T-Cell Dysfunction In CLL Is Mediated Not Only By PD-1/PD-L1 But Also By PD-1/PD-L2 Interactions - Partial Functionality Is Maintained In PD-1 Defined CD8 Subsets and This Can Be Further Promoted By Ibrutinib Treatment

CLL-induced severe T-cell dysfunction and ineffective anti-tumor immune-responses are hallmarks of the disease, but the specific interactions remain poorly understood. The PD-1/PD-L1 axis is an important mediator of T-cell dysfunction in solid tumors, and we have previously demonstrated that T cells from CLL patients exhibit impaired immunological synapse (IS) formation, predominantly mediated by PD-L1 (CD274) expression on CLL cells. We have also shown that the corresponding T-cell ligand PD-1 (CD279) is also upregulated, probably as a result of chronic antigenic stimulation, and that T cells have similarities to exhausted T cells observed in the context of chronic viral infection. Recent studies demonstrated that ibrutinib has impressive clinical activity in CLL, and mechanisms of action include irreversible binding of essential components of both B-cell- and T-cell-receptor signaling and interactions with the tumor microenvironment. Modulation of PD-1/PD-L1 interactions might therefore be an additional potential mode of action of this drug. Using the well-established Eμ-TCL1 (TCL1) mouse model of CLL, our aims were to demonstrate that (1) altered expression of PD-L1 on CLL cells and PD-1 on T cells and CLL are causally related, (2) the second ligand of PD-1, PD-L2, is also involved in mediating T-cell dysfunction, (3) T-cell effector function and IS formation are directly linked to PD-1 expression and (4) PD-1 associated in vivo T-cell responses can be modulated by treatment with ibrutinib.

As we have previously demonstrated that the spleen is the major organ of disease and representative of T-cell changes in peripheral blood and lymph nodes, experiments were performed on spleens from young TCL1 and wild-type (WT) C57Bl/6 mice with established CLL after adoptive transfer (AT) of syngeneic CLL cells (n=10), and on matched litter-mates after AT of healthy mouse B cells (n=10). An additional 12 mice were randomized to treatment with 25 mg/kg/d ibrutinib in 10% HP-β-CD, vehicle control, or sterile water, all administered by gavage, three weeks after AT of syngeneic CLL cells, and sacrificed 20 days later at a pre-defined endpoint. Multicolor flow cytometry was used to characterize T-cell subsets, expression of PD-1, PD-L1 and PD-L2 and T-cell effector function. Entire population CD8 T cells, PD-1+ve and PD-1-ve CD8 T cells were flow-sorted and used in IS formation assays with healthy murine B cells as antigen-presenting cells.

Our previous studies using aged TCL1 mice and age-matched WT controls indicated that CLL-related PD-1 upregulation on antigen-experienced CD44+ CD8 T cells is masked by aging. However, PD-1 expression could also be induced in young TCL1 and WT mice by AT of CLL cells but not healthy B cells, suggesting a causal relationship with disease. Both PD-L1 and PD-L2 surface expression on CLL B cells were significantly increased compared to healthy B cells. Using TCL1 mice at early stages of CLL development when a healthy CD19+ B-cell population is still present, we were able to confirm that PD-L2 expression is a unique feature of CLL cells, with PD-L2 being virtually absent on healthy B cells. We next compared effector function and the ability to form IS of PD-1+ve and PD-1-ve antigen-experienced CD44+ T-cell subsets in mice with CLL. While proliferation was equally impaired in these subsets, they were both able to degranulate but generally failed to localize granzyme B to the IS. Although subsets produced some IL2/TNFα/IFNγ cytokine responses, PD-1+ve cells had significantly impaired TNFα and slightly impaired IL2 and IFNγ production, and a highly significant impaired ability to form IS compared to PD-1-ve cells. Treatment with ibrutinib reduced PD-1 expression on antigen-experienced CD44+ CD8 T cells and promoted stronger IFNγ production of entire population CD8 T cells, but failed to restore proliferation and granzyme B relocation to the IS.

Our in vivo data suggests that CLL and PD-1/PD-L1-mediated T-cell dysfunction are causally related, but that phenotypic and functional T-cell changes are not absolute and might be at least partly reversible by ibrutinib treatment. We also show that the second ligand of PD-1, PD-L2, is also a critical mediator of PD-1 associated T-cell dysfunction in CLL.

Riches:Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.