Immune Checkpoint Blockade with Anti-PD-L1 Prevents Immune Dysfuntion and CLL Development in the TCL1 Adoptive Transfer Mouse Model
Authors: Hanna Bola , McClanahan Fabienne , Clear Andrew James , Miller Shaun , Lichter Peter , Seiffert Martina , Gribben John G. 
Published: October 2019

Abstract
Background: Clinical studies have demonstrated that targeted immunotherapy using PD-1/PD-L1 antibodies induces tumor regression and prolongs disease stabilization in advanced solid cancers. Data on the clinical efficacy in hematological malignancies is largely missing, even though PD-L1/PD-1 interactions have been described as major mediators of immune dysfunction in several leukemias and lymphomas. They are therefore ideal to study if PD-L1/PD-1 blockade has the potential to control disease by restoring anti-tumor immune responses. Several groups showed that chronic lymphocytic leukemia (CLL) provokes immune evasion via PD-L1/PD-1 inhibitory signaling, and that this is very closely mirrored in the Eµ-TCL1 (TCL1) murine model for CLL. Our recent data suggest that in this model, aberrant PD-L1 expression in myeloid cells contributes to the immune defect in CLL. We further demonstrated that the T cell and myeloid cell immune defects in ageing leukemic mice can be induced in young wild-type (WT) mice by adoptive transfer (AT) of murine CLL. In the current study, we used the AT model to test if in vivoPD-L1 blockade corrects leukemia-induced cellular immune dysfunction in myeloid and T cells and enhances anti-tumor immunity.
Methods: WT mice transplanted with 4x107 TCL1 splenocytes were randomized to treatment with 10 mg/kg α-murine-PD-L1 (n=15) or isotype antibody (n=10), which was administered i.p. every 3 days starting 1 day after AT, and sacrificed 31 days later. Matched non-transplanted WT mice (n=6) served as additional controls. Immune cell subsets, expression of immune checkpoint markers and T cell effector functions were analyzed by multicolor flow cytometry using cells isolated form spleen, peripheral blood (PB), bone marrow (BM) and peritoneal cavity (PC). Cell proliferation was measured by EdU incorporation in vivo. Immune synapse (IS) formation was assessed by confocal microscopy. Serum cytokines were quantified by multiplex bead arrays.
Results: We first confirmed successful engraftment and presence of disease by immunohistochemistry. Compared to isotype controls, α-PD-L1 treated mice had significantly lower spleen weights (median 0.2 g vs 0.9 g, p<0.0001) and a highly significant lower relative frequency of CD19+CD5+ CLL lymphocytes in spleen (1.55% vs 71.69%), PB (10.5% vs 63.53%) and BM (0.26% vs 2.74%) demonstrating very effective tumor control. Compared to non-transplanted animals, α-PD-L1 treated mice showed alterations in almost all phenotypical and functional immune cell parameters, especially in regards to immune cell activation, indicating encounter with and immunological challenge by CLL cells. Along with disease control, α-PD-L1 treated mice had improved immune status as multiple inflammatory cytokines in the serum, including IL-10, TNF-α, CCL2 and GM-CSF were decreased and splenic infiltration of monocytes was reduced. While CLL development skewed monocytes towards Ly6Clow patrolling monocytes, α-PD-L1 treatment restored the presence of Ly6Chi inflammatory monocytes and decreased the expression of adhesion molecules ICAM-1 and PECAM-1. These monocytes regained their differentiation capacity as shown by increased numbers of macrophages and mature MHC-IIhi dendritic cells in the spleens of treated mice. In the T cell compartment, in vivo PD-L1 blockade prevented the CLL-induced CD4/CD8 ratio inversion, the loss of naïve CD8 T cells and the shift towards antigen-experienced and terminally differentiated T cells in spleen, BM and PB. Aberrant expression of immune checkpoint markers PD-1, KLRG-1, LAG-3, and 2B4 was also significantly reduced. The CLL-associated loss of intracellular IL-2 and the increased secretion of IL-4 and IFN-γ in CD4 T cells were prevented in α-PD-L1 treated mice. Respective cytokine patterns were observed in the serum. Functionally, PD-L1 blockade restored CD8 degranulation and IS formation to the level of healthy T cells, and significantly improved both ex vivo and in vivoT cell proliferation.
Conclusion: Our in vivodata demonstrate that early PD-L1 blockade very effectively controls CLL development and enables complex effector function of myeloid and T cells, thus restoring anti-tumor immune responses. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies, potentially in combination with novel substances.
BH/FM and MS/JGG contributed equally to first and last authorship.

Disclosures
No relevant conflicts of interest to declare.