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A novel therapy for melanoma developed in mice: transformation of melanoma into dendritic cells with Listeria monocytogenes.

Bronchalo-Vicente L, Rodriguez-Del Rio E, Freire J, Calderon-Gonzalez R, Frande-Cabanes E, Gomez-Roman JJ, Fernández-Llaca H, Yañez-Diaz S, Alvarez-Dominguez C - PLoS ONE (2015)

Bottom Line: Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours.Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination.These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Genómica, Proteómica y Vacunas, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain; Servicio de Dermatología, Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain.

ABSTRACT
Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.

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Efficiency of Listeria vaccination of melanoma is mediated by activation of LLOrec specific CD8+ T cells and inhibition of LLOrec specific CD4+ T cells.C57BL/6 female were inoculated i.p. with 5 x 105 B16F10/mice (n = 5) for 7 days and next injected i.p. or not (NT) with 5 x 103 bc/mice of GFP-LMWT strain for 3 additional days. Mice were bled and sacrificed. A, Immune cells plot (left) corresponds with spleens were homogenized and cell populations were analysed by FACS. Results were expressed as the mean of the percentages of positive cells ± SD. LM growth plot (right) corresponds with spleen homogenates examined for CFU in blood-agar plates. Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (P< 0,05). B, Levels of pro-inflammatory cytokines (MCP-1, TNF-alfa, IFN-gamma, IL-6, IL-10, IL-12) were analysed in sera of mice using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P<0,05). C, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 (5 x 105 cells/mice) for 7 days and vaccination with LMWT for 5 days (LMWT-MEL). Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. LLO-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4+FITC-labeled or anti-CD8+APC-labelled and data gated to include histograms show the percentages of LLO-CD4+ and IFN-gamma producers (lower left) and LLO-CD8+ and IFN-gamma producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (p < 0.05). D, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 pre-infected with LMWT (5 x 105 cells/mice) for 7 days. Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. Procedures were performed as in C and results expressed as the mean ± SD (p < 0.05).
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pone.0117923.g003: Efficiency of Listeria vaccination of melanoma is mediated by activation of LLOrec specific CD8+ T cells and inhibition of LLOrec specific CD4+ T cells.C57BL/6 female were inoculated i.p. with 5 x 105 B16F10/mice (n = 5) for 7 days and next injected i.p. or not (NT) with 5 x 103 bc/mice of GFP-LMWT strain for 3 additional days. Mice were bled and sacrificed. A, Immune cells plot (left) corresponds with spleens were homogenized and cell populations were analysed by FACS. Results were expressed as the mean of the percentages of positive cells ± SD. LM growth plot (right) corresponds with spleen homogenates examined for CFU in blood-agar plates. Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (P< 0,05). B, Levels of pro-inflammatory cytokines (MCP-1, TNF-alfa, IFN-gamma, IL-6, IL-10, IL-12) were analysed in sera of mice using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P<0,05). C, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 (5 x 105 cells/mice) for 7 days and vaccination with LMWT for 5 days (LMWT-MEL). Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. LLO-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4+FITC-labeled or anti-CD8+APC-labelled and data gated to include histograms show the percentages of LLO-CD4+ and IFN-gamma producers (lower left) and LLO-CD8+ and IFN-gamma producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (p < 0.05). D, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 pre-infected with LMWT (5 x 105 cells/mice) for 7 days. Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. Procedures were performed as in C and results expressed as the mean ± SD (p < 0.05).

Mentions: To explore the immune responses generated by melanoma therapy with LMWT, we examined the cell populations in the spleens of mice with LMWT therapy (black bars in Fig. 3A). First, we observed that 96% of the inoculated LMWT localized in MHC-II+ cells, 56% CD11c+ DCs and 40% CD11b+ macrophages (GFP-LM black bars in Fig. 3A). These percentages were similar to the cell populations observed in classical LMWT infections (GFP-LM grey bars in Fig. 3A). These results confirmed LMWT tropism for APC. Second, melanoma therapy with LMWT increased significantly the innate responses in the spleens since we observed amplifications on the percentages of activated DCs, MHC-II+CD11c+CD40+CD83+CD86+ positive cells, CD11b+F4/80+ positive macrophages (data not shown) and NK cells of the tumorigenic CD49b+ phenotype (black bars of Fig. 3A in left graphic). Third, we detected 200-fold increases in the levels of MCP-1, TNF-alfa, IFN-gamma and IL-12, cytokines produced after melanoma therapy with LMWT (black bars in Fig. 3B) compared to non-treated mice (white bars in Fig. 3B). Melanoma therapy with LMWT also altered T cell responses, increasing the percentages of CD8+ T cells and decreasing in the percentages of CD4+ T cells (CD4 and CD8 black bars on left plot of Fig. 3A). The percentages of CD4 and CD8 T cells detected after melanoma therapy with LMWT were similar to the percentages observed after a classical LMWT infection, suggesting that T cells were Listeria driven (Fig. 3A). We next examined LLO specific T cell responses induced by melanoma therapy with LMWT since LLO seemed relevant in this melanoma model and in most reported Listeria-based immune-therapies [4, 5, 15, 24]. Therapy of melanoma with LMWT stimulated higher 1.79 ± 0.03 percentages of recombinant LLO (LLOrec)-specific CD8+ cells and IFN-gamma producers and lower 0.4 ± 0.01 percentages of LLOrec-specific CD4+ cells and IFN-gamma producers (Fig. 3C). While a classical LM infection after 5 days produced similar percentages of LLO190–201 specific CD4+ and LLO296–304 specific CD8+ T cell subsets and IFN-gamma producers, 1.05 ± 0.02 and 1.02 ± 0.01 percentages, respectively (S3 Fig., panel A). Increased cytotoxic T cell responses seemed to explain the accelerate LMWT clearance in the spleens (LM growth plot in Fig. 3A). These results strongly suggested that in vivo effects of melanoma therapy with LMWT implies the transformation of melanoma into DC-like cells that stimulate LLO-specific T cell immune responses and caused melanoma regression. To confirm this hypothesis we first infected in vitro melanoma with LMWT and next i.p inoculated mice with these melanoma-infected cells. Five days post-inoculation, we examined recombinant LLOrec-specific CD4+ and CD8+ T cells producing IFN-gamma. Similar to melanoma therapy with LMWT, we observed low 0.37 ± 0.02 percentages of LLOrec-specific CD4+ T cells and expanded 1.32 ± 0.03 percentages of LLOrec-specific CD8+ T cells producing IFN-gamma (Fig. 3C). We also observed decreased 0.03 ± 0.01 percentages of LLO190–201 specific CD4+ and enhanced 1.76 ± 0.03 percentages of LLO296–304 specific CD8+ T cells producing IFN-gamma (S3 Fig., panel B). We also evaluated immune responses to melanoma antigens using a B16F10 extract (MELext). Control melanoma inoculation produced 0.60 ± 0.02 percentages of MELext-specific CD4+ and 0.65 ± 0.03 percentages of MELext-specific CD8+ T cells producing IFN-gamma (legend of S3 Fig., panel C). Pre-infection of melanoma with LMWT and inoculation into mice presented diminished 0.15 ± 0.01 percentages of MELext-specific CD4+, while no alteration of 0.65 ± 0.02 percentages of MELext-specific CD8+ T cells (S3 Fig., panel C). These results suggested that LMWT therapy induces LLO, but not melanoma-specific CD8+ T cell expansion; while decreases both LLO and melanoma-specific CD4+ T cells. We confirmed the LLO specific CD8+ T cell expansion examining the frequencies of LLO296–304 specific CD8+ T cells (Table 3). Melanoma therapies with LMWT increased LLO296–304 frequencies, from 1.75 values of a classical LMWT infection to 2.25–2.50 values (B16F10→LMWT and B16F10-LMWT→NV rows in Table 3). The increase in LLO specific CD8+ T cell responses, correlated with regression of melanoma size observed in mice inoculated with melanoma pre-infected with LMWT or control melanoma (S3 Fig., panel D). This is a widely common mechanism of several pathogenic bacteria used in cancer therapies [1, 4, 25]. In fact, other Listeria-based vaccines against breast cancer function by similar mechanisms [5].


A novel therapy for melanoma developed in mice: transformation of melanoma into dendritic cells with Listeria monocytogenes.

Bronchalo-Vicente L, Rodriguez-Del Rio E, Freire J, Calderon-Gonzalez R, Frande-Cabanes E, Gomez-Roman JJ, Fernández-Llaca H, Yañez-Diaz S, Alvarez-Dominguez C - PLoS ONE (2015)

Efficiency of Listeria vaccination of melanoma is mediated by activation of LLOrec specific CD8+ T cells and inhibition of LLOrec specific CD4+ T cells.C57BL/6 female were inoculated i.p. with 5 x 105 B16F10/mice (n = 5) for 7 days and next injected i.p. or not (NT) with 5 x 103 bc/mice of GFP-LMWT strain for 3 additional days. Mice were bled and sacrificed. A, Immune cells plot (left) corresponds with spleens were homogenized and cell populations were analysed by FACS. Results were expressed as the mean of the percentages of positive cells ± SD. LM growth plot (right) corresponds with spleen homogenates examined for CFU in blood-agar plates. Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (P< 0,05). B, Levels of pro-inflammatory cytokines (MCP-1, TNF-alfa, IFN-gamma, IL-6, IL-10, IL-12) were analysed in sera of mice using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P<0,05). C, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 (5 x 105 cells/mice) for 7 days and vaccination with LMWT for 5 days (LMWT-MEL). Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. LLO-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4+FITC-labeled or anti-CD8+APC-labelled and data gated to include histograms show the percentages of LLO-CD4+ and IFN-gamma producers (lower left) and LLO-CD8+ and IFN-gamma producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (p < 0.05). D, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 pre-infected with LMWT (5 x 105 cells/mice) for 7 days. Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. Procedures were performed as in C and results expressed as the mean ± SD (p < 0.05).
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pone.0117923.g003: Efficiency of Listeria vaccination of melanoma is mediated by activation of LLOrec specific CD8+ T cells and inhibition of LLOrec specific CD4+ T cells.C57BL/6 female were inoculated i.p. with 5 x 105 B16F10/mice (n = 5) for 7 days and next injected i.p. or not (NT) with 5 x 103 bc/mice of GFP-LMWT strain for 3 additional days. Mice were bled and sacrificed. A, Immune cells plot (left) corresponds with spleens were homogenized and cell populations were analysed by FACS. Results were expressed as the mean of the percentages of positive cells ± SD. LM growth plot (right) corresponds with spleen homogenates examined for CFU in blood-agar plates. Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (P< 0,05). B, Levels of pro-inflammatory cytokines (MCP-1, TNF-alfa, IFN-gamma, IL-6, IL-10, IL-12) were analysed in sera of mice using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P<0,05). C, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 (5 x 105 cells/mice) for 7 days and vaccination with LMWT for 5 days (LMWT-MEL). Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. LLO-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4+FITC-labeled or anti-CD8+APC-labelled and data gated to include histograms show the percentages of LLO-CD4+ and IFN-gamma producers (lower left) and LLO-CD8+ and IFN-gamma producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (p < 0.05). D, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 pre-infected with LMWT (5 x 105 cells/mice) for 7 days. Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. Procedures were performed as in C and results expressed as the mean ± SD (p < 0.05).
Mentions: To explore the immune responses generated by melanoma therapy with LMWT, we examined the cell populations in the spleens of mice with LMWT therapy (black bars in Fig. 3A). First, we observed that 96% of the inoculated LMWT localized in MHC-II+ cells, 56% CD11c+ DCs and 40% CD11b+ macrophages (GFP-LM black bars in Fig. 3A). These percentages were similar to the cell populations observed in classical LMWT infections (GFP-LM grey bars in Fig. 3A). These results confirmed LMWT tropism for APC. Second, melanoma therapy with LMWT increased significantly the innate responses in the spleens since we observed amplifications on the percentages of activated DCs, MHC-II+CD11c+CD40+CD83+CD86+ positive cells, CD11b+F4/80+ positive macrophages (data not shown) and NK cells of the tumorigenic CD49b+ phenotype (black bars of Fig. 3A in left graphic). Third, we detected 200-fold increases in the levels of MCP-1, TNF-alfa, IFN-gamma and IL-12, cytokines produced after melanoma therapy with LMWT (black bars in Fig. 3B) compared to non-treated mice (white bars in Fig. 3B). Melanoma therapy with LMWT also altered T cell responses, increasing the percentages of CD8+ T cells and decreasing in the percentages of CD4+ T cells (CD4 and CD8 black bars on left plot of Fig. 3A). The percentages of CD4 and CD8 T cells detected after melanoma therapy with LMWT were similar to the percentages observed after a classical LMWT infection, suggesting that T cells were Listeria driven (Fig. 3A). We next examined LLO specific T cell responses induced by melanoma therapy with LMWT since LLO seemed relevant in this melanoma model and in most reported Listeria-based immune-therapies [4, 5, 15, 24]. Therapy of melanoma with LMWT stimulated higher 1.79 ± 0.03 percentages of recombinant LLO (LLOrec)-specific CD8+ cells and IFN-gamma producers and lower 0.4 ± 0.01 percentages of LLOrec-specific CD4+ cells and IFN-gamma producers (Fig. 3C). While a classical LM infection after 5 days produced similar percentages of LLO190–201 specific CD4+ and LLO296–304 specific CD8+ T cell subsets and IFN-gamma producers, 1.05 ± 0.02 and 1.02 ± 0.01 percentages, respectively (S3 Fig., panel A). Increased cytotoxic T cell responses seemed to explain the accelerate LMWT clearance in the spleens (LM growth plot in Fig. 3A). These results strongly suggested that in vivo effects of melanoma therapy with LMWT implies the transformation of melanoma into DC-like cells that stimulate LLO-specific T cell immune responses and caused melanoma regression. To confirm this hypothesis we first infected in vitro melanoma with LMWT and next i.p inoculated mice with these melanoma-infected cells. Five days post-inoculation, we examined recombinant LLOrec-specific CD4+ and CD8+ T cells producing IFN-gamma. Similar to melanoma therapy with LMWT, we observed low 0.37 ± 0.02 percentages of LLOrec-specific CD4+ T cells and expanded 1.32 ± 0.03 percentages of LLOrec-specific CD8+ T cells producing IFN-gamma (Fig. 3C). We also observed decreased 0.03 ± 0.01 percentages of LLO190–201 specific CD4+ and enhanced 1.76 ± 0.03 percentages of LLO296–304 specific CD8+ T cells producing IFN-gamma (S3 Fig., panel B). We also evaluated immune responses to melanoma antigens using a B16F10 extract (MELext). Control melanoma inoculation produced 0.60 ± 0.02 percentages of MELext-specific CD4+ and 0.65 ± 0.03 percentages of MELext-specific CD8+ T cells producing IFN-gamma (legend of S3 Fig., panel C). Pre-infection of melanoma with LMWT and inoculation into mice presented diminished 0.15 ± 0.01 percentages of MELext-specific CD4+, while no alteration of 0.65 ± 0.02 percentages of MELext-specific CD8+ T cells (S3 Fig., panel C). These results suggested that LMWT therapy induces LLO, but not melanoma-specific CD8+ T cell expansion; while decreases both LLO and melanoma-specific CD4+ T cells. We confirmed the LLO specific CD8+ T cell expansion examining the frequencies of LLO296–304 specific CD8+ T cells (Table 3). Melanoma therapies with LMWT increased LLO296–304 frequencies, from 1.75 values of a classical LMWT infection to 2.25–2.50 values (B16F10→LMWT and B16F10-LMWT→NV rows in Table 3). The increase in LLO specific CD8+ T cell responses, correlated with regression of melanoma size observed in mice inoculated with melanoma pre-infected with LMWT or control melanoma (S3 Fig., panel D). This is a widely common mechanism of several pathogenic bacteria used in cancer therapies [1, 4, 25]. In fact, other Listeria-based vaccines against breast cancer function by similar mechanisms [5].

Bottom Line: Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours.Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination.These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Genómica, Proteómica y Vacunas, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain; Servicio de Dermatología, Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain.

ABSTRACT
Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.

Show MeSH
Related in: MedlinePlus