Immunomodulatory Effects of Tyrosine Kinase Inhibitor In Vitro and In Vivo Study

Abstract

Pathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined, involving donor-derived CD4 and CD8-positive T lymphocytes as well as B cells. Standard treatment is lacking for steroid-dependent/refractory cases; therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. However, TKIs seem to have pleiotropic activity. We sought to evaluate the in vitro and in vivo impact of different TKIs on lymphocyte phenotype and function. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of increasing concentrations of nilotinib, imatinib, dasatinib, and ponatinib; in parallel, 44 PBMC samples from 15 patients with steroid-dependent/refractory cGVHD treated with nilotinib in the setting of a phase I/II trial were analyzed at baseline, after 90, and after 180 days of therapy. Flow cytometry was performed after labeling lymphocytes with a panel of monoclonal antibodies (CD3, CD4, CD16, CD56, CD25, CD19, CD45RA, FoxP3, CD127, and 7-amino actinomycin D). Cytokine production was assessed in supernatants of purified CD3 T cells and in plasma samples from nilotinib-treated patients. Main T lymphocyte subpopulations were not significantly affected by therapeutic concentrations of TKIs in vitro, whereas proinflammatory cytokine (in particular, IL-2, IFN-γ, tumor necrosis factor-α, and IL-10) and IL-17 production showed a sharp decline. Frequency of T regulatory, B, and natural killer (NK) cells decreased progressively in presence of therapeutic concentrations of all TKIs tested in vitro, except for nilotinib, which showed little effect on these subsets. Of note, naive T regulatory cell (Treg) subset accumulated after exposure to TKIs. Results obtained in vivo on nilotinib-treated patients were largely comparable, both on lymphocyte subset kinetics and on cytokine production by CD3-positive cells. This study underlines the anti-inflammatory and immunomodulatory effects of TKIs and supports their potential usefulness as treatment for patients with steroid-dependent/refractory cGVHD. In addition, both in vitro and in vivo data point out that compared with other TKIs, nilotinib could better preserve the integrity of some important regulatory subsets, such as Treg and NK cells.

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“…Src family tyrosine kinases are important components of the signaling pathways initiated by the TLRs (critical for cytokine production) and many cytokines, such as TNF, use Src family kinases in their own signaling pathways ( 17 , 66 ). In our model, dasatinib reduced the levels of all measured cytokines confirming data from the literature ( 25 , 67 ), except IL-1β. Impairing TLR4-related signaling pathway inhibits cytokine production including both TNF and IL-10 ( 68 ).…”

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“…Nilotinib is now in experimentation also in GVHD, on the basis of data from the preclinical studies that clearly demonstrated its anti-inflammatory power. Indeed, Nilotinib significantly reduced production of pro-inflammatory cytokines (IL-2, IFN-gamma, TNF alpha, IL-17, TGF beta), without losing the lymphocyte immunocompetence [73,74]. Nevertheless, no definitive data on Nilotinib safety in GVHD are still available; consequently, safety profile must be derived from the experience in CML.…”

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“…3 B, 4 , 5 and Table 1 show for both drugs very similar EMF induced reduction of Igk loci rearrangement levels(if not even slightly more pronounced reduction for GSK). IMA although a more potent RAG inducer than GSK has the disadvantage that post recombination blocks cells in Go phase preventing further their division 36 , 47 – 49 . On the contrary, the AKT inhibitor GSK-690693 not only is a weaker RAG induction stimulus (closer to the one physiologically occurring in small pre-B cells) 50 , 51 but also enables cells to divide prior to and after Igk loci rearrangements and protect their progress to the next stage of development 19 .…”