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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.

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Depletion of Tregs 15 days post-tumor implantation induces brain tumor regression.(A) Diagram depicting the different treatments administered to 4 cohorts of tumor bearing mice. GL26 cells were implanted in the brain of all mice on day 0 and were treated by the intratumoral delivery of saline on day 17 with depletion of CD25+ cells using PC61 on day 15 (green) or on day 24 (red). Control groups of tumor bearing mice received rat IgG1 isotype control on day 15 (blue), or no systemic administration of immunoglobulins (Black) as indicated. Mice were used either for survival studies, or euthanized at day 27 post-tumor implantation for analysis of immune cell populations by flow cytometry. (B) Kaplan-Meier curve displaying survival of the 4 groups of mice described in (A). Ten mice were used in each group and a log-rank test was used to calculate significance between untreated group and PC61 treated groups. *p<0.05 compared with untreated mice and isotype control treated mice. (C) DTH test was performed on long-term survivor mice 100 d after tumor implantation. Mice had originally been treated with PC61 on day 15 (green lines). Irradiated GL26 cells in PBS in the right rear footpad (filled box) or PBS control in the left rear footpad (open box) of mice. The thickness of the footpad was determined 0 h, 4 h, 24 h and 48 h after injection of irradiated cells or PBS. Two Way ANOVA with Tukey's post test was used to calculate significant differences. (D–F) Tregs (CD3ε+ CD4+ Foxp3+) infiltrating tumors (D), draining lymph nodes (E) or spleens (F) were quantified in mice administered rat IgG1 isotype control (iso) or PC61 on day 15 (15) or day 24 (24) as indicated in the graphs (left). Dot plots (right) display representative data from 5 mice per group. The percentages of T regs with respect to the total number of CD45+ cells in the tumors, draining lymph nodes (dLN) or spleen are indicated in representative dot plots. One Way ANOVA with Tukey's post test were used to determine significant differences (***p<0.001).
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pone-0001983-g003: Depletion of Tregs 15 days post-tumor implantation induces brain tumor regression.(A) Diagram depicting the different treatments administered to 4 cohorts of tumor bearing mice. GL26 cells were implanted in the brain of all mice on day 0 and were treated by the intratumoral delivery of saline on day 17 with depletion of CD25+ cells using PC61 on day 15 (green) or on day 24 (red). Control groups of tumor bearing mice received rat IgG1 isotype control on day 15 (blue), or no systemic administration of immunoglobulins (Black) as indicated. Mice were used either for survival studies, or euthanized at day 27 post-tumor implantation for analysis of immune cell populations by flow cytometry. (B) Kaplan-Meier curve displaying survival of the 4 groups of mice described in (A). Ten mice were used in each group and a log-rank test was used to calculate significance between untreated group and PC61 treated groups. *p<0.05 compared with untreated mice and isotype control treated mice. (C) DTH test was performed on long-term survivor mice 100 d after tumor implantation. Mice had originally been treated with PC61 on day 15 (green lines). Irradiated GL26 cells in PBS in the right rear footpad (filled box) or PBS control in the left rear footpad (open box) of mice. The thickness of the footpad was determined 0 h, 4 h, 24 h and 48 h after injection of irradiated cells or PBS. Two Way ANOVA with Tukey's post test was used to calculate significant differences. (D–F) Tregs (CD3ε+ CD4+ Foxp3+) infiltrating tumors (D), draining lymph nodes (E) or spleens (F) were quantified in mice administered rat IgG1 isotype control (iso) or PC61 on day 15 (15) or day 24 (24) as indicated in the graphs (left). Dot plots (right) display representative data from 5 mice per group. The percentages of T regs with respect to the total number of CD45+ cells in the tumors, draining lymph nodes (dLN) or spleen are indicated in representative dot plots. One Way ANOVA with Tukey's post test were used to determine significant differences (***p<0.001).

Mentions: After ruling out any direct effect of PC61 on tumor cell growth we studied the effect of T reg depletion in wild type C57/B6 mice bearing intracranial GL26 glioma. The experimental paradigm used is depicted in Fig. 3A. Depletion of Tregs 15 days after tumor cell implantation significantly increased the number of long term survivors with 40% of mice were still alive 150 days later (p<0.05, Fig. 3B). This was more than 5 times longer than the mean survival of control tumor bearing mice (Fig. 3B). No significant increase in long term survival was observed when Tregs were depleted in larger tumors (24 days) (p>0.05) and only 10% of mice survived long term from this group (Fig. 3B). Interestingly, we did not observe any DTH responses when we injected irradiated GL26 cells into the footpad of long term survivors from Treg depleted mice (Fig. 3C). In order to assess the effects of treatment with PC61 in vivo, we analyzed the presence of Foxp3+ CD4+ Tregs infiltrating the GBM mass as well as in the draining lymph nodes and the spleen after intraperitoneal injection of PC61 (days 15 and 24) or the rat IgG1 isotype control (day 15 days) into tumor bearing mice. PC61 injection on day 15 led to a 9 fold reduction (p<0.001) in the number of tumor infiltrating Tregs when measured using flow cytometry 12 days later (day 27), and injection of PC61 on day 24 led to the almost complete depletion of Tregs (p<0.001) in the tumor 3 days later (day 27) (Fig. 3D). As expected, peripheral Treg populations in the draining lymph nodes (Fig. 3E) and spleens (Fig. 3F) were also significantly reduced compared with isotype treated controls (p<0.001). Interestingly, we observed a 3 fold decrease in the absolute percentage of CD4+ T cells (Fig. 4A) and CD8a+ T cells (Fig. 4B) infiltrating into the tumor after administration of PC61. Furthermore, PC61 significantly depleted populations of CD4+ T cells (p<0.05, Fig. 4C) and CD8+ T cells (p<0.01, Fig. 4D) in the tumor draining lymph nodes. There was no change noted in the percentages of CD4+ T cells and CD8a+ T cells in the spleen (p>0.05, Fig. 4E–F) and MΦ and DC remained unchanged in tumors and draining lymph nodes although increased slightly in spleen (Fig. 5).


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)

Depletion of Tregs 15 days post-tumor implantation induces brain tumor regression.(A) Diagram depicting the different treatments administered to 4 cohorts of tumor bearing mice. GL26 cells were implanted in the brain of all mice on day 0 and were treated by the intratumoral delivery of saline on day 17 with depletion of CD25+ cells using PC61 on day 15 (green) or on day 24 (red). Control groups of tumor bearing mice received rat IgG1 isotype control on day 15 (blue), or no systemic administration of immunoglobulins (Black) as indicated. Mice were used either for survival studies, or euthanized at day 27 post-tumor implantation for analysis of immune cell populations by flow cytometry. (B) Kaplan-Meier curve displaying survival of the 4 groups of mice described in (A). Ten mice were used in each group and a log-rank test was used to calculate significance between untreated group and PC61 treated groups. *p<0.05 compared with untreated mice and isotype control treated mice. (C) DTH test was performed on long-term survivor mice 100 d after tumor implantation. Mice had originally been treated with PC61 on day 15 (green lines). Irradiated GL26 cells in PBS in the right rear footpad (filled box) or PBS control in the left rear footpad (open box) of mice. The thickness of the footpad was determined 0 h, 4 h, 24 h and 48 h after injection of irradiated cells or PBS. Two Way ANOVA with Tukey's post test was used to calculate significant differences. (D–F) Tregs (CD3ε+ CD4+ Foxp3+) infiltrating tumors (D), draining lymph nodes (E) or spleens (F) were quantified in mice administered rat IgG1 isotype control (iso) or PC61 on day 15 (15) or day 24 (24) as indicated in the graphs (left). Dot plots (right) display representative data from 5 mice per group. The percentages of T regs with respect to the total number of CD45+ cells in the tumors, draining lymph nodes (dLN) or spleen are indicated in representative dot plots. One Way ANOVA with Tukey's post test were used to determine significant differences (***p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291560&req=5

pone-0001983-g003: Depletion of Tregs 15 days post-tumor implantation induces brain tumor regression.(A) Diagram depicting the different treatments administered to 4 cohorts of tumor bearing mice. GL26 cells were implanted in the brain of all mice on day 0 and were treated by the intratumoral delivery of saline on day 17 with depletion of CD25+ cells using PC61 on day 15 (green) or on day 24 (red). Control groups of tumor bearing mice received rat IgG1 isotype control on day 15 (blue), or no systemic administration of immunoglobulins (Black) as indicated. Mice were used either for survival studies, or euthanized at day 27 post-tumor implantation for analysis of immune cell populations by flow cytometry. (B) Kaplan-Meier curve displaying survival of the 4 groups of mice described in (A). Ten mice were used in each group and a log-rank test was used to calculate significance between untreated group and PC61 treated groups. *p<0.05 compared with untreated mice and isotype control treated mice. (C) DTH test was performed on long-term survivor mice 100 d after tumor implantation. Mice had originally been treated with PC61 on day 15 (green lines). Irradiated GL26 cells in PBS in the right rear footpad (filled box) or PBS control in the left rear footpad (open box) of mice. The thickness of the footpad was determined 0 h, 4 h, 24 h and 48 h after injection of irradiated cells or PBS. Two Way ANOVA with Tukey's post test was used to calculate significant differences. (D–F) Tregs (CD3ε+ CD4+ Foxp3+) infiltrating tumors (D), draining lymph nodes (E) or spleens (F) were quantified in mice administered rat IgG1 isotype control (iso) or PC61 on day 15 (15) or day 24 (24) as indicated in the graphs (left). Dot plots (right) display representative data from 5 mice per group. The percentages of T regs with respect to the total number of CD45+ cells in the tumors, draining lymph nodes (dLN) or spleen are indicated in representative dot plots. One Way ANOVA with Tukey's post test were used to determine significant differences (***p<0.001).
Mentions: After ruling out any direct effect of PC61 on tumor cell growth we studied the effect of T reg depletion in wild type C57/B6 mice bearing intracranial GL26 glioma. The experimental paradigm used is depicted in Fig. 3A. Depletion of Tregs 15 days after tumor cell implantation significantly increased the number of long term survivors with 40% of mice were still alive 150 days later (p<0.05, Fig. 3B). This was more than 5 times longer than the mean survival of control tumor bearing mice (Fig. 3B). No significant increase in long term survival was observed when Tregs were depleted in larger tumors (24 days) (p>0.05) and only 10% of mice survived long term from this group (Fig. 3B). Interestingly, we did not observe any DTH responses when we injected irradiated GL26 cells into the footpad of long term survivors from Treg depleted mice (Fig. 3C). In order to assess the effects of treatment with PC61 in vivo, we analyzed the presence of Foxp3+ CD4+ Tregs infiltrating the GBM mass as well as in the draining lymph nodes and the spleen after intraperitoneal injection of PC61 (days 15 and 24) or the rat IgG1 isotype control (day 15 days) into tumor bearing mice. PC61 injection on day 15 led to a 9 fold reduction (p<0.001) in the number of tumor infiltrating Tregs when measured using flow cytometry 12 days later (day 27), and injection of PC61 on day 24 led to the almost complete depletion of Tregs (p<0.001) in the tumor 3 days later (day 27) (Fig. 3D). As expected, peripheral Treg populations in the draining lymph nodes (Fig. 3E) and spleens (Fig. 3F) were also significantly reduced compared with isotype treated controls (p<0.001). Interestingly, we observed a 3 fold decrease in the absolute percentage of CD4+ T cells (Fig. 4A) and CD8a+ T cells (Fig. 4B) infiltrating into the tumor after administration of PC61. Furthermore, PC61 significantly depleted populations of CD4+ T cells (p<0.05, Fig. 4C) and CD8+ T cells (p<0.01, Fig. 4D) in the tumor draining lymph nodes. There was no change noted in the percentages of CD4+ T cells and CD8a+ T cells in the spleen (p>0.05, Fig. 4E–F) and MΦ and DC remained unchanged in tumors and draining lymph nodes although increased slightly in spleen (Fig. 5).

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