Advanced myelofibrosis is marked by loss of NKG2D and DNAM-1 NK activating signaling and increased TIM-3 T CD8 exhaustion

Abstract Myelofibrosis (MF), driven by JAK/STAT signaling mutations in hematopoietic stem cells, presents the worst prognosis among myeloproliferative neoplasms (MPNs), progressing from pre-fibrotic MF (PFMF) to overt MF (OMF), and potentially to acute leukemia. Although the acquisition of additional mutations contributes to clonal expansion, disease progression can not be fully explained and predicted by the clinical-mutational scores used in clinical practice. It is plausible that the exposure to the leukemic inflammatory microenvironment of MF may impair immune surveillance mediated by cytotoxic cells such as Natural Killer (NK) and T lymphocytes, thus contributing progression. To investigate immune dysfunction across clinical and molecular MF subtypes, we performed immunophenotyping of NK and T cells (frequency, maturation and activation/inhibitory receptors) in peripheral blood mononuclear cells (PBMCs) from untreated MF patients (n=32: 13 PFMF, 16 OMF, 3 not applicable; 13 JAK2 wild-type, 18 JAK2-mutated, 1 not applicable) admitted at Ribeirao Preto Clinical Hospital between August 2022 and December 2024, and from age-matched healthy donors. Patients with MF exhibited increased frequencies of total NK cells (CD45hiCD3-CD19-CD56+) compared to healthy donors, particularly CD56bright and immature subsets (CD27-CD11b-, CD27+CD11b-), and reduced frequencies of CD56dim, cytotoxic (CD27-CD11b+), and hypermature (CD57+NKp80+) NK cells. There was a marked decrease of the inhibitory receptor NKG2A (in total, cytotoxic, and hypermature NK cells) and of the activating receptor NKp46 (in total and hypermature NK cells). T cell maturation was also impaired, with reduced frequencies of stem central memory (TSCM: CD45RO-CCR7+CD62L+CD27+CD45RA+) and central memory (TCM: CD45RO+CCR7+CD62L+CD27+CD45RA-) subsets in CD4+ T cells, and TCM in CD8+ cells. In addition, terminally differentiated effector memory (TEMRA: CD45RO-CCR7-CD62L-CD45RA+) cells increased in both lineages. CD8+ T cells also showed decreased NKG2D expression. No significant differences were observed in Treg cell frequencies. During disease progression from PFMF to OMF, there was a further increase in hypermature NK cells and downregulation of DNAM-1 and NKG2A. JAK2-mutated patients showed decreased expression of KIR2DL1 (total and hypermature NK), reduced CD4+ T cells, increased CD8+ T cells, and decreased CD8+ TCM cells.To further explore how soluble factors from the leukemic microenvironment affect the immune system, we established a chimeric murine model of JAK2V617F-driven MPN. Healthy recipient mice were transplanted with total bone marrow from JAK2V617F donor mice following sublethal irradiation (n=4 MPN-exposed, 6 control-exposed). Four weeks post-transplant, spleen cells were collected, and NK and T cells were immunophenotyped. The MPN-exposed group exhibited reduced frequencies of mature NK cells (CD45hiCD19-CD3-CD122+NK1.1+CD49b+NKp46+), NKG2D expression, and CD3+ T cell frequencies (CD45hiCD19-NK1.1-CD3+), but increased CD8+ T cells, and elevated TIM-3 expression on CD8+ subsets, mirroring the immune alterations observed in human MF. These findings reveal distinct immune signatures associated with MF progression and JAK2V617F mutation. NK cell dysfunction, characterized by impaired maturation, loss of activating receptors, and accumulation of exhausted phenotypes, may compromise immune surveillance and promote leukemic transformation. Concurrently, T cell alterations suggest chronic inflammation and immunesenescence, particularly in JAK2-mutated cases. The murine model supports a direct role for JAK2V617F-mutant hematopoiesis in driving immune exhaustion. Collectively, our data highlights the contribution of both innate and adaptive immune dysfunction to MF pathogenesis and progression, providing insights into how molecular heterogeneity may influence disease evolution and offering potential targets for future immunomodulatory therapies.