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

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/K^b−/− 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 OVA_257–264 peptide stimulation. D, Percentage of annexin V⁺-expressing cells in the total OT-1 population. E, Cytolytic activity of OT-1 T cells toward OVA_257–264-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.

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.

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-A^b−/− 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 OVA_323–339 peptide. C, Percentage of annexin V expressing cells in the total OT-2 population. D, Recall responses to OVA protein and OVA_2323–339 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.

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).

Priming by MCMV-infected DC stunts T cells in vivo. C57BL/6-K^bm1/CD45.2⁺ recipient mice received CFSE-labeled purified OT-1- K^bm1/CD45.1⁺ cells mixed in a 1:1 ratio with CFSE-labeled, nontransgenic K^bm1/CD45.2⁺ CD8 T cells. Twenty-four hours later, naive or MCMV-infected actmOVA-K^b+/+ DC were adoptively transferred. PD-L1-blocking Ab or isotype control was administered daily. Three days after DC transfer, the OT-1-K^bm1/CD45.1⁺ responses were analyzed. A, Schema of the transfer and treatments. B, Proliferation of OT-1-K^bm1/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-K^bm1/CD45.1⁺ cells. C, Expansion of OT-1-K^bm1/CD45.1⁺ T cells in the different groups determined by the ratio of OT-1- K^bm1/CD45.1⁺ T cells compared with the internal control (CFSE-labeled C57BL/6-K^bm1/CD45.2⁺ CD8 T cells). D, IFN-γ production by OT-1-K^bm1/CD45.1⁺ T cells after a 5-h stimulation with OVA_257–264 peptide. Numbers indicate the percentage of IFN-γ-producing cells. E, Cytolytic activity on a per-cell basis of OT-1-K^bm1/CD45.1⁺ T cells toward OVA_257–264-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.