IMMUNE SENESCENCE AND EXHAUSTION PHENOTYPE MARKED BY LOSS OF NKG2D AND DNAM-1 NK ACTIVATING SIGNALING AND INCREASED TIM-3 T CD8 CHARACTERIZES ADVANCED MYELOFIBROSIS

Myelofibrosis (MF), driven by JAK/STAT signaling mutations in hematopoietic stem cells, has the worst prognosis among myeloproliferative neoplasms (MPNs), progressing from pre-fibrotic MF (PFMF) to overt MF (OMF). Although additional mutations promote clonal expansion, progression cannot be fully explained by clinical- mutational scores. Exposure to the leukemic inflammatory microenvironment may impair immune surveillance by cytotoxic cells such as Natural Killer (NK) and T lymphocytes, fostering disease advancement. To investigate immune dysfunction across clinical and molecular MF subtypes, focusing on NK and T lymphocyte phenotype, maturation, and receptor expression, and to examine the leukemic microenvironment effects using a JAK2V617F murine model. We performed immunophenotyping of NK and T cells (frequency, maturation, and activation/inhibitory receptors) in peripheral blood mononuclear cells from untreated MF patients (13 PFMF, 16 OMF; 13 JAK2 wild-type, 18 JAK2-mutated) admitted at Ribeirao Preto Clinical Hospital (August 2022 – December 2024), and from age-matched healthy donors. To further explore the effects of soluble factors from the leukemic microenvironment, 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. MF patients exhibited increased frequencies of 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 decrease of NKG2A (total, cytotoxic, and hypermature NK cells) and NKp46 (total and hypermature NK cells) receptors. T cell maturation was also impaired, with reduced frequencies of stem central memory (CD45RO-CCR7+CD62L+CD27+CD45RA+) and central memory (TCM: CD45RO+CCR7+CD62L+CD27+CD45RA-) subsets in CD4 cells, and TCM in CD8 cells. Terminally differentiated effector memory (CD45RO-CCR7-CD62L-CD45RA+) cells increased in both lineages. CD8 T cells also showed decreased NKG2D expression. 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. The MPN-exposed group exhibited reduced frequencies of mature NK cells (CD45hiCD19-CD3-CD122+NK1.1+CD49b+NKp46+), NKG2D expression, and T cell frequencies (CD45hiCD19- NK1.1-CD3+), but increased CD8 T cells, with elevated TIM-3 expression.