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1.
Figure 6

Figure 6. From: 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.

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.

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
2.
Figure 4

Figure 4. From: 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.

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 (○).

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
3.
Figure 2

Figure 2. From: 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.

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.

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
4.
Figure 3

Figure 3. From: 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.

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.

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
5.
Figure 7

Figure 7. From: 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.

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 (▵).

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
6.
Figure 1

Figure 1. From: 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.

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 (×).

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
7.
Figure 5

Figure 5. From: 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.

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.

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.
8.
Figure 8

Figure 8. From: 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.

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.

Roger P.M. Sutmuller, et al. J Exp Med. 2001 September 17;194(6):823-832.

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