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Dendritic Cell Programming by Cytomegalovirus Stunts Naive T Cell Responses via the PD-L1/PD-1 Pathway

Chris A. Benedict, Andrea Loewendorf, Zacarias Garcia, Bruce R. Blazar and Edith M. Janssen
J Immunol April 1, 2008, 180 (7) 4836-4847; DOI: https://doi.org/10.4049/jimmunol.180.7.4836
Chris A. Benedict
Department of Molecular Immunology and
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Andrea Loewendorf
Department of Molecular Immunology and
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Zacarias Garcia
Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and
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Bruce R. Blazar
Cancer Center and Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455
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Edith M. Janssen
Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and
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  • FIGURE 1.
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    FIGURE 1.

    MCMV selectively down-regulates MHC and costimulatory molecules on DC. DC were isolated from spleens of B6 wild-type mice (ex vivo) or were generated from bone marrow (BM). CD11c+ DC were left untreated (naive), exposed to UV-MCMV, or infected with MCMV-GFP (multiplicity of infection of 1). After 6 (dotted lines) and 24 h (bold lines) the expression of MHC classes I and II, costimulatory molecules CD40, CD80, and CD86, and PD-L1 and PD-L2 was determined in the total DC population (naive and UV-treated MCMV-exposed DC) or within the infected (GFP+) DC. Data are representative of at least five independent experiments.

  • FIGURE 2.
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    FIGURE 2.

    MCMV-infected DC stunt CD8 T cells through engagement of PD-L1/PD-1. Uninfected CD11c+ mDC, MCMV-infected mDC, and UV-MCMV-exposed mDC were incubated with OVA or irradiated actmOVA/Kb−/− T cells. Twenty-four hours later, CD45.1+ CFSE-labeled OT-1 T cells were added with blocking Ab to PD-L1 or isotype control and incubated for 70 h. A, Proliferation of OT-1 T cells (CD8+, CD45.1+, Vα2+, and 7-AAD−) as determined by CFSE dilution. B, Fold expansion of OT-1 T cells. C, IFN-γ production by OT-1 T cells upon OVA257–264 peptide stimulation. D, Percentage of annexin V+-expressing cells in the total OT-1 population. E, Cytolytic activity of OT-1 T cells toward OVA257–264-pulsed EL-4 cells. Background killing was <3% killing in all groups. Data in A, C, and D are representative data of at least three independent experiments. Data in B and E represent mean and individual values (n = 3–4) and are representative of at least two independent experiments.

  • FIGURE 3.
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    FIGURE 3.

    PD-L1-mediated regulation of T cell responses by MCMV-infected DC when presenting “self” Ag. DC were generated from bone marrow of actmOVA B6 mice. Purified untreated DC, MCMV-infected, or UV-MCMV-exposed DC were cultured with CD45.1+ CFSE-labeled OT-1 (A and B) or CD90.1+ CFSE-labeled OT-2 T cells (C and D) in the presence of blocking Ab to PD-L1 or isotype control. A and C, Proliferation of OT-1 and OT-2 T cells was determined 70 h later by analysis of CFSE dilution in combination with 7-AAD staining and Abs to CD4/8, CD45.1/90.1, and Vα2. B and D, Expression of annexin V by total OT-1 and OT-2 T cells upon 70 h of culture under the indicated conditions. Numbers in the upper right corner represent the percentage of annexin V+ OT-2 T cells. Data are representative of three independent experiments.

  • FIGURE 4.
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    FIGURE 4.

    MCMV-infected DC stunt CD4 T cells through engagement of PD-L1/PD-1. Uninfected, MCMV-infected and UV-MCMV-exposed DC were the same as in Fig. 2 and were incubated with irradiated actmOVA/I-Ab−/− T cells. CD90.1+ CFSE-labeled OT-2 T cells were added together with blocking Ab to PD-L1 or isotype control and analyzed after 70 h of culture. A, Proliferation of OT-2 T cells as determined by CFSE dilution. B, IFN-γ production by OT-2 T cells (CD4+, CD90.1+, Vα2+, and 7-AAD−) upon stimulation with OVA323–339 peptide. C, Percentage of annexin V expressing cells in the total OT-2 population. D, Recall responses to OVA protein and OVA2323–339 peptide in the presence of T-depleted splenocytes after a 6-day culture with the different DC. Data in A–C are representative data of at least three independent experiments. Data in D represent mean ± SEM (n = 3–4) and are representative of at least two independent experiments.

  • FIGURE 5.
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    FIGURE 5.

    Stunted CD4 T cells are rescued by IL-2 and do not acquire regulatory function. A, IL-2 restores proliferative capacity in OT-2 T cells activated by MCMV-infected DC. OT-2 T cells were incubated for 3 days with indicated DC and Abs. Subsequently, cells were rested for 3 days and restimulated with OVA and syngeneic T-depleted splenocytes in the presence of absence of recombinant IL-2. B, OT-2 T cells activated by MCMV-infected DC do not express FoxP3 or have the capacity to suppress proliferation in CFSE-labeled naive CD4 T cells upon cross-linking of CD3. Data in A represent mean ± SEM (n = 4) and are representative of at least three independent experiments. Data in B show a representative histogram of two independent experiments (n = 3/group).

  • FIGURE 6.
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    FIGURE 6.

    Priming by MCMV-infected DC stunts T cells in vivo. C57BL/6-Kbm1/CD45.2+ recipient mice received CFSE-labeled purified OT-1- Kbm1/CD45.1+ cells mixed in a 1:1 ratio with CFSE-labeled, nontransgenic Kbm1/CD45.2+ CD8 T cells. Twenty-four hours later, naive or MCMV-infected actmOVA-Kb+/+ DC were adoptively transferred. PD-L1-blocking Ab or isotype control was administered daily. Three days after DC transfer, the OT-1-Kbm1/CD45.1+ responses were analyzed. A, Schema of the transfer and treatments. B, Proliferation of OT-1-Kbm1/CD45.1 as determined by CFSE dilution. Left panels, CFSE vs CD45.1 staining within the CD8+ T cell population. Right panels, CFSE histograms of the OT-1-Kbm1/CD45.1+ cells. C, Expansion of OT-1-Kbm1/CD45.1+ T cells in the different groups determined by the ratio of OT-1- Kbm1/CD45.1+ T cells compared with the internal control (CFSE-labeled C57BL/6-Kbm1/CD45.2+ CD8 T cells). D, IFN-γ production by OT-1-Kbm1/CD45.1+ T cells after a 5-h stimulation with OVA257–264 peptide. Numbers indicate the percentage of IFN-γ-producing cells. E, Cytolytic activity on a per-cell basis of OT-1-Kbm1/CD45.1+ T cells toward OVA257–264-pulsed target cells (•) or control targets (○). Data in B and D are representative data of at least two independent experiments (n = 3–4 in each experiment). Data in C and E represent mean ± SEM (n = 3–4) and are representative of at least two independent experiments.

  • FIGURE 7.
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    FIGURE 7.

    Direct priming by MCMV-infected DC inhibits endogenous T cell responses in vivo. A, B6 wild-type mice were directly infected with MCMV (5 × 104 PFU) and treated for 3 days with anti-PD-L1 (black bars) or isotype (gray bars). Seven days postinfection the frequency of IFN-γ-producing MCMV-specific CD8 T cell responses was determined after in vitro restimulation with the indicated peptides. For B and C, DC from either wild-type or Kb−/−Db−/− mice were infected in vitro for 24 h, and purified infected DC were transferred into Kbm1 recipients. Mice were treated with anti-PD-L1 or isotype control for the first 3 days following transfer, and 4 days later frequencies of MCMV-specific CD8 T cell responses were determined by IFN-γ production upon stimulation with indicated Db-restricted (B) and Kb-restricted peptides (C). Gray bars represent responses to irrelevant peptide. Data are presented as mean ± SEM (n = 5).

Tables

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    Table I.

    Effect of PD-1, PD-L1, and PD-L2 blocking on OT-1 T cell expansion, survival, and IFN-γ productiona

    Cells (×105)bFold Expansion% IFN-γ+c% 7-AAD/Annexin V+
    NO DC
     IgG2a0.7 ± 0.30.1 ± 0.10.1 ± 0.190.3 ± 18.4
     Anti-PD-10.6 ± 0.20.1 ± 0.10.2 ± 0.287.1 ± 9.0
     Anti-PD-L10.7 ± 0.30.1 ± 0.10.1 ± 0.189.5 ± 9.5
     Anti-PD-L20.6 ± 0.30.1 ± 0.10.1 ± 0.182.4 ± 13.3
    Uninfected DC
     IgG2a28.1 ± 4.27.0 ± 1.269.8 ± 6.924.2 ± 8.5
     Anti-PD-132.7 ± 5.78.1 ± 1.473.2 ± 10.722.9 ± 12.4
     Anti-PD-L134.1 ± 6.18.4 ± 1.571.5 ± 5.427.1 ± 8.7
     Anti-PD-L231.6 ± 3.37.9 ± 0.867.9 ± 9.324.6 ± 11.3
    MCMV-infected DC
     IgG2a1.7 ± 0.60.4 ± 0.214.7 ± 4.374.4 ± 11.9
     Anti-PD-18.3 ± 2.7d2.1 ± 0.5d44.3 ± 7.80d40.7 ± 7.7d
     Anti-PD-L18.9 ± 1.3d2.2 ± 0.3d47.1 ± 11.4d43.1 ± 8.8d
     Anti-PD-L21.5 ± 0.40.4 ± 0.118.2 ± 10.181.2 ± 12.1
    • a Purified uninfected and MCMV-infected DC were incubated with irradiated actmOVA-Kb−/− T cells. After an overnight incubation, CD45.1+ CFSE-labeled OT-1 T cells were added to the wells with blocking antibody to PD-1, PD-L1, PD-L2, or isotype control. Proliferation, fold expansion, apoptosis, and IFN-γ production by OT-1 T cells were determined 70 h later.

    • b Number of live cells at the end of the 70-h culture (n = 3/group).

    • c Determined within the 7-AAD/annexin-V-negative population.

    • d , p < 0.05 compared to isotype-treated culture.

    • View popup
    Table II.

    Effect of PD-1, PD-L1, and PD-L2 blocking on OT-2 T cell expansion, survival, and IFN-γ productiona

    Cells (×105)bFold Expansion% IFN-γ+c% 7-AAD/Annexin V+
    NO DC
     IgG2a0.4 ± 0.10.1 ± 0.12.8 ± 0.995.7 ± 8.2
     Anti-PD-10.4 ± 0.30.1 ± 0.12.7 ± 0.896.8 ± 3.5
     Anti-PD-L10.3 ± 0.10.1 ± 0.12.6 ± 0.895.3 ± 4.1
     Anti-PD-L20.4 ± 0.20.1 ± 0.12.7 ± 1.196.8 ± 3.3
    Uninfected DC
     IgG2a49.6 ± 8.312.2 ± 2.189.1 ± 9.222.2 ± 6.5
     Anti-PD-145.8 ± 9.611.7 ± 2.483.2 ± 7.523.0 ± 5.7
     Anti-PD-L154.0 ± 12.413.5 ± 3.191.2 ± 5.423.1 ± 7.8
     Anti-PD-L254.5 ± 8.513.6 ± 2.182.2 ± 11.022.6 ± 3.3
    MCMV-infected DC
     IgG2a2.1 ± 1.30.5 ± 0.316.3 ± 6.486.6 ± 1.9
     Anti-PD-120.7 ± 2.3d5.2 ± 0.6d68.9 ± 11.7d51.4 ± 8.9d
     Anti-PD-L119.7 ± 4.5d4.9 ± 1.1d72.1 ± 10.1d49.1 ± 8.6d
     Anti-PD-L22.6 ± 1.10.7 ± 0.317.9 ± 12.484.2 ± 7.1
    • a Purified uninfected and MCMV-infected DC were incubated with irradiated actmOVA-I-Ab−/− T cells. After an overnight incubation, CD45.1+ CFSE-labeled OT-2 T cells were added to the wells with blocking antibody to PD-1, PD-L1, PD-L2, or isotype control. Proliferation, fold expansion, and apoptosis production by OT-2 T cells were determined 70 h later. IFN-γ production was determined after an additional 20 incubations with OVA323–339peptide.

    • b Number of live cells at the end of the 70-h culture (n = 3 per group).

    • c Determined within the 7-AAD/annexin V-negative population.

    • d , p < 0.05 compared to isotype-treated culture.

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The Journal of Immunology: 180 (7)
The Journal of Immunology
Vol. 180, Issue 7
1 Apr 2008
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Dendritic Cell Programming by Cytomegalovirus Stunts Naive T Cell Responses via the PD-L1/PD-1 Pathway
Chris A. Benedict, Andrea Loewendorf, Zacarias Garcia, Bruce R. Blazar, Edith M. Janssen
The Journal of Immunology April 1, 2008, 180 (7) 4836-4847; DOI: 10.4049/jimmunol.180.7.4836

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Dendritic Cell Programming by Cytomegalovirus Stunts Naive T Cell Responses via the PD-L1/PD-1 Pathway
Chris A. Benedict, Andrea Loewendorf, Zacarias Garcia, Bruce R. Blazar, Edith M. Janssen
The Journal of Immunology April 1, 2008, 180 (7) 4836-4847; DOI: 10.4049/jimmunol.180.7.4836
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