Programmed Death Ligand 1 Is Overexpressed by Neutrophils in the Blood of Immunocompromised Human Immunodeficiency Virus-Negative Patients with Pneumocystis jirovecii Pneumonia
Pneumocystis jirovecii pneumonia (PCP) represents a critical clinical challenge in immunocompromised individuals, particularly those who are human immunodeficiency virus (HIV)-negative. Despite advances in antimicrobial therapies, mortality rates for HIV-negative patients with severe PCP requiring intensive care unit (ICU) admission exceed 50%. Neutrophilic inflammation in the bronchoalveolar lavage (BAL) fluid has been recognized as a marker of poor prognosis in these patients. Recent discoveries highlighting the presence of programmed death ligand 1 (PD-L1) on neutrophils have prompted investigations into their role in modulating immune responses. This study explores the expression of PD-L1 on circulating neutrophils and its association with T-cell dysfunction in HIV-negative patients with PCP, providing novel insights into disease pathogenesis and potential therapeutic targets.
Clinical Characteristics and Study Design
A cohort of 17 immunocompromised, HIV-negative patients diagnosed with PCP and 10 non-PCP controls were enrolled from the China-Japan Friendship Hospital between September 2017 and October 2018. All patients were diagnosed based on PCR detection of Pneumocystis jirovecii DNA in BAL fluid, supplemented by Giemsa and methenamine silver staining. The control group comprised immunocompromised patients undergoing diagnostic evaluations for respiratory symptoms but confirmed to be PCP-negative. Key demographic and clinical parameters, including age, sex, underlying immunosuppressive conditions, and pulmonary coinfections, were comparable between groups.
Common underlying conditions in the PCP cohort included interstitial lung disease (52.9%), systemic inflammatory diseases (35.3%), hematological malignancies (17.6%), solid organ transplants (11.8%), and solid tumors (5.9%). Pulmonary coinfections—such as cytomegalovirus (35.3%), bacterial infections (29.4%), and fungal infections (11.8%)—were prevalent but did not differ significantly from controls. Both groups had similar exposure to immunosuppressive agents, including corticosteroids (88.2% in PCP vs. 70% in controls) and T-cell suppressants (70.6% vs. 30%). Notably, peripheral white blood cell (WBC) and neutrophil counts were elevated in PCP patients (median WBC: 11.07 × 10⁹/L vs. 6.56 × 10⁹/L; median neutrophils: 9.63 × 10⁹/L vs. 6.12 × 10⁹/L). Disease severity, assessed via APACHE II scores, was significantly higher in the PCP group (mean 21.47 ± 7.46 vs. 16.00 ± 4.76).
Increased PD-L1 Expression on Circulating Neutrophils in PCP
Flow cytometry analysis of peripheral blood cells revealed a marked upregulation of PD-L1 on neutrophils in PCP patients compared to controls. Neutrophils were identified using CD11b staining, and PD-L1 expression was quantified as a percentage of positive cells. PCP patients exhibited a median PD-L1 positivity of 8.2% (interquartile range [IQR]: 5.3–12.1%) versus 3.5% (IQR: 1.8–5.2%) in controls (P = 0.0087). A representative flow cytometry plot demonstrated distinct PD-L1+ neutrophil populations in PCP patients (Figure 1A–B).
PD-1 Upregulation on CD4+ T Cells and Correlation with Neutrophil PD-L1
Concurrent analysis of T-cell populations revealed elevated PD-1 expression on CD4+ T cells in PCP patients. The median frequency of PD-1+ CD4+ T cells was 24.6% (IQR: 18.4–32.1%) in the PCP group versus 12.7% (IQR: 8.9–16.5%) in controls (P = 0.0053). In contrast, PD-1 levels on CD8+ T cells were comparable between groups (19.3% vs. 16.8%; P = 0.64) (Figure 1C–D). A significant positive correlation was observed between PD-L1 expression on neutrophils and PD-1 levels on CD4+ T cells (Pearson’s R = 0.549, P = 0.0244), suggesting a potential interaction between these cells in driving immunosuppression.
Phenotypic Heterogeneity of Neutrophil Subsets
Neutrophils were further categorized into three subsets based on surface marker expression:
- Mature neutrophils: CD62LbrightCD16brightCD11bbright
- Activated neutrophils: CD62LdimCD16brightCD11bbright
- Immature neutrophils: CD62LbrightCD16dimCD11bbright
No significant differences in the proportions of these subsets were observed between PCP patients and controls (Supplementary Figure S1B–D). However, activated neutrophils exhibited significantly higher PD-L1 expression in the PCP group (median 15.4% [IQR: 10.2–20.6%] vs. 8.9% [IQR: 5.1–12.3%]; P = 0.0341) (Supplementary Figure S2B). Mature and immature subsets showed minimal PD-L1 expression in both groups.
Association with Disease Severity
PD-L1+ neutrophil levels correlated positively with APACHE II scores (R = 0.5, P = 0.041), indicating a link between neutrophil-driven immunosuppression and disease severity. Furthermore, the proportion of PD-L1+ activated neutrophils correlated with PD-1+ CD4+ T-cell frequencies (R = 0.4869, P = 0.0474) (Supplementary Figure S3B). These findings underscore the clinical relevance of PD-L1+ neutrophils in PCP progression.
Mechanistic Insights and Implications
Neutrophils are dual-edged components of the immune response in PCP. While their recruitment is essential for pathogen clearance, excessive or dysregulated neutrophilic activity can exacerbate tissue damage. Prior studies in murine models found that neutrophil depletion did not ameliorate lung injury, challenging the notion of neutrophils as primary effectors of harm. However, this study introduces a novel paradigm: neutrophils may contribute to immunosuppression via PD-L1/PD-1 signaling, impairing CD4+ T-cell function.
The activated neutrophil subset (CD62LdimCD16bright), which showed the highest PD-L1 expression, likely plays a central role in this process. These cells may migrate to infected tissues and interact with PD-1+ CD4+ T cells, inhibiting their effector functions. This mechanism aligns with observations in tuberculosis, where PD-L1+ neutrophils suppress T-cell responses. The correlation between PD-L1+ neutrophils and APACHE II scores further supports their role in worsening clinical outcomes.
Limitations and Future Directions
This study has limitations, including its small sample size and restriction to ICU patients, which may limit generalizability. The absence of BAL fluid analysis for PD-L1/PD-1 expression precludes direct comparisons between circulating and lung-localized immune cells. Future studies should explore tissue-specific interactions and evaluate the functional consequences of PD-L1/PD-1 blockade in preclinical models.
Conclusion
This study identifies PD-L1 overexpression on circulating neutrophils, particularly activated subsets, as a hallmark of PCP in HIV-negative immunocompromised patients. The correlation between neutrophil PD-L1, CD4+ T-cell PD-1, and disease severity highlights a potential mechanism of immune evasion in PCP. These findings advocate for further investigation into PD-L1/PD-1-targeted therapies to restore antimicrobial immunity and improve outcomes in this high-risk population.
doi.org/10.1097/CM9.0000000000000237
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