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. 2004 Jul 20;101(29):10691-6.
doi: 10.1073/pnas.0307252101. Epub 2004 Jul 12.

PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells

Yvette E Latchman  1 , Spencer C Liang, Yin Wu, Tatyana Chernova, Raymond A Sobel, Martina Klemm, Vijay K Kuchroo, Gordon J Freeman, Arlene H Sharpe
Affiliations

PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells

Yvette E Latchman et al. Proc Natl Acad Sci U S A. .

Abstract

Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1(-/-)) mice. CD4(+) and CD8(+) T cell responses were markedly enhanced in PD-L1(-/-) mice compared with wild-type mice in vitro and in vivo. PD-L1(-/-) dendritic cells stimulated greater wild-type CD4(+) T cell responses than wild-type dendritic cells, and PD-L1(-/-) CD4(+) T cells produced more cytokines than wild-type CD4(+) T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8(+) T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8(+) T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4(+) T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1(-/-) recipients led to exacerbated disease. Disease was even more severe in PD-L1(-/-) recipients of PD-L1(-/-) T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.

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Figures

Fig. 1.
Fig. 1.
Generation of PD-L1–/– mice. (a) The structure of the PD-L1-_targeting vector is shown (Top), and Neo replaced the signal exon and the IgV region (C57BL/6 construct). (Middle) The genomic organization of the PD-L1 gene (not to scale). Exons are open boxes. Homologous recombination of the PD-L1 gene is represented (Bottom). *, the position of the probe. (b) Southern blot analysis of PD-L1–/– mice. Wild-type DNA yields a 15.7-kb band and the _targeted allele yields a 10.1-kb band. (c) Splenocytes were activated for 24 h with anti-CD3 (1 μg/ml) for T cells or LPS (10 μg/ml) for B cells. Flt-3-expanded DCs were isolated and matured overnight on plastic. Cells were stained with anti-PD-L1 FITC and relevant lineage-specific mAb-PE. Graphs are gated on CD4+, CD8+, CD19+, or CD11c+ cells as indicated. PD-L1+/+ mice (thick solid line), PD-L1–/– mice (dotted line), and isotype control (thin solid line).
Fig. 2.
Fig. 2.
PD-L1 deficiency on the T cell and the APC enhanced IFN-γ production by T cells. To evaluate the role of PD-L1 on T cells, purified CD4+ T cells were stimulated with 1 μg/ml anti-CD3 plus 1 μg/ml anti-CD28 (a) and 10 μg/ml anti-CD3 plus 1 μg/ml anti-CD28 (b), and IFN-γ production was analyzed. (c) Purified CD8+ T cells were stimulated as in b, and IFN-γ production was analyzed. To examine the role of PD-L1 on the APC, C57BL/6 PD-L1+/+ or PD-L1–/– DC were compared as stimulators in a mixed lymphocyte reaction with BALB/c CD4+ T cells. DCs were expanded in vivo by Flt-3 ligand, isolated, matured overnight on plastic, irradiated, and cultured with BALB/c CD4+ T cells. IFN-γ (d), IL-2 (e), and IL-4 (f) were assayed by ELISA. These data are representative of three to six independent experiments.
Fig. 3.
Fig. 3.
Augmented CD8+ T cell clonal expansion and cytotoxic T lymphocyte responses in PD-L1–/– mice. (a) Mice were immunized with OVA in CFA, and 10 days later CD8+ T cells from LN cells were purified and restimulated with EL4-OVA or EL4 cells, and IFN-γ production assayed at days 1, 2, and 3. (b) LN cells were restimulated with EL4-OVA or EL4 cells and stained with CD8-FITC and Kb SIINFEKL tetramer-PE. Numbers in the upper right corner represent percent of CD8+ that was tetramer-positive. (c) Mice were immunized with OVA in CFA, and 10 days later splenocytes were restimulated with EL4-OVA cells for 5 days. Effector cells were recovered and plated with 51Cr-labeled SIINFEKL-pulsed EL4 at the indicated effector/_target ratios. (d) Experiments were set up as in c, except CD8+ T cell numbers were normalized according to Kb SIINFEKL tetramer staining. These data are representative of four independent experiments.
Fig. 4.
Fig. 4.
Increased susceptibility to EAE in PD-L1–/– mice. (a) 129Sv PD-L1–/– (○) and PD-L1+/+ (•) mice were immunized with MOG33–55 and mice were scored daily. (b–f) To assess MOG-specific responses, mice were immunized with MOG33–55, and 10 days later draining LN cells were harvested and restimulated with MOG33–55. (b) Proliferation was measured at day 2, and IL-2 (c), IFN-γ (d), and IL-10 (e) were assayed from days 0 to 4 by ELISA. To determine the number of antigen-specific IFN-γ producing cells, draining LN cells were restimulated with MOG33–55 for 24 h, and (f) enzyme-linked immunospot assays were performed. These data are representative of three to four independent experiments.
Fig. 5.
Fig. 5.
Effector phase of EAE is exacerbated in the absence of PD-L1. Mice were immunized with MOG33–55. LN cells were harvested 10 days later, restimulated with MOG33–55 for 4 days, and transferred into PD-L1+/+ or PD-L1–/– recipients as indicated. These data are representative of four independent experiments.
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