Acentral role of transforming growth factor β (TGF-β) in the pathobiology of myelofibrosis has long been recognized, but surprisingly little is known about the cell types that mediate its activation. In a recent study by Lecomte and colleagues, a role for regulatory T cells (Tregs) and the transmembrane protein GARP in activation of TGF-β is described, with promising in vivo efficacy shown for an anti-GARP:TGF-β monoclonal antibody in a mouse model of myelofibrosis.
Among the many inflammatory and profibrotic cytokines that are deranged in myelofibrosis, TGF-β is the most notorious, with many lines of evidence pointing to a central role in fibrosis and megakaryocytes as a major cellular source.1 However, TGF-β is secreted in a latent, inactive form, due to an association of the TGF-β protein dimer with a latency-associated protein (LAP) that masks it from its cognate receptors until the LAP is cleaved and TGF-β is released.
The transmembrane protein GARP, expressed in the transmembrane of Tregs, binds to LAP, releasing TGF-β and enabling its activity. The authors of the present study had previously generated and tested monoclonal antibodies that block TGF-β activation by GARP-expressing cells, showing that inhibition of TGF-β improves responses to immune checkpoint blockade in murine tumor models.2 In their recent paper in Blood, they explore a similar mechanism in myelofibrosis, with unexpected findings regarding the cellular mediator of TGF-β activation. They used a murine model of myelofibrosis induced by expression of a mutant form of Mpl in hematopoietic cells, which causes a rapidly progressive, myeloproliferative phenotype including leukocytosis, thrombocytosis, splenomegaly, fibrosis, and lethality within two to four weeks.3 GARP protein was significantly increased in the bone marrow and spleen of mice with myelofibrosis and exclusively expressed by megakaryocytes and Tregs.
Administration of an inhibitory monoclonal anti-GARP:TGF-β antibody, with treatments starting prior to the myeloproliferative neoplasm induction, abrogated the disease phenotype. The researchers observed a selective reduction in mutant cells but no impact on wild-type hematopoiesis, suggesting that the treatment allowed healthy, wild-type hematopoiesis to dominate. This effect was preserved using an Fc-neutralized form of the therapeutic antibody, confirming that activity was not due to Fc-mediated cytotoxicity against GARP+ cells or their phagocytosis. The authors used the cytokine-dependent murine cell line Ba/F3, transduced to express either a mutant or wild-type Mpl receptor, to show that TGF-β suppressed the proliferation of Mpl mutant but not wild-type cells, indicating that the constitutive and cytokine-independent JAK-STAT signaling conferred by the Mpl mutation resulted in resistance of the Ba/F3 cells to the inhibitory effects of TGF-β.
By deleting GARP exclusively in Tregs or megakaryocytes using lineage-specific promoters, Dr. Lecomte and colleagues showed that only the loss of GARP on Tregs prevented the myeloproliferative phenotype, with no impact following GARP knockout in megakaryocytes alone. In further support of their hypothesis that the mechanism of activity of the antibody was mediated by increased T-cell anticancer cytotoxicity, RNA-sequencing of spleen cells showed positive enrichment of interferon alfa and interferon gamma signaling in responding mice. Depletion of CD8+ T cells also abolished the treatment effect of the anti-GARP:TGF-β antibody.
This study describes a previously unexplored axis for TGF-β activation in which GARP+ Tregs play a central role in preventing cytotoxic attack of the myeloproliferative neoplasm clone. Potential caveats to this study’s findings exist. While GARP was expressed on T cells and platelets in patients with myelofibrosis, the level of expression was no higher than in controls, in contrast to the increased expression observed in the mouse model. Additional TGF-β activators were not explored. Also, further work will be required to determine the potential toxicities of repressing TGF-β activation and whether the disease phenotype can be ameliorated when treatment is initiated at later stages of the disease. Nonetheless, the paper contributes to the field by highlighting that, although megakaryocytes may be a major cellular source for TGF-β, they are not necessarily responsible for its activation. Understanding the immunoregulatory mechanisms in patients with myelofibrosis is of high importance, particularly in the present era when vaccine and antibody therapies are now in clinical development.
Dr. Psaila indicated no relevant conflicts of interest.