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.

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.

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.

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).

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.

Prochazka:Roche: Travel grants Other.