Chronic Lymphocytic Leukemia Cells Exhibit Inhibitory Properties of Regulatory B Cells
Authors: Ringelstein-Harlev Shimrit , Avivi Irit , Bisharat Lina , Katz Tamar 
Published: October 2019

Abstract
Abstract 3874

Background:
Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy, characterized by distinct immune suppression rendering both tumor cells and invading pathogens invisible to the immune system. However, CLL cells also display profound immune sensitivity as proven by long-term remissions achieved with allogeneic bone marrow transplantation. Many phenotypic properties of B-CLL cells resemble a subset of B-cells, studied mostly in autoimmunity and termed regulatory B cells (Bregs). Bregs are thought to suppress CD4+ T-cell mediated immune responses, directly through cell contact and indirectly through inhibitory cytokines. This study aims to define whether malignant B-CLL cells exhibit Breg suppressive properties, contributing to immune dysfunction in this disease.

Methods:
B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV) using immunomagnetic separation (STEMCELL technologies). Naïve cells and those stimulated with B-cell activators TLR-9 agonist or CD40Ligand (CD40L) were analyzed by FACS for Breg phenotypic markers and intracellular IL-10. Additionally, B-CLL cell effects on autologous CD4+ T cells (isolated by immunomagnetic beads; Miltenyi Biotec) were studied. T-cells were stimulated with anti-CD3/CD28 antibodies and IL-2, and exposed to B-cells either directly or through hanging cell culture inserts (Millipore) preventing physical cell-cell contact. T-cell proliferation was assessed using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method and phenotype was analyzed by FACS.

Results:
B-cell phenotype was studied in 11 patients. Breg markers (CD5, CD38, CD25 and intracellular IL-10) as well as inhibitory molecules PD-1 and PDL-1 were expressed at high levels on B-CLL cells (62%, 37%, 50%, 52%, 29%, 61%, respectively), although not every patient expressed all markers. These expression levels were higher than those reported for normal peripheral blood B-cells. TLR-9 stimulation of B-CLL cells resulted in a 5.7-fold increase in expression of CD25 in 77% of patients. Increments were also observed in IL-10 (1.9-fold; 62% of patients), PDL-1 (1.96-fold; 83% of patients) and PD-1 (2.19-fold; 57% of patients). Of 13 patients whose T-cell proliferation potential was evaluated after exposure to B-CLL cells, proliferation was induced in only 69%; in the other 31% (4 patients) no proliferation was observed; moreover, inhibition was demonstrated in one of them. Among the former group only 33% of patients expressed CD25 on their B-cells, whereas within the latter group, 75% of patients' B-cells were CD25-positive. Stimulation of B-CLL cells with TLR-9 markedly increased their inhibitory capacity (72% of 11 patients tested), while CD40L stimulation caused a weaker effect (50% of 6 patients tested). T-cell proliferation remained unchanged when evaluated using a Transwell system versus a contact system, as demonstrated in 3 of 4 experiments. T-cells exposed to B-CLL cells altered the ratio of CD25high vs. CD25low T-cells in favor of CD25 high cells (2.44-fold increase for stimulation with naïve B-CLL cells, 4.94-fold increase with TLR-9 stimulated cells; in all the 5 tested patients).

Conclusions:
Previously identified Breg markers as well as PD-1 and PDL-1 were highly expressed in B-CLL cells, supporting the role of these cells in shaping an immune tolerant environment, enabling tumor growth. Stimulation of B-CLL cells with TLR-9 agonist enhanced this phenotype and resulted in consistent inhibition of T-cell proliferation, likely to be independent of cell-to-cell contact. These findings demonstrate the presence of Breg features within the CLL clone. The observed alterations in CD4+CD25+ T-cell populations after exposure to B-CLL cells suggest induction of T-regulatory cells, another mechanism supposedly used by Bregs for immune suppression. The enhancement of Breg properties in B-CLL cells following B-cell activation can serve as a platform for further studies of the innate regulatory mechanisms utilized by tumor cells.

Disclosures:
No relevant conflicts of interest to declare.