T-Cell Memory Compartment Downregulation and NK Cell Activation Are Associated with Myelofibrosis Progression
Authors: Medeiros Mariana , Garcia Camila Araújo Bernardino , Binelli Larissa Sarri , Carvalho Allana Guimarães de , Marani Leticia Olops , Scheucher Priscila Santos , Schiavinato Josiane Lillian , Palma Leonardo Carvalho , Garibaldi Pedro Manoel Marques , Welner Robert , Figueiredo-Pontes Lorena Lobo 
Published: December 2024

Myelofibrosis (MF), driven by JAK/STAT signaling mutations in hematopoietic stem cells, has the worst survival rates among myeloproliferative neoplasms (MPNs), worsening from pre-fibrotic MF (PFMF) to overt MF (OMF) that often precedes acute myeloid leukemia. We previously demonstrated that Natural Killer (NK) cells have phenotypical and functional alterations that predominate in MF when compared to other classical MPNs, suggesting that the immune content may contribute to disease burden. We hypothesized that the cytotoxic immune cell profile, along with fibrosis and mutations, drives MF progression. Aiming to characterize the cytotoxic immune cells of MF patients, peripheral blood mononuclear cells (PBMC) from 9 PFMF and 9 OMF, and 9 healthy donors (controls: CT) were analyzed by multiparametric flow cytometry to profile: NK cell frequency and subtypes (CD45hiCD3-CD19-CD56brightCD16-; CD45hiCD3-CD19-CD56dimCD16+; NK maturation stages [CD57+ hypermature, CD11b- immature, CD11b+ mature; CD11b-CD27- tolerant (DN), CD27+CD11b- immature secretory (IS), CD27+CD11b+ mature secretory (MS), CD11b+CD27- cytotoxic]; T lymphocytes (CD45hiCD3+) frequency and subtypes (CD4, CD8) and maturation [CD45RO-CD45RA+CD27+ Naïve (N), CD45RO+CD45RA+CD27+ intermediate (INT), CD45RO+CD45RA+CD27- Central Effector (CE), CD45RO+CD45RA-CD27+ Central Memory (CM), CD45RO-CD45RA+CD27- End Stage (ES)]. Immune evasion was assessed by CD62L, and Treg cells (CD4+CD25hiCD127lowFOxp3+) were also quantified. NK cytotoxic capacity was measured by quantifying dead/K562 target cells, NK activation (CD69) and degranulation (CD107a) after PBMC co-culture.Top of FormBottom of Form Total lymphocyte frequency was reduced in MF vs CT (CT 53.3±19.4%, MF 40.9±24.2%, p=0.23) as well as in OMF vs PFMF (OMF 53.1±22%, PFMF 28.6±20.5%, p=0.02). CD56bright NK cells were increased and CD56dim decreased in MF vs CT (CD56bright: CT 3.4±2.6%, MF 17.7±17.9%, p&lt;0.001; CD56dim: CT 82.7±7.6%, MF 53.4±25%, p&lt;0.001). NK maturation profiles in MF showed increased immature NK cells (CT 4.7±3.7%, MF 15.1±8.9%, p=0.001), with higher DN (CT 4.5±3.5%, MF 13.9±8.7%, p=0.002) and IS (CT 0.2±0.3%, MF 1.2±1.2%, p=0.03), and lower cytotoxic NK cells (CT 92.1±3.8%, MF 77.3±11.2%, p&lt;0.001). Of note, when compared to PFMF, OMF samples presented higher frequency of cytotoxic NK cells (PFMF 72.2±12%, OMF 82.4±7.9%, p=0.09), and a tendency to lower DN and MS frequencies. Similarly, hypermature NK frequency was slightly lower in MF compared to CT (CT 60.5±15.3%, MF 53.5±26.4%, p=0.4) but higher in OMF compared to PFMF (PFMF 45.5±25.7%, OMF 61.5±25.9%, p=0.1). In agreement, reduced NK cytotoxic capacity was observed despite increased degranulation in MF (death: CT 81.8±4.2%, MF 30.1±7.76%, p=0.02; CD107a: CT 11±5.2%, MF 21.1±14.7%, p=0.4) and slightly increased cytotoxicity and degranulation accompanied fibrosis (death: PFMF 28±6.4%, OMF 31.4±9.9%, p=0.7; CD107a: PFMF 10.1±8.8%, OMF 32.17±9.9%, p=0.1). No changes in total T lymphocytes or CD4/CD8 frequencies were seen between CT and MF or PFMF and OMF. However, CD8 effector compartment was reduced in MF (INT and CE subtypes: CT 19.8±9.9%, MF 7.3±12.2%, p&lt;0.001; CT 19.1±16.4%, MF 8.1±13.8%, p=0.002) and decreased CD8 memory compartment (CM) was observed in OMF as compared to PFMF (PFMF 18.9±11.9%, OMF 10.6±9.5%, p=0.02). CD62L+ CM decreased in MF (CT 21.5±12.8%, MF 6.8±6.8%, p=0.006) and in OMF (PFMF 9.4±8%, OMF 3.9±3.7%, p=0.2), with a modest rise in CD62L- CM (PFMF 6.5±8.4%, OMF 13.1±9.9%, p=0.3). Treg frequency decreased in MF (CT 1.9±1.7%, MF 0.25±0.22%, p=0.01) but increased in OMF (PFMF 0.2±0.13%, OMF 0.3±0.2%, p=0.5). More advanced MF has a higher degree of fibrosis, which leads to an inflammatory profile in the disease. This, in turn, may activate NK cells and result in a more mature but cytotoxic-deficient profile probably due to exhaustion. On the other hand, increased Treg cells found in OMF negatively regulate cytotoxic T cells and its memory compartment, specifically leading to immune evasion via a decrease in CD62L+ CM, which is associated with a better anti-tumor response. The results shed light on the mechanisms involved in MF progression, suggesting that changes in the maturation profile of cytotoxic cells is associated with disease progression, thus inspiring new therapies that target antitumoral cells from the leukemic environment.