Number of PD-1+/CD8+ Cells in Peripheral Blood of Patients with Lymphoma Reflects Tumor Burden, Lymphoma Subtype, Disease Phase and Is Significantly Higher Compared to Healthy Volunteers.
Authors: Prochazka Vit , Novák Martin , Pikalova Zuzana , Papajik Tomas , Indrak Karel , Divoky Vladimir 
Published: October 2019

Abstract
Abstract 2670

Background:
Programmed death-1 (PD-1) and programmed death-1 ligand (PD-L) signaling pathways are involved in the functional impairment and “exhaustion” of cytotoxic CD8+ T cells in conditions such as chronic viral infection and in tumor immune evasion. The interaction of PD-1 with its ligand PD-L suppresses antitumor T cell function and indirectly stimulates Treg population. We investigated a hypothesis of whether examining PD-1 expression in peripheral T cells of patients with different lymphoma subtypes reflects tumor subtype or stage and compared results with healthy volunteers.

Methods:
Patients were assessed prior to their treatment or at the time of disease relapse or progression. We analyzed 5 patients with HL and 30 patients with NHL (T-cell n=6, diffuse large B-cell n=12, follicular lymphoma n=9, marginal zone lymphoma n=3). Twelve of the patients had relapsed or refractory diseases (B-NHL n=6, T-NHL n=2, HL n=4). Eleven patients (32%) had advanced (III/IV) disease stages. Data were compared with samples obtained from 12 healthy blood donors. Peripheral blood samples were stained with anti-CD3 FITC (Exbio), PD-1 (CD279) PE (BioLegend), anti-CD8 PerCP (Exbio), CD4 APC (Exbio), anti-CD25 FITC (BD), and anti-CD127 PE (BioLegend) using a lyse/no-wash protocol. Stained cells were acquired using the FACSCalibur cytometer (BD). Analysis of immunocompetent subpopulations was performed using the CellQuest Pro (BD) software. PD-1 (CD279) population was gated from CD3-positive T cells; minimal acquisition was designated as 10,000 CD3+ events. The percentage of PD-1+ cells within the live CD3+CD4+ and CD3+CD8+ populations was compared to isotype controls to establish baseline values. Absolute numbers were expressed as number of cells*10exp6 per liter. Population of Tregs was defined as CD4+/CD25int-hi / CD127low cells. Tregs were gated from CD4+ lymphocytes with minimal acquisition of 5,000 CD4+ cells.

Results:
Proportion of PD-1+/CD8+ of CD3+/CD8+ cells was significantly higher in patients with lymphoma than in healthy subjects: healthy volunteers (HV) 8.8%, B-NHL 16.0% (p=0.02), HL 21.8% (p<0.01), and T-NHL 30.8% (p<0.01). In absolute numbers of PD-1+/CD8+ cells, no significant difference was found when comparing healthy subjects and B-NHL: HV 0.23, B-NHL 0.56 (p=0.21), T-NHL 0.93 (p<0.01), and HL 1.51 (p<0.01). When analyzing the proportion of PD-1+/CD8+ cells according to disease phases, the highest numbers were found in patients with refractory/relapsed lymphoma as compared to patients with untreated disease and healthy subjects: HV 8.8%, untreated 14.6% (p=0.04), and relapsed 28.6% (p<0.01). Untreated patients had a significantly lower proportion of PD-1+/CD8+ cells than relapsed patients (p<0.01). Similar results were obtained with absolute numbers: HV 0.22, untreated 0.55 (p=0.03), and relapsed 1.24 (p=0.03). Untreated vs. relapsed patients p=0.05. Patients with limited disease stages had almost the same proportion of PD-1+/CD8+ lymphocytes compared to HV: HV 8.8%, limited stage 11% (p=0.21), and advanced stage 24.3% (p<0.01). In absolute numbers, HV had much less PD-1+/CD8+ cells in PB: HV 0.22, limited stage 0.49 (p<0.01), and advanced stage 0.97 (p<0.01). When analyzing the population of PD-1+/CD4+ cells, differences were only found in absolute numbers between HV (0.35) and HL (1.34; p<0.01), and between B-NHL (0.54) and HL (p=0.01). Regarding the population of Tregs, statistical differences were found between HV and B-NHL, HL or T-NHL in either relative or absolute numbers. On the other hand, there was a close correlation between absolute numbers of Tregs and PD-1+/CD4+ cells (p<0.01, correlation 0.73), and between Tregs and PD-1+/CD8+ cells (p<0.01, correlation 0.53).

Conclusion:
PD-1 expression in peripheral blood CD4+ and CD8+ cells is markedly different between lymphoma subtypes and compared with healthy subjects. The highest numbers of PD-1+/CD8+ are in patients with advanced lymphoma and at the time of disease relapse. This fact support the hypothesis that tumor clones actively switch effector CD8+ cells through the PD1L/PD-1 pathway into an immunotolerant state. PD-1 may be a potential marker of systemic immune dysregulation in lymphoma patients and further exploration of T cell subpopulations may define its role as a potential biomarker. Supported by grants: MSM 6198959205, LF-2012-007 and MZ ÈR IGA NT 11103.

Disclosures:
Prochazka: Roche: Travel grants Other.