Role of PD-1 and its ligand, B7-H1, in early fate decisions of CD8 T cells

Abstract

Expression of the PD-1 receptor on T cells has been shown to provide an important inhibitory signal that down-modulates peripheral effector responses in normal tissues and tumors. Furthermore, PD-1 up-regulation on chronically activated T cells can maintain them in a partially reversible inactive state. The function of PD-1 in the very early stages of T-cell response to antigen in vivo has not been fully explored. In this study, we evaluate the role of PD-1 and its 2 B7 family ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), in early fate decisions of CD8 T cells. We show that CD8 T cells specific for influenza hemagglutinin (HA) expressed as a self-antigen become functionally tolerized and express high levels of surface PD-1 by the time of their first cell division. Blockade of PD-1 or B7-H1, but not B7-DC, at the time of self-antigen encounter mitigates tolerance induction and results in CD8 T-cell differentiation into functional cytolytic T lymphocytes (CTLs). These findings demonstrate that, in addition to modulating effector functions in the periphery, B7-H1:PD-1 interactions regulate early T-cell-fate decisions.

Figure 1

Early induction of PD-1 on CD8 T cells responding to self-antigen. (A, B) Unsorted Thy1.1-marked HA-specific CD8 T cells from clone 4 TCR transgenic mice were CFSE-labeled and adoptively transferred into animals that express HA as a self-antigen in different amounts (C3-HA^low and C3-HA^high) or to WT mice infected with an attenuated L monocytogenes strain that expresses HA (LM-HA). Plots shown are gated on CD8⁺ Thy1.1⁺ lymphocytes (clone 4 donor cells). (A) PD-1 expression on clone 4 CD8 T cells as a function of division (CFSE dilution). Horizontal line represents isotype control. (B) PD-1 expression on clone 4 T cells transferred to mice that express high levels (C3-HA^high, inverted triangles) or low levels (C3-HA^low, squares) of HA. Mean ± SEM shown. (C) Unlabeled clone 4 T cells transferred as above, total cell number in spleen is shown. For a-c, 5 animals per group, data representative at least 2 experiments. (D) Adoptive transfer into C3-HA^low mice was performed in the presence of 100 μg of the indicated blocking antibody (administered intraperitoneally on day 0). Total number of clone 4 cells per spleen on day 4 is shown. n = 5.

Figure 2

Blockade of PD-1 and its ligands at the time of antigen recognition renders self-antigen-specific T cells competent to produce effector cytokines. (A) Thy1.1-marked clone 4 cells were adoptively transferred into indicated hosts and harvested on day 4. Intracellular cytokine staining for IFN-γ was performed after 5-hour in vitro stimulation with the HA class I peptide in the presence of a blocking antibody cocktail (α-PD-1, α-B7-H1, and α-B7-DC, 30 μg/mL each, middle row) or isotype antibodies (top row). Gated on Thy1.1, 5 animals per group. (B,C) Clone 4 cells were adoptively transferred into C3-HA^low animals as above and PD-1, B7-H1 or B7-DC were blocked in vivo with 100 μg of indicated antibody administered at the time of adoptive transfer. Intracellular staining for IFN-γ was performed on day 4 after transfer. (B) Representative FACS plots, gated on CD8⁺ Thy1.1⁺ clone 4 lymphocytes. (C) Summary data of panel B, mean ± SEM n = 5, representative of 2 experiments.

Figure 3

In vivo blockade of PD-1 and B7-H1 at the time of antigen encounter by self-antigen-specific CD8 T cells results in the development of functional CTL. (A) Clone 4 cells were adoptively transferred into C3-HA^low mice with the indicated blocking antibodies administered intraperitoneally on day 0. Specific lysis in vivo was assayed by transfer of CFSE- or PKH26-labeled, HA peptide-pulsed targets on day 6. Targets from WT, B7-H1 KO, and B7-DC KO animals were differentially labeled (see “Materials and methods”) and injected simultaneously. Percentage specific lysis calculated as described previously, n = 5. No significant differences in target lysis within the antibody treatment groups were detected by ANOVA. (B) In vitro CTL. clone 4 T cells were adoptively transferred as above, harvested on day 4 and sorted by FACS to < 95% purity. CTL were coincubated with HA peptide pulsed targets for at 37° for 4 hours, and percentage target lysis was calculated as described previously.

Figure 4

In vivo blockade of PD-1 and B7-H1 at the time of antigen encounter abrogates tolerance and results in autoimmunity. (A-D) C3-HA^high mice were adoptively transferred with clone 4 cells and anti-PD-1 or anti-B7-H1 blocking antibodies or isotype control on day 0. (A) Survival of antibody-treated treated C3-HA^high mice, n = 5. (B) Mean body weight relative to isotype control. Data are mean ± SEM. (C) FACS analysis of lung-infiltrating lymphocytes, gated on lymphocytes. Percentage shows the gated clone 4 T cells per total lymphocytes. (D) Pulmonary histology: 8-μm frozen sections were stained with hematoxylin and eosin, 100× magnification shown.