Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro

Huixia Cui, Wenlu Zhang, Wei Hu, Kun Liu, Tong Wang, Nan Ma, Xiaohui Liu, Yunpeng Liu, Youhong Jiang, Huixia Cui, Wenlu Zhang, Wei Hu, Kun Liu, Tong Wang, Nan Ma, Xiaohui Liu, Yunpeng Liu, Youhong Jiang

Abstract

Mammaglobin A (MGBA) is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC)-induced tumor antigen specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad) vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs) of two HLA-A33(+) healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA). After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+) CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12), but down-regulated interleukin-10 (IL-10) secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+)CTLs displayed the highest cytotoxicity towards HLA-A33(+)/MGBA(+) breast cancer MDA-MB-415 cells compared with other CD8(+)CTL populations, and compared with the cytotoxicity towards HLA-A33(-)/MGBA(+) breast cancer HBL-100 cells and HLA-A33(-)/MGBA(-) breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+) CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+)/MGBA(+) breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-)/MGBA(+) HBL-100 and HLA-A33(-)/MGBA(-)MDA-MB-231 cells. In addition, killing of CD8(+)CTLs against breast cancer was in a major histocompability complex (MHC)-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity against MGBA(+) breast cancers, which could provide a novel strategy in the immunotherapy of breast cancer.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of MGBA protein detected…
Figure 1. Expression of MGBA protein detected by Western blot in breast cancer cell lines.
The 18 KD MGBA protein was detected in MDA-MB-415 and HBL-100, but not in MDA-MB-231.
Figure 2. FACS analysis of the changed…
Figure 2. FACS analysis of the changed gene expressions in different DC populations.
A-H displays the data of from one volunteer. (A) 2 day culture; (B) 5 day culture; (C) 7 day culture (not stimulated); (D) 7 day culture and 48 h after Ad-MGBA transfection; (E)7 day culture and 48 h after TNF-α addition; (F) 7 day culture, 72 h after recombinant MGBA protein-pulse and 48 h after TNF-α addition; (G) 7 day culture and 48 h after Ad-null transfection and TNF-α addition; (H) 7 day culture and 48 h after Ad-MGBA transfection and TNF-α addition; (I) The histogram shows the data of six independent experiments from two volunteers as mean ± SE. The cultured DCs were collected and stained with PE anti-CD80, APC anti-CD83, FITC anti-CD86, and Percp anti-HLA-DR antibody, respectively. Isotype control for each group is indicated by blue/grey. * indicates that this group has statistically significant differences compared to the others (p

Figure 3. Western blot detection of MGBA…

Figure 3. Western blot detection of MGBA protein expression in DCs after Ad-MGBA viral infection.

Figure 3. Western blot detection of MGBA protein expression in DCs after Ad-MGBA viral infection.
DCs were grown and infected with Ad-MGBA or Ad-null at an MOI of 200 for 48 h. Total cellular protein was extracted and subjected to Western blot analysis of MGBA protein expression. The expression of the MGBA protein was detected in Ad-MGBA-transfected DCs (Lane 4), while there was no expression of the MGBA protein in DCs infected with Ad-null (Lane 1) and non-transfected DCs (Lane 2). Lane 3, MGBA protein expressing MDA-MB-415 cells (as a positive control).

Figure 4. ELISA detection of IL-10 and…

Figure 4. ELISA detection of IL-10 and IL-12 expression in DCs.

The supernatants of 7-day-old…

Figure 4. ELISA detection of IL-10 and IL-12 expression in DCs.
The supernatants of 7-day-old DCs (5×105/ml) including DCs without any stimulation, DCs transfected only with Ad-MGBA, DCs added with TNF-α, DCs added with Ad-null and TNF-α, DCs added with recombinant MGBA protein and TNF-α, DCs added with Ad-MGBA and TNF-α were collected and assayed in triplicate for levels of IL-10 and IL-12. * indicates that this group has statistically significant differences compared to the others, directed by a short line (p<0.05).

Figure 5. The cytotoxic effects of CD8…

Figure 5. The cytotoxic effects of CD8 + CTLs against breast cancer MDA-MB-415 cells.

The five types…

Figure 5. The cytotoxic effects of CD8+CTLs against breast cancer MDA-MB-415 cells.
The five types of CD8+CTLs, i.e., DC-CTL, Ad-null-CTL, MGBAp-CTL, and Ad-MGBA-CTL and Ad-MGBA-CTL (without TNF-α ) were added into MDA-MB-415 cell cultures at an E:T ratio of 10∶1, 20∶1, and 40∶1. 12 h later, breast cancer cell apoptosis rates were analyzed by FACS. The data showed that Ad-MGBA infected DCs-stimulated CD8+CTLs had the highest cytotoxic effect among these five types of CD8+CTLs. Ad-MGBA-CD8+CTL could efficiently lyse MDA-MB-415 cells at an E:T ratio of 20∶1.

Figure 6. The cytotoxic effects of MGBA…

Figure 6. The cytotoxic effects of MGBA specifically stimulated CD8 + CTLs on breast cancer…

Four…

Figure 6. The cytotoxic effects of MGBA specifically stimulated CD8+CTLs on breast cancer cells.
Four CD8+CTLs co-cultured with different DCs treated with TNF-α (i.e., DC-CTL, Ad-null-CTL, MGBAp-CTL, and Ad-MGBA-CTL) and CD8+CTLs co-cultured with DCs transfected only with Ad-MGBA (i.e., Ad-MGBA-CTL without TNF-α ) were added into breast cancer cell cultures at an E:T ratio of 20∶1. 12 h later, apoptosis rates of these breast cancer cells were analyzed by FACS. (A) DC-CD8+CTLs; (B) Ad-null infected DC-CD8+CTLs; (C) MGBAp-CD8+CTL; (D) Ad-MGBA infected DC-CD8+CTLs (without TNF-α); (E) Ad-MGBA infected DC-CD8+CTLs; (F) Non-FITC-CD8-conjugated breast cancer cells in co-culture cells gated; (G) Histogram summarizing the data from six independent experiments from two volunteers. The apoptosis rate in HLA-A33+/MGBA+ MDA-MB 415 cells induced by Ad-MGBA-CD8+CTL was the highest among these five CTLs (p<0.01). * indicates that this group has statistically significant differences compared to the others (p<0.05). ** indicates that this group has statistically significant differences compared to the others (p<0.01).

Figure 7. The cytotoxic effects of MGBA…

Figure 7. The cytotoxic effects of MGBA specifically stimulated CD8 + CTLs on breast cancer…

Figure 7. The cytotoxic effects of MGBA specifically stimulated CD8+CTLs on breast cancer MDA-MB 415 cells and MDA-MB 231 cells by a fluorescence microscope(x 400).
The Ad-MGBA-CD8+CTLs were added into breast cancer MDA-MB 415 cell and MDA-MB 231 cells culture at an E:T ratio of 20∶1. 12 h later, the most of MDA-MB 415 cells appeared apoptosis (B, green color) and necrosis (B, red color) while only a few MDA-MB 231 cells appeared apoptosis and necrosis (D). (A) MDA-MB 415 cells on bright field; (B) MDA-MB 415 cells on fluorescence field; (C) MDA-MB 231 cells on bright field; (D) MDA-MB 231 cells on fluorescence field.

Figure 8. Different CD8 + CTL populations…

Figure 8. Different CD8 + CTL populations were measured for interferon-γ (IFNγ) produce when stimulated…

Figure 8. Different CD8+ CTL populations were measured for interferon-γ (IFNγ) produce when stimulated with three breast cancer cells by ELISPOT assay.
1×105 CD8+ CTLs were cultured in triplicate wells in 100 µl of complete medium containing 1×103 breast cancer cells in 96-well anti-IFNγ antibody precoated plates. After 20 h incubation, IFNγ spots were analyzed. (A) displayed partly spots from a experiment; (B) The histogram shows the data of six independent experiments from two volunteers as mean ± SE. **indicates that this group has statistically significant differences compared to the others (p<0.01). * indicates that there are statistically significant differences compared to the groups without * (p<0.01).
All figures (8)
Figure 3. Western blot detection of MGBA…
Figure 3. Western blot detection of MGBA protein expression in DCs after Ad-MGBA viral infection.
DCs were grown and infected with Ad-MGBA or Ad-null at an MOI of 200 for 48 h. Total cellular protein was extracted and subjected to Western blot analysis of MGBA protein expression. The expression of the MGBA protein was detected in Ad-MGBA-transfected DCs (Lane 4), while there was no expression of the MGBA protein in DCs infected with Ad-null (Lane 1) and non-transfected DCs (Lane 2). Lane 3, MGBA protein expressing MDA-MB-415 cells (as a positive control).
Figure 4. ELISA detection of IL-10 and…
Figure 4. ELISA detection of IL-10 and IL-12 expression in DCs.
The supernatants of 7-day-old DCs (5×105/ml) including DCs without any stimulation, DCs transfected only with Ad-MGBA, DCs added with TNF-α, DCs added with Ad-null and TNF-α, DCs added with recombinant MGBA protein and TNF-α, DCs added with Ad-MGBA and TNF-α were collected and assayed in triplicate for levels of IL-10 and IL-12. * indicates that this group has statistically significant differences compared to the others, directed by a short line (p<0.05).
Figure 5. The cytotoxic effects of CD8…
Figure 5. The cytotoxic effects of CD8+CTLs against breast cancer MDA-MB-415 cells.
The five types of CD8+CTLs, i.e., DC-CTL, Ad-null-CTL, MGBAp-CTL, and Ad-MGBA-CTL and Ad-MGBA-CTL (without TNF-α ) were added into MDA-MB-415 cell cultures at an E:T ratio of 10∶1, 20∶1, and 40∶1. 12 h later, breast cancer cell apoptosis rates were analyzed by FACS. The data showed that Ad-MGBA infected DCs-stimulated CD8+CTLs had the highest cytotoxic effect among these five types of CD8+CTLs. Ad-MGBA-CD8+CTL could efficiently lyse MDA-MB-415 cells at an E:T ratio of 20∶1.
Figure 6. The cytotoxic effects of MGBA…
Figure 6. The cytotoxic effects of MGBA specifically stimulated CD8+CTLs on breast cancer cells.
Four CD8+CTLs co-cultured with different DCs treated with TNF-α (i.e., DC-CTL, Ad-null-CTL, MGBAp-CTL, and Ad-MGBA-CTL) and CD8+CTLs co-cultured with DCs transfected only with Ad-MGBA (i.e., Ad-MGBA-CTL without TNF-α ) were added into breast cancer cell cultures at an E:T ratio of 20∶1. 12 h later, apoptosis rates of these breast cancer cells were analyzed by FACS. (A) DC-CD8+CTLs; (B) Ad-null infected DC-CD8+CTLs; (C) MGBAp-CD8+CTL; (D) Ad-MGBA infected DC-CD8+CTLs (without TNF-α); (E) Ad-MGBA infected DC-CD8+CTLs; (F) Non-FITC-CD8-conjugated breast cancer cells in co-culture cells gated; (G) Histogram summarizing the data from six independent experiments from two volunteers. The apoptosis rate in HLA-A33+/MGBA+ MDA-MB 415 cells induced by Ad-MGBA-CD8+CTL was the highest among these five CTLs (p<0.01). * indicates that this group has statistically significant differences compared to the others (p<0.05). ** indicates that this group has statistically significant differences compared to the others (p<0.01).
Figure 7. The cytotoxic effects of MGBA…
Figure 7. The cytotoxic effects of MGBA specifically stimulated CD8+CTLs on breast cancer MDA-MB 415 cells and MDA-MB 231 cells by a fluorescence microscope(x 400).
The Ad-MGBA-CD8+CTLs were added into breast cancer MDA-MB 415 cell and MDA-MB 231 cells culture at an E:T ratio of 20∶1. 12 h later, the most of MDA-MB 415 cells appeared apoptosis (B, green color) and necrosis (B, red color) while only a few MDA-MB 231 cells appeared apoptosis and necrosis (D). (A) MDA-MB 415 cells on bright field; (B) MDA-MB 415 cells on fluorescence field; (C) MDA-MB 231 cells on bright field; (D) MDA-MB 231 cells on fluorescence field.
Figure 8. Different CD8 + CTL populations…
Figure 8. Different CD8+ CTL populations were measured for interferon-γ (IFNγ) produce when stimulated with three breast cancer cells by ELISPOT assay.
1×105 CD8+ CTLs were cultured in triplicate wells in 100 µl of complete medium containing 1×103 breast cancer cells in 96-well anti-IFNγ antibody precoated plates. After 20 h incubation, IFNγ spots were analyzed. (A) displayed partly spots from a experiment; (B) The histogram shows the data of six independent experiments from two volunteers as mean ± SE. **indicates that this group has statistically significant differences compared to the others (p<0.01). * indicates that there are statistically significant differences compared to the groups without * (p<0.01).

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