Abstract B041: Ibrutinib treatment counteracts the immunosuppressive activity of malignant B cells
Authors: Long Meixiao , Beckwith Kyle A. , Do Priscilla , Gordon Amber , Lehman Amy , Maddocks Kami , Cheney Carolyn , Jones Jeffrey , Andritsos Leslie , Awan Farrukh , Woyach Jennifer , Amy Johnson J. , Muthusamy Natarajan , Byrd John 
Published: November 2016

Abstract
Introduction: Regulatory B cells (B-reg cells) and cytokine producing B cells have been increasingly recognized as key regulators of adaptive and innate immunity. Malignant B cells, such as chronic lymphocytic leukemia (CLL) cells share many features of B-reg cells and suppress host adaptive immunity via multiple mechanisms, including production of suppressive cytokines such as IL-10, surface expression of checkpoint molecules such as PD-L1 and CD200, as well as inducing expansion of regulatory T cells and Myeloid-derived Suppressor Cells (MDSC). Ibrutinib is an orally available irreversible BTK inhibitor that has been proved to be a safe and effective therapy for CLL. Recently we and others have demonstrated favorable cellular immunomodulatory effects of Ibrutinib through inhibition of an alternative target interleukin-2 induced T-cell kinase (ITK) that promotes Th1/Tc1 polarization. Herein, we explore the influence of Ibrutinib on the immune suppressive features and the B-reg phenotype associated with malignant B cells.
Methods: PBMCs were collected from CLL patients treated with Ibrutinib at the time of pretreatment, cycle 3 day 1 (8 weeks into treatment) and cycle 6 day 1(20 weeks into treatment). Surface expression of checkpoint molecules and immunosuppressive capability of CLL cells were evaluated. For brief stimulation (“B10” condition), PBMCs were stimulated with Phorbol Myristate Acetate (PMA)/Ionomycin/Golgi-stop plus CpG for 5 hours. For prolonged stimulation (“B10-Pro” condition), PBMCs were stimulated with CpG plus CD40L for 48 hours, PMA / Ionomycin / Golgi-stop were added for final 5 hours.
Results: CLL cells collected from patients treated with Ibrutinib in vivo were significantly impaired in their capability to make IL-10 after in vitro stimulation. On average, there is about a threefold reduction (P&lt; 0.001) in the frequency of cells producing IL-10 by cycle 3, and a fivefold reduction (P&lt; 0.001) by cycle 6. We have also shown that during the first two cycles of Ibrutinib, patients' plasma levels of IL-10 decreased. The impaired IL-10 production by CLL cells collected from post-Ibrutinib treatment patients is likely to due to an intrinsic change of the malignant B cells, rather than from acute inhibition of BTK; since resting these CLL cells in vitro for 48 hours (which allows for regeneration of BTK) did not lead to a recovery of IL-10 production. Interestingly, IFNγ production after in vitro re-stimulation is increased in CLL cells collected post Ibrutinib treatment. The analysis of potential immunosuppressive molecules revealed 3-5 fold reduction in surface expression of CD200 and BTLA(P&lt;0.001 for both), but no significant change in the expression of PD-L1 or B7-H3 in CLL cells collected post Ibrutinib treatment compared to pre-treatment samples. Given that B-regs can influence both T-regs and MDSCs, we exammined these and found the percentage of CD4+/CD25+/Foxp3+ regulatory T cells and CD14+/HLADRlow Myeloid-derived Suppressor Cells were significantly reduced after Ibrutinib treatment(P&lt;0.001 and P&lt;0.05).
Conclusion: CLL cells cause both global and tumor specific immunosuppression via multiple mechanism recapitulating regulatory B cells including production of IL-10, expression of checkpoint molecules such as CD200, and induction of suppressive/regulatory cells such as Treg cells and MDSC cells. Here we demonstrate that Ibrutinib treatment has a powerful in vivo immunomodulatory effect on the malignant B cells by counteracting all the aforementioned B-reg like activities, which makes it a promising immunotherapeutic agent.
Note: This abstract was not presented at the conference.
Citation Format: Meixiao Long, Kyle A. Beckwith, Priscilla Do, Amber Gordon, Amy Lehman, Kami Maddocks, Carolyn Cheney, Jeffrey Jones, Leslie Andritsos, Farrukh Awan, Jennifer Woyach, Johnson J. Amy, Natarajan Muthusamy, John Byrd. Ibrutinib treatment counteracts the immunosuppressive activity of malignant B cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B041.