Characterization of Soluble Immune Checkpoint Protein PD-1, PD-L1 and PD-L2 Levels in Different Types of Lymphoid Malignancies
Authors: Mortensen Julie Bondgaard , Yoanna Vladimirova , Hansen Ida Monrad , Bjerre Mette , d'Amore Francesco 
Published: October 2019

Abstract
Background
Checkpoint proteins regulate the immune system and their down-regulation of effector mechanisms is exploited by malignant cells to evade antitumor immune response. Membrane bound programmed death 1 protein (PD-1) and its ligands 1 and 2 (PD-L1 and PD-L2) are pivotal members of the immune checkpoint family. The significance of the PD-1 pathway have been demonstrated in a variety of malignant diseases and suppressing the pathway with targeting antibodies can re-establish antitumor immunity.
Soluble forms of the proteins (sPD-1, sPD-L1 and sPD-L2) are detectable in the peripheral blood, but their biological and clinical significance are still largely unknown. To our knowledge, this is the first study to simultaneously characterize sPD-1, sPD-L1 and sPD-L2 in different types of lymphoma.
Aims
The aim of the present study was to measure the pre-therapeutic sPD-1, sPD-L1 and sPD-L2 levels in different lymphoid malignancies and correlate them with clinico-pathological features and outcome.
Methods
We established and validated a Time Resolved Immunoflourometric assay (TRIFMA) to determine levels of sPD-1 and sPD-L2. Soluble PD-L1 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) applied according to manufacturer's instructions.
In total, archival serum samples from 102 patients were analysed in duplicate for each protein. This single-institution study population consisted of patients with chronic lymphocytic leukemia (CLL; n=42), diffuse large B cell lymphoma (DLBCL; n=33), follicular lymphoma (FL; 15) and Hodgkin lymphoma (HL; n=12). In addition, 22 and 17 healthy controls were included for analysis of sPD-1/sPD-L1 and sPD-L2, respectively. Information on clinical parameters, response to treatment and outcome was obtained from medical records. Measured levels were compared between patient subgroups and controls using a non-parametric test (Mann-Whitney). P-values below 0.05 were considered statistically significant.
Results
Soluble PD-1 levels were higher in patients, taken as one group, compared to controls (p=0.0002). When analyzed according to lymphoma type, patients with DLBCL, CLL and FL still had significantly elevated sPD-1 levels as compared to controls (p=0.0089, p=0.0008 and p<0.0001, respectively). Highest sPD-1 levels were observed in FL and these were also significantly higher than in any of the other lymphoma subtypes analyzed.
Soluble PD-L1 levels were similar in patients, taken as one group, and controls. Interestingly, patients with FL had lower sPD-L1 levels than patients from all the other lymphoma subtypes and it was the only histology whose sPD-L1 levels differed significantly (lower values) from healthy controls (p=0.0017, see table 1). Moreover, we identified a reverse expression pattern of sPD-1/sPD-L1 in FL as compared to other lymphoma subtypes, as FL had significantly higher sPD-1 and lower sPD-L1 than all other remaining patients (see table 1).
Soluble PD-L2 levels were generally higher than those of sPD-1 and sPD-L1, and the highest values were found in healthy controls. When analyzed according to histological subtype, patients with CLL, DLBCL and FL had all significantly lower sPD-L2 levels than healthy controls (see table 1).
Interestingly, the sPD-1, sPD-L1 and sPD-L2 levels measured in HL patients did not differ significantly from controls, however, this may be due to limited sample size.
Conclusion
We simultaneously characterized sPD-1, sPD-L1 and sPD-L2 in selected lymphomas subtypes and found varying levels across the investigated lymphoma entities. In particular, we identified a reverse expression pattern of sPD-1 and sPD-L1 in FL compared to other lymphoid malignancies. We were the first to also characterize sPD-L2 levels in the same patients and found its levels to be generally high throughout lymphoma entities, but highest in healthy controls. A correlation analysis with outcome-related parameters is ongoing.

Disclosures
No relevant conflicts of interest to declare.