NK Cells and KIR+ / NKG2A+ Innate like CD8+ T Lymphocytes Features in the Bone Marrow of Multiple Myeloma Patients
Authors: Gardeney Helene , Narbeburu Emma , Barbarin Alice , Moya Niels , Bobin Arthur , Levy Anthony , Gruchet Cecile , Leleu Xavier , Herbelin Andre , Gombert Jean-Marc 
Published: December 2024

Background: Multiple myeloma (MM) is characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM), and consequently the dysregulation of the bone marrow (BM) niche, including composition of the resident immune cells. The tumor microenvironment (TME), which includes immune cells, offers both tumor promoting and tumor restrictive properties. A role in cancer immune surveillance has been attributed to the newly described subset of CD8+ T-cells expressing inhibitory NK cell receptors (KIR/NKG2A), called innate CD8 T cells (CD8 ITC). Yet, the interactions between tumor PCs and the BM TME, especially resident and non-resident immune cells, should be further elucidated. We sought to describe the phenotype of NK cells and CD8 ITC in the BM and its evolution with the spread of clonal BM PCs.
Methods: We analyzed NK and CD8 ITC subsets among living cells in BM samples from newly diagnosed MM (NDMM, n=13), relapsed/refractory MM (RRMM, n=8) patients and healthy donors (HD n=10) using spectral flow cytometry. CD8 ITC were separated into four distinct contingents based on NKG2A vs. KIR (KIR2D and/or KIR3DL1/2) expression. NK cells were identified based on CD56 and CD16 expression. CD69 was used as a BM-resident marker. We focused our analysis on activation/exhaustion (CD39, TIM-3, TIGIT and PD-1) and metabolic parameters: glucose uptake (2-NBDG labeling) and mitochondrial mass (MitoTracker-Deep-Red labeling).
Results:NK cells and CD8 ITC with a resident (CD69+) phenotype were present in the BM of HD and MM patients. Fewer CD56 dim NK cells were observed in the BM of NDMM, compared to HD (3% vs. 15%, respectively), while no difference was observed for CD56 bright NK cells. We observed a significative augmentation of the relative proportion of the KIR3DL1/2+ ITC CD8 contingent among both total and resident CD8+ T-cells, while no difference was observed for KIR2D+ and NKG2A+ CD8 ITC contingents.
CD56 dim NK were preferentially KIR+ CD39- both in MM patients and HD. No difference between HD and MM patients was evidenced in exhaustion and metabolism phenotypes of these cells, regardless of their resident status. On the other hand, CD56 bright NK cells were mainly NKG2A+ CD39+ both in MM patients and HD and their metabolic status was modified in NDMM with a decrease level of glucose uptake. Resident CD56 bright NK cells showed a greater co-expression of exhaustion markers PD-1, TIM-3 and TIGIT in MM compared to HD (40% vs. 10%, respectively). Overall, the higher expression of TIGIT on resident CD56 bright NK cells was positively correlated with the percentage of PCs BM infiltration in MM patients, to the contrary of their non-resident (CD69-) counteract.
Among CD8 ITC, TIM-3 expression level was very low in both HD and MM patients, compared to the other exhaustion markers. Considering TIGIT+ PD-1+ cells, no difference was observed between KIR (KIR3DL1/2+ and/or KIR2D+) CD8 ITC in MM and HD patients. A greater proportion of NKG2A+ CD8 ITC were TIGIT+ PD-1+ in NDMM when compared to HD, regardless of their resident status.
Conclusion:These results suggest that NK cells and CD8 ITC are mainly resident, activated and exhausted in the BM niche of MM. Overall, exhaustion of CD56 bright NK cells (NKG2A+) and effector NKG2A+ CD8 ITC seem to be closely associated with PCs infiltration. To the contrary, cytotoxic CD56 dim NK cells and regulatory KIR3DL1/2+ CD8 ITC don't seem to be affected by PCs infiltration, while only their proportion in the BM is altered in MM patients. All in all, these finding suggest a possible anti-tumoral response of NK cells and CD8 ITC in MM patients, but with probable less effective (exhaustion of NKG2A+ CD8 ITC, decreased proportion of CD56dim NK cells) and augmented regulatory (expansion of KIR3DL1/2+ CD8 ITC) functions.