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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.

Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes RE, Offringa R, Melief CJ - J. Exp. Med. (2001)

Bottom Line: Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Tumor Immunology Lab, E3-Q, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.

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 2(180-188)-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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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.
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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.

Mentions: The synergistic effect of CTLA-4 blockade and CD25 depletion on CTL induction and antitumor response described here could in theory still be caused by differences in biokinetics of the two antibodies used. To prove that CTLA-4 blockade and Treg cell function are indeed alternative pathways for regulation of autoreactive CTL responses, we separated the anti–CTLA-4 and anti-CD25 antibody treatments in space and time and analyzed the effects with regard to CTL induction in vitro as well as in vivo. For this purpose we used CD25-depleted splenocytes. CD25 depletion was accomplished by injection of 450 μg of anti-CD25 antibody on 2 d consecutively and harvesting the splenocytes 3 d later. Flow cytometry analysis with noncrossreactive anti-CD25 (7D4) confirmed >95% depletion of CD25+ T cells. The CD25− splenocytes were then used in an in vitro priming assay to induce in vitro TRP-2180–188–specific CTLs. Fig. 8 A shows that CD25− splenocytes stimulated with TRP-2180–188 peptide and cultured for 1 wk specifically recognize TRP-2180–188–loaded target cells. CTLA-4 blockade during the first 3 d of culturing markedly increased the induction of the TRP-2180–188–specific response. We also used 5 × 107 CD25− splenocytes to reconstitute T cell–deficient C57BL/6 Nude (nu/nu) recipients. The nude recipients were subsequently vaccinated with GM-CSF–producing B16 with or without CTLA-4–blocking antibodies. 3 wk after the last vaccination, the spleens were harvested and the splenocyte cultures were analyzed for the presence of TRP-2180–188–specific CTLs. In T cell cultures from recipients reconstituted with complete splenocytes and vaccinated with or without CTLA-4 blockade no TRP-2180–188–specific reactivity was demonstrable (Fig. 8 B). This is probably due to the decreased T cell numbers in reconstituted nude recipients compared with normal C57BL/6 mice rendering it more difficult to initiate a detectable CTL response. However, when nude mice were reconstituted with CD25− splenocytes, specific IFN-γ release in response to TRP-2180–188 was detected, but only if the GM-CSF–producing B16 vaccine was combined with CTLA-4 blockade. These in vitro and in vivo results show that CTLA-4 blockade acts in the complete absence of CD25+ T cells, probably by directly releasing the brakes on costimulation in responding T cells, including effector CTLs.


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.

Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes RE, Offringa R, Melief CJ - J. Exp. Med. (2001)

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.
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Related In: Results  -  Collection

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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.
Mentions: The synergistic effect of CTLA-4 blockade and CD25 depletion on CTL induction and antitumor response described here could in theory still be caused by differences in biokinetics of the two antibodies used. To prove that CTLA-4 blockade and Treg cell function are indeed alternative pathways for regulation of autoreactive CTL responses, we separated the anti–CTLA-4 and anti-CD25 antibody treatments in space and time and analyzed the effects with regard to CTL induction in vitro as well as in vivo. For this purpose we used CD25-depleted splenocytes. CD25 depletion was accomplished by injection of 450 μg of anti-CD25 antibody on 2 d consecutively and harvesting the splenocytes 3 d later. Flow cytometry analysis with noncrossreactive anti-CD25 (7D4) confirmed >95% depletion of CD25+ T cells. The CD25− splenocytes were then used in an in vitro priming assay to induce in vitro TRP-2180–188–specific CTLs. Fig. 8 A shows that CD25− splenocytes stimulated with TRP-2180–188 peptide and cultured for 1 wk specifically recognize TRP-2180–188–loaded target cells. CTLA-4 blockade during the first 3 d of culturing markedly increased the induction of the TRP-2180–188–specific response. We also used 5 × 107 CD25− splenocytes to reconstitute T cell–deficient C57BL/6 Nude (nu/nu) recipients. The nude recipients were subsequently vaccinated with GM-CSF–producing B16 with or without CTLA-4–blocking antibodies. 3 wk after the last vaccination, the spleens were harvested and the splenocyte cultures were analyzed for the presence of TRP-2180–188–specific CTLs. In T cell cultures from recipients reconstituted with complete splenocytes and vaccinated with or without CTLA-4 blockade no TRP-2180–188–specific reactivity was demonstrable (Fig. 8 B). This is probably due to the decreased T cell numbers in reconstituted nude recipients compared with normal C57BL/6 mice rendering it more difficult to initiate a detectable CTL response. However, when nude mice were reconstituted with CD25− splenocytes, specific IFN-γ release in response to TRP-2180–188 was detected, but only if the GM-CSF–producing B16 vaccine was combined with CTLA-4 blockade. These in vitro and in vivo results show that CTLA-4 blockade acts in the complete absence of CD25+ T cells, probably by directly releasing the brakes on costimulation in responding T cells, including effector CTLs.

Bottom Line: Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Tumor Immunology Lab, E3-Q, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.

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 2(180-188)-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.

Show MeSH
Related in: MedlinePlus