal in keeping with findings from our other studies (21). This is also true for combined treatment of irradiation and PS-341, which did not further inhibit proteasome function.



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  		FIGURE 7. Irradiation (10 Gy) of DCs inhibits proteasome activity, as does treatment with the reversible proteasome inhibitor, PS-341, or combined treatment of 100 nM PS-341 (3 h), followed by irradiation. Chymotrypsin-like activity was measured in cellular extracts by the rate of degradation of fluorogenic substrate. Results are shown as percentage of relative fluorescence units of corresponding control cells.

 
To determine whether proteasome inhibition could modulate Ag presentation by the endogenous pathway and exogenously loaded DCs in the same way as was seen for radiation, DCs were treated with 100 nM PS-341 for 3 h before AdVMART1 transduction or MART-127–35 peptide pulsing and injected into wild-type or HLA-A2.1/Kb transgenic mice as before. Lymphocytes expressing IFN-{gamma}, as measured by ELISPOT, were >50% reduced if the DCs had been treated with PS-341 before AdVMART1 transduction (Fig. 8A). In contrast, the number of IFN-{gamma}-producing lymphocytes in spleens of mice immunized with PS-341-treated MART-1-pulsed DCs was increased compared with untreated DCs (Fig. 8B). Taken together, these results suggest that inhibition of proteasome function with PS-341 affects the ability of DCs to present peptides in a manner similar to irradiation, and this depends on how the Ags are generated.



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  		FIGURE 8. Proteasome inhibition by PS-341 affects the ability of DCs to generate MART-1-specific lymphocyte responses. A, C57BL/6 mice were immunized twice with DC treated with 100 nM PS-341 for 3 h before AdVMART1 transduction or were left unimmunized. DCs (5 x 105) were injected s.c. into C57BL/6 mice twice with a weekly interval. Splenocytes were harvested 10–14 days after the last immunization and were restimulated in vitro for 48 h with EL4 ({square}), or EL4(MART-1) (), or without restimulation (control; {blacksquare}). IFN-{gamma}-producing lymphocytes were detected by ELISPOT (*, p < 0.05; t test). B, HLA-A2/Kb mice were unimmunized or immunized (s.c.) with 5 x 105 irradiated (10 Gy) or nonirradiated DC that had been pulsed with MART-127–35. Splenocytes were harvested after 10–14 days and restimulated in vitro with MART-127–35 (), or nonspecific TYP1–9 peptide ({square}), or left without restimulation (control; {blacksquare}). ELISPOT assays were performed to assess the frequency of splenocytes producing IFN-{gamma} at 48 h (**, p < 0.01; t test). Results are the mean number of spots ± 1 SEM of triplicate data of one representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The immunosuppressive effects of ionizing radiation are well known and are generally ascribed to killing of radiosensitive lymphocytes. In contrast, even local radiotherapy can be immunosuppressive (42, 43), leading one to wonder whether mechanisms other than cell death might participate. Furthermore, there is little evidence that radiation-induced cell death translates efficiently into the development of tumor-specific immunity, suggesting Ag presentation might be compromised, although few studies have examined this topic in any depth. In this study, we describe the effects of irradiation on DC function as it relates to MHC class I-restricted Ag presentation. DCs are not particularly sensitive to the cytotoxic effects of radiation, as is shown in this study, presumably because they are largely nonproliferative and, unlike lymphocytes, are relatively resistant to radiation-induced apoptosis.

We have shown that radiation can have divergent effects on DC function depending on the pathway that is being used. Immune responses to hMART-1 produced endogenously from an adenoviral vector were decreased in DCs irradiated before transduction, as was their ability to generate protective immunity against tumor growth. We are not aware of many studies on the effect of ionizing radiation on Ag presentation. However, markers of Langerhans cells, which are skin DCs, have been reported to be lacking late after irradiation in humans (3) and mice (44). Several mechanisms could be responsible, but the finding that low dose irradiation of allografts often decreases their immunogenicity (45, 46) is hard to explain other than by an effect on functional Ag presentation by passenger leukocytes.

We found that expression of MHC class II and CD86 on DCs decreased marginally over a 24-h culture period after irradiation, although viability was not significantly affected. Maturation of DCs under the influence of CD40L and IFN-{gamma} was also not greatly affected after irradiation. Radiation-induced alteration in MHC class I molecule expression has been reported in the B16 melanoma cell line. H-2Db expression was enhanced, implying that irradiated tumor cells may be more susceptible to MHC class I-restricted CTLs (47). The costimulatory molecule CD80 (B7.1) was also found to be up-regulated by radiation of B cell lymphoma (48) and myeloid leukemic cells (49). We observed no consistent change in MHC class I, CD80, or CD40 expression by DCs after irradiation in four separate experiments and under a variety of experimental conditions. The discrepancies may be due to differences in the pathways activated by radiation in different cell types.

Radiation has been reported to alter tumor Ag expression. Ciernik et al. (50) showed ionizing radiation enhanced killing of fibrosarcoma cells by increasing expression of tumor-associated mutant p53 epitopes, although the mechanistic basis for this effect is unclear. Our attempts to measure MART-1 expression by AdVMART1/DCs by immunohistochemistry showed no effect of irradiation. In short, we have evidence that radiation can affect DC phenotype, but the magnitude of the effects is small and unlikely to account for loss of the ability of irradiated DCs to present endogenous Ag.

In addition, perhaps the best argument against phenotypic changes being responsible for the observed immunosuppressive effect of radiation is the finding that presentation of an MHC class I-restricted exogenous peptide was enhanced in irradiated DC, as was the ability of these DCs to generate protective immunity. The fact that irradiation of DCs enhanced responses to exogenous MART-127–35 peptide Ag and decreased responses to endogenously processed MART-1 argues strongly against a radiation-induced deficit in presentation per se and the phenotypic changes being critical to the observed altered responsiveness. Furthermore, it suggests that radiation switches the direction of immunity away from endogenous Ag processing toward exogenous peptide presentation pathways.

Our working hypothesis is that the mechanism underlying these effects is at the level of the proteasome. Proteolytic cleavage by the proteasome is in most cases essential for production of antigenic peptides for loading onto MHC class I molecules. Several studies have demonstrated that proteasome inhibitors block proteasome degradation processes, inhibiting MHC class I Ag presentation (51, 52), something we demonstrate in this study for AdVMART1 using the proteasome chymotryptic inhibitor PS-341. We have shown that the proteasome is a direct target for radiation in tumor cells, resulting in a decrease in function by ~50–60% (21). In this study, we show the same for DCs following irradiation, and it seems highly possible that this affects endogenous Ag processing. In keeping with the suggestion that the proteasome is involved in the effects we observed of irradiation on DC function, PS-341 had the same enhancing effect on presentation of MART-127–35 peptide by pulsed DCs as did radiation. Although the hypothesis that radiation affects Ag presentation by affecting proteasome function has yet to be proved, it seems possible that proteasome inhibition could enhance peptide presentation by preventing processing of endogenous self Ags, freeing MHC class I molecules for more efficient exogenous peptide loading. Wiertz et al. (53) showed that the proteasome inhibitor, lactacystin, interfered with the recycling of MHC class I molecules. It seems possible that by inhibiting proteasome activity and the recycling of MHC class I molecules, irradiation might increase the t1/2 of peptide/MHC class I complexes on the cell surface.

The major argument against the proteasome hypothesis is the fact that we have shown that inhibition of proteasome functions at doses as low as 25 cGy (21). We have no data on the effect of such low doses of radiation on DC function; however, our preliminary data suggest that, while a single clinically relevant (2 Gy) fraction of radiation enhances peptide presentation, less consistent effects are seen with endogenously processed Ag. Further studies over a wider range of single and fractionated doses might elucidate whether the dose-response relationships for effects on proteasome inhibition and Ag presentation are similar. Alternative hypotheses might involve radiation-induced costimulatory cytokine molecules such as TNF-{alpha} (54, 55), IL-1 (56), IL-6 (57, 58), and IL-10 (59).

These studies add further evidence to the emerging notion that radiation has much more to offer than being a powerful cytotoxic agent. It has profound effects in changing cellular biology. Radiation has definitive immunodulatory properties and is a potential adjuvant for cancer immunotherapy, if we knew how to best harness its cytotoxic potential to the generation of tumor-specific immunity. Several studies with tumor irradiation followed by DC administration showed enhanced antitumor effects in mouse models (60, 61). Within an appropriate danger milieu, DC vaccines might recruit tumor Ags more efficiently, and local infiltrating DCs might up-regulate their abilities for Ag processing and presentation following irradiation treatment. DCs may serve as targets for immunomodulation in combination with radiotherapy for cancer. One might expect that providing more effective danger signals than is provided by radiation (62, 63, 64, 65) alone might overcome some of its inhibitory effects and increase the generation of immunity by tumor cells, allowing increased control of local and micrometastatic disease. An appropriate understanding of the effect of radiation on DC function may therefore allow for new strategies in cancer therapy.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Institutes of Health/National Cancer Institute Grants RO1 CA87887:01A1 and RO1 CA101752:01. Back

2 Address correspondence and reprint requests to Dr. William H. McBride, Department of Radiation Oncology, School of Medicine, University of California, B3-109 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095-1714. E-mail address: wmcbride{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; AdV, adenovirus; h, human; MART, melanoma Ag recognized by T cells. Back

Received for publication February 19, 2004. Accepted for publication June 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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