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Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus

Presentation of adenoviral antigens required JNK expression and activation.(a) JNK wt and JNK1/2-/- MEFs were mock-infected or infected with AdWT (100 MOI) for 48 h and incubated with two combined sets of anti-adenoviral antibodies (a blend of adenoviral coat proteins and adenoviral fiber). They were then incubated with APC-conjugated secondary antibody and analyzed by flow cytometry. IgG isotype was used as a control. Propidium iodide was used to assess cell viability and to analyze live cells. Data represent the percentage of positive cells as mean ± SD. *** P < 0.001 (AdWT-infected JNK1/2-/- MEFs versus AdWT-infected JNK wt MEFs) (unpaired, two-tailed Student t-test). (b) Splenocytes were isolated from naive and Δ24RGD-infected C57BL/6 mice and co-cultured with JNK wt or JNK1/2-/- MEFs that were mock-infected or infected with Δ24RGD adenovirus (100 MOI) for 24 h before co-culture. After 48 h of co-culture, IFN-γ levels (pg/mL) in the conditioned media were assessed by ELISA. Data represent IFN-γ levels (pg/mL) as mean ± SD from three independent co-cultures. ** P < 0.01 (unpaired, two-tailed Student t-test). (c) U87 MG cells were pretreated with DMSO, SP600125 (25 μM), or bafilomycin A1 (BA1, 10 nM) 30 min before infection with Δ24RGD adenovirus (50 MOI) for 48 h. Live cells were incubated with antibodies raised against adenoviral capsid proteins, or IgG isotype as control, and analyzed by flow cytometry. The percentage of APC-positive cells were quantified and represented as mean ± SD (n = 3). * P < 0.05 (percent of positive cells after treatment with AdWT and SP600125 vs. AdWT and BA1) (unpaired, two-tailed Student t-test). (d) Significant increase in the presentation of adenoviral antigens in cells infected with Δ24RGD when rapamycin was added to the cultures. U87 MG cells were mock-infected, infected with Δ24RGD, or infected with Δ24RGD and rapamycin (100 nM/48 h) for 48 h and then examined for adenoviral antigens by flow cytometry.
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pone.0153814.g001: Presentation of adenoviral antigens required JNK expression and activation.(a) JNK wt and JNK1/2-/- MEFs were mock-infected or infected with AdWT (100 MOI) for 48 h and incubated with two combined sets of anti-adenoviral antibodies (a blend of adenoviral coat proteins and adenoviral fiber). They were then incubated with APC-conjugated secondary antibody and analyzed by flow cytometry. IgG isotype was used as a control. Propidium iodide was used to assess cell viability and to analyze live cells. Data represent the percentage of positive cells as mean ± SD. *** P < 0.001 (AdWT-infected JNK1/2-/- MEFs versus AdWT-infected JNK wt MEFs) (unpaired, two-tailed Student t-test). (b) Splenocytes were isolated from naive and Δ24RGD-infected C57BL/6 mice and co-cultured with JNK wt or JNK1/2-/- MEFs that were mock-infected or infected with Δ24RGD adenovirus (100 MOI) for 24 h before co-culture. After 48 h of co-culture, IFN-γ levels (pg/mL) in the conditioned media were assessed by ELISA. Data represent IFN-γ levels (pg/mL) as mean ± SD from three independent co-cultures. ** P < 0.01 (unpaired, two-tailed Student t-test). (c) U87 MG cells were pretreated with DMSO, SP600125 (25 μM), or bafilomycin A1 (BA1, 10 nM) 30 min before infection with Δ24RGD adenovirus (50 MOI) for 48 h. Live cells were incubated with antibodies raised against adenoviral capsid proteins, or IgG isotype as control, and analyzed by flow cytometry. The percentage of APC-positive cells were quantified and represented as mean ± SD (n = 3). * P < 0.05 (percent of positive cells after treatment with AdWT and SP600125 vs. AdWT and BA1) (unpaired, two-tailed Student t-test). (d) Significant increase in the presentation of adenoviral antigens in cells infected with Δ24RGD when rapamycin was added to the cultures. U87 MG cells were mock-infected, infected with Δ24RGD, or infected with Δ24RGD and rapamycin (100 nM/48 h) for 48 h and then examined for adenoviral antigens by flow cytometry.

Mentions: Pathogen-derived antigens may be processed in the autophagolysosomal compartment via autophagy,[10] but the role of autophagy in the presentation of adenoviral epitopes has not yet been determined. With the use of a cocktail of anti-adenoviral protein antibodies, we screened infected cells for the presence of adenovirus-derived peptides on the surface of live, nonpermeabilized, infected cells. For these studies, we took advantage of the fact that JNK- cells are deficient for autophagy. [15, 16] We observed that whereas the majority (>77%) of JNK wt MEFs infected with AdWT expressed adenoviral proteins on their surface, as detected by the FACS analyses (Fig 1A), the genetic ablation of JNK1 and JNK2 isoforms resulted in a significant decrease in the percentage of cells testing positive for adenoviral proteins. Because T-cell activation, evidenced by the synthesis and secretion of IFN-γ, is the hallmark method for confirming antigen presentation through interactions between epitope-containing MHC molecules and the T-cell receptor,[26] we substantiated the immune relevance of our data by quantifying the secretion of IFN-γ by splenocytes from uninfected mice (naïve) or adenovirus-treated mice (primed) in co-culture with JNK wt or JNK1/2-/- MEFs infected with adenovirus. As expected, co-cultures of naïve splenocytes with adenovirus-infected JNK wt, or JNK1/2-/- cells did not elicit significant secretion of IFN-γ. However, wild-type MEFs infected with adenovirus prompted IFN-γ production when co-cultured with primed splenocytes (Fig 1B). In contrast, cultures of autophagy-deficient JNK1/2-/- MEFs with adenovirus-primed splenocytes contained significantly lower secreted IFN-γ levels (Fig 1B). These data showed that autophagy-impaired cells are deficient for the presentation of adenovirus-derived antigens to the immune system.


Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Presentation of adenoviral antigens required JNK expression and activation.(a) JNK wt and JNK1/2-/- MEFs were mock-infected or infected with AdWT (100 MOI) for 48 h and incubated with two combined sets of anti-adenoviral antibodies (a blend of adenoviral coat proteins and adenoviral fiber). They were then incubated with APC-conjugated secondary antibody and analyzed by flow cytometry. IgG isotype was used as a control. Propidium iodide was used to assess cell viability and to analyze live cells. Data represent the percentage of positive cells as mean ± SD. *** P < 0.001 (AdWT-infected JNK1/2-/- MEFs versus AdWT-infected JNK wt MEFs) (unpaired, two-tailed Student t-test). (b) Splenocytes were isolated from naive and Δ24RGD-infected C57BL/6 mice and co-cultured with JNK wt or JNK1/2-/- MEFs that were mock-infected or infected with Δ24RGD adenovirus (100 MOI) for 24 h before co-culture. After 48 h of co-culture, IFN-γ levels (pg/mL) in the conditioned media were assessed by ELISA. Data represent IFN-γ levels (pg/mL) as mean ± SD from three independent co-cultures. ** P < 0.01 (unpaired, two-tailed Student t-test). (c) U87 MG cells were pretreated with DMSO, SP600125 (25 μM), or bafilomycin A1 (BA1, 10 nM) 30 min before infection with Δ24RGD adenovirus (50 MOI) for 48 h. Live cells were incubated with antibodies raised against adenoviral capsid proteins, or IgG isotype as control, and analyzed by flow cytometry. The percentage of APC-positive cells were quantified and represented as mean ± SD (n = 3). * P < 0.05 (percent of positive cells after treatment with AdWT and SP600125 vs. AdWT and BA1) (unpaired, two-tailed Student t-test). (d) Significant increase in the presentation of adenoviral antigens in cells infected with Δ24RGD when rapamycin was added to the cultures. U87 MG cells were mock-infected, infected with Δ24RGD, or infected with Δ24RGD and rapamycin (100 nM/48 h) for 48 h and then examined for adenoviral antigens by flow cytometry.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836716&req=5

pone.0153814.g001: Presentation of adenoviral antigens required JNK expression and activation.(a) JNK wt and JNK1/2-/- MEFs were mock-infected or infected with AdWT (100 MOI) for 48 h and incubated with two combined sets of anti-adenoviral antibodies (a blend of adenoviral coat proteins and adenoviral fiber). They were then incubated with APC-conjugated secondary antibody and analyzed by flow cytometry. IgG isotype was used as a control. Propidium iodide was used to assess cell viability and to analyze live cells. Data represent the percentage of positive cells as mean ± SD. *** P < 0.001 (AdWT-infected JNK1/2-/- MEFs versus AdWT-infected JNK wt MEFs) (unpaired, two-tailed Student t-test). (b) Splenocytes were isolated from naive and Δ24RGD-infected C57BL/6 mice and co-cultured with JNK wt or JNK1/2-/- MEFs that were mock-infected or infected with Δ24RGD adenovirus (100 MOI) for 24 h before co-culture. After 48 h of co-culture, IFN-γ levels (pg/mL) in the conditioned media were assessed by ELISA. Data represent IFN-γ levels (pg/mL) as mean ± SD from three independent co-cultures. ** P < 0.01 (unpaired, two-tailed Student t-test). (c) U87 MG cells were pretreated with DMSO, SP600125 (25 μM), or bafilomycin A1 (BA1, 10 nM) 30 min before infection with Δ24RGD adenovirus (50 MOI) for 48 h. Live cells were incubated with antibodies raised against adenoviral capsid proteins, or IgG isotype as control, and analyzed by flow cytometry. The percentage of APC-positive cells were quantified and represented as mean ± SD (n = 3). * P < 0.05 (percent of positive cells after treatment with AdWT and SP600125 vs. AdWT and BA1) (unpaired, two-tailed Student t-test). (d) Significant increase in the presentation of adenoviral antigens in cells infected with Δ24RGD when rapamycin was added to the cultures. U87 MG cells were mock-infected, infected with Δ24RGD, or infected with Δ24RGD and rapamycin (100 nM/48 h) for 48 h and then examined for adenoviral antigens by flow cytometry.
Mentions: Pathogen-derived antigens may be processed in the autophagolysosomal compartment via autophagy,[10] but the role of autophagy in the presentation of adenoviral epitopes has not yet been determined. With the use of a cocktail of anti-adenoviral protein antibodies, we screened infected cells for the presence of adenovirus-derived peptides on the surface of live, nonpermeabilized, infected cells. For these studies, we took advantage of the fact that JNK- cells are deficient for autophagy. [15, 16] We observed that whereas the majority (>77%) of JNK wt MEFs infected with AdWT expressed adenoviral proteins on their surface, as detected by the FACS analyses (Fig 1A), the genetic ablation of JNK1 and JNK2 isoforms resulted in a significant decrease in the percentage of cells testing positive for adenoviral proteins. Because T-cell activation, evidenced by the synthesis and secretion of IFN-γ, is the hallmark method for confirming antigen presentation through interactions between epitope-containing MHC molecules and the T-cell receptor,[26] we substantiated the immune relevance of our data by quantifying the secretion of IFN-γ by splenocytes from uninfected mice (naïve) or adenovirus-treated mice (primed) in co-culture with JNK wt or JNK1/2-/- MEFs infected with adenovirus. As expected, co-cultures of naïve splenocytes with adenovirus-infected JNK wt, or JNK1/2-/- cells did not elicit significant secretion of IFN-γ. However, wild-type MEFs infected with adenovirus prompted IFN-γ production when co-cultured with primed splenocytes (Fig 1B). In contrast, cultures of autophagy-deficient JNK1/2-/- MEFs with adenovirus-primed splenocytes contained significantly lower secreted IFN-γ levels (Fig 1B). These data showed that autophagy-impaired cells are deficient for the presentation of adenovirus-derived antigens to the immune system.

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus