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. 2010 Feb;84(4):2078-89.
doi: 10.1128/JVI.01579-09. Epub 2009 Dec 2.

Tissue-specific differences in PD-1 and PD-L1 expression during chronic viral infection: implications for CD8 T-cell exhaustion

Shawn D Blackburn  1 , Alison Crawford, Haina Shin, Antonio Polley, Gordon J Freeman, E John Wherry
Affiliations

Tissue-specific differences in PD-1 and PD-L1 expression during chronic viral infection: implications for CD8 T-cell exhaustion

Shawn D Blackburn et al. J Virol. 2010 Feb.

Abstract

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8(+) T cells. We examined the expression of PD-1 and its ligands, PD-L1 and PD-L2, in multiple tissues during the course of chronic viral infection and determined how the amount of PD-1 expressed, as well as the anatomical location, influenced the function of exhausted CD8 T cells. The amount of PD-1 on exhausted CD8 T cells from different anatomical locations did not always correlate with infectious virus but did reflect viral antigen in some tissues. Moreover, lower expression of PD-L1 in some locations, such as the bone marrow, favored the survival of PD-1(Hi) exhausted CD8 T cells, suggesting that some anatomical sites might provide a survival niche for subpopulations of exhausted CD8 T cells. Tissue-specific differences in the function of exhausted CD8 T cells were also observed. However, while cytokine production did not strictly correlate with the amount of PD-1 expressed by exhausted CD8 T cells from different tissues, the ability to degranulate and kill were tightly linked to PD-1 expression regardless of the anatomical location. These observations have implications for human chronic infections and for therapeutic interventions based on blockade of the PD-1 pathway.

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Figures

FIG. 1.
FIG. 1.
Kinetics and pattern of PD-1 expression by virus specific CD8+ T cells in different anatomical locations. (A) After infection with LCMV clone 13, lymphocytes were isolated from the eight tissues indicated, and PD-1 expression was determined on DbGP33 tetramer-positive CD8 T cells at the indicated times postinfection or on naive (CD44Lo) CD8 T cells by flow cytometry. The numbers on the right for each histogram series indicate the MFI of the corresponding histogram. (B) Examples of DbGP33 tetramer versus PD-1 staining for the spleen, liver, and BM on day 90 p.i. (C) Summary data from panel A. PD-1 expression was examined on DbGP33 CD8+ T cells isolated from eight anatomical locations of LCMV Armstrong and LCMV clone 13-infected mice on days 8, 30, and 90 p.i. (n = 3 to 6 mice/tissue/time point).
FIG. 2.
FIG. 2.
Kinetics of control of viral replication varies by anatomical location. (A) Viral load was determined by plaque assay in the indicated tissues of LCMV clone 13-infected mice on day 8, 30, and 90 p.i. Dashed line indicates the approximate limit of detection determined by serum volume, tissue weight, or by assaying the BM from an entire femur. (B) Heat map visualization of the comparison between tissue PD-1 MFI on DbGP33+ CD8 T cells (see Fig. 1) and tissue viral load from panel A. Some Arm viral load data are derived from an earlier study (39). Tissues where PD-1 expression remains high and the viral load is low are highlighted in yellow.
FIG. 3.
FIG. 3.
Persistent expression of viral proteins in LCMV clone 13-infected mice in the absence of plaque-forming virus. (A) Cells were isolated from the spleen and BM of LCMV clone 13-infected mice at day 90 p.i. and stained for intracellular LCMV NP expression. (B) The relative frequency of LCMV NP expressing cells in the spleen and BM on days 8, 30, and 90 p.i. is indicated. The data represent three mice/time point and are representative of two (days 8 and 30) or three (day 90) independent experiments.
FIG. 4.
FIG. 4.
PD-ligand expression varies by anatomical location and time p.i. (A) PD-L1 expression on phenotypically defined cell types from the spleens and the BM of clone 13-infected mice at day 30 p.i. FMO, fluorescence minus one negative control. (B) Longitudinal analysis of PD-L1 expression in the spleen and BM expressed as the MFI. (C) PD-L2 expression on phenotypically defined cell types from the spleen and BM of clone 13-infected mice at day 30 p.i. (D) Longitudinal analysis of PD-L2 expression in the BM and the spleen expressed as MFI. (E) PD-L1 expression on virus-infected cells (anti-LCMV NP+, primarily CD4− B220− CD8aLo CD11bHi CD11cInt) in the spleen and BM (n = 3 to 5 for each time point for panels A to E).
FIG. 5.
FIG. 5.
Survival of PD-1Hi exhausted CD8+ T cells was influenced by tissue-resident APCs. (A) PD-1Hi exhausted CD8+ T cells from the BM of LCMV clone 13-infected mice (∼3 months p.i.) were cultured for 5 h with BM-derived CD8-depleted “APCs” alone or in combination with splenic CD8-depleted APC- from the same infected mice. The percentage of dead/dying PD-1Hi CD8+ T cells was determined by using an amine reactive dye (vital dye). (B) The assay in panel A was repeated with or without the addition of 10 μg of αPD-L1/ml. Similar results were observed using vital dye exclusion and staining for active caspase-3 (data not shown). The data represent the findings in two to four independent experiments.
FIG. 6.
FIG. 6.
PD-1Lo and PD-1Hi subsets of virus-specific CD8+ T-cell differ in their functional capacity during chronic LCMV infection. (A) CD8+ T cells from the spleen (PD-1Lo/Int) and from the BM (PD-1Hi) from clone 13-infected mice day 45 p.i. were stimulated for 5 h with the GP33-41 peptide. IFN-γ, TNF, and MIP-1α production were examined by flow cytometry. The numbers indicate the percentages of responding cells coproducing the two indicated cytokines. (B) Summary data on the coproduction of IFN-γ and TNF for multiple mice (n = 3). (C) Lymphocytes from the spleens, livers, and BM isolated from clone 13-infected mice at day 45 p.i. were stimulated for 5 h with the GP33-41 peptide and stained for PD-1 and IFN-γ. The numbers indicate the percentages of IFN-γ+ cells that were PD-1Lo or PD-1Hi. (D) The MFI of IFN-γ for PD-1Lo or PD-1Hi virus-specific CD8 T cells from the spleen, liver, or BM on day 45 p.i.
FIG. 7.
FIG. 7.
Anatomical differences in functional capacity are not dramatically influenced by resident APCs. (A) Splenocytes from LCMV clone 13-infected CD45.1 mice (day 90 p.i.) were mixed with BM lymphocytes from LCMV clone 13-infected CD45.2 mice (day 90 p.i.), stimulated with GP33 peptide, and the intracellular cytokines were examined after 5 h as in Fig. 6. (B) Splenocytes and BM lymphocytes from LCMV clone 13-infected mice (day 90 p.i.) were stimulated with GP33-41 peptide in the presence or absence of αPD-L1 antibody, and the production of IFN-γ was assessed by ICS after 5 h.
FIG. 8.
FIG. 8.
PD-1Hi CD8+ T cells have diminished cytotoxic potential despite high expression of granzyme B. (A) A VITAL killing assay was performed using GP33-41-pulsed targets and purified CD8+ T cells from the spleens, livers, or BM of LCMV clone 13-infected mice at day 90 p.i. Splenocytes from LCMV Arm-infected mice (day 10 p.i.) served as a positive control. The assay was performed in the presence or absence of αPD-L1 antibody with an E:T for DbGP33-specific CD8 T cells of 0.5 for 4, 12, or 20 h (20-h time point shown; data are representative of three independent experiments). (B and C) DbGP33+ CD8+ T cells from the spleens, livers, and BM isolated from clone 13-infected mice at day 45 p.i were stained for granzyme B and PD-1. The MFI values of granzyme B for DbGP33+ CD8 T cells from the indicated tissues are shown. (D and E) Lymphocytes from the spleens, livers, and BM were stimulated in vitro with GP33-41 peptide, and CD107a degranulation was assessed in combination with staining for PD-1. Degranulation for DbGP33+ CD8 T cells from the three tissues is indicated.
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