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Observational Study
. 2016 Dec;100(6):1239-1254.
doi: 10.1189/jlb.4HI0616-255R. Epub 2016 Sep 26.

Frontline Science: Defects in immune function in patients with sepsis are associated with PD-1 or PD-L1 expression and can be restored by antibodies targeting PD-1 or PD-L1

Andriani C Patera  1 , Anne M Drewry  2 , Katherine Chang  2 , Evan R Beiter  2 , Dale Osborne  2 , Richard S Hotchkiss  2
Affiliations
Observational Study

Frontline Science: Defects in immune function in patients with sepsis are associated with PD-1 or PD-L1 expression and can be restored by antibodies targeting PD-1 or PD-L1

Andriani C Patera et al. J Leukoc Biol. 2016 Dec.

Abstract

Sepsis is a heterogeneous syndrome comprising a highly diverse and dynamic mixture of hyperinflammatory and compensatory anti-inflammatory immune responses. This immune phenotypic diversity highlights the importance of proper patient selection for treatment with the immunomodulatory drugs that are entering clinical trials. To better understand the serial changes in immunity of critically ill patients and to evaluate the potential efficacy of blocking key inhibitory pathways in sepsis, we undertook a broad phenotypic and functional analysis of innate and acquired immunity in the same aliquot of blood from septic, critically ill nonseptic, and healthy donors. We also tested the ability of blocking the checkpoint inhibitors programmed death receptor-1 (PD-1) and its ligand (PD-L1) to restore the function of innate and acquired immune cells. Neutrophil and monocyte function (phagocytosis, CD163, cytokine expression) were progressively diminished as sepsis persisted. An increasing frequency in PD-L1+-suppressor phenotype neutrophils [low-density neutrophils (LDNs)] was also noted. PD-L1+ LDNs and defective neutrophil function correlated with disease severity, consistent with the potential importance of suppressive neutrophil populations in sepsis. Reduced neutrophil and monocyte function correlated both with their own PD-L1 expression and with PD-1 expression on CD8+ T cells and NK cells. Conversely, reduced CD8+ T cell and NK cell functions (IFN-γ production, granzyme B, and CD107a expression) correlated with elevated PD-L1+ LDNs. Importantly, addition of antibodies against PD-1 or PD-L1 restored function in neutrophil, monocyte, T cells, and NK cells, underlining the impact of the PD-1:PD-L1 axis in sepsis-immune suppression and the ability to treat multiple deficits with a single immunomodulatory agent.

Keywords: immunosuppression; monocytes; neutrophils; programmed cell death.

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Figures

Figure 1.
Figure 1.. Neutrophil and monocyte function during stays in the ICU.
The function of neutrophils and monocytes are shown during the time in the ICU. Function was determined by flow cytometric methods using whole blood. Phagocytic activity of neutrophils and monocytes in CINS, healthy, or septic donor whole blood was determined by measuring fluorescence emitted by ingested pHrodo Red-labeled E. coli particles (A and B). Surface expression of phagocyte maturation-associated marker CD163 was determined separately in whole blood following LPS stimulation (C and D). Comparison among CINS, healthy, and septic donor samples are shown as the percentage of positive cells. Septic donor sample data are presented as all time points together (Septic All) or separated by the time in the ICU: d 1–3 (Septic A), d 4–7 (Septic B), and d 8–12 and 13–21 (Septic C and D). Panels E to H show progrssion during the time in the ICU for individual septic donors for whom multiple, sequential blood draws were available. Data are presented as means ± sem. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (P > 0.05), ND = not determined. Means ± sem of the MFI phagocytosis by neutrophils was 385.2 ± 55.14 (healthy, P = 0.096 compared with all septic), 367.3 ± 118.6 (CINS, P = 0.708 compared with all septic), 234.9 ± 38.41 (all septic), 330.3 ± 58.7 (Septic A, ICU d 1–3), 126.2 ± 39.6 (Septic B, ICU d 4–7, P = 0.0101 compared with Septic A), and 99.75 ± 33.1 (Septic C and D, ICU d 8–21, P = 0.0011 compared with Septic A). Means ± sem for the percentage of phagocytosis by neutrophils was 32.49 ± 5.52% (Septic A, ICU d 1–3), 14.99 ± 6.28% (Septic B, ICU d 4–7, P = 0.0180 compared with Septic A), and 9.99 ± 5.14% (Septic C and D, ICU d 8–21, P = 0.0151 compared with Septic A). Neutrophil means ± sem for the percentage of CD163 over time in the ICU was 52.29 ± 6.83% (Septic A), 14.66 ± 4.89% (Septic B, P = 0.0003 compared with Septic A), and 16.14 ± 7.60% (Septic C and D, P = 0.0148 compared with Septic A. Phagocytosis and CD163 expression by whole blood neutrophils and monocytes from patients with CINS compared with healthy donors: CINS 32.83 ± 6.77% neutrophil phagocytosis compared with healthy volunteers, 42.28 ± 4.62%, P = 0.253; CINS 49.60 ± 7.57% neutrophil CD163 compared with healthy controls, 51.84 ± 4.85%, P = 0. 985.
Figure 2.
Figure 2.. Comparison of neutrophil and monocyte functions.
Function of neutrophils and monocytes were determined by flow cytometric methods using whole blood. Phagocytic activity of neutrophils and monocytes in whole blood was determined by measuring the fluorescence emitted by ingested pHrodo Red-labeled E. coli particles. Surface expression of phagocyte maturation-associated marker CD163 and intracellular expression of TNF-α, MPO, and IL-10 were determined separately in whole blood after LPS stimulation. Graphs presented show comparisons of 2 neutrophil (A–C) or 2 monocytes (D–F) functions and comparisons of neutrophil function with monocyte function (G–I) by the percentage of positive cells (A and D–H) or MFI (B–C and I). Correlation plots are shown with the linear regression line of fit. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (P > 0.05).
Figure 3.
Figure 3.. Frequency of neutrophils, monocytes, LDNs, and MDSCs during time in the ICU.
Absolute granulocyte and monocyte counts were determined by standard hospital procedures. Populations of low-density, suppressor-phenotype neutrophils LDNs (SSChighCD16+CD15+CD33+CD66bhighCD114+CD11b+/low) and MDSCs (SSClowCD14+CD11b+CD16−CD15+) were determined by flow cytometric methods and sequential gating (as shown in Supplemental Fig. 1A and B, respectively) using unstimulated CINS, healthy, and septic donor whole blood. Absolute granulocyte and monocyte numbers (in thousands per cubic millimeter [K/mm3] (A and B) and the percentage of LDNs and MDSCs (C and D) are shown. Comparisons among CINS, healthy, and septic donor samples are shown. Septic donor-sample data are presented as all time points together (Septic All), or separated by time in the ICU: d 1–3 (Septic A), d 4–7 (Septic B), and d 8–12 and 13–21 (Septic C and D). Comparisons between septic donor samples at each time point in the ICU are also shown for LDNs and MDSCs (C and D). Data are presented as means ± sem. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (P > 0.05), ND = not determined.
Figure 4.
Figure 4.. PD-L1 expression on neutrophils and LDNs during time in the ICU.
Expression of PD-L1 on neutrophils and LDNs was determined by flow cytometric methods using unstimulated septic donor whole blood. The expression of PD-L1 is shown as MFI (A and B) and as the percentage of positive cells (C and D). Comparison among CINS, healthy, and septic donor samples are shown. Septic donor sample data are presented at all time points together (Septic All) or separated by time in the ICU: d 1–3 (Septic A), d 4–7 (Septic B), and d 8–12 and 13–21 (Septic C and D). Data are presented as means ± sem. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (P > 0.05).
Figure 5.
Figure 5.. PD-1 and PD-L1 expression on neutrophils, monocytes, and suppressor phenotypes LDN and MDSC.
Expression levels of PD-1 (A and B) and PD-L1 (C and D) on neutrophil and monocyte subsets were determined by flow cytometric methods using unstimulated whole blood from septic donors at all time points of their stays in the ICU. Graphs presented show comparison of expression on neutrophils and LDNs and comparisons of the expression on monocytes and MDSCs by MFI. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 6.
Figure 6.. Reduced neutrophil and monocyte function correlates with PD-L1 expression.
Expression of PD-L1 on neutrophils and monocytes was determined by flow cytometric methods using unstimulated whole blood from all time points of stays in the ICU. The function of neutrophils (A and B) and monocytes (C and D) were determined by flow cytometric methods using whole blood. Phagocytic activity was determined by measuring fluorescence emitted by ingested pHrodo Red-labeled E. coli particles. Correlation plots are shown with the linear regression line of fit. Graphs presented show comparisons of neutrophil and monocyte functions with PD-L1 expression on neutrophils and monocytes as the percentage of positive cells. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7.
Figure 7.. Reduced neutrophil and monocyte functions correlate with PD-1 expression on CD8 T cells and NK cells.
PD-1 expression on CD3+8+ T cells and CD3−56+ NK cells was determined by flow cytometric methods using unstimulated whole blood. The functions of neutrophils and monocytes were determined separately by flow cytometric methods using whole blood. Phagocytic activity of the neutrophils and monocytes in whole blood was determined by measuring the fluorescence emitted by ingested pHrodo Red-labeled E. coli particles. Graphs presented show comparisons of neutrophil and monocyte functions with PD-1 expression on CD8 T cells and NK cells as MFIs. Correlation plots are shown with the linear regression line of fit (A–D). (E and F) Graphs compare phagocytic activity of neutrophils and monocytes in septic donor samples with PD-1 levels (MFI) on CD8+ T cells below or above the MFI PD-1 on healthy donor CD8 T cells (healthy donor mean = 17.71). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 8:.
Figure 8:.. Reduced CD8+ cytotoxic T cell and NK cell functions correlate with PD-L1 expression on LDNs.
Expression of PD-L1 on LDNs was determined by flow cytometric methods using unstimulated whole blood from all time points of stays in the ICU. Markers of CD3+8+ T cell (A–C) and CD3-56+ NK cell (D–F) functions were determined by flow cytometric methods using whole blood. Intracellular expression of IFN-γ and granzyme B and surface expression of degranulation marker CD107a were determined separately in whole blood after 5 h stimulation with PMA/ionomycin. Correlation plots are shown with the linear regression line of fit. Graphs presented show comparisons of CD8 T cell and NK cell functions as the percentage of positive cells with PD-L1 expression on LDNs as MFIs. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 9:.
Figure 9:.. Phagocytic function is restored by anti-PD1 and anti-PDL1 Abs.
The function of neutrophils (A) and monocytes (B) was determined by flow cytometric methods after overnight incubation of whole blood with anti–PD-1, anti–PD-L1, or isotype control mAbs. Phagocytic activity was determined by measuring fluorescence emitted by ingested pHrodo Red-labeled E. coli particles. Graphs presented show comparison of phagocytic function of neutrophils and monocytes as the percentage of positive cells per individual donor with control mAbs, anti–PD-1 mAbs, or anti–PD-L1 mAbs. Arrows indicate an example of a subject with intermediate baseline function who is responding to a mAb. (A) Letters in the lower-right panel show time in the ICU (A, ICU d 1–3; B, ICU d 4–7; C, ICU d 8–12; and D, ICU d 13–21) of specific septic donors who responded to mAbs. The means per group are indicated by the circular symbols, and means ± sem values are shown for each treatment group. P values for pairwise comparisons of isotype vs. antibody treatment are shown. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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