Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis

Abstract

Introduction: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients.

Methods: Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry.

Results: Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01).

Conclusions: In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.

Figure 1

PD-1, PD-L1 and HLA-DR expression in septic and non-septic patients. Septic and non-septic patients were identified and heparinized blood samples obtained at a maximum of four time points during their septic course. Peripheral blood mononuclear cells were stained for lymphocyte (CD4, CD8) and monocyte markers (CD14). Immunostaining was also performed for programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1) and human leukocyte antigen-DR (HLA-DR). Flow cytometry revealed an increase in PD-1 and PD-L1 expression in CD8 T cells and monocytes from septic versus non-septic patients. HLA-DR expression was decreased in monocytes from septic versus non-septic patients as well. Data are from 43 septic (70 data points) and 16 non-septic patients (16 data points). Septic and non-septic patients had up to four serial blood samples obtained depending upon the duration of their illness and/or discharge from the ICU; - first draw = days 1 to 3 after admission to the ICU, days 4 to 7 (second blood draw), days 8 to 12 (third blood draw), and days 13 to 21 (fourth blood draw) after sepsis onset. Most non-septic patients were discharged from the ICU within four to five days and, therefore, they had less serial blood samples obtained compared to septic patients.

Figure 2

Markers of immune exhaustion increase with protracted sepsis. Flow cytometry revealed an increase in expression of immune exhaustion markers over time in the ICU. A) Monocyte PD-L1 expression was increased in septic patients compared to critically-ill non-septic patients (CINS) patients. In contrast to changes in programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression on CD8 T cells during sepsis, there was no change in the expression of PD-L1 during the septic time period. B) Comparison of monocyte PD-L1 expression and human leukocyte antigen-DR (HLA-DR) expression did not show any correlation in patients with sepsis. (C) PD-1 expression increased as PD-L1 expression decreased on CD8 T cells in samples from septic patients over the course of their sepsis, that is, time points A (days 1 to 3), B (days 4 to 7), C (days 8 to 12) and D (days 13 to 21). CD8 T cell PD-1 expression was higher in septic versus critically-ill non-septic patients (CINS). CD8 T cell expression of PD-L1 fell to very low levels at time point D in septic patients compared to CINS patients and at time points A-C in septic patients. P-values in 2A and 2C are comparison of septic samples with CINS for each draw.

Figure 3

Representative flow histograms of lymphocyte gating strategy and detection of apoptosis and IFN-γ. Peripheral blood mononuclear cells (PBMCs) from a septic patient were plated overnight with isotype control antibody, anti-programmed cell death 1 (PD-1) or anti-programmed cell death ligand 1 (PD-L1) antibody. A: Cells were immunostained for CD4 and CD8 T cells and TUNEL assay performed. The lymphocyte fraction in the PBMCs was identified by characteristic forward and side scatter properties and CD4 and CD8 T cells identified by cell-specific antibodies. Apoptosis in CD4 T cells incubated in inactive isotype control antibody was 7.5%. Anti-PD-1 and anti-PD-L1 decreased CD4 apoptosis to 1.25 and 2.0%, respectively. A similar protective effect was seen in CD8 T cells. B: PBMCs from a septic patient were incubated overnight with isotype control antibody, anti-PD-1 or anti-PD-L1 antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 h, washed, immunostained with phenotypic markers to CD3 and CD56, fixed and stained for intracellular interferon (IFN)-γ. The lymphocyte gate was identified by characteristic forward and side scatter properties. Natural killer (NK) cells were identified as CD3 negative CD56 positive. The percentage of NK cells that were positive for IFN-γ that were incubated in inactive isotype control antibody was 9.9%. Treatment with anti-PD-1 and anti-PD-L1 increased the percentage of NK cells that were IFN-γ positive to 34.8 and 21.8%, respectively.

Figure 4

Anti-PD-1 and anti-PD-L1 decrease lymphocyte apoptosis in sepsis. Peripheral blood mononuclear cells (PBMCs) from septic or critically-ill non-septic (CINS) patients were incubated overnight in media containing isotype control antibody, anti-programmed cell death 1 (−PD-1) or anti-programmed cell death ligand 1 (−PD-L1) antibody. The following morning, cells were washed and underwent immunostaining followed by detection of apoptosis via TUNEL assay. Compared to treatment with inactive isotype control antibody, incubation with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis in total lymphocytes, CD4 and CD8 T cells from septic patients; P <0.001. There was no significant effect of anti-PD-1 or anti-PD-L1 in CINS patients, possibly due to the fact that baseline apoptosis in the non-septic patients was so low. Values shown are the mean ± SEM values for all time points for 19 septic and 7 CINS patients. Circles indicate mean per group.

Figure 5

Sepsis impairs lymphocyte IFN-γ and IL-2 production. Peripheral blood mononuclear cells (PBMCs) from septic or critically-ill non-septic (CINS) patients were incubated overnight in media containing isotype control antibody. Blood from septic patients was obtained at three time points during the sepsis, that is, days 1 to 3 (Septic A), days 4 to 7 (Septic B) and days 8 to 12 (Septic C). Samples from days 13 to 21 (Septic D) were not tested. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 h, washed, immunostained with phenotypic markers to CD3 and CD56, fixed and stained for intracellular interferon (IFN)-γ or interleukin (IL)-2. Flow cytometric analysis revealed a persistent decrease in the percentage of total lymphocytes and natural killer T (NKT) cells that were IFN-γ positive in septic compared to CINS patients throughout most of the septic duration. The difference in IFN-γ production in septic versus CINS patients did not quite reach statistical significance for CD3 T cells, P = 0.07. A similar pattern of decreased IL-2 production in septic versus CINS patients occurred at all septic time points. Data are from 15 septic (21 data points) and 7 CINS patients (7 data points) obtained during their illness. P-values shown are comparison of septic samples with CINS for each draw.

Figure 6

Anti-PD-1 and anti-PD-L1 antibodies increase IFN-γ production in sepsis. Peripheral blood mononuclear cells (PBMCs) from septic or critically-ill non-septic (CINS) patients were incubated overnight in media containing isotype control antibody, anti- programmed cell death 1 (−PD-1) or anti- programmed cell death ligand 1 (−PD-L1) antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 h, washed, immunostained with phenotypic markers to CD3 and CD56, fixed and stained for intracellular interferon (IFN)-γ. Flow cytometric analysis revealed that, compared to inactive isotype control antibody, both anti-PD-1 and anti-PD-L1 antibody caused an increase in the percentage of total lymphocytes and natural killer T (NKT) cells that were IFN-γ positive. Anti-PD-1 had no significant effect in CINS patients while anti-PD-L1 increased IFN-γ in total lymphocytes only. Note that IFN-γ production was higher in CINS patients compared to septic patients when incubated with inactive isotype control antibody. Data are from 15 septic patients (21 data points) throughout their illness - all blood draws. Values shown are mean ± SEM values for all time points. Circles indicate mean per group.

Figure 7

Anti-PD-1 and anti-PD-L1 antibodies increase IL-2 production in sepsis. Peripheral blood mononuclear cells (PBMCs) from septic or critically-ill non-septic (CINS) patients were incubated overnight in media containing isotype control antibody, anti- programmed cell death 1 (PD-1) or anti- programmed cell death ligand 1 (PD-L1) antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 h, washed, immunostained with phenotypic markers to CD3 and CD56, fixed and stained for intracellular interleukin (IL)-2. Flow cytometric analysis revealed that, compared to inactive isotype control antibody, both anti-PD-1 and anti-PD-L1 antibody caused an increase in the percentage of CD3 and natural killer T (NKT) cells that were IL-2 positive. Anti-PD-L1 had an effect to increase IL-2 production in total lymphocytes and NKT cells in CINS patients. Note that IL-2 production was higher in CINS patients compared to septic patients when incubated with inactive isotype control antibody. Data are from 15 septic patients (21 data points) throughout their illness. Values shown are the mean ± SEM values for all time points. Circles indicate mean per group.

Figure 8

Correlation of absolute lymphocyte count and apoptosis. A low absolute lymphocyte count (ALC) is a frequent finding in sepsis and correlates with outcome (see Discussion). We examined the relationship between ALC and apoptosis as determined by the Tunel assay. There was no relationship of apoptosis (percent Tunel positive cells) and the ALC in septic patient considered as a whole (A). If the correlation of apoptosis and ALC was restricted to septic patients who had an ALC below the lower limit of normal at Barnes Jewish Hospital (1.2 × 10³ lymphocytes/μL blood), there was a correlation such that patients with the lowest ALCs had the highest percent of Tunel + (apoptotic) lymphocytes. (B) There was a statistical correction between ALC and lymphocyte apoptosis in septic patients who had an ALC less than 1.2 × 10³ cells/μL blood (the lower limit of normal at our hospital).

Figure 9

Correlation of PD-1 expression and absolute cell counts and apoptosis. (A and B): Since anti-programmed cell death 1 (PD-1) antibody decreased lymphocyte apoptosis (Figure 4), we investigated the correlation between PD-1 expression on CD4 and CD8 T cells and absolute CD4 and CD8 T cells, respectively. Neither CD4 nor CD8 PD-1 expression correlated with absolute (total) CD4 or CD8 T cell counts respectively. (C): We also investigated the correlation between PD-1 expression and apoptosis by examining the relationship between percent PD-1 + CD4 T cells and the percent Tunel + CD4 T cells. At the time of the first blood draw in septic patients (days 1 to 3), there was a positive correlation between PD-1 expression on CD4 T cells and the percent of apoptotic cells (Tunel+).