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The stimulation of PD-L1-specific cytotoxic T lymphocytes can both directly and indirectly enhance antileukemic immunity.

Ahmad SM, Svane IM, Andersen MH - Blood Cancer J (2014)

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Immune Therapy (CCIT), Department of Hematology and Oncology, Copenhagen University Hospital, Herlev, Herlev, Denmark.

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from acute myeloid leukemia (AML) as well as myelodysplastic syndrome patients, blasts have been found to be positive for PD-L1, whereas stroma/non-blast cellular No severe toxicity was reported and the treatment seemed to induce clinical effects... addition of PD-L1-specific CTLs 1 week after stimulation of PBMCs with viral epitopes Hence, PD-L1-specific CTLs may effectively enhance the effector phase of the immune response... AML cells were killed, as the PD-L1-specific CTLs were not able to kill SET-2 cells... PBMCs with SET-2 cells either in co-culture with the PD-L115–23 epitope PD-L115–23 epitope more efficiently lysed SET-2 cells compared with Hence, although PD-L115–23-specific CTLs do not recognize SET-2 cells, the principal difference between therapeutically induced T cells and surface blockade by antibodies is that the former reduces not only the target protein-mediated immune

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PD-L1-specific CTLs directly kill AML cells and enhance additional antileukemicimmunity. (a) Functional capacity of I PD-L115–23-specificCTLs assayed by 51Cr release assay. Specific lysis of thePD-L1+, HLA-A2+ AML cells UKE-1 (black) andSET-2 (gray). (b) Functional capacity of IPD-L115–23-specific CTLs assayed by 51Cr releaseassay. Specific lysis of PD-L1+, HLA-A2+ AML cellsTHP-1 (black) and the EBV-positive B-lymphoblastoid cell line RPMI6666 (gray).(c) Specificity of PD-L115–23-specific CTLs assayed by51Cr release assay. Lysis of the TAP-deficient T2 cell line eitherin the presence of PD-L115–23 peptide (black) or irrelevantcontrol peptide HIV-1 pol476–484 (gray). (d) PBMCs fromthree HLA-A2+ donors were stimulated in vitro withirradiated SET-2 cells (at a PBMC:SET-2 ratio of 10:1) every week for 4 weekseither in co-culture with HIV-1 pol476–484 peptide orPD-L115–23 peptide. To avoid binding of the peptides to thesurface of SET-2 cells, the peptides were added to the cultures 3 days after SET-2stimulation. (e) Three 51Cr release assays examining the lysisof SET-2 cells by SET-2-stimulated T cells from three different donors that hadeither been co-stimulated with HIV-1 pol476–484 peptide (gray) orPD-L115–23 peptide (black).
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fig2: PD-L1-specific CTLs directly kill AML cells and enhance additional antileukemicimmunity. (a) Functional capacity of I PD-L115–23-specificCTLs assayed by 51Cr release assay. Specific lysis of thePD-L1+, HLA-A2+ AML cells UKE-1 (black) andSET-2 (gray). (b) Functional capacity of IPD-L115–23-specific CTLs assayed by 51Cr releaseassay. Specific lysis of PD-L1+, HLA-A2+ AML cellsTHP-1 (black) and the EBV-positive B-lymphoblastoid cell line RPMI6666 (gray).(c) Specificity of PD-L115–23-specific CTLs assayed by51Cr release assay. Lysis of the TAP-deficient T2 cell line eitherin the presence of PD-L115–23 peptide (black) or irrelevantcontrol peptide HIV-1 pol476–484 (gray). (d) PBMCs fromthree HLA-A2+ donors were stimulated in vitro withirradiated SET-2 cells (at a PBMC:SET-2 ratio of 10:1) every week for 4 weekseither in co-culture with HIV-1 pol476–484 peptide orPD-L115–23 peptide. To avoid binding of the peptides to thesurface of SET-2 cells, the peptides were added to the cultures 3 days after SET-2stimulation. (e) Three 51Cr release assays examining the lysisof SET-2 cells by SET-2-stimulated T cells from three different donors that hadeither been co-stimulated with HIV-1 pol476–484 peptide (gray) orPD-L115–23 peptide (black).

Mentions: We have described that natural existing PD-L1-specific cytotoxic T lymphocytes (CTLs)are able to recognize and kill both malignant lymphoma cells as well as normalPD-L1-expressing immune cells.10, 11 Furthermore, we recently described that theaddition of PD-L1-specific CTLs 1 week after stimulation of PBMCs with viral epitopesfrom Epstein–Barr virus (EBV) or cytomegalovirus (CMV) resulted in an immenseincrease in the number of virus-specific CD8+ T cells invitro.12 Hence, PD-L1-specific CTLsmay effectively enhance the effector phase of the immune response. To further examinethe potential of using PD-L1-specific T cells in the treatment of hematologicalmalignancies, we here stimulated PBMCs from 14 human leukocyte antigen(HLA)-A2+ donors with a well-known HLA-A2-restricted CMV epitopeeither in co-culture with the HLA-A2-restricted epitope PD-L115–23 oran irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, as depictedin Figure 1a. After four in vitro stimulations theT-cell reactivity toward the CMV epitope was examined for each donor by the use ofHLA-A2/CMV tetramers (Figure 1b). Notably, we observed asignificant increase in the numbers of virus-specific T cells in the cultures that hadbeen co-stimulated with the PD-L115–23 peptide epitope. Examples ofthree donors, where co-activation of PD-L1-specific T cells significantly boosted T-cellimmunity toward CMV are illustrated in Figure 1c. Thus, thestimulation of PD-L1-specific CTLs by vaccination may additionally boost other effectorT cells by removing PD-L1-positive, immune-suppressive cells that inhibit the activationand proliferation of PD-1-positive T cells. Next, to examine how PD-L1-specific CTLs caninfluence antileukemia immunotherapy in general, we examined the ability ofPD-L115–23-specific CTLs11to kill well-characterized PD-L1+ AML cells—UKE-1,13 SET-2 (ref. 13) andTHP-1 (ref. 14)—in standard 51Cr releaseassays. PD-L115–23-specific CTLs efficiently killed UKE-1 and THP-1cells (Figures 2a and b). In contrast, the SET-2 cells werenot killed by the PD-L115–23-specific CTLs (Figure2a). Likewise, a control EBV-positive B-lymphoblastoid cell line RPMI6666(ref. 15) was not killed by the PD-L1-specific CTLs(Figure 2b). As a further control, thePD-L115–23-specific CTLs efficiently lysed TAP-deficient T2 cellspulsed with PD-L115–23 efficiently, whereas no cytotoxicity wasobserved against T2 cells pulsed with an irrelevant peptide from HIV (Figure 2c). Our observations on one hand show that PD-L1-specific CTLs areable to react directly toward AML cells and kill the malignant cells. However, not allAML cells were killed, as the PD-L1-specific CTLs were not able to kill SET-2 cells. Toexamine whether the activation of PD-L1-specific CTLs could have an indirect effect onthe immunity against SET-2 cells, we stimulated PBMCs from the three donors in which wehad observed an increased CMV response after co-stimulation with thePD-L115–23 peptide (as depicted in Figure2d) with SET-2 cells. Thus, after four in vitro stimulations ofPBMCs with SET-2 cells either in co-culture with the PD-L115–23 epitopeor an irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, weexamined the ability of the resulting T-cell cultures to recognize and kill SET-2 cellsin standard 51Cr release assays. As illustrated in Figure2e the T-cell cultures from all three donors co-stimulated withPD-L115–23 epitope more efficiently lysed SET-2 cells compared withthe cultures co-stimulated with an irrelevant HIV epitope. Hence, althoughPD-L115–23-specific CTLs do not recognize SET-2 cells, theactivation of these by stimulation boosted additional T-cell immunity toward SET-2cells. This could point to a scenario were PD-L1-based vaccination might be beneficialeven in leukemia patients where PD-L1-specific CTLs do not react toward the leukemiacells themselves. Thus, the enhancement of PD-L1-specific CTLs in patients might bevaluable both by the direct killing of leukemia cells as well as indirectly by thereinforcement of antileukemic T cells. The addition of PD-L1 vaccination should beeasily implementable and highly synergistic with other immune-based therapies. Theinduction of specific T cells represents a new and attractive immune therapeuticapproach, in which the specific depletion of target cells is not limited to targetingproteins that are expressed on the cell surface. This is important, as the PD-L1 epitopeused in this study is located near the N-terminal of the PD-L1 sequence as part of thesignal peptide, and is therefore not part of the extracellular domain. An additionalprincipal difference between therapeutically induced T cells and surface blockade byantibodies is that the former reduces not only the target protein-mediated immunesuppression but also other immune-suppressive effects mediated by the target cells.Taken together, vaccination against PD-L1 and antibody-mediated PD-1/PD-L1 blockadeshould therefore be considered complementary rather than combative. In fact, an excitingtherapeutic strategy would be to combine anti-PD-L1 vaccination with, for example,anti-CTLA4- or anti-LAG3-blocking antibodies. Taken together, we believe that thefindings justify and warrant clinical testing to evaluate the efficiency and safety ofPD-L1-based vaccinations in hematological malignancies. Hence, we are in the process ofinitiating a phase I vaccination study at the Center for Caner Immune Therapy,Copenhagen University Hospital, Herlev.


The stimulation of PD-L1-specific cytotoxic T lymphocytes can both directly and indirectly enhance antileukemic immunity.

Ahmad SM, Svane IM, Andersen MH - Blood Cancer J (2014)

PD-L1-specific CTLs directly kill AML cells and enhance additional antileukemicimmunity. (a) Functional capacity of I PD-L115–23-specificCTLs assayed by 51Cr release assay. Specific lysis of thePD-L1+, HLA-A2+ AML cells UKE-1 (black) andSET-2 (gray). (b) Functional capacity of IPD-L115–23-specific CTLs assayed by 51Cr releaseassay. Specific lysis of PD-L1+, HLA-A2+ AML cellsTHP-1 (black) and the EBV-positive B-lymphoblastoid cell line RPMI6666 (gray).(c) Specificity of PD-L115–23-specific CTLs assayed by51Cr release assay. Lysis of the TAP-deficient T2 cell line eitherin the presence of PD-L115–23 peptide (black) or irrelevantcontrol peptide HIV-1 pol476–484 (gray). (d) PBMCs fromthree HLA-A2+ donors were stimulated in vitro withirradiated SET-2 cells (at a PBMC:SET-2 ratio of 10:1) every week for 4 weekseither in co-culture with HIV-1 pol476–484 peptide orPD-L115–23 peptide. To avoid binding of the peptides to thesurface of SET-2 cells, the peptides were added to the cultures 3 days after SET-2stimulation. (e) Three 51Cr release assays examining the lysisof SET-2 cells by SET-2-stimulated T cells from three different donors that hadeither been co-stimulated with HIV-1 pol476–484 peptide (gray) orPD-L115–23 peptide (black).
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fig2: PD-L1-specific CTLs directly kill AML cells and enhance additional antileukemicimmunity. (a) Functional capacity of I PD-L115–23-specificCTLs assayed by 51Cr release assay. Specific lysis of thePD-L1+, HLA-A2+ AML cells UKE-1 (black) andSET-2 (gray). (b) Functional capacity of IPD-L115–23-specific CTLs assayed by 51Cr releaseassay. Specific lysis of PD-L1+, HLA-A2+ AML cellsTHP-1 (black) and the EBV-positive B-lymphoblastoid cell line RPMI6666 (gray).(c) Specificity of PD-L115–23-specific CTLs assayed by51Cr release assay. Lysis of the TAP-deficient T2 cell line eitherin the presence of PD-L115–23 peptide (black) or irrelevantcontrol peptide HIV-1 pol476–484 (gray). (d) PBMCs fromthree HLA-A2+ donors were stimulated in vitro withirradiated SET-2 cells (at a PBMC:SET-2 ratio of 10:1) every week for 4 weekseither in co-culture with HIV-1 pol476–484 peptide orPD-L115–23 peptide. To avoid binding of the peptides to thesurface of SET-2 cells, the peptides were added to the cultures 3 days after SET-2stimulation. (e) Three 51Cr release assays examining the lysisof SET-2 cells by SET-2-stimulated T cells from three different donors that hadeither been co-stimulated with HIV-1 pol476–484 peptide (gray) orPD-L115–23 peptide (black).
Mentions: We have described that natural existing PD-L1-specific cytotoxic T lymphocytes (CTLs)are able to recognize and kill both malignant lymphoma cells as well as normalPD-L1-expressing immune cells.10, 11 Furthermore, we recently described that theaddition of PD-L1-specific CTLs 1 week after stimulation of PBMCs with viral epitopesfrom Epstein–Barr virus (EBV) or cytomegalovirus (CMV) resulted in an immenseincrease in the number of virus-specific CD8+ T cells invitro.12 Hence, PD-L1-specific CTLsmay effectively enhance the effector phase of the immune response. To further examinethe potential of using PD-L1-specific T cells in the treatment of hematologicalmalignancies, we here stimulated PBMCs from 14 human leukocyte antigen(HLA)-A2+ donors with a well-known HLA-A2-restricted CMV epitopeeither in co-culture with the HLA-A2-restricted epitope PD-L115–23 oran irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, as depictedin Figure 1a. After four in vitro stimulations theT-cell reactivity toward the CMV epitope was examined for each donor by the use ofHLA-A2/CMV tetramers (Figure 1b). Notably, we observed asignificant increase in the numbers of virus-specific T cells in the cultures that hadbeen co-stimulated with the PD-L115–23 peptide epitope. Examples ofthree donors, where co-activation of PD-L1-specific T cells significantly boosted T-cellimmunity toward CMV are illustrated in Figure 1c. Thus, thestimulation of PD-L1-specific CTLs by vaccination may additionally boost other effectorT cells by removing PD-L1-positive, immune-suppressive cells that inhibit the activationand proliferation of PD-1-positive T cells. Next, to examine how PD-L1-specific CTLs caninfluence antileukemia immunotherapy in general, we examined the ability ofPD-L115–23-specific CTLs11to kill well-characterized PD-L1+ AML cells—UKE-1,13 SET-2 (ref. 13) andTHP-1 (ref. 14)—in standard 51Cr releaseassays. PD-L115–23-specific CTLs efficiently killed UKE-1 and THP-1cells (Figures 2a and b). In contrast, the SET-2 cells werenot killed by the PD-L115–23-specific CTLs (Figure2a). Likewise, a control EBV-positive B-lymphoblastoid cell line RPMI6666(ref. 15) was not killed by the PD-L1-specific CTLs(Figure 2b). As a further control, thePD-L115–23-specific CTLs efficiently lysed TAP-deficient T2 cellspulsed with PD-L115–23 efficiently, whereas no cytotoxicity wasobserved against T2 cells pulsed with an irrelevant peptide from HIV (Figure 2c). Our observations on one hand show that PD-L1-specific CTLs areable to react directly toward AML cells and kill the malignant cells. However, not allAML cells were killed, as the PD-L1-specific CTLs were not able to kill SET-2 cells. Toexamine whether the activation of PD-L1-specific CTLs could have an indirect effect onthe immunity against SET-2 cells, we stimulated PBMCs from the three donors in which wehad observed an increased CMV response after co-stimulation with thePD-L115–23 peptide (as depicted in Figure2d) with SET-2 cells. Thus, after four in vitro stimulations ofPBMCs with SET-2 cells either in co-culture with the PD-L115–23 epitopeor an irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, weexamined the ability of the resulting T-cell cultures to recognize and kill SET-2 cellsin standard 51Cr release assays. As illustrated in Figure2e the T-cell cultures from all three donors co-stimulated withPD-L115–23 epitope more efficiently lysed SET-2 cells compared withthe cultures co-stimulated with an irrelevant HIV epitope. Hence, althoughPD-L115–23-specific CTLs do not recognize SET-2 cells, theactivation of these by stimulation boosted additional T-cell immunity toward SET-2cells. This could point to a scenario were PD-L1-based vaccination might be beneficialeven in leukemia patients where PD-L1-specific CTLs do not react toward the leukemiacells themselves. Thus, the enhancement of PD-L1-specific CTLs in patients might bevaluable both by the direct killing of leukemia cells as well as indirectly by thereinforcement of antileukemic T cells. The addition of PD-L1 vaccination should beeasily implementable and highly synergistic with other immune-based therapies. Theinduction of specific T cells represents a new and attractive immune therapeuticapproach, in which the specific depletion of target cells is not limited to targetingproteins that are expressed on the cell surface. This is important, as the PD-L1 epitopeused in this study is located near the N-terminal of the PD-L1 sequence as part of thesignal peptide, and is therefore not part of the extracellular domain. An additionalprincipal difference between therapeutically induced T cells and surface blockade byantibodies is that the former reduces not only the target protein-mediated immunesuppression but also other immune-suppressive effects mediated by the target cells.Taken together, vaccination against PD-L1 and antibody-mediated PD-1/PD-L1 blockadeshould therefore be considered complementary rather than combative. In fact, an excitingtherapeutic strategy would be to combine anti-PD-L1 vaccination with, for example,anti-CTLA4- or anti-LAG3-blocking antibodies. Taken together, we believe that thefindings justify and warrant clinical testing to evaluate the efficiency and safety ofPD-L1-based vaccinations in hematological malignancies. Hence, we are in the process ofinitiating a phase I vaccination study at the Center for Caner Immune Therapy,Copenhagen University Hospital, Herlev.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Immune Therapy (CCIT), Department of Hematology and Oncology, Copenhagen University Hospital, Herlev, Herlev, Denmark.

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from acute myeloid leukemia (AML) as well as myelodysplastic syndrome patients, blasts have been found to be positive for PD-L1, whereas stroma/non-blast cellular No severe toxicity was reported and the treatment seemed to induce clinical effects... addition of PD-L1-specific CTLs 1 week after stimulation of PBMCs with viral epitopes Hence, PD-L1-specific CTLs may effectively enhance the effector phase of the immune response... AML cells were killed, as the PD-L1-specific CTLs were not able to kill SET-2 cells... PBMCs with SET-2 cells either in co-culture with the PD-L115–23 epitope PD-L115–23 epitope more efficiently lysed SET-2 cells compared with Hence, although PD-L115–23-specific CTLs do not recognize SET-2 cells, the principal difference between therapeutically induced T cells and surface blockade by antibodies is that the former reduces not only the target protein-mediated immune

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Related in: MedlinePlus