Limits...
Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials.

Curtin JF, Candolfi M, Fakhouri TM, Liu C, Alden A, Edwards M, Lowenstein PR, Castro MG - PLoS ONE (2008)

Bottom Line: Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors.We conclude that this approach will be useful in a setting of minimal residual disease.Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT

Background: Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients.

Findings: Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs present in tumors, draining lymph nodes and spleen and improved long-term survival (50% of mice survived >150 days). No improvement in survival was observed when Tregs were depleted 24 days after tumor implantation, suggesting that tumor burden is an important factor for determining efficacy of Treg depletion in clinical trials. In a T cell dependent model of brain tumor regression elicited by intratumoral delivery of adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Herpes Simplex Type 1-Thymidine Kinase (TK) with ganciclovir (GCV), we demonstrate that administration of PC61 24 days after tumor implantation (7 days after treatment) inhibited T cell dependent tumor regression and long term survival. Further, depletion with PC61 completely inhibited clonal expansion of tumor antigen-specific T lymphocytes in response to the treatment.

Conclusions: Our data demonstrate for the first time, that although Treg depletion inhibits the progression/eliminates GBM tumors, its efficacy is dependent on tumor burden. We conclude that this approach will be useful in a setting of minimal residual disease. Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.

Show MeSH

Related in: MedlinePlus

Intracranial GBM tumors are densely infiltrated with immune cells.(A) GL26 tumor volume 7, 14 and 21 days after implantion into the brain striatum of C57BL/6 mice was determined. Reactive astrocytic staining (GFAP+) was used to define the border of the tumor with non-malignant brain tissue. Representative sections of mouse brains bearing tumors implanted 7, 14 and 21 days previously and stained with GFAP are shown on the left. Tumor growth kinetics were essentially exponential over the time period analyzed with a doubling time of approximately 1.8 days. 5 mice were used to determine tumor volume at each time point. (B) Infiltration of immune cells in intracranial GL26 tumors. Brain sections from tumor bearing mice were stained with antibodies against CD45 (leukocytes) or F4/80 (macrophages/activated microglia). Representative Confocal images show immune cells (green) within the tumor mass and in the tumor borders. DAPI (blue) was used to stain nuclei. Yellow arrows indicate immunoreactive cells. T: tumor. (C) Tumor infiltrating immune cells were isolated from tumors 24 days after tumor implantation and analyzed using flow cytometry. (i) Dot plot of CD11c against I-Ab that was first gated for live CD45+ leukocytes. DC (CD11c+ CD45+ I-Ab+) are shown in the red box. (ii) Macrophages (MΦ) were assessed by gating live leukocytes with CD45, then plotting CD11b against I-Ab. The red box outlines the population of tumor infiltrating macrophages (CD11b+ CD45+ I-Ab+). (iii) T cells were stained with CD3ε-PE, CD4-PerCP and CD8a-FITC. The plot displays CD4 against CD8a when cells were first gated for CD3ε+ live leukocytes. CD4+ T cells and CD8a+ T cells are shown in red boxes. (iv) Tumor infiltrating Tregs were observed by staining with CD3ε-PE, CD4-PerCP and Foxp3-FITC. CD3ε+ live leukocytes were gated and dot plots display Foxp3 against CD4 staining. The population of Tregs (CD4+ Foxp3+ CD3ε+) are shown in the red box. (v) NK and NK-T cells were visualized by gating live CD45+ leukocytes, then displaying NK1.1 against CD3ε. The population of tumor infiltrating NK cells (CD3ε- CD45+ NK1.1+) and NK-T cells (CD3ε+ CD45+ NK1.1+) are shown in red boxes. (vi) Tumor infiltrating B cells are visualized by gating for live leukocytes, then plotting CD45 against CD19. The population of B cells (CD19+ CD45+) is shown in a red box. The percentages of each immune cell population infiltrating the tumor with respect to the total number of tumor CD45+ cells is indicated in representative dot plots. (D) Nissl staining was used to visualize tumors in the brain. Tumors are dense in Nissl substance, so stain darker than normal brain tissue. Representative Confocal images show tumor infiltrating CD4+ T cells, CD8+ T cells, CD205+ mDCs, CD19+ B cells, and CD25+ immune cells (seen in green) and DAPI (blue) was used to visualize the nuclei. Yellow arrows indicate immunoreactive cells. (E) Flow cytometric analysis of the percentage of T cells that express CD25 in spleens, dLN and tumors in mice bearing intracranial GL26 brain tumors 24 days after tumor implantation. (F) Representative dot plots of CD25 vs CD3ε in immune cells isolated from the spleen, LN and tumor. The percentages of CD25+ cells population with respect to the total number of CD3ε+ cells in the tumor, draining lymph nodes or spleen are indicated in representative dot plots.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2291560&req=5

pone-0001983-g001: Intracranial GBM tumors are densely infiltrated with immune cells.(A) GL26 tumor volume 7, 14 and 21 days after implantion into the brain striatum of C57BL/6 mice was determined. Reactive astrocytic staining (GFAP+) was used to define the border of the tumor with non-malignant brain tissue. Representative sections of mouse brains bearing tumors implanted 7, 14 and 21 days previously and stained with GFAP are shown on the left. Tumor growth kinetics were essentially exponential over the time period analyzed with a doubling time of approximately 1.8 days. 5 mice were used to determine tumor volume at each time point. (B) Infiltration of immune cells in intracranial GL26 tumors. Brain sections from tumor bearing mice were stained with antibodies against CD45 (leukocytes) or F4/80 (macrophages/activated microglia). Representative Confocal images show immune cells (green) within the tumor mass and in the tumor borders. DAPI (blue) was used to stain nuclei. Yellow arrows indicate immunoreactive cells. T: tumor. (C) Tumor infiltrating immune cells were isolated from tumors 24 days after tumor implantation and analyzed using flow cytometry. (i) Dot plot of CD11c against I-Ab that was first gated for live CD45+ leukocytes. DC (CD11c+ CD45+ I-Ab+) are shown in the red box. (ii) Macrophages (MΦ) were assessed by gating live leukocytes with CD45, then plotting CD11b against I-Ab. The red box outlines the population of tumor infiltrating macrophages (CD11b+ CD45+ I-Ab+). (iii) T cells were stained with CD3ε-PE, CD4-PerCP and CD8a-FITC. The plot displays CD4 against CD8a when cells were first gated for CD3ε+ live leukocytes. CD4+ T cells and CD8a+ T cells are shown in red boxes. (iv) Tumor infiltrating Tregs were observed by staining with CD3ε-PE, CD4-PerCP and Foxp3-FITC. CD3ε+ live leukocytes were gated and dot plots display Foxp3 against CD4 staining. The population of Tregs (CD4+ Foxp3+ CD3ε+) are shown in the red box. (v) NK and NK-T cells were visualized by gating live CD45+ leukocytes, then displaying NK1.1 against CD3ε. The population of tumor infiltrating NK cells (CD3ε- CD45+ NK1.1+) and NK-T cells (CD3ε+ CD45+ NK1.1+) are shown in red boxes. (vi) Tumor infiltrating B cells are visualized by gating for live leukocytes, then plotting CD45 against CD19. The population of B cells (CD19+ CD45+) is shown in a red box. The percentages of each immune cell population infiltrating the tumor with respect to the total number of tumor CD45+ cells is indicated in representative dot plots. (D) Nissl staining was used to visualize tumors in the brain. Tumors are dense in Nissl substance, so stain darker than normal brain tissue. Representative Confocal images show tumor infiltrating CD4+ T cells, CD8+ T cells, CD205+ mDCs, CD19+ B cells, and CD25+ immune cells (seen in green) and DAPI (blue) was used to visualize the nuclei. Yellow arrows indicate immunoreactive cells. (E) Flow cytometric analysis of the percentage of T cells that express CD25 in spleens, dLN and tumors in mice bearing intracranial GL26 brain tumors 24 days after tumor implantation. (F) Representative dot plots of CD25 vs CD3ε in immune cells isolated from the spleen, LN and tumor. The percentages of CD25+ cells population with respect to the total number of CD3ε+ cells in the tumor, draining lymph nodes or spleen are indicated in representative dot plots.

Mentions: Accumulation of Foxp3+ Tregs in human gliomas correlates with the grade of the tumor and patient survival [24]. Accumulation of Foxp3+ Tregs in syngeneic mouse gliomas has also been described and depletion of Tregs with CD25-specific immunoglobulins can induce tumor regression and improve survival in these preclinical models [25], [32], [33]. In order to investigate whether Treg depletion could improve therapeutic outcome in combination with immunotherapy/gene therapy, we first established whether Tregs infiltrated into the syngeneic mouse glioma arising from the intracranial implantation of GL26 cells into the brain striatum. Implantation of syngeneic GL26 cells reproducibly led to the linear growth of tumors (R2>0.99) with a mean tumor volume of approximately 0.5mm3 at day 15 and 30 times larger (15mm3) at day 24 (Fig. 1A). These tumors exhibit profuse infiltration of CD45+ and F4/80+ cells, which are evident throughout the tumor mass and also at the borders (Fig 1B). Immune cell infiltration into the tumors was also assessed by flow cytometry 24 days after tumor implantation (Fig. 1 C). We detected tumor infiltrating mDC (CD11c+ CD45+ I-Ab+, Fig. 1Ci) and MΦ (CD11b+ CD45+ I-Ab+, Fig. 1Cii) that represented 3.5% and 19% of the total number of CD45+ immune cells present in the tumor respectively. Lymphocytes were also identified and constituted ∼80% of the total number of CD45+ immune cells infiltrating the tumors (Fig. 1 C iii-vi). These included CD4+ T cells (CD3ε+ CD4+ CD8a−, 26%), CD8a+ T cells (CD3ε+ CD4− CD8a+, 18%), Foxp3+ Tregs (CD3ε+ CD4+ Foxp3+, 8.4%), NK cells (CD3ε− NK1.1+ CD45+, 18%), NK-T cells (CD3ε+ NK1.1+ CD45+, 7%) and B cells (CD3ε− CD19+ CD45+, 5%). The presence of CD4+, CD8a+, CD19+ lymphocytes as well as CD205 mDC were also observed by immunofluorescence (Fig. 1 D). In addition, large numbers of lymphocytes that were immunoreactive for CD25 were observed within the tumor using confocal microscopy (Fig. 1 D) and flow cytometry (CD3ε+ CD25+ CD45+) (Fig. 1 E, F). Moreover, the percentage of CD3ε+ T cells that expressed cell surface CD25 was ∼40% of the total number of tumor infiltrating T cells and was greatly elevated in the draining (cervical) lymph node (dLN) (p<0.05) and tumor (p<0.01) when compared with the spleen of tumor bearing mice (Fig. 1 E).


Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials.

Curtin JF, Candolfi M, Fakhouri TM, Liu C, Alden A, Edwards M, Lowenstein PR, Castro MG - PLoS ONE (2008)

Intracranial GBM tumors are densely infiltrated with immune cells.(A) GL26 tumor volume 7, 14 and 21 days after implantion into the brain striatum of C57BL/6 mice was determined. Reactive astrocytic staining (GFAP+) was used to define the border of the tumor with non-malignant brain tissue. Representative sections of mouse brains bearing tumors implanted 7, 14 and 21 days previously and stained with GFAP are shown on the left. Tumor growth kinetics were essentially exponential over the time period analyzed with a doubling time of approximately 1.8 days. 5 mice were used to determine tumor volume at each time point. (B) Infiltration of immune cells in intracranial GL26 tumors. Brain sections from tumor bearing mice were stained with antibodies against CD45 (leukocytes) or F4/80 (macrophages/activated microglia). Representative Confocal images show immune cells (green) within the tumor mass and in the tumor borders. DAPI (blue) was used to stain nuclei. Yellow arrows indicate immunoreactive cells. T: tumor. (C) Tumor infiltrating immune cells were isolated from tumors 24 days after tumor implantation and analyzed using flow cytometry. (i) Dot plot of CD11c against I-Ab that was first gated for live CD45+ leukocytes. DC (CD11c+ CD45+ I-Ab+) are shown in the red box. (ii) Macrophages (MΦ) were assessed by gating live leukocytes with CD45, then plotting CD11b against I-Ab. The red box outlines the population of tumor infiltrating macrophages (CD11b+ CD45+ I-Ab+). (iii) T cells were stained with CD3ε-PE, CD4-PerCP and CD8a-FITC. The plot displays CD4 against CD8a when cells were first gated for CD3ε+ live leukocytes. CD4+ T cells and CD8a+ T cells are shown in red boxes. (iv) Tumor infiltrating Tregs were observed by staining with CD3ε-PE, CD4-PerCP and Foxp3-FITC. CD3ε+ live leukocytes were gated and dot plots display Foxp3 against CD4 staining. The population of Tregs (CD4+ Foxp3+ CD3ε+) are shown in the red box. (v) NK and NK-T cells were visualized by gating live CD45+ leukocytes, then displaying NK1.1 against CD3ε. The population of tumor infiltrating NK cells (CD3ε- CD45+ NK1.1+) and NK-T cells (CD3ε+ CD45+ NK1.1+) are shown in red boxes. (vi) Tumor infiltrating B cells are visualized by gating for live leukocytes, then plotting CD45 against CD19. The population of B cells (CD19+ CD45+) is shown in a red box. The percentages of each immune cell population infiltrating the tumor with respect to the total number of tumor CD45+ cells is indicated in representative dot plots. (D) Nissl staining was used to visualize tumors in the brain. Tumors are dense in Nissl substance, so stain darker than normal brain tissue. Representative Confocal images show tumor infiltrating CD4+ T cells, CD8+ T cells, CD205+ mDCs, CD19+ B cells, and CD25+ immune cells (seen in green) and DAPI (blue) was used to visualize the nuclei. Yellow arrows indicate immunoreactive cells. (E) Flow cytometric analysis of the percentage of T cells that express CD25 in spleens, dLN and tumors in mice bearing intracranial GL26 brain tumors 24 days after tumor implantation. (F) Representative dot plots of CD25 vs CD3ε in immune cells isolated from the spleen, LN and tumor. The percentages of CD25+ cells population with respect to the total number of CD3ε+ cells in the tumor, draining lymph nodes or spleen are indicated in representative dot plots.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2291560&req=5

pone-0001983-g001: Intracranial GBM tumors are densely infiltrated with immune cells.(A) GL26 tumor volume 7, 14 and 21 days after implantion into the brain striatum of C57BL/6 mice was determined. Reactive astrocytic staining (GFAP+) was used to define the border of the tumor with non-malignant brain tissue. Representative sections of mouse brains bearing tumors implanted 7, 14 and 21 days previously and stained with GFAP are shown on the left. Tumor growth kinetics were essentially exponential over the time period analyzed with a doubling time of approximately 1.8 days. 5 mice were used to determine tumor volume at each time point. (B) Infiltration of immune cells in intracranial GL26 tumors. Brain sections from tumor bearing mice were stained with antibodies against CD45 (leukocytes) or F4/80 (macrophages/activated microglia). Representative Confocal images show immune cells (green) within the tumor mass and in the tumor borders. DAPI (blue) was used to stain nuclei. Yellow arrows indicate immunoreactive cells. T: tumor. (C) Tumor infiltrating immune cells were isolated from tumors 24 days after tumor implantation and analyzed using flow cytometry. (i) Dot plot of CD11c against I-Ab that was first gated for live CD45+ leukocytes. DC (CD11c+ CD45+ I-Ab+) are shown in the red box. (ii) Macrophages (MΦ) were assessed by gating live leukocytes with CD45, then plotting CD11b against I-Ab. The red box outlines the population of tumor infiltrating macrophages (CD11b+ CD45+ I-Ab+). (iii) T cells were stained with CD3ε-PE, CD4-PerCP and CD8a-FITC. The plot displays CD4 against CD8a when cells were first gated for CD3ε+ live leukocytes. CD4+ T cells and CD8a+ T cells are shown in red boxes. (iv) Tumor infiltrating Tregs were observed by staining with CD3ε-PE, CD4-PerCP and Foxp3-FITC. CD3ε+ live leukocytes were gated and dot plots display Foxp3 against CD4 staining. The population of Tregs (CD4+ Foxp3+ CD3ε+) are shown in the red box. (v) NK and NK-T cells were visualized by gating live CD45+ leukocytes, then displaying NK1.1 against CD3ε. The population of tumor infiltrating NK cells (CD3ε- CD45+ NK1.1+) and NK-T cells (CD3ε+ CD45+ NK1.1+) are shown in red boxes. (vi) Tumor infiltrating B cells are visualized by gating for live leukocytes, then plotting CD45 against CD19. The population of B cells (CD19+ CD45+) is shown in a red box. The percentages of each immune cell population infiltrating the tumor with respect to the total number of tumor CD45+ cells is indicated in representative dot plots. (D) Nissl staining was used to visualize tumors in the brain. Tumors are dense in Nissl substance, so stain darker than normal brain tissue. Representative Confocal images show tumor infiltrating CD4+ T cells, CD8+ T cells, CD205+ mDCs, CD19+ B cells, and CD25+ immune cells (seen in green) and DAPI (blue) was used to visualize the nuclei. Yellow arrows indicate immunoreactive cells. (E) Flow cytometric analysis of the percentage of T cells that express CD25 in spleens, dLN and tumors in mice bearing intracranial GL26 brain tumors 24 days after tumor implantation. (F) Representative dot plots of CD25 vs CD3ε in immune cells isolated from the spleen, LN and tumor. The percentages of CD25+ cells population with respect to the total number of CD3ε+ cells in the tumor, draining lymph nodes or spleen are indicated in representative dot plots.
Mentions: Accumulation of Foxp3+ Tregs in human gliomas correlates with the grade of the tumor and patient survival [24]. Accumulation of Foxp3+ Tregs in syngeneic mouse gliomas has also been described and depletion of Tregs with CD25-specific immunoglobulins can induce tumor regression and improve survival in these preclinical models [25], [32], [33]. In order to investigate whether Treg depletion could improve therapeutic outcome in combination with immunotherapy/gene therapy, we first established whether Tregs infiltrated into the syngeneic mouse glioma arising from the intracranial implantation of GL26 cells into the brain striatum. Implantation of syngeneic GL26 cells reproducibly led to the linear growth of tumors (R2>0.99) with a mean tumor volume of approximately 0.5mm3 at day 15 and 30 times larger (15mm3) at day 24 (Fig. 1A). These tumors exhibit profuse infiltration of CD45+ and F4/80+ cells, which are evident throughout the tumor mass and also at the borders (Fig 1B). Immune cell infiltration into the tumors was also assessed by flow cytometry 24 days after tumor implantation (Fig. 1 C). We detected tumor infiltrating mDC (CD11c+ CD45+ I-Ab+, Fig. 1Ci) and MΦ (CD11b+ CD45+ I-Ab+, Fig. 1Cii) that represented 3.5% and 19% of the total number of CD45+ immune cells present in the tumor respectively. Lymphocytes were also identified and constituted ∼80% of the total number of CD45+ immune cells infiltrating the tumors (Fig. 1 C iii-vi). These included CD4+ T cells (CD3ε+ CD4+ CD8a−, 26%), CD8a+ T cells (CD3ε+ CD4− CD8a+, 18%), Foxp3+ Tregs (CD3ε+ CD4+ Foxp3+, 8.4%), NK cells (CD3ε− NK1.1+ CD45+, 18%), NK-T cells (CD3ε+ NK1.1+ CD45+, 7%) and B cells (CD3ε− CD19+ CD45+, 5%). The presence of CD4+, CD8a+, CD19+ lymphocytes as well as CD205 mDC were also observed by immunofluorescence (Fig. 1 D). In addition, large numbers of lymphocytes that were immunoreactive for CD25 were observed within the tumor using confocal microscopy (Fig. 1 D) and flow cytometry (CD3ε+ CD25+ CD45+) (Fig. 1 E, F). Moreover, the percentage of CD3ε+ T cells that expressed cell surface CD25 was ∼40% of the total number of tumor infiltrating T cells and was greatly elevated in the draining (cervical) lymph node (dLN) (p<0.05) and tumor (p<0.01) when compared with the spleen of tumor bearing mice (Fig. 1 E).

Bottom Line: Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors.We conclude that this approach will be useful in a setting of minimal residual disease.Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT

Background: Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients.

Findings: Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs present in tumors, draining lymph nodes and spleen and improved long-term survival (50% of mice survived >150 days). No improvement in survival was observed when Tregs were depleted 24 days after tumor implantation, suggesting that tumor burden is an important factor for determining efficacy of Treg depletion in clinical trials. In a T cell dependent model of brain tumor regression elicited by intratumoral delivery of adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Herpes Simplex Type 1-Thymidine Kinase (TK) with ganciclovir (GCV), we demonstrate that administration of PC61 24 days after tumor implantation (7 days after treatment) inhibited T cell dependent tumor regression and long term survival. Further, depletion with PC61 completely inhibited clonal expansion of tumor antigen-specific T lymphocytes in response to the treatment.

Conclusions: Our data demonstrate for the first time, that although Treg depletion inhibits the progression/eliminates GBM tumors, its efficacy is dependent on tumor burden. We conclude that this approach will be useful in a setting of minimal residual disease. Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.

Show MeSH
Related in: MedlinePlus