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Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

Goudin N, Chappert P, Mégret J, Gross DA, Rocha B, Azogui O - PLoS ONE (2016)

Bottom Line: These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs.These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites.They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Plateau technique de Cytometrie et d'Imagerie Cellulaire, Structure Fédérative de Recherche Necker, INSERM US 24-CNRS, UMS 3633, Paris, France.

ABSTRACT
Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

No MeSH data available.


Related in: MedlinePlus

Treg depletion results in increased infiltration of CD8+CD11c+ effector T cells.(A) Representative dot plots show tumour infiltrating lymphocytes analysed by flow cytometry for cell surface CD4 and CD8β expression gated on CD3 T cells in untreated and treated tumour infiltrates (day 14). (B) Percentages of tumour infiltrating CD8 T cells in untreated (n = 4) and treated (n = 4) mice at different time points after tumour inoculation. (C) Representative dot plots of cell surface expression of CD8+CD11c+ gated on CD8 T cells. (D) Histogram shows frequencies of CD8+CD11c+ T cells in indicated groups of mice (n = 3). (E) Flow cytometry analysing proliferating cells (KI67+) in CD8+CD11c+ and CD8+CD11c- T cells in the indicated groups of mice. A representative experiment of at least two independent experiments is shown, n = 3 mice per group. (F,left) Graph shows percentages of CD8+CD11c+ T cells expressing each gene and sorted from both group of untreated (119 cells) and PC61-treated (107 cells). (F,right) Graph shows percentages of CD8+CD11c+ T cells co-expressing the four genes (right). Data are obtained from two different cell-sorting (G) Frozen tumor sections obtained 14 days after tumor injection from treated and untreated mice were examined by confocal microscopy after staining with CD8β (red), CD11c (green) specific antibodies and Dapi (gray, left panels). Direct photography acquired with a 63x objective show co-staining of CD8 and CD11c antibodies on CD8 T cells. Cell surface co-expression of CD8β and CD11c (circles) and contact between CD8 T cells and dendritic cells (rectangle) are shown in white color. Data are representative of at least two independent experiments. (H) The tumour vasculature was examined by confocal microscopy following staining with CD31 specific antibody. Representative tumour sections from indicated group are shown by (A) 20x magnification; scale bar: 100 μm and (B) 63x magnification; scale bar: 100 μm. These data are representative of three independent experiments. *,p<0.05 **,p<0.001. P values were calculated using Student’s t test and (a) by Fisher’s exact test p<0.05.
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pone.0157822.g004: Treg depletion results in increased infiltration of CD8+CD11c+ effector T cells.(A) Representative dot plots show tumour infiltrating lymphocytes analysed by flow cytometry for cell surface CD4 and CD8β expression gated on CD3 T cells in untreated and treated tumour infiltrates (day 14). (B) Percentages of tumour infiltrating CD8 T cells in untreated (n = 4) and treated (n = 4) mice at different time points after tumour inoculation. (C) Representative dot plots of cell surface expression of CD8+CD11c+ gated on CD8 T cells. (D) Histogram shows frequencies of CD8+CD11c+ T cells in indicated groups of mice (n = 3). (E) Flow cytometry analysing proliferating cells (KI67+) in CD8+CD11c+ and CD8+CD11c- T cells in the indicated groups of mice. A representative experiment of at least two independent experiments is shown, n = 3 mice per group. (F,left) Graph shows percentages of CD8+CD11c+ T cells expressing each gene and sorted from both group of untreated (119 cells) and PC61-treated (107 cells). (F,right) Graph shows percentages of CD8+CD11c+ T cells co-expressing the four genes (right). Data are obtained from two different cell-sorting (G) Frozen tumor sections obtained 14 days after tumor injection from treated and untreated mice were examined by confocal microscopy after staining with CD8β (red), CD11c (green) specific antibodies and Dapi (gray, left panels). Direct photography acquired with a 63x objective show co-staining of CD8 and CD11c antibodies on CD8 T cells. Cell surface co-expression of CD8β and CD11c (circles) and contact between CD8 T cells and dendritic cells (rectangle) are shown in white color. Data are representative of at least two independent experiments. (H) The tumour vasculature was examined by confocal microscopy following staining with CD31 specific antibody. Representative tumour sections from indicated group are shown by (A) 20x magnification; scale bar: 100 μm and (B) 63x magnification; scale bar: 100 μm. These data are representative of three independent experiments. *,p<0.05 **,p<0.001. P values were calculated using Student’s t test and (a) by Fisher’s exact test p<0.05.

Mentions: Similarly to the DLN, the frequency of total CD8 T cells infiltrating the tumours was much higher in treated than untreated mice (Fig 4A and 4B). Among these, the compartment of CD8+CD11c+ T cells increased progressively between days 7–14 after tumour inoculation (Fig 4C and 4D). Moreover, CD8+CD11c+ T cells displayed functional characteristics. Based on the expression of KI67, both CD8+CD11c+ and CD8+CD11c- T cell subsets proliferated twice as much in treated mice compared with untreated mice (Fig 4E), indicating that Treg depletion prevents all tumour infiltrating CD8 T cells from exhaustion. Next we examined the cytotoxic function using a method of single cell RT/PCR multiplex already described [18]. Single-cell RT-PCR is an appropriate tool to resolve « co-expression of the three genes associated with the cytotoxic function » (Grzb, IFNγ, Prf), and to assess the frequency of the cytotoxic T cells in DLNs and at the tumour sites. We observed that the proportion of single cells expressing the cytotoxic associated genes IFNγ, granzyme B (Grzb), and perforin (Prf) genes was similar in both groups at day 7 but however increased in Grzb at day 14 in treated mice. In contrast, co-expression of the three genes was markedly augmented at Day 14 from regressive tumours (Fig 4F).


Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

Goudin N, Chappert P, Mégret J, Gross DA, Rocha B, Azogui O - PLoS ONE (2016)

Treg depletion results in increased infiltration of CD8+CD11c+ effector T cells.(A) Representative dot plots show tumour infiltrating lymphocytes analysed by flow cytometry for cell surface CD4 and CD8β expression gated on CD3 T cells in untreated and treated tumour infiltrates (day 14). (B) Percentages of tumour infiltrating CD8 T cells in untreated (n = 4) and treated (n = 4) mice at different time points after tumour inoculation. (C) Representative dot plots of cell surface expression of CD8+CD11c+ gated on CD8 T cells. (D) Histogram shows frequencies of CD8+CD11c+ T cells in indicated groups of mice (n = 3). (E) Flow cytometry analysing proliferating cells (KI67+) in CD8+CD11c+ and CD8+CD11c- T cells in the indicated groups of mice. A representative experiment of at least two independent experiments is shown, n = 3 mice per group. (F,left) Graph shows percentages of CD8+CD11c+ T cells expressing each gene and sorted from both group of untreated (119 cells) and PC61-treated (107 cells). (F,right) Graph shows percentages of CD8+CD11c+ T cells co-expressing the four genes (right). Data are obtained from two different cell-sorting (G) Frozen tumor sections obtained 14 days after tumor injection from treated and untreated mice were examined by confocal microscopy after staining with CD8β (red), CD11c (green) specific antibodies and Dapi (gray, left panels). Direct photography acquired with a 63x objective show co-staining of CD8 and CD11c antibodies on CD8 T cells. Cell surface co-expression of CD8β and CD11c (circles) and contact between CD8 T cells and dendritic cells (rectangle) are shown in white color. Data are representative of at least two independent experiments. (H) The tumour vasculature was examined by confocal microscopy following staining with CD31 specific antibody. Representative tumour sections from indicated group are shown by (A) 20x magnification; scale bar: 100 μm and (B) 63x magnification; scale bar: 100 μm. These data are representative of three independent experiments. *,p<0.05 **,p<0.001. P values were calculated using Student’s t test and (a) by Fisher’s exact test p<0.05.
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pone.0157822.g004: Treg depletion results in increased infiltration of CD8+CD11c+ effector T cells.(A) Representative dot plots show tumour infiltrating lymphocytes analysed by flow cytometry for cell surface CD4 and CD8β expression gated on CD3 T cells in untreated and treated tumour infiltrates (day 14). (B) Percentages of tumour infiltrating CD8 T cells in untreated (n = 4) and treated (n = 4) mice at different time points after tumour inoculation. (C) Representative dot plots of cell surface expression of CD8+CD11c+ gated on CD8 T cells. (D) Histogram shows frequencies of CD8+CD11c+ T cells in indicated groups of mice (n = 3). (E) Flow cytometry analysing proliferating cells (KI67+) in CD8+CD11c+ and CD8+CD11c- T cells in the indicated groups of mice. A representative experiment of at least two independent experiments is shown, n = 3 mice per group. (F,left) Graph shows percentages of CD8+CD11c+ T cells expressing each gene and sorted from both group of untreated (119 cells) and PC61-treated (107 cells). (F,right) Graph shows percentages of CD8+CD11c+ T cells co-expressing the four genes (right). Data are obtained from two different cell-sorting (G) Frozen tumor sections obtained 14 days after tumor injection from treated and untreated mice were examined by confocal microscopy after staining with CD8β (red), CD11c (green) specific antibodies and Dapi (gray, left panels). Direct photography acquired with a 63x objective show co-staining of CD8 and CD11c antibodies on CD8 T cells. Cell surface co-expression of CD8β and CD11c (circles) and contact between CD8 T cells and dendritic cells (rectangle) are shown in white color. Data are representative of at least two independent experiments. (H) The tumour vasculature was examined by confocal microscopy following staining with CD31 specific antibody. Representative tumour sections from indicated group are shown by (A) 20x magnification; scale bar: 100 μm and (B) 63x magnification; scale bar: 100 μm. These data are representative of three independent experiments. *,p<0.05 **,p<0.001. P values were calculated using Student’s t test and (a) by Fisher’s exact test p<0.05.
Mentions: Similarly to the DLN, the frequency of total CD8 T cells infiltrating the tumours was much higher in treated than untreated mice (Fig 4A and 4B). Among these, the compartment of CD8+CD11c+ T cells increased progressively between days 7–14 after tumour inoculation (Fig 4C and 4D). Moreover, CD8+CD11c+ T cells displayed functional characteristics. Based on the expression of KI67, both CD8+CD11c+ and CD8+CD11c- T cell subsets proliferated twice as much in treated mice compared with untreated mice (Fig 4E), indicating that Treg depletion prevents all tumour infiltrating CD8 T cells from exhaustion. Next we examined the cytotoxic function using a method of single cell RT/PCR multiplex already described [18]. Single-cell RT-PCR is an appropriate tool to resolve « co-expression of the three genes associated with the cytotoxic function » (Grzb, IFNγ, Prf), and to assess the frequency of the cytotoxic T cells in DLNs and at the tumour sites. We observed that the proportion of single cells expressing the cytotoxic associated genes IFNγ, granzyme B (Grzb), and perforin (Prf) genes was similar in both groups at day 7 but however increased in Grzb at day 14 in treated mice. In contrast, co-expression of the three genes was markedly augmented at Day 14 from regressive tumours (Fig 4F).

Bottom Line: These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs.These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites.They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Plateau technique de Cytometrie et d'Imagerie Cellulaire, Structure Fédérative de Recherche Necker, INSERM US 24-CNRS, UMS 3633, Paris, France.

ABSTRACT
Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

No MeSH data available.


Related in: MedlinePlus