In Chronic Lymphocytic Leukemia PD-1 Is Expressed Independently From PDCD1 Gene Polymorphisms and Does Not Influence BCR Signaling
Authors: Giannopoulos Krzysztof , Grzywnowicz Maciej , Karabon Lidia , Piechnik Agnieszka , Zajac Malgorzata , Skorka Katarzyna , Zaleska Joanna , Tomczak Waldemar , Wlasiuk Paulina , Frydecka Irena , Dmoszynska Anna 
Published: October 2019

Abstract

Background
The programmed death-1 (PD-1, CD279) is a negative lymphocytic immunoreceptor responsible for peripheral tolerance. Through inhibition of TCR/BCR signaling PD-1 impairs activation and effectors functions of T and B lymphocytes, where it is expressed upon activation. PD-1 was described on exhausted T cells and it is aberrantly expressed in several malignancies, where a PD-1 pathway was related to a potential tumor escape mechanism from the immunosurveillance. Previously, we characterized PD-1 as a novel phenotypic feature of chronic lymphocytic leukemia (CLL) cells. PD-1 was up-regulated on both transcript and surface protein levels in CLL patients in comparison to healthy volunteers (HVs). Moreover, in vitro activated CLL cells in conditions mimicking microenvironment of proliferation centres up-regulated PD-1 on their surface.

Aims
Despite aberrant expression in CLL patients, PD-1 mechanism and function in CLL is unclear. To investigate PD-1 influence on BCR signaling in CLL cells we analyzed expression and phosphorylation of SYK, LYN, and ZAP-70 tyrosine kinases. To verify a relation of PD-1 expression to microenvironment in vivo, we analyzed PD-1 expression in mononuclears from 149 peripheral blood (PBMC) and 140 bone marrow (BMMC) CLL samples, representing accumulation and proliferation disease compartment respectively and 30 healthy PBMC samples. Finally, we characterized polymorphism of PDCD1 gene in 114 CLL patients and 150 HVs.

Methods
Quantitative reverse transcriptase PCR was performed for the PD-1 transcript assessment in 182 CLL patients (148 PBMC, 140 BMMC). Expression and phosphorylation of tyrosine kinases were analyzed with western blot (for SYK and LYN) and flow cytometric methods (for ZAP-70 and PD-1) in CD19+ magnetically separated cells from 12 CLL patients. For each kinase two separate phosphorylation sites were analyzed, responsible either for activation or inhibition respectively: Y525/526 and Y323 for SYK, Y507 and Y396 for LYN, Y315/319 and Y292 for ZAP-70. Five single nucleotide polymorphism sites (SNPs) were analyzed by restriction fragments length polymorphism (RFLP-PCR) in group of 114 CLL patients and 150 HVs.

Results
The median level of PD-1 transcripts in CLL patients was higher in comparison with HVs (p<0.0001), but there was no difference between blood and bone marrow compartments. The expression and phosphorylation of LYN and SYK were not dependent on PD-1 expression in 12 CLL patients, with PD-1 range from 4.2%. to 67.53%. Flow cytometric analysis showed that ZAP-70 expression was not related to the PD-1 level. Median percentages of cells with phosphorylated ZAP-70 activation/inhibition sites were low (Y315/319: 0.62%, Y292: 0.5%). Phosphorylation of Y292 showed strong negative correlation with PD-1 level (r2=-0.8, p=0.0019). Analysis of PDCD1 polymorphism showed that neither of SNPs frequency (loci: rs36084323, rs11568821, rs2227981, rs2227982, rs41386349) was different in CLL group compared to HVs. Particular substitutions were not related to the PD-1 expression level. Frequencies of haplotypes in CLL and HVs showed no significant difference. Linkage disequilibrium comparison of CLL and HVs revealed complete dependency of rs2227981/rs2227982 (p=0.03) and rs2227981/rs36084323 (p=0.03) loci in CLL patients.

Summary / Conclusion
PD-1 expression is strongly up-regulated in CLL patients regardless microenvironment of leukemic cells. No PD-1 influence on expression and activation of BCR related kinases might suggest impaired or alternative function of PD-1 pathway in CLL cells. Characterization of PDCD1 polymorphism revealed no association of PD-1 SNPs with CLL, therefore, PD-1 up-regulation might not be driven by genetic alternations.

Disclosures:
No relevant conflicts of interest to declare.