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Cited by 1,008T-cell and B-cell ImmunologyCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

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Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses

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Sutmuller, Roger P.M., van Duivenvoorde, Leonie M., van Elsas, Andrea, Schumacher, Ton N.M., Wildenberg, Manon E., Allison, James P., Toes, Rene E.M., Offringa, Rienk, Melief, Cornelis J.M.·2001·The Journal of Experimental Medicine
DOI 10.1084/jem.194.6.823PMC PMC2195955
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Abstract

Therapeutic efficacy of a tumor cell–based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte–associated antigen (CTLA)-4 pathway or the CD25+ regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25+ Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2180–188–specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ Treg cells indicates that CD25+ Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.

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Figures

Figure 1 CD4+ T cell depletion before vaccination enhances efficacy of B16 treatment. Survival data of mice challenged subcutaneously (day 0) with 5 × 103 B16-BL6 tumor cells. Mice received either no treatment (n = 25, ♦) or B16-GM-CSF tumor cell vaccine in combination with anti–CTLA-4–blocking Ab on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab on days −1, 0, 7, and 14: depleting anti-CD8 Ab (n = 25, □), depleting anti-CD4 Ab (n = 25, ×), or isotype matched control Ab (n = 25, •). Details on experimental procedure and Ab are described in Materials and Methods. The graph indicates the percentage of surviving mice over time. Significant (P = 0.04, log rank test) difference was found between B16-GM-CSF and anti–CTLA-4–treated mice injected with control antibody (•) and mice injected with CD4-depleting Ab (×).
Figure 1 CD4+ T cell depletion before vaccination enhances efficacy of B16 treatment. Survival data of mice challenged subcutaneously (day 0) with 5 × 103 B16-BL6 tumor cells. Mice received either no treatment (n = 25, ♦) or B16-GM-CSF tumor cell vaccine in combination with anti–CTLA-4–blocking Ab on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab on days −1, 0, 7, and 14: depleting anti-CD8 Ab (n = 25, □), depleting anti-CD4 Ab (n = 25, ×), or isotype matched control Ab (n = 25, •). Details on experimental procedure and Ab are described in Materials and Methods. The graph indicates the percentage of surviving mice over time. Significant (P = 0.04, log rank test) difference was found between B16-GM-CSF and anti–CTLA-4–treated mice injected with control antibody (•) and mice injected with CD4-depleting Ab (×).CC BY-NC-SA · PubMed Central
Figure 2 Characterization of blood lymphocytes in CD25-depleted C57BL/6 mice. (A and B) Blood lymphocytes from a naive (A) or a CD25-depleted (B) mouse were stained 21 d after CD25 depletion, with APC-conjugated anti-CD4 and PE-conjugated anti-CD25. Numbers in the upper right quadrant indicate the percentage of CD25+/CD4+ T cells of total CD4+ T cells. (C and D) Blood lymphocytes from a naive (C) or a CD25-depleted/B16-GM-CSF/anti–CTLA-4 vaccinated mouse (D) were stained on day 17 after vaccination for the presence of TRP-2180-188–specific CD8+ T cells using an APC-conjugated TRP-2180–188 Kb-tetramer and anti–CD8-FITC. Numbers in the upper right quadrant indicate the percentage of TRP-2180–188–specific CD8+ T cells of total CD8+ T cells. Representative stainings of three independent experiments are shown.
Figure 2 Characterization of blood lymphocytes in CD25-depleted C57BL/6 mice. (A and B) Blood lymphocytes from a naive (A) or a CD25-depleted (B) mouse were stained 21 d after CD25 depletion, with APC-conjugated anti-CD4 and PE-conjugated anti-CD25. Numbers in the upper right quadrant indicate the percentage of CD25+/CD4+ T cells of total CD4+ T cells. (C and D) Blood lymphocytes from a naive (C) or a CD25-depleted/B16-GM-CSF/anti–CTLA-4 vaccinated mouse (D) were stained on day 17 after vaccination for the presence of TRP-2180-188–specific CD8+ T cells using an APC-conjugated TRP-2180–188 Kb-tetramer and anti–CD8-FITC. Numbers in the upper right quadrant indicate the percentage of TRP-2180–188–specific CD8+ T cells of total CD8+ T cells. Representative stainings of three independent experiments are shown.CC BY-NC-SA · PubMed Central
Figure 3 CD25+ T cell depletion before vaccination enhances efficacy of treatment. (A) Survival data of mice challenged subcutaneously with 2.5 × 103 B16-BL6 tumor cells. Mice received either no treatment (n = 6, ▪), or depleting anti-CD25 on day −4 (n = 6, ⋄) or vaccination with GM-CSF–producing B16 on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab: CTLA-4 blocking Ab on days 0, 3, and 6 (n = 8, •); depleting anti-CD25 Ab on day −4 (n = 8, ×); or depleting anti-CD25 Ab on day −4 plus CTLA-4–blocking Ab on days 0, 3, and 6 (n = 8, ▴). (B) Survival data of mice challenged subsutaneously (day 0) with 5 × 103 B16-BL6 tumor cells. Mice received either depleting anti-CD25 Ab on day −4 (n = 6, ⋄) or were vaccinated on days 0, 3, and 6 with GM-CSF–producing B16. The vaccinated mice were divided over two groups that received the following Ab: blocking anti–CTLA-4 Ab on days 0, 3, and 6 (n = 9, •); or depleting anti-CD25 Ab on day −4 combined with blocking anti-CTLA-4 Ab on days 0, 3, and 6 (n = 9, ▴). See Materials and Methods for details. Significant (A, P = 0.025; B, P = 0.0004, log rank test) differences were found between B16-GM-CSF vaccinated mice that received either anti–CTLA-4 Ab (•) or were injected with anti-CD25 Ab plus anti-CTLA-4 Ab (▴). Representative results from two independent experiments are shown.
Figure 3 CD25+ T cell depletion before vaccination enhances efficacy of treatment. (A) Survival data of mice challenged subcutaneously with 2.5 × 103 B16-BL6 tumor cells. Mice received either no treatment (n = 6, ▪), or depleting anti-CD25 on day −4 (n = 6, ⋄) or vaccination with GM-CSF–producing B16 on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab: CTLA-4 blocking Ab on days 0, 3, and 6 (n = 8, •); depleting anti-CD25 Ab on day −4 (n = 8, ×); or depleting anti-CD25 Ab on day −4 plus CTLA-4–blocking Ab on days 0, 3, and 6 (n = 8, ▴). (B) Survival data of mice challenged subsutaneously (day 0) with 5 × 103 B16-BL6 tumor cells. Mice received either depleting anti-CD25 Ab on day −4 (n = 6, ⋄) or were vaccinated on days 0, 3, and 6 with GM-CSF–producing B16. The vaccinated mice were divided over two groups that received the following Ab: blocking anti–CTLA-4 Ab on days 0, 3, and 6 (n = 9, •); or depleting anti-CD25 Ab on day −4 combined with blocking anti-CTLA-4 Ab on days 0, 3, and 6 (n = 9, ▴). See Materials and Methods for details. Significant (A, P = 0.025; B, P = 0.0004, log rank test) differences were found between B16-GM-CSF vaccinated mice that received either anti–CTLA-4 Ab (•) or were injected with anti-CD25 Ab plus anti-CTLA-4 Ab (▴). Representative results from two independent experiments are shown.CC BY-NC-SA · PubMed Central
Figure 4 Depigmentation of mice vaccinated with GM-CSF–producing B16. Groups of mice were untreated (n = 12, ⋄), treated with B16-GM-CSF and anti–CTLA-4 Ab alone (n = 16, ▵) or in combination with prior CD25 depletion (n = 20, ○) as described in Material and Methods. Each marker represents the average depigmentation score for a single mouse from three independent (blinded) scorings. For each group the mean is shown ± SEM. Significant (P < 0.02, Student's t test) difference was found between B16-GM-CSF vaccinated mice that received either anti–CTLA-4 Ab alone (▵) or in combination with anti-CD25 Ab (○).
Figure 4 Depigmentation of mice vaccinated with GM-CSF–producing B16. Groups of mice were untreated (n = 12, ⋄), treated with B16-GM-CSF and anti–CTLA-4 Ab alone (n = 16, ▵) or in combination with prior CD25 depletion (n = 20, ○) as described in Material and Methods. Each marker represents the average depigmentation score for a single mouse from three independent (blinded) scorings. For each group the mean is shown ± SEM. Significant (P < 0.02, Student's t test) difference was found between B16-GM-CSF vaccinated mice that received either anti–CTLA-4 Ab alone (▵) or in combination with anti-CD25 Ab (○).CC BY-NC-SA · PubMed Central
Figure 5 Analysis of TRP-2180–188–specific CD8+ T cells in vaccinated mice. (A) Blood lymphocytes were stained on day 17 after tumor challenge for the presence of TRP-2180–188–specific CD8+ T cells using an APC-conjugated TRP-2180–188 Kb-tetramer and anti–CD8-FITC. The percentage of TRP-2180–188–specific CD8+ T cells of total CD8+ T cells is indicated. Treatments of mice were as follows: group 1, naive mice; groups 2–8, B16-BL6 tumor challenge on day 0; groups 3–7, vaccination on days 0, 3, and 6 with GM-CSF–producing B16. If indicated, 200 μg blocking anti–CTLA-4 Ab was injected on days 0, 3, and 6, depleting anti-CD25 Ab (450 μg) was injected at day −4. Significant difference (P = 0.03, Student's t test) was found between mice from groups 3 and 4. Error bars indicate the SD of three measurements from one experiment. One representative experiment of two is shown. (B) Lymph node cells were stained on day 17 after tumor challenge for the presence of TRP-2–specific IFN-γ–producing CD8+ T cells. Results indicate the percentage of TRP-2–specific CD8+ T cells from the total CD8 population calculated as (percentage of TRP-2 responders) − (percentage of E1A responders). A representative result from two experiments is shown.
Figure 5 Analysis of TRP-2180–188–specific CD8+ T cells in vaccinated mice. (A) Blood lymphocytes were stained on day 17 after tumor challenge for the presence of TRP-2180–188–specific CD8+ T cells using an APC-conjugated TRP-2180–188 Kb-tetramer and anti–CD8-FITC. The percentage of TRP-2180–188–specific CD8+ T cells of total CD8+ T cells is indicated. Treatments of mice were as follows: group 1, naive mice; groups 2–8, B16-BL6 tumor challenge on day 0; groups 3–7, vaccination on days 0, 3, and 6 with GM-CSF–producing B16. If indicated, 200 μg blocking anti–CTLA-4 Ab was injected on days 0, 3, and 6, depleting anti-CD25 Ab (450 μg) was injected at day −4. Significant difference (P = 0.03, Student's t test) was found between mice from groups 3 and 4. Error bars indicate the SD of three measurements from one experiment. One representative experiment of two is shown. (B) Lymph node cells were stained on day 17 after tumor challenge for the presence of TRP-2–specific IFN-γ–producing CD8+ T cells. Results indicate the percentage of TRP-2–specific CD8+ T cells from the total CD8 population calculated as (percentage of TRP-2 responders) − (percentage of E1A responders). A representative result from two experiments is shown.CC BY-NC-SA · PubMed Central
Figure 6 IFN-γ release in response to TRP-2180–188 peptide. Splenocytes from mice were in vitro restimulated with irradiated B16/B7.1 and tested for recognition of TRP-2180–188 peptide-loaded target cells in an IFN-γ release assay 1 wk later. Treatments of mice are described in legend to Fig. 5. Values indicate average from three measurements with SD indicated. One representative experiment of two is shown.
Figure 6 IFN-γ release in response to TRP-2180–188 peptide. Splenocytes from mice were in vitro restimulated with irradiated B16/B7.1 and tested for recognition of TRP-2180–188 peptide-loaded target cells in an IFN-γ release assay 1 wk later. Treatments of mice are described in legend to Fig. 5. Values indicate average from three measurements with SD indicated. One representative experiment of two is shown.CC BY-NC-SA · PubMed Central
Figure 7 Analysis of in vivo anti-CD25 mAb administration. (A) Prophylactic vaccination efficiency is decreased by administration of anti-CD4 mAb or anti-CD25 mAb. Mice received either no treatment (n = 6, ▪) or were vaccinated with B16-GM-CSF and blocking anti-CTLA-4 Ab on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab in combination with CD4 depletion (n = 8, ×), CD25 depletion (n = 8, ▴), or without depletion (n = 8, •). Administration of depleting mAb was performed on day 17. All mice were challenged with 5 × 103 B16-BL6 tumor cells on day 21. (B) Therapeutic vaccination in CD25-depleted mice is dependent of CD4+ as well as CD8+ T cells. Therapeutic treatment with irradiated GM-CSF–producing B16 cells plus CTLA-4 blockade was performed in mice depleted for CD25+ T cells (n = 8, ▴), in mice depleted for CD25+ T cells and CD8+ T cells (n = 8, ♦), or in mice depleted for CD25+ T cells and CD4+ T cells (n = 8, ▵). Significant (P = 0.025, log rank test) difference was found between CD25 depleted/vaccinated mice (▴) and CD4 plus CD25 depleted/vaccinated mice (▵).
Figure 7 Analysis of in vivo anti-CD25 mAb administration. (A) Prophylactic vaccination efficiency is decreased by administration of anti-CD4 mAb or anti-CD25 mAb. Mice received either no treatment (n = 6, ▪) or were vaccinated with B16-GM-CSF and blocking anti-CTLA-4 Ab on days 0, 3, and 6. The vaccinated mice were divided over three groups that received the following Ab in combination with CD4 depletion (n = 8, ×), CD25 depletion (n = 8, ▴), or without depletion (n = 8, •). Administration of depleting mAb was performed on day 17. All mice were challenged with 5 × 103 B16-BL6 tumor cells on day 21. (B) Therapeutic vaccination in CD25-depleted mice is dependent of CD4+ as well as CD8+ T cells. Therapeutic treatment with irradiated GM-CSF–producing B16 cells plus CTLA-4 blockade was performed in mice depleted for CD25+ T cells (n = 8, ▴), in mice depleted for CD25+ T cells and CD8+ T cells (n = 8, ♦), or in mice depleted for CD25+ T cells and CD4+ T cells (n = 8, ▵). Significant (P = 0.025, log rank test) difference was found between CD25 depleted/vaccinated mice (▴) and CD4 plus CD25 depleted/vaccinated mice (▵).CC BY-NC-SA · PubMed Central
Figure 8 CTLA-4 blockade enhances CTL induction in the absence of CD25+ Treg cells. CD25− splenocytes were used to analyze the effect of CTLA-4 blockade on the induction of effector CTL in vitro (A) and in vivo (B). (A) Naive CD25− splenocytes were, if indicated, stimulated with TRP-2 peptide loaded target cells and anti–CTLA-4 (50 μg/ml) during the first 3 d of culture. At day 7, specific IFN-γ release in response to TRP-2 peptide was measured. (B) C57BL/6 nude recipients were reconstituted with 5 × 107 splenocytes from wild-type C57BL/6 mice on day 0 and vaccinated with GM-CSF–producing B16 cells on days 4, 7, and 10. If indicated, CD25− splenocytes were used to reconstitute the recipients and 200 μg of CTLA-4–blocking Ab was administered on days 4, 7, and 10. 3 wk after the last vaccination splenocytes from mice were in vitro restimulated with irradiated B16/B7.1 and tested for recognition of TRP-2180–188 peptide-loaded target cells in an IFN-γ release assay 1 wk later. Values indicate average from three measurements with SD indicated. Representative results from two independent experiments are shown. Significant difference (A, P < 0.03; B, P < 0.01, Student's t test) was found between treatments no. 3 and 4.
Figure 8 CTLA-4 blockade enhances CTL induction in the absence of CD25+ Treg cells. CD25− splenocytes were used to analyze the effect of CTLA-4 blockade on the induction of effector CTL in vitro (A) and in vivo (B). (A) Naive CD25− splenocytes were, if indicated, stimulated with TRP-2 peptide loaded target cells and anti–CTLA-4 (50 μg/ml) during the first 3 d of culture. At day 7, specific IFN-γ release in response to TRP-2 peptide was measured. (B) C57BL/6 nude recipients were reconstituted with 5 × 107 splenocytes from wild-type C57BL/6 mice on day 0 and vaccinated with GM-CSF–producing B16 cells on days 4, 7, and 10. If indicated, CD25− splenocytes were used to reconstitute the recipients and 200 μg of CTLA-4–blocking Ab was administered on days 4, 7, and 10. 3 wk after the last vaccination splenocytes from mice were in vitro restimulated with irradiated B16/B7.1 and tested for recognition of TRP-2180–188 peptide-loaded target cells in an IFN-γ release assay 1 wk later. Values indicate average from three measurements with SD indicated. Representative results from two independent experiments are shown. Significant difference (A, P < 0.03; B, P < 0.01, Student's t test) was found between treatments no. 3 and 4.CC BY-NC-SA · PubMed Central

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Authors
Sutmuller, R. P. M., van Duivenvoorde, L. M., van Elsas, A., Schumacher, T. N. M., Wildenberg, M. E., Allison, J. P., Toes, R. E. M., Offringa, R., & Melief, C. J. M.
Affiliations
  • Leiden University Medical Center (NL)
  • aDepartment of Immunohematology and Blood Transfusion, Tumor Immunology Lab, E3-Q, Leiden University Medical Center, 2300 RC Leiden, Netherlands
  • The Netherlands Cancer Institute (NL)
  • University of California, Berkeley (US)
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2001-09-17
Volume
194
Issue
6
Pages
823-832
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.194.6.823
PMC
PMC2195955
OpenAlex
W2148911309
Cited by
1,008
References
44
Citations by year
2026: 3 · 2025: 7 · 2024: 8 · 2023: 12 · 2022: 15 · 2021: 15 · 2020: 18 · 2019: 11 · 2018: 12 · 2017: 25 · 2016: 22 · 2015: 36 · 2014: 26 · 2013: 48 · 2012: 41
FWCI
13.30
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • Pure Amsterdam UMC (submittedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
CTLA-4 blockade, CD25+ regulatory T cells, Treg depletion, antitumor immunity, B16 melanoma, CD8+ T cells, tyrosinase-related protein 2, immune regulation, tumor rejection
MeSH terms
Abatacept, Animals, Antibodies, Monoclonal, Antigens, Differentiation, Antigens, Neoplasm, Female, Immunotherapy, Lymphocyte Depletion, Melanoma, Experimental, Mice, Inbred C57BL, Mice, Nude, T-Lymphocytes, Cytotoxic, Tumor Cells, Cultured, Receptors, Interleukin-2, Biomarkers, CD4-Positive T-Lymphocytes, Antigens, CD, Immunoconjugates, Intramolecular Oxidoreductases, Mice, CTLA-4 Antigen

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