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Comparative Study
. 2007 Jun;81(11):5819-28.
doi: 10.1128/JVI.00024-07. Epub 2007 Mar 21.

Elevated expression levels of inhibitory receptor programmed death 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination

Vijayakumar Velu  1 , Sunil Kannanganat, Chris Ibegbu, Lakshmi Chennareddi, Francois Villinger, Gordon J Freeman, Rafi Ahmed, Rama Rao Amara
Affiliations
Comparative Study

Elevated expression levels of inhibitory receptor programmed death 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination

Vijayakumar Velu et al. J Virol. 2007 Jun.

Abstract

Here, we study the temporal expression of the inhibitory receptor programmed death 1 (PD-1) on simian immunodeficiency virus (SIV) Gag-specific T cells following pathogenic SIV infection or following vaccination with a DNA/modified vaccinia virus Ankara (DNA/MVA) vaccine and simian/human immunodeficiency virus (SHIV) challenge in macaques. Following infection, the majority (>95%) of Gag-specific CD8 T cells expressed PD-1, and the level of PD-1 expression per cell increased over time. The level of PD-1 expression in lymph nodes and rectal mucosal tissue, the major sites of virus replication, was higher compared to blood. In vitro blockade of PD-1 resulted in enhanced proliferation of SIV-specific CD8 as well as CD4 T cells. In contrast, following vaccination, the majority of peak effector Gag-specific CD8 T cells expressed low levels of PD-1, and these levels decreased further as the cells differentiated into memory cells. In addition, following SHIV challenge of these vaccinated macaques, the level of PD-1 expression on Gag-specific CD8 T cells correlated positively with plasma viremia. These results demonstrate that SIV-specific CD8 T cells express PD-1 after exposure to antigen but downregulate expression under conditions of antigen clearance and enhance expression under conditions of antigen persistence. They also demonstrate that the level of PD-1 expression per cell rather than the presence or absence of expression plays an important role in regulating CD8 T-cell dysfunction in pathogenic SIV infection. In addition, they demonstrate that similar to HIV infection, the PD-1:PD-1 ligand inhibitory pathway is operational in pathogenic SIV infection, and the macaque/SIV model would be ideal to test the safety and therapeutic benefit of blocking this pathway in vivo.

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Figures

FIG. 1.
FIG. 1.
PD-1 expression on total and SIV Gag-specific CD8 T cells following pathogenic SIV or SHIV infection. (A) Fluorescence-activated cell sorter (FACS) plots representing the expression of PD-1 on total and SIV Gag CM9 tetramer-specific CD8 T cells in blood from a SIV251-infected macaque. PBMC were stained on the surface with antibodies to human CD3, CD8 and PD-1, and SIV Gag-CM9 tetramer. CD8-positive cells (CD3+, CD8+) and tetramer-positive cells were analyzed for expression of PD-1. The open and filled histograms represent PD-1 expression on total and tetramer-specific CD8 T cells, respectively. The numbers on the FACS plots represent the frequency of tetramer-positive cells as a percentage of total CD8 T cells. (B) Summary of frequency of PD-1-positive cells and the MFI of PD-1 on total CD8 T cells at various times following SIV251 infection. (C) Summary of frequency of PD-1-positive cells and the MFI of PD-1 on tetramer-positive cells at various times following SIV251 infection. The data represent the mean values for four macaques. Error bars represent standard deviations. (D) Summary of the frequency of PD-1-positive cells as a percentage of tetramer-positive CD8 T cells at 12 weeks following infection. (E) Fold increase in the MFI of PD-1 on Gag-CM9 tetramer-positive cells over the MFI for total CD8 T cells in respective macaques at 12 weeks after infection. Each symbol represents an individual macaque.
FIG. 2.
FIG. 2.
PD-1 expression on total and SIV Gag-specific CD8 T cells in lymph node and rectal mucosal tissue. (A) Fluorescence-activated cell sorter (FACS) plots representing the expression of PD-1 on total (open histograms) and SIV Gag CM9 tetramer-specific (filled histograms) CD8 T cells in blood, lymph node, and rectal mucosal tissue from a SHIV-infected macaque. Cells were stained on the surface with antibodies to human CD3, CD8 and PD-1, and SIV Gag-CM9 tetramer. CD8-positive cells and tetramer-positive cells were analyzed for expression of PD-1. The numbers on the FACS plots represent the frequencies of tetramer-positive cells as a percentage of total CD8 T cells. (B) Summary of PD-1 expression on total and tetramer-positive CD8 T cells in blood, peripheral lymph node, and rectal mucosal tissue from normal and SHIV-infected macaques. For SHIV-infected macaques, analysis was performed at 12 weeks after infection. Each symbol represents an individual macaque.
FIG. 3.
FIG. 3.
Effect of in vitro blockade of PD-1 on SIV-specific CD8 T cells in SIV- or SHIV-infected macaques. (A) Fluorescence-activated cell sorter plots representing the frequency of Gag-CM9 tetramer-positive cells following stimulation in vitro. PBMC were prestained with CFSE and stimulated for 6 days with P11C peptide in the absence and presence of anti-PD-1 blocking antibody. Unstimulated cells (Nostim) in the presence and absence of blocking antibody served as negative controls. At the end of 6 days, cells were stained on the surface for CD3, CD8, and Gag-CM9 tetramer, acquired on a FACSCalibur, and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA). Cells were gated on CD3 and analyzed for expression of CD8 and tetramer binding. The numbers on the plots represent the frequency of tetramer-positive cells as a percentage of total CD8 T cells. (B) Summary of proliferation data for SIV- or SHIV-infected macaques. Fold increase (frequency of tetramer-positive cells in stimulated cultures over unstimulated cultures) in the frequency of tetramer-positive cells is plotted for each macaque. Each symbol represents an individual macaque. (C) Granzyme B and perforin expression on tetramer-positive cells following in vitro stimulation in the presence and absence of blocking antibody. PBMC were stimulated for 6 days with P11C peptide in the absence and presence of anti-PD-1 blocking antibody. Unstimulated cells (Nostim) served as negative controls. At the end of 6 days cells, were stained on the surface for CD3, CD8, and Gag-CM9 tetramer. Cells were then fixed, permeabilized, and stained for intracellular perforin and granzyme B and acquired on an LSRII apparatus. Cells were gated on CD3, CD8, and tetramer and analyzed for expression of granzyme B or perforin. The gray filled histograms and black open histograms represent expression on P11C-stimulated cells in the absence and presence of anti-PD-1 blocking antibody, respectively. The gray open histograms represent expression on total CD8 T cells in unstimulated cultures. (D) Summary of granzyme B and perforin data for three SHIV-infected macaques.
FIG. 4.
FIG. 4.
PD-1 expression on total and SIV Gag-specific CD8 T cells following vaccination with a DNA/MVA SIV vaccine. (A) Fluorescence-activated cell sorter plots representing the expression of PD-1 on total and SIV Gag CM9 tetramer-specific CD8 T cells in blood from a macaque vaccinated with DNA/MVA vaccine. PBMC were stained on the surface with antibodies to human CD3, CD8 and PD-1, and SIV Gag-CM9 tetramer. CD8-positive cells and tetramer-positive cells were analyzed for expression of PD-1. The gray filled histograms and black open histograms represent PD-1 expression on total and tetramer-specific CD8 T cells, respectively. (B) Summary of PD-1 expression on tetramer-positive CD8 T cells at the peak (1 week) and memory (6 months) phases following the MVA boost. (C) Summary of MFI of PD-1 on tetramer-positive CD8 T cells at the peak (1 week) and memory (6 months) phases following the MVA boost. (D) Effect of in vitro blockade of PD-1 on the proliferative capacity of SIV Gag CM9 tetramer-specific memory cells. Cells were stimulated, stained, and analyzed as described for Fig. 3. Fold increase (frequency of tetramer-positive cells in stimulated cultures over unstimulated cultures) in the frequency of tetramer-positive cells is plotted for each macaque. Each symbol represents an individual macaque.
FIG. 5.
FIG. 5.
PD-1 expression following SHIV challenge in unvaccinated and DNA/MVA-vaccinated macaques. (A) Viral RNA levels in plasma following SHIV challenge. (B) Fluorescence-activated cell sorter plots representing expression of PD-1 on total and SIV Gag CM9 tetramer-specific CD8 T cells in blood from unvaccinated (control) and DNA/MVA-vaccinated macaques at 2 and 12 weeks after SHIV 89.6P challenge. PBMC were stained on the surface with antibodies to human CD3, CD8 and PD-1, and SIV Gag-CM9 tetramer. CD8-positive cells and tetramer-positive cells were analyzed for expression of PD-1. The contour plots and black dots represent PD-1 expression on total and tetramer-specific CD8 T cells, respectively. The boxes on the plots represent PD-1lo (left) and PD-1hi (right) cells. The numbers on the plots represent the frequency of PD-1hi cells as a percentage of total tetramer-positive cells. (C) Summary of the frequency of PD-1-positive cells as a percentage of tetramer-positive CD8 T cells at 12 weeks following SIV or SHIV infection in unvaccinated SIV-infected (control SIV), unvaccinated SHIV-infected (control SHIV), and DNA/MVA-vaccinated SHIV-infected (vaccinated SHIV) macaques. (D) Summary of frequency of PD-1hi cells on tetramer-positive CD8 T cells at 12 weeks after SIV or SHIV infection. (E) Correlation between the frequency of PD-1hi cells and plasma viral load at 12 weeks after infection in unvaccinated SIV-infected, unvaccinated SHIV-infected, and DNA/MVA-vaccinated SHIV-infected macaques. The MFI of PD-1 on tetramer-positive cells could not be used for comparisons between groups because these analyses were performed using different batches of antibody that resulted in differences in staining intensity. Each symbol represents an individual macaque.
FIG. 6.
FIG. 6.
Effect of in vitro blockade of PD-1 on SIV-specific CD4 T cells in SIV-infected macaques. (A) Fluorescence-activated cell sorter (FACS) plots representing the expression of PD-1 on total CD4 T cells in blood from a SIV-infected macaque. PBMC were stained on the surface with antibodies to human CD3, CD4, CD8, and PD-1. CD3+, CD4+, and CD8− cells were analyzed for expression of PD-1. (B) FACS plots representing the frequency of Gag-specific CD4 T cells following stimulation in vitro. PBMC were prestained with CFSE and stimulated for 6 days with Gag peptide pool in the absence and presence of anti-PD-1 blocking antibody. Unstimulated cells (Nostim) served as negative controls. At the end of 6 days cells were stained on the surface for CD3, CD8, and intracellular Ki-67, acquired on a FACSCalibur, and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA). CD4 cells (CD3+, CD8−) were analyzed for CFSE dilution and Ki-67 expression. The numbers on the plots represent the frequency of CFSE−, Ki-67+ cells as a percentage of total CD4 T cells. (C) Summary of proliferation data for SIV-infected macaques. Fold increase (frequency of CFSE-negative, Ki-67-positive cells in stimulated cultures over unstimulated cultures) in the frequency of Gag-specific cells is plotted for each macaque. Each symbol represents an individual macaque.
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