Ph.D Thesis

Ph.D StudentHarlev Shimrit
SubjectChronic Lymphocytic Leukemia Cell Exhibit Regulatory B-cell
Properties as a Mechanism of Immune Evasion
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Benjamin Brenner
DR. Irit Avivi
PROF. Tamar Katz
Full Thesis textFull thesis text - English Version


Chronic lymphocytic leukemia (CLL) is the most common hematologic malignancy in the Western world. The cells are CD19 B-cells, displaying a phenotype characterized by CD5, CD23, CD38, with elevated serum levels of IL10. Cells also express PD1 and PDL1, molecules involved in immune modulation. One of the hallmarks of CLL is an altered immune milieu characterized by abnormal T-cell subtype frequency and cytokine secretion, defective immune synapse formation, and enhanced frequencies of regulatory T-cells (Tregs). These properties suggest that CLL cells are involved in immune modulation, representing a clone of "regulatory B-cells". 

Regulatory B-cells (Bregs) can attenuate effector T-cell responses and modify myeloid cell immune responses. They have been studied mostly in autoimmunity models, but have recently been implicated in promoting cancer immune evasion. Although no specific markers were defined, the major subtypes are found within the IL10 producing B-cell population. Bregs require various signals in order to mature and function, including TLR9 and CD40 stimulation.

To determine whether CLL represents a clone of Bregs, we investigated the cells' phenotype and characterized their inhibitory function in response to TLR9 agonist (ODN) and CD40Ligand (CD40L).

CLL cells, known to be exceptionally sensitive to TLR9, were found to enhance a Breg phenotype in response to ODN by up-regulating CD25, IL10 and PD1, and to a lesser extent CD38, CD23 and PDL1. Cytokine array studies have demonstrated that TLR9 and CD40L stimulation lead to secretion of IL8, RANTES, MCP2 and thrombopoietin, substances that may contribute to improving regulatory properties of the clone along with IL10.

Two functional regulatory effects imposed by CLL cells were found. Firstly, under TLR9 stimulation, the cells inhibit autologous CD4T-cell proliferation, and secondly, TLR9 and CD40L activated CLL cells induced Tregs from autologous CD4 T-cells. Treg induction required cell-cell contact while proliferation inhibition did not, suggesting that each stimulator activates different pathways, allowing immune evasion even if certain environmental stimulants are lacking.

Further investigation demonstrated that disruption of early TLR9 signaling through IRAK4 inhibition caused down-regulation of IL10 and PDL1 expression, supporting their role in the clone's regulatory properties.
The modulation of PD1 levels induced by TLR9 prompted investigation of this pathway in Breg activity.  Blocking the PD1 receptor on CLL cells interferes with the inhibitory effect of TLR9 on T-cell proliferation and CD40L-related Treg induction, indicating involvement of the PD1/PDL1 axis in promoting CLL Breg behavior.  PD1 blockage also reversed ODN-induced down-regulation of genes, demonstrating cross-talk between these signaling cascades.

Data also suggest that Breg capacities are linked to genetic events in the Immunoglobulin heavy chain variable region ( IgHV) genes, with mutated cells enhancing Breg activity and responding more uniformly to TLR9-originated signals.

In conclusion, CLL B-cells demonstrate a Breg profile, utilizing TLR9 and CD40 activation to promote regulatory behavior through collaboration with the PD1/PDL1 pathway. Differences in Breg features observed in the cohort may be related to genetic differences represented by IgHV mutations, suggesting that certain genetic combinations incur Breg abilities, possibly providing the malignant clone with a survival advantage.